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EGFL7 regulates the collective migration of endothelial cells by restricting their spatial distribution
Abstract
During sprouting angiogenesis, groups of endothelial cells (ECs) migrate together in units called sprouts. In this study, we demonstrate that the vascular-specific secreted factor EGFL7 regulates the proper spatial organization of ECs within each sprout and influences their collective movement. In the homozygous Egfl7-knockout mice, vascular development is delayed in many organs despite normal EC proliferation, and 50% of the knockout embryos die in utero. ECs in the mutant vasculatures form abnormal aggregates and the vascular basement membrane marker collagen IV is mislocalized, suggesting that ECs fail to recognize the proper spatial position of their neighbors. Although the migratory ability of individual ECs in isolation is not affected by the loss of EGFL7, the aberrant spatial organization of ECs in the mutant tissues decreases their collective movement. Using in vitro and in vivo analyses, we showed that EGFL7 is a component of the interstitial extracellular matrix deposited on the basal sides of sprouts, a location suitable for conveying positional information to neighboring ECs. Taken together, we propose that EGFL7 defines the optimal path of EC movement by assuring the correct positioning of each EC in a nascent sprout.
... • Cluster 0: Endothelial cells Markers: Pecam1, Ushbp1, Oit3, F8, Bmp2, Mmrn2, Pcdh12, Dpp4, Tek, S100a1, Scarf1, Stab2, Lyve1, Icam2, Sox18, Egfl7, Flt4, Nr2f2, Tie1 [59,22,26,53,57,62,28,43,54,30,18,17,15]. ...
... • Cluster 0: Endothelial cells Markers: Pecam1, Ushbp1, Oit3, F8, Bmp2, Pcdh12, Dpp4, Tek, S100a1, Scarf1, Stab2, Lyve1, Icam2, Sox18, Egfl7, Flt4, Nr2f2, Tie1 [59,22,26,53,57,62,28,43,54,30,18,17,15]. ...
... • Cluster 1: Endothelial cells Markers: Pecam1, Ushbp1, Oit3, F8, Bmp2, Mmrn2, Pcdh12, Dpp4, Tek, S100a1, Stab2, Lyve1, Icam2, Sox18, Egfl7, Flt4, Nr2f2, Tie1 [59,22,26,53,57,62,28,43,54,30,18,17,15]. ...
Assessing similarity is highly important for bioinformatics algorithms to determine correlations between biological information. A common problem is that similarity can appear by chance, particularly for low expressed entities. This is especially relevant in single cell RNA-seq (scRNA-seq) data because read counts are much lower compared to bulk RNA-seq.
Recently, a Bayesian correlation scheme, that assigns low similarity to genes that have low confidence expression estimates, has been proposed to assess similarity for bulk RNA-seq. Our goal is to extend the properties of the Bayesian correlation in scRNA-seq data by considering 3 ways to compute similarity. First, we compute the similarity of pairs of genes over all cells. Second, we identify specific cell populations and compute the correlation in those populations. Third, we compute the similarity of pairs of genes over all clusters, by considering the total mRNA expression.
We demonstrate that Bayesian correlations are more reproducible than Pearson correlations. Compared to Pearson correlations, Bayesian correlations have a smaller dependence on the number of input cells. We show that the Bayesian correlation algorithm assigns high similarity values to genes with a biological relevance in a specific population.
We conclude that Bayesian correlation is a robust similarity measure in scRNA-seq data.
... Indeed, through a regulation of its expression by CASZ1, Egfl7 controls RhoA expression in endothelial cells leading to blood vessel assembly and morphogenesis [13]. In mammals, the generation of specific Egfl7-deficient mice has been hindered by the presence of miR-126 microRNA in the egfl7 gene, leading to some confusion in the understanding of Egfl7 functions in vivo [14]. The first report describing the inactivation of egfl7 without affecting expression of miR-126 found no vascular defect in the mutant mice [15]. ...
... Several lines of evidence support the idea that EGFL7 is an ECM protein. Indeed, EGFL7 is abundantly found in the ECM and in the cellular extracts of EGFL7-producing cells, whereas it is only faintly detected in their supernatant [14,18]. In vivo, endogenous Egfl7 is also detected around blood vessels, associated with several matrix components such as fibronectin and elastin [8,14,18]. ...
... Indeed, EGFL7 is abundantly found in the ECM and in the cellular extracts of EGFL7-producing cells, whereas it is only faintly detected in their supernatant [14,18]. In vivo, endogenous Egfl7 is also detected around blood vessels, associated with several matrix components such as fibronectin and elastin [8,14,18]. To date, the exact mechanisms which promote the deposition of EGFL7 into the ECM and their consequences on EGFL7 endothelial functions are unknown. ...
The extracellular matrix (ECM) is essential to provide mechanical support to tissues but is also a bioactive edifice which controls cell behavior. Cell signaling generated by ECM components through integrin‐mediated contacts, modulates cell biological activity. In addition, by sequestrating or releasing growth factors the ECM is an active player of physiological and pathological processes such as vascular development. EGFL7 is mainly expressed during blood vessel development and is deposited in the ECM after secretion by endothelial cells. While EGFL7 is known to control various endothelial cell molecular mechanisms (i.e. the repression of endothelial‐derived lysyl oxidase (LOX) enzyme, the regulation of the Notch pathway and the expression of leucocyte adhesion molecules and of RHOA by endothelial cells), it is not established whether EGFL7 functions when bound to the ECM. Here, we show that microfibrillar associated glycoprotein‐1 (MAGP‐1) and fibronectin drive the deposition of EGFL7 into both fibers and individual aggregates in endothelial ECM. Whereas EGFL7 does not need to be docked into the ECM to control endothelial adhesion molecule expression, the ECM accumulation of EGFL7 is required for its regulation of LOX activity and of HEY2 expression along the Notch pathway. The interaction of EGFL7 with MAGP‐1 is necessary for LOX activity repression by EGFL7 while it does not participate in the control of the Notch pathway by this protein. Altogether, this study highlights the roles played by EGFL7 in controlling various endothelial molecular mechanisms upon its localization and shows how the ECM can modulate its functions. This article is protected by copyright. All rights reserved.
... Some studies have proved that it plays an important role in the tube formation process of embryonic vascular development. Recent studies have found that it can significantly enhance the movement and migration ability of endothelial cells and fibroblasts, suggesting that it has the biological function of regulating cell movement and migration [7,8]. However, EGFL7 has not been reported in the invasion and metastasis of colon cancer cells. ...
... EGFL7 is expressed in a variety of tumors, including liver cancer, malignant glioma, breast cancer, lung cancer, and pancreatic cancer. EGFL7 may be involved in the following two aspects of the process of tumor metastasis: (1) EGFL7 protein stimulates the movement and migration ability of tumor cells by autocrine or paracrine and participates in the invasion 8 Journal of Oncology and metastasis of tumor from the primary site to the surrounding areas; (2) EGFL7 protein secreted by tumor endothelial cells facilitates the infiltration of tumor cells into the bloodstream from the primary site, leading to the formation of tumor organ-specific metastases. Neovascularization plays a very important role in tumor growth and metastasis. ...
In this study, we studied the therapeutic potential of PF in mouse models of CAC. PF could inhibit the proliferation, migration, invasion, and clone formation and promote the apoptosis of colon cancer cells. Furthermore, PF showed a good effect on inhibiting the aggregation and infiltration of inflammatory cells, protecting the intestinal mucosal barrier, and inhibiting the growth of colon tumors in the AOM/DSS-induced CAC model. PF also significantly inhibited the expression of TNF-α, IL-1β, IL-6, IL-13, and CEA-related inflammatory factors in the AOM/DSS-induced CAC model by inhibiting the TLR4/NF-κB signaling pathway and EGFL7 expression. Therefore, PF showed a potential therapeutic effect on mice in the CAC model by inhibitingTLR4/NF-κB mediated inflammatory response and EGFL7 expression.
... In contrast to the C2 cluster, the TCF21+ C3 cell cluster was mostly TBX18− and WT1− (Supplementary Fig 3b, Fig. 2b). Interestingly, these C3 cells expressed high levels of extracellular matrix transcripts implicated in cell migration, including fibronectin (FN1), periostin (POSTN), epidermal growth factor-like domain multiple 7 (EGFL7) 17,18 , and agrin (AGRN) [19][20][21][22] ( Supplementary Fig. 3b, Supplementary Fig. 3g-h and Fig. 2b). This cluster also had upregulated expression of the well-known EMT regulator snail-1 (SNAI1) 23 and was TEK− which is a TIE2 ortholog and an endocardial-derived TCF21+ fibroblast marker 24 (Supplementary Fig. 3b, Fig. 1d). ...
... Previous studies have also reported upregulation of POSTN and FN1 in EPDCs undergoing EMT in mice 46,47 . EGFL7 is a secreted growth factor known to be expressed by endothelial cells and critical to angiogenesis 18 but has not been implicated in epicardial development. Interestingly, EGFL7 transcripts have been shown to be present in a subset of murine neonatal fibroblasts that sharply declines during the murine neonatal heart regenerative window 49 . ...
Single-cell RNA sequencing is a powerful tool to study developmental biology but does not preserve spatial information about tissue morphology and cellular interactions. Here, we combine single-cell and spatial transcriptomics with algorithms for data integration to study the development of the chicken heart from the early to late four-chambered heart stage. We create a census of the diverse cellular lineages in developing hearts, their spatial organization, and their interactions during development. Spatial mapping of differentiation transitions in cardiac lineages defines transcriptional differences between epithelial and mesenchymal cells within the epicardial lineage. Using spatially resolved expression analysis, we identify anatomically restricted expression programs, including expression of genes implicated in congenital heart disease. Last, we discover a persistent enrichment of the small, secreted peptide, thymosin beta-4, throughout coronary vascular development. Overall, our study identifies an intricate interplay between cellular differentiation and morphogenesis.
... Studies in zebrafish have also uncovered a functional role for the epidermal growth factor like domain 7 (Egfl7), a conserved vascular-endothelial specific secreted growth factor in vertebrates (Parker et al., 2004). Egfl7 is required for the processes of tubulogenesis, lumenization, EC migration and polarity (Parker et al., 2004;Schmidt et al., 2007;De Mazière et al., 2008). Recently, a functional role for Egfl7 in neurovascular stabilization has been shown via the use of TAL effector nucleases (TALENs) designed against egfl7. ...
... Injection of TALENs RNA into fertilized zebrafish embryos induces ICH in some of the embryos by 2 dpf (Rossi et al., 2015). The effects of Egfl7 knockdown on neurovascular stability are likely due to aberrantly distributed EC adherens and tight junctions, as evidenced by previous findings (De Mazière et al., 2007). ...
Intracerebral hemorrhage (ICH) is the most severe subtype of stroke. Treatment options are scarce and given the high morbidity and mortality, relatively ineffective. Since patients with ICH may have an unknown heritable component, the need to identify potential risk factors necessitates the use of animal models to elucidate the genetic underpinnings of neurovascular development and, thereby, identify candidate regulatory pathways that are likely to be disrupted in patients with ICH. Zebrafish (Danio rerio) exhibits the anatomical and physiological complexity of a closed circulatory system observed in all vertebrates (with arteries, veins and capillaries). Moreover, studies over the last decade, aided by the application of chemical mutagenesis screens, morpholino mediated knockdown approaches and tissue-specific transgenic markers, have paved the way for the identification of several genes and signaling pathways that regulate developmental neurovascular stabilization. We hypothesize that mutations in these genes or pharmacological perturbations of these gene-products may account, at least in part, for the etiology of some forms of spontaneous ICH in humans.
... (legend continued on next page) interstitial ECM. Moreover, fibronectin (FN) facilitates EGFL7 deposition (Schmidt et al., 2007). We investigated the FN expression in conditions of EGFL7 deficiency and BMP9 induction. ...
... In addition, secreted EGFL7 is involved in overall ECM composition and remodeling by direct binding of other ECM proteins in ECs (Schmidt et al., 2007). Here, we demonstrate a dramatic decrease in the expression of FN in EGFL7 À/À HUVECs and an induction of FN expression upon BMP9 treatment. ...
Human embryonic stem cells (hESCs) are instrumental in characterizing the molecular mechanisms of human vascular development and disease. Bone morphogenetic proteins (BMPs) play a pivotal role in cardiovascular development in mice, but their importance for vascular cells derived from hESCs has not yet been fully explored. Here, we demonstrate that BMP9 promotes, via its receptor ALK1 and SMAD1/5 activation, sprouting angiogenesis of hESC-derived endothelial cells. We show that the secreted angiogenic factor epidermal growth factor-like domain 7 (EGFL7) is a downstream target of BMP9-SMAD1/5-mediated signaling, and that EGFL7 promotes expansion of endothelium via interference with NOTCH signaling, activation of ERK, and remodeling of the extracellular matrix. CRISPR/Cas9-mediated deletion of EGFL7 highlights the critical role of EGFL7 in BMP9-induced endothelial sprouting and the promotion of angiogenesis. Our study illustrates the complex role of the BMP family in orchestrating hESC vascular development and endothelial sprouting. : In this article, Valdimarsdottir and colleagues substantiate the importance of BMP9/ALK1 in vascular commitment of hESCs and endothelial sprouting via the secreted extracellular matrix protein EGFL7. They demonstrate the connection of EGFL7 with the NOTCH and ERK pathways and reveal its role in remodeling of the extracellular matrix using CRISPR/Cas9-mediated deletion of EGFL7. Keywords: BMP signaling, ALK1, EGFL7, human embryonic stem cells, endothelial development, angiogenic sprouting
... Given the involvement of EGFL7/miR-126 locus in regulating angiogenesis, we focused on lncEG-FL7OS, which partially overlaps with EGFL7/miR-126 gene but is transcribed in opposite direction ( Figure 2A) (Fish et al., 2008;Wang et al., 2008a;Kuhnert et al., 2008;Durrans and Stuhlmann, 2010;Parker et al., 2004;Schmidt et al., 2007). The existence of lncEGFL7OS was confirmed by RT-PCR cloning using human placental RACE-ready cDNAs and subsequent sequencing, and the size of lncEGFL7OS is consistent with deposited gene AF161442 (Figure 2-figure supplement 1A). ...
... Loss-of-function studies in mice and zebrafish revealed an important function of miR-126 in governing vascular integrity and angiogenesis (Fish et al., 2008;Wang et al., 2008a). Egfl7 -/mice display similar vascular abnormalities to MiR126 -/mice, including edema, defective cranial vessel and retinal vascularization (Schmidt et al., 2007). However, an independent study suggests that the vascular phenotype of Egfl7 -/mice could be attributed to the MiR126 deletion (or downregulation) in the mice (Kuhnert et al., 2008). ...
In an effort to identify human endothelial cell (EC)-enriched lncRNAs,~500 lncRNAs were shown to be highly restricted in primary human ECs. Among them, lncEGFL7OS, located in the opposite strand of the EGFL7/miR-126 gene, is regulated by ETS factors through a bidirectional promoter in ECs. It is enriched in highly vascularized human tissues, and upregulated in the hearts of dilated cardiomyopathy patients. LncEGFL7OS silencing impairs angiogenesis as shown by EC/fibroblast co-culture, in vitro/in vivo and ex vivo human choroid sprouting angiogenesis assays, while lncEGFL7OS overexpression has the opposite function. Mechanistically, lncEGFL7OS is required for MAPK and AKT pathway activation by regulating EGFL7/miR-126 expression. MAX protein was identified as a lncEGFL7OS-interacting protein that functions to regulate histone acetylation in the EGFL7/miR-126 promoter/enhancer. CRISPR-mediated targeting of EGLF7/miR-126/lncEGFL7OS locus inhibits angiogenesis, inciting therapeutic potential of targeting this locus. Our study establishes lncEGFL7OS as a human/primate-specific EC-restricted lncRNA critical for human angiogenesis.
... EGFL7 gene codifies a secreted angiogenic protein, mainly expressed by endothelial cells in vasculogenesis during embryonic period [54,55]. The relation between EGFL7 and the PI3K/Akt pathway has been observed in endothelial regeneration subsequent to vascular damage, with enhanced cell proliferation and invasion [56][57][58] and contributing to evidence that such interaction is also associated with physiological and pathological angiogenesis [54,59,60]. ...
Ischemic stroke (IS) is one of the most impairing complications of sickle cell anemia (SCA), responsible for 20% of mortality in patients. Rheological alterations, adhesive properties of sickle reticulocytes, leukocyte adhesion, inflammation and endothelial dysfunction are related to the vasculopathy observed prior to ischemic events. The role of the vascular endothelium in this complex cascade of mechanisms is emphasized, as well as in the process of ischemia-induced repair and neovascularization. The aim of the present study was to perform a comparative transcriptomic analysis of endothelial colony-forming cells (ECFCs) from SCA patients with and without IS. Next, to gain further insights of the biological relevance of differentially expressed genes (DEGs), functional enrichment analysis, protein–protein interaction network (PPI) construction and in silico prediction of regulatory factors were performed. Among the 2469 DEGs, genes related to cell proliferation (AKT1, E2F1, CDCA5, EGFL7), migration (AKT1, HRAS), angiogenesis (AKT1, EGFL7) and defense response pathways (HRAS, IRF3, TGFB1), important endothelial cell molecular mechanisms in post ischemia repair were identified. Despite the severity of IS in SCA, widely accepted molecular targets are still lacking, especially related to stroke outcome. The comparative analysis of the gene expression profile of ECFCs from IS patients versus controls seems to indicate that there is a persistent angiogenic process even after a long time this complication has occurred. Thus, this is an original study which may lead to new insights into the molecular basis of SCA stroke and contribute to a better understanding of the role of endothelial cells in stroke recovery.
... Nonetheless, when the specification process does go awry in the absence of Mir-126 activity, the phenotypic consequences are profound with most mice dying from severe anemia (Fish et al., 2008;Kuhnert et al., 2008;Wang et al., 2008). Although initial knock-out studies incorrectly attributed this vascular defect to the host protein-encoding gene Egfl7 (Schmidt et al., 2007), careful dissection of this two-gene locus whereby the knock-out of Egfl7 without the perturbation of Mir-126 results in a phenotypically normal mouse (Fish et al., 2008;Kuhnert et al., 2008;Wang et al., 2008). Further, the introduction of a frame-shift mutation that F I G U R E 6 Cellular evolution of endothelial cells and thrombocytes. ...
The evolution of specialized cell-types is a long-standing interest of biologists, but given the deep time-scales very difficult to reconstruct or observe. microRNAs have been linked to the evolution of cellular complexity and may inform on specialization. The endothelium is a vertebrate-specific specialization of the circulatory system that enabled a critical new level of vasoregulation. The evolutionary origin of these endothelial cells is unclear. We hypothesized that Mir-126, an endothelial cell-specific microRNA may be informative. We here reconstruct the evolutionary history of Mir-126. Mir-126 likely appeared in the last common ancestor of vertebrates and tunicates, which was a species without an endothelium, within an intron of the evolutionary much older EGF Like Domain Multiple (Egfl) locus. Mir-126 has a complex evolutionary history due to duplications and losses of both the host gene and the microRNA. Taking advantage of the strong evolutionary conservation of the microRNA among Olfactores, and using RNA in situ hybridization, we localized Mir-126 in the tunicate Ciona robusta. We found exclusive expression of the mature Mir-126 in granular amebocytes, supporting a long-proposed scenario that endothelial cells arose from hemoblasts, a type of proto-endothelial amoebocyte found throughout invertebrates. This observed change of expression of Mir-126 from proto-endothelial amoebocytes in the tunicate to endothelial cells in vertebrates is the first direct observation of the evolution of a cell-type in relation to microRNA expression indicating that microRNAs can be a prerequisite of cell-type evolution.
... Constitutive EGFL7-/-mice were generated and kindly provided by Weilan Ye, (Genentech, San Francisco, USA) and applied as previously described [53]. KO was achieved by insertion of a retroviral gene trap vector upstream of intron 2 of the Egfl7 gene. ...
Neural stem cells reside in the subgranular zone, a specialized neurogenic niche of the hippocampus. Throughout adulthood, these cells give rise to neurons in the dentate gyrus, playing an important role in learning and memory. Given that these core cognitive processes are disrupted in numerous disease states, understanding the underlying mechanisms of neural stem cell proliferation in the subgranular zone is of direct practical interest. Here, we report that mature neurons, neural stem cells and neural precursor cells each secrete the neurovascular protein epidermal growth factor-like protein 7 (EGFL7) to shape this hippocampal niche. We further demonstrate that EGFL7 knock-out in a Nestin-CreERT2-based mouse model produces a pronounced upregulation of neurogenesis within the subgranular zone. RNA sequencing identified that the increased expression of the cytokine VEGF-D correlates significantly with the ablation of EGFL7. We substantiate this finding with intraventricular infusion of VEGF-D upregulating neurogenesis in vivo and further show that VEGF-D knock-out produces a downregulation of neurogenesis. Finally, behavioral studies in EGFL7 knock-out mice demonstrate greater maintenance of spatial memory and improved memory consolidation in the hippocampus by modulation of pattern separation. Taken together, our findings demonstrate that both EGFL7 and VEGF-D affect neurogenesis in the adult hippocampus, with the ablation of EGFL7 upregulating neurogenesis, increasing spatial learning and memory, and correlating with increased VEGF-D expression.
... The copyright holder for this preprint this version posted November 24, 2022. ; https://doi.org/10.1101/2022.11.23.517662 doi: bioRxiv preprint encoding gene Egfl7 (Schmidt et al., 2007), careful dissection of this two-gene locus whereby the knock-out of Egfl7 without the perturbation of Mir-126 results in a phenotypically normal mouse (Fish et al., 2008;Kuhnert et al., 2008;Wang et al., 2008). Further, the introduction of a frame-shift mutation that results in the non-sense mediated decay of the egfl7 transcript in the zebrafish again results in a phenotypically normal fish (Rossi et al. 2015). ...
The evolution of specialized cell-types is a long-standing interest of biologists but given the deep time-scales very difficult to reconstruct or observe. microRNAs have been linked to the evolution of cellular complexity and may inform on specialization. The endothelium is a vertebrate specific specialization of the circulatory system that enabled a critical new level of vasoregulation. The evolutionary origin of these endothelial cells is unclear. We hypothesized that Mir-126, an endothelial cell-specific microRNA may be informative.
We here reconstruct the evolutionary history of Mir-126. Mir-126 likely appeared in the last common ancestor of vertebrates and tunicates, a species without an endothelium, within an intron of the evolutionary much older EGF Like Domain Multiple (Egfl) locus. Mir-126 has a complex evolutionary history due to duplications and losses of both the host gene and the microRNA. Taking advantage of the strong evolutionary conservation of the microRNA among Olfactores, and using RNA in situ hybridization (RISH), we localized Mir-126 in the tunicate Ciona robusta. We found exclusive expression of the mature Mir-126 in granular amebocytes, supporting a long-proposed scenario that endothelial cells arose from hemoblasts, a type of proto-endothelial amoebocyte found throughout invertebrates.
This observed change of expression of Mir-126 from proto-endothelial amoebocytes in the tunicate to endothelial cells in vertebrates is the first direct observation of the evolution of a cell-type in relation to microRNA expression indicating that microRNAs can be a prerequisite of cell-type evolution.
... As reported, the high expression of e-cadherin (E-cad) in villous tissue affects the invasion of trophoblast, making it difficult for placenta implantation, resulting in spontaneous abortion (Li et al., 2017). MiR126, located in the region of epidermal growth factor-like domain 7 (EGFL7), negatively regulates vascular endothelial growth factor (VEGF), which reduces shallow implantation of trophoblasts, and finally leads to spontaneous abortion (Schmidt et al., 2007). ...
A growing number of studies have demonstrated that N6 methyladenine (m⁶A) acts as an important role in the pathogenesis of reproductive diseases. Therefore, it is essential to profile the genome-wide m⁶A modifications such as in spontaneous abortion. In this study, due to the trace of human villi during early pregnancy, we performed high-throughput sequencing in villous tissues from spontaneous abortion (SA group) and controls with induced abortion (normal group) in the first trimester. Based on meRIP-seq data, 18,568 m⁶A peaks were identified. These m⁶A peaks were mainly located in the coding region near the stop codon and were mainly characterized by AUGGAC and UGGACG motif. Compared with normal group, the SA group had 2,159 significantly upregulated m⁶A peaks and 281 downregulated m⁶A peaks. Biological function analyses revealed that differential m⁶A-modified genes were mainly involved in the Hippo and Wnt signaling pathways. Based on the conjoint analysis of meRIP-seq and RNA-seq data, we identified thirty-five genes with differentially methylated m⁶A peaks and synchronously differential expression. And these genes were mainly involved in the Wnt signaling pathway, phosphatase activity regulation, protein phosphatase inhibitor activity, and transcription inhibitor activity. This study is the first to profile the transcriptome-wide m⁶A methylome in spontaneous abortion during early pregnancy, which provide novel insights into the pathogenesis and treatment of spontaneous abortion in the first trimester.
... Zhen et al.(Zhen et al., 2016) established a rat model of MI and found that the treatment of Huoxue Yiqi decoction (1 g/g −1 /d −1 ) in rats by gavage decreased the expression of miR-126, inhibited the regulation of VEGF by miR-126, and increased the expression of VEGF, thereby promoting angiogenesis. Other studies have shown that miR-126 can also regulate endothelial cell responses to VEGF (Schmidt et al., 2007). ...
Atherosclerotic cardiovascular diseases (ASCVDs) are the most important diseases that endanger people’s health, leading to high morbidity and mortality worldwide. In addition, various thrombotic events secondary to cardiovascular and cerebrovascular diseases need must be considered seriously. Therefore, the development of novel anti-platelet drugs with high efficiency, and fewer adverse effects has become a research focus for preventing of cardiovascular diseases (CVDs). Blood-activation and stasis-removal from circulation have been widely considered as principles for treating syndromes related to CVDs. Blood-activating Chinese (BAC botanical drugs, as members of traditional Chinese medicine (TCM), have shown to improve hemodynamics and hemorheology, and inhibit thrombosis and atherosclerosis. Modern medical research has identified that a combination of BAC botanical drugs and anti-platelet drugs, such as aspirin or clopidogrel, not only enhances the anti-platelet effects, but also reduces the risk of bleeding and protects the vascular endothelium. The anti-platelet mechanism of Blood-activating Chinese (BAC) botanical drugs and their compounds is not clear; therefore, their potential targets need to be explored. With the continuous development of bioinformatics and “omics” technology, some unconventional applications of BAC botanical drugs have been discovered. In this review, we will focus on the related targets and signaling pathways of anti-atherosclerotic treatments involving a combination of BAC botanical drugs and anti-platelet drugs reported in recent years.
... Egfl7 was originally identified as an endothelial restricted gene, showing high expression levels in proliferating endothelial cells during embryogenesis and physiologic and pathologic angiogenesis 7,8,[12][13][14] . A role for EGFL7 has been demonstrated in vascular development, where it promotes endothelial cell proliferation, adhesion, migration, sprouting, and invasion [12][13][14][15][16] . More recently, other sources of EGFL7 have been identified, such as primordial germ cells, adult neural stem cells, cancer cells of various human tumors and trophoblast cells [17][18][19][20][21] . ...
Isolated intrauterine growth restriction (IUGR) and preeclampsia (PE) share common placental pathogenesis. Differently from IUGR, PE is a systemic disorder which may also affect liver and brain. Early diagnosis of these conditions may optimize maternal and fetal management. Aim of this study was to assess whether Epidermal Growth Factor-Like domain 7 (EGFL7) dosage in maternal blood discriminates between isolated IUGR and PE. A total of 116 women were enrolled in this case–control study: 12 non-pregnant women, 34 healthy pregnant women, 34 women presenting with isolated IUGR and 36 presenting with PE. Levels of circulating EGFL7 and other known pro- and anti-angiogenic factors were measured by ELISA at different gestational ages (GA). Between 22–25 weeks of gestation, EGFL7 levels in early-onset PE (e-PE) plasma samples were significantly higher than those measured in controls or isolated IUGR samples (69.86 ± 6.17 vs. 19.8 ± 2.5 or 18.8 ± 2.8 µg/ml, respectively). Between 26–34 weeks, EGFL7 levels remained significantly higher in e-PE compared to IUGR. At term, circulating and placental EGFL7 levels were comparable between IUGR and late-onset PE (l-PE). In contrast, circulating levels of PlGF were decreased in both IUGR- and PE- complicated pregnancies, while levels of both sFLT-1 and sENDOGLIN were increased in both conditions. In conclusion, EGFL7 significantly discriminates between isolated IUGR and PE.
... Epidermal growth factor-like domain 7 (Egfl7) is a recently identified secreted protein which binds the extracellular matrix surrounding the blood vessels and it appears to act in an autocrine manner to promote angiogenesis [26][27][28]. Egfl7 is expressed at high levels in early embryos of mice, but only high expressed in a few organs in adult mice such as the lung, heart and kidney [29]. However, Egfl7 is strongly upregulated in tumors [30]. ...
Abstract Background Epidermal growth factor-like domain 7 (Egfl7), a recently identified secreted protein, was significantly increased in patients with HCC by our previous studies. However, its efficacy in the diagnosis of early HCC remains unknown. In this study, we therefore evaluate the efficacy of serum Egfl7 for early HCC diagnosis and compare it with alpha-fetoprotein (AFP). Methods Serum Egfl7 levels in testing cohort (1081 participants) and validation cohort (476 participants) were measured by a sandwich enzyme-linked immunoassay (ELISA). The cut-off value of Egfl7 was determined by Youden’s index and the efficacies of Egfl7 and AFP in diagnosing early HCC were estimated by receiver operating characteristic (ROC). Results Serum Egfl7 was significantly elevated in patients with early HCC than all non-HCC controls in whatever Testing Cohort or Validation Cohort. In the Testing Cohort, ROC curves showed the optimum cut-off value of Egfl7 was 2610 ng/mL and Egfl7 showed a significantly higher sensitivity than AFP in discriminating early HCC from healthy individuals (77.4% vs. 65.3%, P = 0.0013) but the area under ROC (AUROC) and accuracy of Egfl7 and AFP were similar (0.860 vs. 0.868, P = 0.704; 80.2% vs. 83.8%, P = 0.184). In distinguishing patients with early HCC from patients with chronic liver disease (CLD), the AUROC, sensitivity, specificity and accuracy of Egfl7 were 0.800, 75.2, 71.7 and 73.5%, which were all significantly higher than AFP (0.675, 61.8, 62.0 and 61.9% in order). Egfl7 also showed a significant higher sensitivity and accuracy than AFP (76.6% vs. 64.0%, P = 0.0031; 79.9% vs. 66.1%, P
... Unlike most secreted angiogenic signaling molecules such as vascular endothelial growth factor and fibroblast growth factor-2, which are mainly expressed by non-endothelial cell types, EGFL7 is almost exclusively expressed and secreted by endothelial cells, it binds to components of the extracellular matrix and acts as a chemoattractant for endothelial cells (Nichol and Stuhlmann, 2012). EGFL7 also regulates the collective migration of endothelial cells and controls their spatial distribution (Schmidt et al., 2007). EGFL7 expression is highest when the endothelium is in an active and proliferating state (Nichol and Stuhlmann, 2012). ...
The advances in single-cell RNA sequencing technologies and the development of bioinformatics pipelines enable us to more accurately define the heterogeneity of cell types in a selected tissue. In this report, we re-analyzed recently published single-cell RNA sequencing data sets and provide a rationale to redefine the heterogeneity of cells in both intact and injured mouse peripheral nerves. Our analysis showed that, in both intact and injured peripheral nerves, cells could be functionally classified into four categories: Schwann cells, nerve fibroblasts, immune cells, and cells associated with blood vessels. Nerve fibroblasts could be sub-clustered into epineurial, perineurial, and endoneurial fibroblasts. Identified immune cell clusters include macrophages, mast cells, natural killer cells, T and B lymphocytes as well as an unreported cluster of neutrophils. Cells associated with blood vessels include endothelial cells, vascular smooth muscle cells, and pericytes. We show that endothelial cells in the intact mouse sciatic nerve have three sub-types: epineurial, endoneurial, and lymphatic endothelial cells. Analysis of cell type-specific gene changes revealed that Schwann cells and endoneurial fibroblasts are the two most important cell types promoting peripheral nerve regeneration. Analysis of communication between these cells identified potential signals for early blood vessel regeneration, neutrophil recruitment of macrophages, and macrophages activating Schwann cells. Through this analysis, we also report appropriate marker genes for future single cell transcriptome data analysis to identify cell types in intact and injured peripheral nerves. The findings from our analysis could facilitate a better understanding of cell biology of peripheral nerves in homeostasis, regeneration, and disease.
... The fifth group is involved in the internalization (of ligands from the extracellular matrix to be recycled back to the endosomal compartment), endocytosis, migratory and/or invasive capacity, and motility. It comprises urokinase plasminogen activator (uPA), urokinase plasminogen activator receptor (uPAR), urokinase plasminogen activator receptorassociated protein (uPARAP), tissue-type plasminogen activator (tPA), Neuropilin-1 NRP1, Neuropilin-2 NRP2, VEGF R1, 2 and 3, PECAM-1, ICAM-1, VE-cadherin, Ephrin-B2, EphB4, and EGFL7 [46][47][48][49][50][51][52][53][54][55][56][57]. ...
Background
Endothelial progenitor cells (EPCs) are promising candidates for the cellular therapy of peripheral arterial and cardiovascular diseases. However, hitherto there is no specific marker(s) defining precisely EPCs. Herein, we are proposing a new in silico approach for finding novel EPC markers.
Methods
We assembled five groups of chosen EPC-related genes/factors using PubMed literature and Gene Ontology databases. This shortened database of EPC factors was fed into publically published transcriptome matrix to compare their expression between endothelial colony-forming cells (ECFCs), HUVECs, and two adult endothelial cell types (ECs) from the skin and adipose tissue. Further, the database was used for functional enrichment on Mouse Phenotype database and protein-protein interaction network analyses. Moreover, we built a digital matrix of healthy donors’ PBMCs (33 thousand single-cell transcriptomes) and analyzed the expression of these EPC factors.
Results
Transcriptome analyses showed that BMP2, 4, and ephrinB2 were exclusively highly expressed in EPCs; the expression of neuropilin-1 and VEGF-C were significantly higher in EPCs and HUVECs compared with other ECs; Notch 1 was highly expressed in EPCs and skin-ECs; MIR21 was highly expressed in skin-ECs; PECAM-1 was significantly higher in EPCs and adipose ECs. Moreover, functional enrichment of EPC-related genes on Mouse Phenotype and STRING protein database has revealed significant relations between chosen EPC factors and endothelial and vascular functions, development, and morphogenesis, where ephrinB2, BMP2, and BMP4 were highly expressed in EPCs and were connected to abnormal vascular functions. Single-cell RNA-sequencing analyses have revealed that among the EPC-regulated markers in transcriptome analyses, (i) ICAM1 and Endoglin were weekly expressed in the monocyte compartment of the peripheral blood; (ii) CD163 and CD36 were highly expressed in the CD14+ monocyte compartment whereas CSF1R was highly expressed in the CD16+ monocyte compartment, (iii) L-selectin and IL6R were globally expressed in the lymphoid/myeloid compartments, and (iv) interestingly, PLAUR/UPAR and NOTCH2 were highly expressed in both CD14+ and CD16+ monocytic compartments.
Conclusions
The current study has identified novel EPC markers that could be used for better characterization of EPC subpopulation in adult peripheral blood and subsequent usage of EPCs for various cell therapy and regenerative medicine applications.
... The collective migration of cells is a form of harmonized behavior required for critical life activities such as development [1,2] and regeneration [3] as well as for cancer metastasis [4]. Tissues and organs consist of physically connected cells that move by exerting mechanical forces in response to their extracellular environment, which includes mechanical stimuli [3,5,6]. One type of mechanical stimulus is fluid flow that modulates the functions of cells and organs in the body. ...
The movement of collective cells is affected through changes in physical interactions of cells in response to external mechanical stimuli, including fluid flow. Most tissues are affected by fluid flow at the interstitial level, but few studies have investigated the physical effects in collective cells affected by a low flow rate. In this study, collective cell migration of Madin–Darby canine kidney (MDCK) epithelial cells was investigated under static or interstitial flow (0, 0.1, and 1 μL/min) using a traction microfluidic device. The optimization of calculation of cellular traction forces was first achieved by changing interrogation window size from the fluorescent bead images. Migration analysis of cell collectives patterned with a 700 μm circular shape reveals that cells under the slow flow (0.1 and 1 μL/min) showed the inhibitory migration by decreasing cell island size and cellular speed compared to that of static condition. Analysis of cellular forces shows that level of traction forces was lower in the slow flow condition (~20 Pa) compared to that of static condition (~50 Pa). Interestingly, the standard deviation of traction force of cells was dramatically decreased as the flow rate increased from 0 to 1 μL/min, which indicates that flow affects the distribution of cellular traction forces among cell collectives. Cellular tension was increased by 50% in the cells under the fluid flow rate of 1 μL/min. Treatment of calcium blocker increased the migratory speed of cells under the flow condition, whereas there is little change of cellular forces. In conclusion, it has been shown that the interstitial flow inhibited the collective movement of epithelial cells by decreasing and re-distributing cellular forces. These findings provide insights into the study of the effect of interstitial flow on cellular behavior, such as development, regeneration, and morphogenesis.
... It is secreted by endothelial cells and plays a regulatory role in the recruitment of smooth muscle cells, migration and sprouting of endothelial cells during formation lumen of the new vessel. EGF-L7 is present in the environment of EC surrounding sprouting vessel and affects optimal assurance microenvironment for migrating ECs (Schmidt, 2007). Chicken ovalbumin upstream promotertrascription factor II (Coup TFII) gene is essential during physiological vasculogenesis and lymphangiogenesis. ...
According to the World Health Organization, up to 30% of cancer diseases may be related to nutritional factors. Although many dietary ingredients with protective (anti-cancer) effects are known, the health-promoting impact of spices remains underestimated. Rich in the bioactive compounds, they are essential ingredients in the diet of people with cancer disease around the world. Spices have antioxidant, anti-cancer, anti-inflammatory, antifungal, antibacterial action. They inhibit the bioactivation of carcinogens in the body. Attention should be paid to natural antioxidants –cloves, oregano, thyme, cinnamon, marjoram, cumin, and basil. Their chemopreventive properties have been extensively investigated and well documented. Curcumin, rosemary, and bay leaf show anti-inflammatory effects. Ginger, infusions of mint, chamomile, sage, and lemon balm help with ailments associated with cancer therapy. Herbs increase appetite and dishes' attractiveness in patients with a lack of appetite or impaired sense of taste caused by cancer therapy. Researchers suggest that phenolic acids in spices and herbs can increase the antioxidant capacity, affecting the bioactivity of the food consumed. The purpose of the article was to show the role of spices in the diet of cancer patients generally. At first, the role of oxidative stress and chronic inflammation in cancer formation was described. Then, recommendations for dietotherapy and the role of dietary counselling were presented, focusing on selected health problems arising as a result of cancer treatment. In the following, the chemopreventive properties of bioactive ingredients of selected spices were shown. Their impact on patients' health has been described through multidirectional mechanisms of influence, paying attention to practical (culinary) use.
... The fth group is involved in the internalization (of ligands from the extracellular matrix to be recycled back to the endosomal compartment), endocytosis, migratory and/or invasive capacity and motility. It comprises urokinase plasminogen activator (uPA), urokinase plasminogen activator receptor(uPAR), urokinase plasminogen activator receptor associated protein(uPARAP), tissue-type plasminogen activator (tPA), Neuropilin-1 NRP1, Neuropilin-2 NRP2, VEGFR1, 2 & 3, PECAM-1, ICAM-1, VE-cadherin , Ephrin-B2, EphB4 and EGFL7 [46][47][48][49][50][51][52][53][54][55][56][57]. ...
Background: Endothelial progenitor cells (EPCs) are promising candidates for the cellular therapy of peripheral arterial & cardiovascular diseases. However, hitherto there is no specific marker(s) defining precisely EPCs. Herein, we are proposing a new in-silico approach for finding novel EPCs markers.
Methods: we assembled five groups of chosen EPCs-related genes/factors using Pubmed literature & gene ontology databases. This shortened database of EPCs factors was fed into publically-published transcriptome matrix to compare their expression between endothelial colony-forming cells (ECFCs), HUVECs and two adult endothelial cell types (ECs) from skin and adipose tissue. Further, the database was used for functional enrichment on mouse phenotype database and protein-protein interaction network analyses. Moreover, we built a digital matrix of healthy donors' PBMCs (33 thousand single cell transcriptomes) and analyzed the expression of these EPCs factors
Results: Transcriptome analyses showed that BMP2,4 & ephrinB2 were exclusively highly expressed in EPCs; the expression of neuropilin-1 & VEGF-C were significantly higher in EPCs & HUVECs compared with other ECs; Notch 1 was highly expressed in EPCs & skin-ECs; MIR21 was highly expressed in skin-ECs; PECAM-1 were significantly higher in EPCs & adipose ECs. Moreover, functional enrichment of EPCs-related genes on mouse phenotype and STRING protein database has revealed significant relations between chosen EPCs factors and endothelial & vascular functions, development and morphogenesis, where ephrinB2, BMP2 and BMP4 were highly expressed in EPCs and were connected to abnormal vascular functions. Single cell RNA-sequencing analyses has revealed that among the EPCs regulated markers in transcriptome analyses: i-ICAM1 & Endoglin were weekly expressed in the monocyte compartment of peripheral blood; ii-CD163 & CD36 were highly expressed in CD14+ monocyte compartment whereas CSF1R was highly expressed in CD16+ monocyte compartment; iii-L-selectin & IL6R were globally expressed in the lymphoid/myeloid compartments; iv-interestingly, PLAUR/UPAR & NOTCH2 were highly expressed in both CD14+ & CD16+ monocytic compartments.
Conclusions: The current study has identified novel EPCs markers that could be used for better characterization of EPCs sub-population in adult peripheral blood and subsequent usage of EPCs for various cell therapy and regenerative medicine applications.
... It is secreted by endothelial cells and plays a regulatory role in the recruitment of smooth muscle cells, migration and sprouting of endothelial cells during formation lumen of the new vessel. EGF-L7 is present in the environment of EC surrounding sprouting vessel and affects optimal assurance microenvironment for migrating ECs (Schmidt, 2007). Chicken ovalbumin upstream promotertrascription factor II (Coup TFII) gene is essential during physiological vasculogenesis and lymphangiogenesis. ...
The next book in the series Advances in Biomedical Research edited by I. Mlynarczuk-Bialy and L. Bialy. This book contains chapters dealing with cancer research.
TABLE OF CONTENTS
Assessment of dietary intake of polyphenols in the prevention of cancer; Is regular physical activity an effective method of cancer prevention?; Psychiatric disorders in cancer patients ; Female internet blogs as (self) help in the fight against malignant cancer ; Personalized Medicine: from molecular methods to targeted therapy in cancer; Spices – bioactive compounds in the diet of patients with cancer; Computational analysis of AP-2γ role in bladder cancer; Burkitt-like lymphoma with 11q aberration a provisional entity in the 2016 WHO classification of lymphoid malignancies;. Relationship between redox status in blood of patients with chronic myeloid leukemia (CML) and values of selected inflammatory markers - preliminary studies; Patient eligibility for chemotherapy in the geriatric population – how to best balance benefits and risks?; Hormonotherapy in gynecological cancers; Sinonasal malignancies- clinicopathological characteristics and difficulties in diagnostics; Antiemetics in oncology – current knowledge and problems with effectiveness; Contemporary sonography and ultrasound-based systems used for the discrimination of the benign and malignant endometrial lesions; Antimicrobial peptides and their bactericidal, immunomodulating and anticancer activities – literature mini-review; A short guide on the selection of melanocytes and melanoma cells’ isolation procedures for cancer research; Application of Clostridium spp. and their toxins/enzymes in treatment of oncologic and other pathologies; Tumor angiogenesis – therapeutic approach; Photodynamic therapy as alternative therapy for prostate cancer and colorectal carcinoma as well as an antimicrobial treatment – a systematic review
... The members of the EGFL family have symbolic homology with EGF and can be attributed to EGF-related proteins because they have highly homologous EGFL domains. In addition, they have common functional characteristics (Soncin et al., 2003;Parker et al., 2004;Schmidt et al., 2007). When EGFL proteins bind to their receptors, they can perform functions such as proliferation, differentiation, apoptosis, adhesion, and migration (Kang et al., 2020), indicating that the EGFL family has a crucial significance in regulating cell metastasis, tumor growth, and progression (Delfortrie et al., 2011;An et al., 2019). ...
With highly homologous epidermal growth factor (EGF)-like (EGFL) domains, the members of the EGFL family play crucial roles in growth, invasion, and metastasis of tumors and are closely associated with the apoptosis of tumor cells and tumor angiogenesis. Furthermore, their contribution to immunoreaction and tumor microenvironment is highly known. In this study, a comprehensive analysis of EGFL6, −7, and −8 was performed on the basis of their expression profiles and their relationship with the rate of patient survival. Through a pan-cancer study, their effects were correlated with immune subtypes, tumor microenvironment, and drug resistance. Using The Cancer Genome Atlas pan-cancer data, expression profiles of EGFL6, −7, and −8, and their association with the patient survival rate and tumor microenvironment were analyzed in 33 types of cancers. The expression of the EGFL family was different in different cancer types, revealing the heterogeneity among cancers. The results showed that the expression of EGFL8 was lower than EGFL6 and EGFL7 among all cancer types, wherein EGFL7 had the highest expression. The univariate Cox proportional hazard regression model showed that EGFL6 and EGFL7 were the risk factors to predict poor prognosis of cancers. Survival analysis was then used to verify the relationship between gene expression and patient survival. Furthermore, EGFL6, EGFL7, and EGFL8 genes revealed a clear association with immune infiltrate subtypes; they were also related to the infiltration level of stromal cells and immune cells with different degrees. Moreover, they were negatively correlated with the characteristics of cancer stem cells measured by DNAs and RNAs. In addition, EGFL6, −7, and −8 were more likely to contribute to the resistance of cancer cells. Our systematic analysis of EGFL gene expression and their correlation with immune infiltration, tumor microenvironment, and prognosis of cancer patients emphasized the necessity of studying each EGFL member as a separate entity within each particular type of cancer. Simultaneously, EGFL6, −7, and −8 signals were verified as promising targets for cancer therapies, although further laboratory validation is still required.
... It is secreted by endothelial cells and plays a regulatory role in the recruitment of smooth muscle cells, migration and sprouting of endothelial cells during formation lumen of the new vessel. EGF-L7 is present in the environment of EC surrounding sprouting vessel and affects optimal assurance microenvironment for migrating ECs (Schmidt, 2007). Chicken ovalbumin upstream promotertrascription factor II (Coup TFII) gene is essential during physiological vasculogenesis and lymphangiogenesis. ...
Hormones play a significant role in the functioning of the human organism, by regulating a number of processes, including tissue activity, cell proliferation and division. Disorders in hormonal balance may promote the development of so-called hormone-dependent or hormone-sensitive cancers.
Many gynecological malignancies are dependent on estrogen and/or progesterone (e.g. epithelial and stromal ovarian carcinomas or endometrial carcinomas and sarcomas). The discovery of the relationship between hormonal stimulation and tumour progression has led to the development of new therapeutic strategy, known as the antihormone therapy (AHT) for cancer. AHT is one of the type of hormone therapy (HT) and it is based on the supressing the activity of selected hormones and/or lowering these hormones levels in the human body. The use of the phrase 'hormonal therapy' in this case seems to be incorrect as the term is associated with hormone replacement therapy, which encourages hormone activity.
Three main courses of action can be distinguished in antihormone therapy:
1.) the reduction in secretion of sex hormones, as a result of the agonism of pituitary gonadotropin-releasing hormone (GnRH) receptors,
2.) the inhibition of the aromatase enzyme activity, based on conversion of androgens into estrogens,
3.) the inhibition of proliferations of cells containing estrogen receptors (ERs) by the prevention of estrogen binding to ERs located on the cancer cells surface. Main advantage of antihormone therapy is the fact that it is better tolerated by patients compared to conventional chemotherapy. However, the limitations of this approach should be remembered, e.g. interactions with other drugs and late therapeutic response. In this article authors present a review of achievements to date of antihormone therapy in the context of some oncological gynaecology and describe the AHT strategies listed above.
... Epidermal growth factor-like domain 7 (Eg 7) is a recently identi ed secreted protein which binds the extracellular matrix surrounding the blood vessels and it appears to act in an autocrine manner to promote angiogenesis [27][28][29]. Eg 7 is expressed at high levels in early embryos of mice, but only high expressed in a few organs in adult mice such as the lung, heart and kidney [30]. However, Eg 7 is strongly upregulated in tumors [31]. ...
Background: Epidermal growth factor-like domain 7 (Egfl7), a recently identified secreted protein, was significantly increased in patients with HCC by our previous studies. However, its efficacy in diagnosis of early HCC remains unknown. In this study we evaluate the efficacy of serum Egfl7 for early HCC diagnosis and compare it with alpha-fetoprotein (AFP).
Methods: Serum Egfl7 levels in testing cohort (1065 participants) and validation cohort (464 participants) were measured by a sandwich enzyme-linked immunoassay (ELISA). The efficacies of Egfl7 and AFP in diagnosing early HCC were estimated by receiver operating characteristic (ROC).
Results: Serum Egfl7 was significantly elevated in patients with early HCC than all non-HCC controls. At the cut-off value of 2610 ng/mL, the area under ROC, sensitivity, specificity and accuracy of Egfl7 were 0.800, 75.2 %, 71.7% and 73.5% in discriminating early HCC from chronic liver disease (CLD), compared with 0.675, 61.8%, 62.0% and 61.9% for AFP using 20 ng/mL as a cut-off value. Egfl7 also exhibited a significant higher sensitivity and accuracy than AFP in differentiating early HCC patients from non-HCC individuals including healthy individuals and patients with CLD (76.6% vs. 64.0%, 79.9% vs. 66.1%). Additionally, 70.8% of early HCC patients with negative AFP could be diagnosed by Egfl7 and the combined use of Egfl7 and AFP increased the sensitivity to 91.0%. These results were confirmed by a validation cohort.
Conclusion: Egfl7 is a valuable serum marker in early diagnosis of HCC with a better sensitivity and accuracy than AFP.
... & 3, PECAM-1, ICAM-1, VE-cadherin, Ephrin-B2, EphB4 and EGFL7[46][47][48][49][50][51][52][53][54][55][56][57].Herein, our main objective is to search for novel markers of EPCs in peripheral blood. Thus, we have created a short list divided into ve groups of EPCs factors/molecules using pubmed literature, gene ...
Background: Endothelial progenitor cells (EPCs) are promising candidates for the cellular therapy of peripheral arterial & cardiovascular diseases. However, hitherto there is no specific marker(s) defining precisely EPCs. Herein, we are proposing a new in-silico approach for finding novel EPCs markers.
Methods: we assembled five groups of chosen EPCs-related genes/factors using Pubmed literature & gene ontology databases. This shortened database of EPCs factors was fed into publically-published transcriptome matrix to compare their expression between endothelial colony-forming cells (ECFCs), HUVECs and two adult endothelial cell types (ECs) from skin and adipose tissue. Further, the database was used for functional enrichment on mouse phenotype database and protein-protein interaction network analyses. Moreover, we built a digital matrix of healthy donors' PBMCs (33 thousand single cell transcriptomes) and analyzed the expression of these EPCs factors
Results: Transcriptome analyses showed that BMP2,4 & ephrinB2 were exclusively highly expressed in EPCs; the expression of neuropilin-1 & VEGF-C were significantly higher in EPCs & HUVECs compared with other ECs; Notch 1 was highly expressed in EPCs & skin-ECs; MIR21 was highly expressed in skin-ECs; PECAM-1 were significantly higher in EPCs & adipose ECs. Moreover, functional enrichment of EPCs-related genes on mouse phenotype and STRING protein database has revealed significant relations between chosen EPCs factors and endothelial & vascular functions, development and morphogenesis, where ephrinB2, BMP2 and BMP4 were highly expressed in EPCs and were connected to abnormal vascular functions. Single cell RNA-sequencing analyses has revealed that among the EPCs regulated markers in transcriptome analyses: i-ICAM1 & Endoglin were weekly expressed in the monocyte compartment of peripheral blood; ii-CD163 & CD36 were highly expressed in CD14+ monocyte compartment whereas CSF1R was highly expressed in CD16+ monocyte compartment; iii-L-selectin & IL6R were globally expressed in the lymphoid/myeloid compartments; iv-interestingly, PLAUR/UPAR & NOTCH2 were highly expressed in both CD14+ & CD16+ monocytic compartments.
Conclusions: The current study has identified novel EPCs markers that could be used for better characterization of EPCs sub-population in adult peripheral blood and subsequent usage of EPCs for various cell therapy and regenerative medicine applications.
... However, it also revealed that most zebrafish egfl7 mutants, unlike the morphants, do not display an obvious phenotype, with less than 5% exhibiting brain hemorrhage . Similarly, most mouse Egfl7 mutants do not exhibit an obvious phenotype either (Schmidt et al., 2007;Kuhnert et al., 2008). Based on these and other observations , it was hypothesized that the phenotypic differences between egfl7 mutants and morphants were due to genetic compensation in the mutants but not the morphants. ...
Mutations play a crucial role in evolution as they provide the genetic variation that allows evolutionary change. Although some mutations in regulatory elements or coding regions can be beneficial, a large number of them disrupt gene function and reduce fitness. Organisms utilize several mechanisms to compensate for the damaging consequences of genetic perturbations. One such mechanism is the recently identified process of transcriptional adaptation (TA): during this event, mutations that cause mutant mRNA degradation trigger the transcriptional modulation of so-called adapting genes. In some cases, for example when one (or more) of the upregulated genes is functionally redundant with the mutated gene, this process compensates for the loss of the mutated gene's product. Notably, unlike other mechanisms underlying genetic robustness, TA is not triggered by the loss of protein function, an observation that has prompted studies into the machinery of TA and the contexts in which it functions. Here, we review the discovery and current understanding of TA, and discuss how its main features appear to be conserved across species. In light of these findings, we also speculate on the importance of TA in the context of human disease, and provide some recommendations for genome-editing strategies that should be more effective.
... Previous studies have shown that EGFL7 is a secretory factor derived from vascular endothelial cells that plays a role in regulating vascular endothelial cell adhesion and migration and inhibits vascular smooth muscle cell migration [16]. EGFL7 is important for regulating tubulogenesis in zebrafish and for controlling vascular patterning and integrity in mice [17][18][19][20][21][22]. Its function in blood vessel development is mediated, at least in part, through modulation of Notch signaling and Akt/ERK activation [23]. ...
Epidermal growth factor-like domain-containing protein 6 (EGFL6) belongs to the epidermal growth factor (EGF) superfamily. EGFL6 is expressed at higher levels in embryos and various malignant tumors than in normal tissues. Recent studies suggest that EGFL6 participates in the development of a variety of tumors. In this review, we summarize findings that support the role for EGFL6 in tumor proliferation, invasion and migration. Furthermore, our review results indicate the mechanism of EGFL6 activity angiogenesis. We also describe work toward the preparation of monoclonal antibodies against EGFL6. Altogether, the work of this review promotes understanding of the role of EGFL6 in tumor development, the mechanism of that action, and the potential of EGFL6 as a therapeutic target for tumor prevention and treatment.
... miR-126 resides in the intron of EGFL7 gene. miR-126 −/− mice display similar vascular abnormalities to the Egfl7 −/− mice, including edema, defective cranial vessel and retinal vascularization, raising a question as to which molecule is responsible for the observed phenotype 3,47 . Shortly after the report of the miR-126 −/− mouse phenotype, floxed alleles of Egfl7 (Egfl7 Δ/Δ ) and miR-126 (miR-126 Δ/Δ ) were generated 5 . ...
microRNA miR-126 was among the early discovered miRNAs that are expressed specifically in the vasculature and have critical functions in vascular development. Recent studies have started to unveil potentially important function of miR-126 in vascular diseases, including atherosclerosis, coronary artery disease, stroke and diabetic vasculopathy. The action of miR-126 reflects its function in angiogenesis and inflammation. The expression of miR-126 is downregulated in a variety of vascular diseases, and miR-126 overexpression appears to beneficial for most vascular disease models. In the minireview, we summarize the historic and current research regarding miR-126 function and mechanisms in the vascular system, its link to long noncoding RNAs (lncRNA), as well as the potential of miR-126-based therapeutics for vascular diseases. To explain the seemingly conflicting function of miR-126 from different studies, an integrated hypothesis is proposed that miR-126 has strand- and cell type-specific functions in angiogenesis and inflammation, making it beneficial in many different vascular disease models.
... The second phase consists of the reconstruction of the basal membrane around the new capillary [6]. These cellular activities involve numerous molecular interactions, growth factors, cytokines, chemical signals, and the production of free radicals [7]. ...
The aim of the present study was to investigate the influence of low-level red (660 nm) and infrared (780 nm) laser with four different radiance exposures on human umbilical vein endothelial cells (HUVECs) in vitro. HUVECs (1.5 × 104) were incubated in 96-well culture plates. The cells were maintained in M199 medium supplemented with 20% fetal bovine serum, 1% antibiotic (penicillin), 1% anti-mycotic (Fungizone), and 1% endothelial cell growth supplement. After centrifugation, irradiations (660/780 nm, 40 mW, 1, 5, 10, and 20 J/cm2, 1 s, 5 s, 10 s, and 20 s, respectively, total energy 0.4 J, 2 J, 4 J, and 8 J, and beam spot size at target 0.04 cm2) were performed at the bottom of Falcon tubes such that the laser beam directly reached the cell without passing through the culture medium. The cells were divided into groups based on radiant exposures. Cell viability and protein concentration were verified after 1, 2, 3, 6, 8, and 10 days. Red laser increased the cell viability and protein concentration in all groups (three-way ANOVA, p < 0.05) beginning on the second day. The greatest peak compared with the control was found when the radiant exposure was 5 J/cm2 and 10 J/cm2. Infrared laser inhibited cell viability and modulated the protein concentration in the cells, with the highest peak protein concentration found on the second day in the group with radiant exposure of 1 J/cm2 and 10 J/cm2 (three-way ANOVA, p < 0.05). Red laser increased the viability and concentration of total proteins in HUVECs, whereas infrared laser had an inhibitory effect on cell viability, while maintaining the total protein concentration similar to that found in the control group.
... Different mechanisms are contributing to sprout elongation, such as cell positional rearrangements, (stalk) cell elongation and division. While tip cell pulling on stalk cells, stalk cell pushing on tip cells, or both, have previously been postulated as mechanical forces that underlie sprout elongation [4][5][6][7][8][9][10], forces during sprout progression have never been measured, so that the nature and role of physical forces during sprout elongation remain unclear. While the measurement of cell-cell forces is far from trivial, cell-ECM forces can be quantified by means of Traction Force Microscopy (TFM). ...
Angiogenesis is the formation of new blood vessels from the pre-existing vasculature. It is essential for normal tissue growth and regeneration, and also plays a key role in many diseases [Carmeliet in Nat Med 9:653–660, 2003]. Cytoskeletal components have been shown to be important for angiogenic sprout initiation and maintenance [Kniazeva and Putnam in Am J Physiol 297:C179–C187, 2009] as well as endothelial cell shape control during invasion [Elliott et al. in Nat Cell Biol 17:137–147, 2015]. The exact nature of cytoskeleton-mediated forces for sprout initiation and progression, however, remains poorly understood. Questions on the importance of tip cell pulling versus stalk cell pushing are to a large extent unanswered, which among others has to do with the difficulty of quantifying and resolving those forces in time and space. We developed methods based on time-lapse confocal microscopy and image processing—further termed 4D displacement microscopy—to acquire detailed, spatially and temporally resolved extracellular matrix (ECM) deformations, indicative of cell-ECM mechanical interactions around invading sprouts. We demonstrate that matrix deformations dependent on actin-mediated force generation are spatio-temporally correlated with sprout morphological dynamics. Furthermore, sprout tips were found to exert radially pulling forces on the extracellular matrix, which were quantified by means of a computational model of collagen ECM mechanics. Protrusions from extending sprouts mostly increase their pulling forces, while retracting protrusions mainly reduce their pulling forces. Displacement microscopy analysis further unveiled a characteristic dipole-like deformation pattern along the sprout direction that was consistent among seemingly very different sprout shapes—with oppositely oriented displacements at sprout tip versus sprout base and a transition zone of negligible displacements in between. These results demonstrate that sprout-ECM interactions are dominated by pulling forces and underline the key role of tip cell pulling for sprouting angiogenesis.
... Previous reports have shown that tumor invasion and metastasis are related to cell adhesion, migration, invasion, and angiogenesis [25][26][27][28][29][30]. In addition, the EGFL6 protein has been reported to induce cell migration of endothelial cells [31][32][33][34][35]. These findings suggested that EGFL6 may promote colorectal cancer tumor invasion and metastasis by promoting cell migration and angiogenesis. ...
To explore the expression levels of EGFL6 in colorectal cancer and the association between EGFL6 and its clinicopathological parameters in patients. Immunohistochemistry assay was used to detect the expression levels of EGFL6 in cancer tissues of 42 colorectal cancer cases and corresponding adjacent normal tissues. The associations between protein expression levels of EGFL6 and the clinicopathological features, such as age, gender, smoking status, drinking status, TNM stage, Tumor T status, lymph node status, distant metastasis, and tumor diameter were also analyzed. We measured the plasma EGFL6 levels of colorectal cancer patients by using a commercial enzyme-linked immunosorbent assay. The positive rate of EGFL6 in cancer tissues was significantly higher than that of cancer-adjacent tissues (P < 0.05). Correlation was found between the protein expression of EGFL6 and the TNM stage (P < 0.05), the tumor T status (P < 0.05), distant metastasis (P < 0.05) and tumor diameter (P < 0.05). The plasma EGFL6 levels were significantly higher in patients with colorectal cancer than in healthy controls (P < 0.001). Moreover, plasma EGFL6 levels were significantly higher in the patients with higher TNM stage (P = 0.024), tumor T status (P = 0.021), distant metastasis (P < 0.001), and tumor diameter (P = 0.049). Therefore, these results demonstrated that EGFL6 expression was correlated with the genesis and development of colorectal cancer.
... miR-126 gene is located on epidermal growth factor like 7 (EGFL7) intron of humans, and was primarily discovered in human breast cancer tissue [20,21]. There have been many reports that claim that miR-126 is involved in cancer tumorigenesis, such as colorectal cancer [22][23][24], pancreatic cancer [25], breast cancer [26], stomach cancer [27], prostate cancer [28], liver cancer [29], lung cancer [30] and so on. ...
Background:
Ovarian cancer (OC) is the gynecologic malignancy with the highest mortality rate (70%), and it is urgent to find out a powerful prognostic marker for OC patients. LncRNAs are recently thought to be oncogenes in various cancers, and its expression levels are validated that can be inhibited by miRNAs. There are several studies indicating that sponging miRNAs will contribute to the tumorigenesis of cancers.
Methods:
In the present study, bioinformatics analysis is used to explore the potential oncogene and its target miRNAs; QRT-PCR is performed to count the expression level of several genes; Flow cytometric analysis is conducted to assess the apoptosis rate of several cell lines; Western blot assays are used to evaluate the expression levels of several proteins; Cells proliferation, migration and invasion abilities are detected by CCK-8 assay, Wound scratch assay and Transwell invasion assay, respectively. In vivo experiments are performed to assess the influence of LNC01133 on the formation of tumor.
Results:
We found LNC01133 was related to poor survival of OC patients, and identified that LNC01133 had significant influence on OC cells' apoptosis, proliferation, migration and invasion abilities. Furthermore, we observed miR-126 could target LNC01133 and decreased the expression level of LNC01133 in OC cells. Therefore, we sponged miR-126 to further study the molecular mechanism of OC tumorigenesis, and found an elevation in proliferation, migration and invasion abilities of OC cells, which suggested that miR-126 could serve as a powerful prognostic marker for OC patients, and had great clinical significance on OC diagnosis and treatment.
Conclusion:
We found LNC01133 was an oncogene in OC, which is targeted by miR-126. miR-126 served as a powerful prognostic marker for OC patients because of its ability of promoting OC tumorigenesis after sponging.
... This sheet migration results in epidermal wound closure 15,18,19 . In the regeneration of endothelial cells, collective strands of endothelial cells migrate as vascular sprouting with a leading sprout, and penetrate the provisional wound matrix to regenerate vessels 3,7, 20 . Researchers have been working on modeling the collective migration during wound healing using continuum model and free boundary system 21 . ...
Cell migration is essential for regulating several biological processes in physiological or pathological conditions, including embryonic development and cancer invasion. As in vitro studies and in silico simulations suggest, collective cell migration implies specific biomechanical markers. Therefore, the phenomenon could be understood by an in depth study of cells’ behavior determinants, including but not limited to mechanical cues from the environment and from fellow “travelers”. This review article aims to cover the recent development of experimental and computational methods for studying the biomechanics of collective cell migration during cancer progression and invasion. We also provided the tested hypotheses regarding the mechanism underlying collective cell migration enabled by these methods. Together, the paper enables a broad overview on the methods and tools currently available to unravel the biophysical mechanisms pertinent to cell collective migration, as well as providing perspectives on future development towards eventually deciphering the key mechanisms behind the most lethal feature of cancer.
... This sheet migration results in epidermal wound closure 15,18,19 . In the regeneration of endothelial cells, collective strands of endothelial cells migrate as vascular sprouting with a leading sprout, and penetrate the provisional wound matrix to regenerate vessels 3,7,20 . Researchers have been working on modeling the collective migration during wound healing using continuum model and free boundary system 21 . ...
Cell migration is essential for regulating many biological processes in physiological or pathological conditions, including embryonic development and cancer invasion. In vitro and in silico studies suggest that collective cell migration is associated with some biomechanical particularities, such as restructuring of extracellular matrix, stress and force distribution profiles, and reorganization of cytoskeleton. Therefore, the phenomenon could be understood by an in-depth study of cells' behavior determinants, including but not limited to mechanical cues from the environment and from fellow travelers. This review article aims to cover the recent development of experimental and computational methods for studying the biomechanics of collective cell migration during cancer progression and invasion. We also summarized the tested hypotheses regarding the mechanism underlying collective cell migration enabled by these methods. Together, the paper enables a broad overview on the methods and tools currently available to unravel the biophysical mechanisms pertinent to cell collective migration, as well as providing perspectives on future development towards eventually deciphering the key mechanisms behind the most lethal feature of cancer.
Background
Folliculogenesis is an intricate process that involves the development and maturation of ovarian follicles in females. During folliculogenesis, multiple factors including hormones, growth factors, and signaling pathways regulate the growth and maturation of follicles. In recent years, microRNA, short non-coding RNA molecules, has gained attention due to its roles in the physiology and pathophysiology of various diseases in humans. It is known to have an important part in ovarian health and illness and its functions extend to several cellular processes.
Main body
In this overview, we look at the importance of microRNAs in ovarian illnesses and how they function during follicle growth in the ovaries. Short RNA molecules (22 nucleotides) called microRNAs may influence several mRNA targets in different biological processes. The expression patterns of these small non-coding RNAs undergo dynamic changes during the several phases of follicular development; they play a function in post-transcriptional gene regulation. Follicle development, follicular atresia (regression of the follicles), and ovulation are all intricately regulated by the dynamic expression of distinct miRNAs throughout the various phases of folliculogenesis.
The role of microRNAs (miRNAs), which are known to regulate gene expression, has recently come to light as crucial in the development and advancement of a number of ovarian diseases. Abnormalities of the human ovary, such as ovarian cancer, polycystic ovary syndrome (PCOS), and endometriosis, have prompted extensive research into the dysregulation of microRNAs. Endometriosis is associated with miRNAs that are known to have a role in processes such as invasion, cell growth, cell adhesion, angiogenesis, and epithelial-mesenchymal transition. The disturbance of target gene expression resulting from abnormal miRNA production is a potential factor contributing to cancer development. Some microRNAs (miRNAs) differ in expression levels between women with polycystic ovary syndrome and healthy controls, indicating that miRNAs may play a role in the development of PCOS.
Conclusion
Extensive research carried out over the last 20 years has illuminated the roles of microRNAs (miRNAs), demonstrating their critical importance in controlling gene expression and the cell cycle. Changes in the quantities of microRNAs (miRNAs) may affect the aggressiveness of cancer and contribute to a variety of gynecological disorders. It appears that microRNAs hold potential as diagnostic biomarkers and treatment potential for various ovarian diseases.
Study question:
Could epidermal growth factor-like domain 7 (EGFL7) be a factor involved in the preparation of the endometrium for implantation and could its dysregulation be implicated in poor reproductive outcomes?
Summary answer:
EGFL7 is highly expressed in the endothelium and glandular epithelium throughout the menstrual cycle; it is upregulated by stromal cells in secretory phase and appears strongly reduced in endometrial biopsies and isolated stromal cells of women with unexplained recurrent pregnancy loss (uRPL) and recurrent implantation failure (RIF).
What is known already:
The secreted factor EGFL7, originally identified as a gene primarily expressed in endothelial cells, is also expressed by the mouse blastocyst and by mouse and human trophoblast cells. It regulates trophoblast migration and invasion by activating NOTCH1 signaling. NOTCH1 has been demonstrated to play a fundamental role in endometrial receptivity and its dysregulation may be involved in selected pregnancy complications characterized by altered endometrial receptivity, such as uRPL.
Study design, size, duration:
This is an exploratory study for which 84 endometrial biopsies were collected from normally fertile women, as well as from women with uRPL and RIF.
Participants/materials, setting, methods:
Samples were collected from women in both the proliferative and secretory phases of the menstrual cycle and stratified into three sub-groups according to the patient clinical history: 20 fertile women (8 in proliferative and 12 in secretory phase), 41 women with uRPL (6 in proliferative and 35 in secretory phase), and 27 women with RIF (8 in proliferative and 19 in secretory phase). Immunohistochemistry, real-time PCR, and western blot analyses were performed to study the expression of EGFL7 and NOTCH1, as well as the NOTCH target genes.
Main results and the role of chance:
Analysis of spatial and temporal distribution of EGFL7 in endometrial biopsies from fertile women revealed higher levels of EGFL7 in samples from the secretory phase compared to proliferative phase. The expected expression of EGFL7 in endothelial cells was shown as well as the novel, not previously reported, expression in endometrial glands and stromal cells. EGFL7 was significantly reduced in the endometrium of women with uRPL and RIF in the secretory phases and this was associated with a downregulation of the NOTCH1 signaling pathway. Human recombinant EGFL7 was able to activate the NOTCH1 signaling pathway in endometrial stromal cells (EndSCs) obtained from fertile women but not in cells from uRPL or RIF patients. EndSCs from fertile women and decidualized in vitro for three days showed an upregulation of EGFL7 expression, whereas cells obtained from women with uRPL and RIF and decidualized in vitro did not.
Limitations, reasons for caution:
This study was conducted with a relatively small number of patient samples. Although results are highly reproducible and consistent, additional observations from multicentric cohorts would strengthen the relevance of the data. Moreover, this is an in vitro study, which might only partially represent the in vivo conditions.
Wider implications of the findings:
Our results demonstrate for the first time that EGFL7 is new player involved in decidualization and provide new insights into the pathophysiology of selected implantation defects and early pregnancy complications. Our studies have revealed that alterations in EGFL7 expression and the consequent dysregulation of NOTCH signaling are potential underlying causes of RIF and uRPL. Our results might have therapeutic relevance, as the EGFL7/NOTCH pathway may represent a potential target for medical intervention.
Study funding/competing interest(s):
This study has been supported by the Grant for Fertility Innovation 2017 (Merck KGaA). There are no competing interests to disclose.
Trial registration number:
Not applicable.
Aim: This study aimed to explore the role of EGFL7 in the healing process of refractory diabetic wounds. Methods: Epidermal stem cells (ESCs) were isolated from healthy mice and diabetic mice, identified by immunofluorescence, transfected with EGFL7 overexpression and silencing lentiviral vectors, and treated with Notch pathway inhibitor (DAPT). Results: SiEGFL7 significantly inhibited the proliferation, invasion and migration of ESCs of healthy mice. DAPT prominently inhibited the expressions of Notch1, Notch2, Hes1 and Jag1 in ESCs of healthy mice induced by overexpressed EGFL7. Overexpressed EGFL7 promoted wound healing in diabetic mice with refractory wounds. Conclusion: EGFL7 affects the proliferation and migration of ESCs in refractory diabetic wounds by regulating the Notch signaling pathway.
Actin, one of the most abundant intracellular proteins in mammalian cells, is a critical regulator of cell shape and polarity, migration, cell division, and transcriptional response. Angiogenesis, or the formation of new blood vessels in the body is a well-coordinated multi-step process. Endothelial cells lining the blood vessels acquire several new properties such as front–rear polarity, invasiveness, rapid proliferation and motility during angiogenesis. This is achieved by changes in the regulation of the actin cytoskeleton. Actin remodelling underlies the switch between the quiescent and angiogenic state of the endothelium. Actin forms endothelium-specific structures that support uniquely endothelial functions. Actin regulators at endothelial cell–cell junctions maintain the integrity of the blood–tissue barrier while permitting trans-endothelial leukocyte migration. This review focuses on endothelial actin structures and less-recognised actin-mediated endothelial functions. Readers are referred to other recent reviews for the well-recognised roles of actin in endothelial motility, barrier functions and leukocyte transmigration. Actin generates forces that are transmitted to the extracellular matrix resulting in vascular matrix remodelling. In this review, we attempt to synthesize our current understanding of the roles of actin in vascular morphogenesis. We speculate on the vascular bed specific differences in endothelial actin regulation and its role in the vast heterogeneity in endothelial morphology and function across the various tissues of our body.
Histopathological studies of multiple sclerosis (MS), a demyelinating disease of the central nervous system (CNS), and its animal model experimental autoimmune encephalomyelitis (EAE) have found newly formed leaky vessels in demyelinated acute and chronic plaques, in addition to blood-brain barrier damage in existing vessels, that exacerbate disease pathology by increasing CNS infiltration of immune cells. However, which vessel subtypes and signaling pathways generate these aberrant vessels is poorly understood. Using single-cell RNA sequencing and in vivo validation, we find that transcriptome signatures of neo-angiogenesis arise in venous endothelial cells in both acute and chronic EAE, and correlate with upregulation in VEGF-A signaling. These neo-angiogenic markers are also increased in human MS lesions. Treatment with a VEGF-A blocking antibody ameliorates EAE pathology by reducing neo-angiogenic transcriptomic signatures in vivo, suggesting that VEGF-A drives angiogenesis from vein ECs in EAE. Our findings suggest novel therapies targeting the vasculature that could benefit chronic MS.
Impaired angiogenesis in scleroderma (SSc) is a critical component of SSc pathology. MicroRNA‐126 (miR‐126) is expressed in endothelial cells (MVECs) where it regulates VEGF responses by repressing the negative regulators of VEGF, including the sprouty‐related protein‐1 (SPRED1), and phosphoinositide‐3 kinase regulatory subunit 2 (PIK3R2). MVECs were isolated from SSc skin and matched subjects (n = 6). MiR‐126 expression was measured by qPCR and in situ hybridization. Matrigel‐based tube assembly was used to test angiogenesis. MiR‐126 expression was inhibited by hsa‐miR‐126 inhibitor and enhanced by hsa‐miR‐126 Mimic. Epigenetic regulation of miR‐126 expression was examined by the addition of epigenetic inhibitors (Aza and TSA) to MVECs and by bisulphite genomic sequencing of DNA methylation of the miR‐126 promoter region. MiR‐126 expression, as well as EGFL7 (miR‐126 host gene), in SSc‐MVECs and skin, was significantly down‐regulated in association with increased expression of SPRED1 and PIK3R2 and diminished response to VEGF. Inhibition of miR‐126 in NL‐MVECs resulted in reduced angiogenic capacity, whereas overexpression of miR‐126 in SSc‐MVECs resulted in enhanced tube assembly. Addition of Aza and TSA normalized miR‐126 and EGFL7 expression levels in SSc‐MVECs. Heavy methylation in miR‐126/EGFL7 gene was noted. In conclusion, these results demonstrate that the down‐regulation of miR‐126 results in impaired VEGF responses.
Fluid-to-solid phase transition in multicellular assembly is crucial in many developmental biological processes, such as embryogenesis and morphogenesis. However, biomechanical studies in this area are limited, and little is known about factors governing the transition and how cell behaviors are regulated. Due to different stresses present, cells could behave distinctively depending on the nature of tissue. Here we report a fluid-to-solid transition in geometrically confined multicellular assemblies. Under circular confinement, Madin-Darby canine kidney (MDCK) monolayers undergo spatiotemporally oscillatory motions that are strongly dependent on the confinement size and distance from the periphery of the monolayers. Nanomechanical mapping reveals that epithelial tensional stress and traction forces on the substrate are both dependent on confinement size. The oscillation pattern and cellular nanomechanics profile appear well correlated with stress fiber assembly and cell polarization. These experimental observations imply that the confinement size-dependent surface tension regulates actin fiber assembly, cellular force generation, and cell polarization. Our analyses further suggest a characteristic confinement size (approximates to MDCK's natural correlation length) below which surface tension is sufficiently high and triggers a fluid-to-solid transition of the monolayers. Our findings may shed light on the geometrical and nanomechanical control of tissue morphogenesis and growth.
The formation of new blood vessels is a critical process in several physiological and pathological contexts. The complexity of the angiogenic process is not completely reproducible using standard two-dimensional (2D) culture models. Three-dimensional (3D) models have emerged as effective tools for deciphering molecular mechanisms controlling endothelial cell sprouting and vascular morphogenesis. These models allow dynamic and detailed analysis of the distinct angiogenic steps, as well as molecular manipulation. The “state-of-the-art” in 3D culture models developed for the study of angiogenesis has been review. The potential for 3D cell and tissue culture models to fill the gap between 2D models and clinical application has been assessed. Advances in combining engineering, materials science, and cell biology to highlight the most promising device and microfluidic systems for 3D angiogenesis have also been reported.
During sprouting angiogenesis – the growth of blood vessels from the existing vasculature ‐ endothelial cells adopt an elongated invasive form and exert forces at cell–cell and cell‐matrix interaction sites. These cell shape changes and cellular tractions require extensive reorganizations of the acto‐myosin network. However, the respective roles of actin and myosin for endothelial sprouting are not fully elucidated. In this study, we further investigate these roles by treating 2D migrating and 3D sprouting endothelial cells with chemical compounds targeting either myosin or actin. These treatments affected the endothelial cytoskeleton drastically and reduced the invasive response in a compound‐specific manner; pointing towards a tight control of the actin and myosin activity during sprouting. Clusters in the data further illustrate that endothelial sprout morphology is sensitive to the in vitro model mechanical micro‐environment, and directs future research towards mechanical substrate guidance as a strategy for promoting engineered tissue vascularization. In summary, our results add to a growing corpus of research highlighting a key role of the cytoskeleton for sprouting angiogenesis.
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Dengue virus (DENV) causes an estimated 390 million infections worldwide annually, with severe forms of disease marked by vascular leakage. Endothelial cells (EC) are directly responsible for vascular homeostasis and are highly responsive to circulating mediators but are not commonly infected. DENV encodes seven non-structural (NS) proteins; with only one of those, NS1, secreted from infected cells and accumulating in the blood of patients. NS1 has been implicated in the pathogenesis of vascular permeability, but the mechanism is not completely understood. Here we used primary endothelial cells and an array of in vitro approaches to study the effect of NS1 in disease-relevant human ECs. Confocal microscopy demonstrated rapid NS1 internalization by ECs into endosomes with accumulation over time. Transcriptomic and pathway analysis showed significant changes in functions associated with EC homeostasis and vascular permeability. Functional significance of this activation was assessed by trans-endothelial electrical resistance and showed that NS1 induced rapid and transient loss in EC barrier function within 3 h post-treatment. To understand the molecular mechanism by which NS1 induced EC activation, we evaluated the stress-sensing p38 MAPK pathway known to be directly involved in EC permeability and inflammation. WB analysis of NS1-stimulated ECs showed clear activation of p38 MAPK and downstream effectors MAPKAPK-2 and HSP27 with chemical inhibition of the p38 MAP kinase pathway restoring barrier function. Our results suggest that DENV NS1 may be involved in the pathogenesis of severe dengue by activating the p38 MAPK in ECs, promoting increased permeability that characterizes severe disease.
Assessing similarity is highly important for bioinformatics algorithms to determine correlations between biological information. A common problem is that similarity can appear by chance, particularly for low expressed entities. This is especially relevant in single-cell RNA-seq (scRNA-seq) data because read counts are much lower compared to bulk RNA-seq. Recently, a Bayesian correlation scheme that assigns low similarity to genes that have low confidence expression estimates has been proposed to assess similarity for bulk RNA-seq. Our goal is to extend the properties of the Bayesian correlation in scRNA-seq data by considering three ways to compute similarity. First, we compute the similarity of pairs of genes over all cells. Second, we identify specific cell populations and compute the correlation in those populations. Third, we compute the similarity of pairs of genes over all clusters, by considering the total mRNA expression. We demonstrate that Bayesian correlations are more reproducible than Pearson correlations. Compared to Pearson correlations, Bayesian correlations have a smaller dependence on the number of input cells. We show that the Bayesian correlation algorithm assigns high similarity values to genes with a biological relevance in a specific population. We conclude that Bayesian correlation is a robust similarity measure in scRNA-seq data.
The role of vessel wall-resident stem cells in the generation of microglia and angiogenisis in the adult CNS Das Zentralnervensystem (ZNS) wird kontinuierlich durch ein eigenes Immunsystem überwacht. Die Mikroglia sind ein wichtiger Vertreter dieses Immunsystems und ein besonderes Charakteristikum des ZNS. Für die Aufrechterhaltung der Hämostase im ZNS spielen die Mikroglia eine zentrale Rolle. Die Herkunft der Mikroglia war für lange Zeit Gegenstand der kontroversen wissenschaftlichen Diskussion. Zusammengefasst wurde deren Ursprung als hämatopoetisch, mesodermal und neuroektodermal beschrieben. Allerdings überwiegt derzeit die Meinung, dass die Mikroglia von Vorläuferzellen geliefert wird, die während der Embryonalentwicklung aus der Dottersackwand ins Gehirn migrieren, dort bis zum Erwachsenenalter persistieren und immer wieder zur Erneuerung der Mikroglia herangezogen werden. Wo genau im Hirngewebe derartige oder andere potenzielle Mikrogliavorläuferzellen im ZNS residieren, ist bis heute nicht abschließend geklärt. In der vorliegenden Arbeit konnte gezeigt werden, dass bereits die frisch präparierten Hirngefäße sowohl CD44+ als auch CD45+ Zellen in ihren Wänden aufweisen. Außerdem ließ sich beobachten, dass die CD44+ Zellen im BRA nach außen wanderten und sich zu Perizyten-ähnlichen und glatten Muskelzellen differenzierten. Diese Befunde ließen darauf schließen, dass die CD44+ Zellen mit diesen Eigenschaften das Potenzial haben, zur Gefäßneubildung beizutragen. Darüber hinaus konnten CD45+ Zellen in der Adventitia frisch isolierter Hirngefäße nachgewiesen werden, die im BRA teilweise für F4/80 und/oder Iba-1 positiv wurden. Dies wiederum lässt vermuten, dass aus der Wand der Hirngefäße Mikroglia- und Makrophagen-ähnliche Zellen generiert werden können. Es blieb jedoch offen, ob diese CD45+ Vorläuferzellen dauerhaft in der Adventitia der Hirngefäße residieren oder aber immer wieder durch im Blut zirkulierende Monozyten erneuert werden. Diese Frage zu klären, ist von klinischer Relevanz, bleibt jedoch zukünftigen Arbeiten überlassen. Das hier etablierte BRA könnte auch bei solchen Analysen hilfreich sein.
Collective cell migration underlies various events in development, regeneration and disease. In most cases in vivo, collective migration occurs by collective cell chemotaxis, the directed migration of cell groups along gradients of soluble chemical cues. Despite this, the mechanisms of collective cell chemotaxis are poorly understood. Here I use Xenopus and zebrafish neural crest, a highly migratory embryonic stem cell population whose behaviour has been likened to malignant invasion, to study collective chemotaxis ex vivo and in vivo. I show that the neural crest exhibits a tensile actomyosin ring at the edge of the migratory cell group that contracts in a supracellular fashion. This contractility is polarized during collective cell chemotaxis: it is inhibited at the front but persists at the rear of the cell cluster. Combining computational simulations with optogenetic and laser ablation experiments, I show that this differential contractility drives directed collective cell migration ex vivo and in vivo through intercalation of rear cells. Rear cell intercalation triggers a wave of cellular anterograde movement through the middle of the cluster that drives the whole group forward. The mechanically coupled cells at the edge of the group are pulled to the rear by the actomyosin contractility. This novel mechanism of collective cell chemotaxis can be conceptually visualized as squeezing a tube of tooth paste at the back, which pushes the toothpaste forward. Thus, in neural crest cells, the engine for collective chemotaxis is at the rear of the cell group. Surprisingly, many of the cell behaviours that I describe here for the whole cell cluster have an equivalent in directional migration of single cells. For example, contraction of rear cells within a cluster is equivalent to actomyosin contraction at the back of single cells during directional migration; retrograde movement of cells in the periphery of the cluster could correspond to the surface/cortex retrograde flow observed in single cells; and rear intercalation and forward movement of cells at the centre of the cluster is analogous to rear endocytosis and forward movement of vesicles in single cells. In summary, I have discovered a novel mechanism of collective cell migration in which we can consider the whole cluster as a single ‘supracell’.
Elucidating the mechanisms underlying sprouting angiogenesis and permeability should enable the development of more effective therapies for various diseases, including retinopathy, cancer, and other vascular disorders. We focused on epidermal growth factor-like domain 7 (EGFL7) which plays an important role in NOTCH signaling and in the organization of angiogenic sprouts. We developed an EGFL7-knockdown in vitro microvessel model and investigated the effect of EGFL7 at a tissue level. We found EGFL7 knockdown suppressed VEGF-A-induced sprouting angiogenesis accompanied by an overproduction of endothelial filopodia and reduced collagen IV deposition at the basal side of endothelial cells. We also observed impaired barrier function which reflected an inflammatory condition. Furthermore, our results showed that proper formation of adherens junctions and phosphorylation of VE-cadherin was disturbed. In conclusion, by using a 3D microvessel model we identified novel roles for EGFL7 in endothelial function during sprouting angiogenesis.
We have studied the role of the hypoxia-inducible angiogenic growth factor vascular endothelial growth factor (VEGF) in the induction and control of vessel growth in the developing retina of rats and cats, using in situ hybridization techniques. VEGF is expressed successively in two layers of neural retina, the innermost (axon) layer and the inner nuclear layer (INL). In the axon layer, VEGF is expressed transiently by astrocytes as they spread across the layer, closely preceding the formation of superficial vessels. In the INL, VEGF is expressed transiently by somas at the middle of the layer (presumably Muller cells), closely preceding the formation of the deep layer of retinal vessels. We propose that hypoxia caused by the onset of neuronal activity is detected by strategically located populations of neuroglia, first astrocytes, then Muller cells. In response they secrete VEGF, inducing formation of the superficial and deep layers of retinal vessels, respectively. As the vessels become patent, they relieve the hypoxic stimulus, so vessel formation is matched to oxygen demand. This hypothesis was tested experimentally in three ways. Expression of the high affinity flk-1 receptor for VEGF was demonstrated in newly formed retinal vessels, confirming that the secreted VEGF acts on the vessels, in a paracrine fashion. Direct hypoxic regulation of VEGF expression by macroglia was demonstrated in primary cultures of astrocytes and in cells of a glioma line. Hypoxic regulation of VEGF expression in the intact developing retina was demonstrated by showing that oxygen-enriched atmospheres that inhibit vessel formation also suppress endogenous VEGF production.
A recent explosion in newly discovered vascular growth factors has coincided
with exploitation of powerful new genetic approaches for studying vascular
development. An emerging rule is that all of these factors must be used in
perfect harmony to form functional vessels. These new findings also demand
re-evaluation of therapeutic efforts aimed at regulating blood vessel growth
in ischaemia, cancer and other pathological settings.
Little is known about how the initial endothelial plexus is remodelled into a mature and functioning vascular network. Studying postnatal remodelling of the retina vasculature, we show that a critical step in vascular maturation, namely pericyte recruitment, proceeds by outmigration of cells positive for (alpha)-smooth muscle actin from arterioles and that coverage of primary and smaller branches lags many days behind formation of the endothelial plexus. The transient existence of a pericyte-free endothelial plexus coincides temporally and spatially with the process of hyperoxia-induced vascular pruning, which is a mechanism for fine tuning of vascular density according to available oxygen. Acquisition of a pericyte coating marks the end of this plasticity window. To substantiate that association with pericytes stabilizes the vasculature, endothelial-pericyte associations were disrupted by intraocular injection of PDGF-BB. Ectopic PDGF-BB caused the detachment of PDGF-beta receptor-positive pericytes from newly coated vessels, presumably through interference with endogenous cues, but had no effect on mature vessels. Disruption of endothelial-pericyte associations resulted in excessive regression of vascular loops and abnormal remodelling. Conversely, intraocular injection of VEGF accelerated pericyte coverage of the preformed endothelial plexus, thereby revealing a novel function of this pleiotropic angiogenic growth factor. These findings also provide a cellular basis for clinical observations that vascular regression in premature neonates subjected to oxygen therapy [i.e. in retinopathy of prematurity] drops precipitously upon maturation of retina vessels and a mechanistic explanation to our previous findings that VEGF can rescue immature vessels from hyperoxia-induced regression.
We have studied the role of the hypoxia-inducible angiogenic growth factor vascular endothelial growth factor (VEGF) in the induction and control of vessel growth in the developing retina of rats and cats, using in situ hybridization techniques. VEGF is expressed successively in two layers of neural retina, the innermost (axon) layer and the inner nuclear layer (INL). In the axon layer, VEGF is expressed transiently by astrocytes as they spread across the layer, closely preceding the formation of superficial vessels. In the INL, VEGF is expressed transiently by somas at the middle of the layer (presumably Müller cells), closely preceding the formation of the deep layer of retinal vessels. We propose that hypoxia caused by the onset of neuronal activity is detected by strategically located populations of neuroglia, first astrocytes, then Müller cells. In response they secrete VEGF, inducing formation of the superficial and deep layers of retinal vessels, respectively. As the vessels become patent, they relieve the hypoxic stimulus, so vessel formation is matched to oxygen demand. This hypothesis was tested experimentally in three ways. Expression of the high affinity flk-1 receptor for VEGF was demonstrated in newly formed retinal vessels, confirming that the secreted VEGF acts on the vessels, in a paracrine fashion. Direct hypoxic regulation of VEGF expression by macroglia was demonstrated in primary cultures of astrocytes and in cells of a glioma line. Hypoxic regulation of VEGF expression in the intact developing retina was demonstrated by showing that oxygen-enriched atmospheres that inhibit vessel formation also suppress endogenous VEGF production.
The recruitment and proliferation of smooth muscle cells and pericytes are two key events for the stabilization of newly formed capillaries during angiogenesis and, when out of control in the adult, are the main causes of arteriosclerosis. We have identified a novel gene, named VE-statin for vascular endothelial-statin, which is expressed specifically by endothelial cells of the developing mouse embryo and in the adult, and in early endothelial progenitors. The mouse and human VE-statin genes have been located on chromosome 2 and 9, respectively, they span >10 kbp and are transcribed in two major variants arising from independent initiation sites. The VE-statin transcripts code for a unique protein of 30 kDa that contains a signal peptide and two epidermal growth factor (EGF)-like modules. VE-statin is found in the cellular endoplasmic reticulum and secreted in the cell supernatant. Secreted VE-statin inhibits platelet-derived growth factor (PDGF)-BB-induced smooth muscle cell migration, but has no effects on endothelial cell migration. VE-statin is the first identified inhibitor of mural cell migration specifically produced by endothelial cells.
The availability of both the mouse and human genome sequences allows for the systematic discovery of human gene function through the use of the mouse as a model system. To accelerate the genetic determination of gene function, we have developed a sequence-tagged gene-trap library of >270,000 mouse embryonic stem cell clones representing mutations in approximately 60% of mammalian genes. Through the generation and phenotypic analysis of knockout mice from this resource, we are undertaking a functional screen to identify genes regulating physiological parameters such as blood pressure. As part of this screen, mice deficient for the Wnk1 kinase gene were generated and analyzed. Genetic studies in humans have shown that large intronic deletions in WNK1 lead to its overexpression and are responsible for pseudohypoaldosteronism type II, an autosomal dominant disorder characterized by hypertension, increased renal salt reabsorption, and impaired K+ and H+ excretion. Consistent with the human genetic studies, Wnk1 heterozygous mice displayed a significant decrease in blood pressure. Mice homozygous for the Wnk1 mutation died during embryonic development before day 13 of gestation. These results demonstrate that Wnk1 is a regulator of blood pressure critical for development and illustrate the utility of a functional screen driven by a sequence-based mutagenesis approach.
Branching morphogenesis in the mammalian lung and Drosophila trachea relies on the precise localization of secreted modulators of epithelial growth to select branch sites and direct branch elongation, but the intercellular signals that control blood vessel branching have not been previously identified. We found that VEGF(120/120) mouse embryos, engineered to express solely an isoform of VEGF-A that lacks heparin-binding, and therefore extracellular matrix interaction domains, exhibited a specific decrease in capillary branch formation. This defect was not caused by isoform-specific differences in stimulating endothelial cell proliferation or by impaired isoform-specific signaling through the Nrp1 receptor. Rather, changes in the extracellular localization of VEGF-A in heparin-binding mutant embryos resulted in an altered distribution of endothelial cells within the growing vasculature. Instead of being recruited into additional branches, nascent endothelial cells were preferentially integrated within existing vessels to increase lumen caliber. The disruption of the normal VEGF-A concentration gradient also impaired the directed extension of endothelial cell filopodia, suggesting that heparin-binding VEGF-A isoforms normally provide spatially restricted stimulatory cues that polarize and thereby guide sprouting endothelial cells to initiate vascular branch formation. Consistent with this idea, we found opposing defects in embryos harboring only a heparin-binding isoform of VEGF-A, including excess endothelial filopodia and abnormally thin vessel branches in ectopic sites. We conclude that differential VEGF-A isoform localization in the extracellular space provides a control point for regulating vascular branching pattern.
Semaphorin 4A (Sema4A) is a member of the transmembrane class 4 family of semaphorins. It has recently been shown to participate in cell-cell communication in the immune system. High levels of sema4A are also present in brain and eye, but its function in the central nervous system has not been studied. To investigate the function of Sema4A, we generated mice deficient in this transmembrane signaling molecule.
An embryonic stem (ES) cell clone with a retroviral gene-trap insertion in the sema4A gene was used to generate mice lacking this transmembrane semaphorin. Fundus photography, fluorescein angiography, and electroretinography were used to evaluate retinal anatomy and physiology in mice lacking Sema4A. Electron microscopy and immunohistochemistry with cell-type-specific markers were used to characterize retinal development. In situ hybridization with sema4A-specific riboprobes was used to localize expression of this gene in the developing and adult eye.
Fundus photography performed at 14 weeks of age revealed severe retinal degeneration, attenuated retinal vessels, and depigmentation in mice lacking Sema4A. At this age, the outer nuclear layer was reduced to a single row of photoreceptor cells, and the outer plexiform layer was thin and disorganized. Disruption of Sema4A also compromised the physiological function of both rod and cone photoreceptors. Developmental studies in Sema4A-deficient mice revealed abnormal morphology of photoreceptor outer segments during the time at which they establish contacts with apical microvilli of the retinal pigment epithelium (RPE). Sema4A is expressed in the inner retina and RPE during the time at which photoreceptor outer segments elongate.
These findings identify a previously unknown function of Sema4A in the developing visual system and provide a useful model for understanding cell-cell interactions that occur between photoreceptors and the RPE.
The development of the embryonic blood vascular and lymphatic systems requires the coordinated action of several transcription factors and growth factors that target endothelial and periendothelial cells. However, according to recent studies, the precise "wiring" of the vascular system does not occur without an ordered series of guidance decisions involving several molecules initially discovered for axons in the nervous system, including ephrins, netrins, slits, and semaphorins. Here, we summarize the new advances in our understanding of the roles of these axonal pathfinding molecules in vascular remodeling and vessel guidance, indicating that neuronal axons and vessel sprouts use common molecular mechanisms for navigation in the body.
The intricate patterning processes that establish the complex vascular system during development depend on a combination of intrinsic pre-patterning and extrinsic responses to environmental parameters. Mutational studies in mice and fish have shown that the vascular system is highly sensitive to genetic disruption and have identified potential targets for therapeutic interventions. New insights into non-vascular roles of vascular endothelial growth factor and the requirement for endothelial cells in adult organs and stem-cell niches highlight possible side effects of anti-angiogenic therapy and the need for new targets.
Myocardial infarction and ischemic heart disease are the leading cause of death in the industrial world. Therapies employed for treating these diseases are aimed at promoting increased blood flow to cardiac tissue. Pharmacological induction of new coronary growth has recently been explored, however, clinical trials with known proangiogenic factors have been disappointing. To identify novel therapeutic targets, we have explored signaling pathways that govern embryonic coronary development. Using a combination of genetically engineered mice and an organ culture system, we identified novel roles for fibroblast growth factor (FGF) and Hedgehog (HH) signaling in coronary vascular development. We show that FGF signals promote coronary growth indirectly by signaling to the cardiomyoblast through redundant function of Fgfr1 and Fgfr2. Myocardial FGF signaling triggers a wave of HH activation that is essential for vascular endothelial growth factor (Vegf)-A, Vegf-B, Vegf-C, and angiopoietin-2 (Ang2) expression. We demonstrate that HH is necessary for coronary vascular development and activation of HH signaling is sufficient to promote coronary growth and to rescue coronary defects due to loss of FGF signaling. These studies implicate HH signaling as an essential regulator of coronary vascular development and as a potential therapeutic target for coronary neovascularization. Consistent with this, activation of HH signaling in the adult heart leads to an increase in coronary vessel density.
Vascular endothelial growth factor (VEGF-A) is a major regulator of blood vessel formation and function. It controls several processes in endothelial cells, such as proliferation, survival, and migration, but it is not known how these are coordinately regulated to result in more complex morphogenetic events, such as tubular sprouting, fusion, and network formation. We show here that VEGF-A controls angiogenic sprouting in the early postnatal retina by guiding filopodial extension from specialized endothelial cells situated at the tips of the vascular sprouts. The tip cells respond to VEGF-A only by guided migration; the proliferative response to VEGF-A occurs in the sprout stalks. These two cellular responses are both mediated by agonistic activity of VEGF-A on VEGF receptor 2. Whereas tip cell migration depends on a gradient of VEGF-A, proliferation is regulated by its concentration. Thus, vessel patterning during retinal angiogenesis depends on the balance between two different qualities of the extracellular VEGF-A distribution, which regulate distinct cellular responses in defined populations of endothelial cells.
The cellular mechanisms underlying organ formation are largely unknown. We visualized early vertebrate eye morphogenesis at single-cell resolution by in vivo imaging in medaka (Oryzias latipes). Before optic vesicle evagination, retinal progenitor cells (RPCs) modulate their convergence in a fate-specific manner. Presumptive forebrain cells converge toward the midline, whereas medial RPCs remain stationary, predetermining the site of evagination. Subsequent optic vesicle evagination is driven by the active migration of individual RPCs. The analysis of mutants demonstrated that the retina-specific transcription factor rx3 determines the convergence and migration behaviors of RPCs. Hence, the migration of individual cells mediates essential steps of organ morphogenesis.
Collagen I provokes endothelial cells to assume a spindle-shaped morphology and to align into solid cord-like assemblies. These cords closely imitate the solid pre-capillary cords of embryonic angiogenesis, raising interesting questions about underlying mechanisms. Studies described here identify a critical mechanism beginning with collagen I ligation of integrins alpha(1)beta(1) and alpha(2)beta(1), followed by suppression of cyclic AMP and cyclic AMP (cAMP)-dependent protein kinase A, and marked induction of actin polymerization to form prominent stress fibers. In contrast to collagen I, laminin-1 neither suppressed cAMP nor protein kinase A activity nor induced actin polymerization or changes in cell shape. Moreover, fibroblasts did not respond to collagen I with changes in cAMP, actin polymerization, or cell shape, thus indicating that collagen signaling, as observed in endothelial cells, does not extend to all cell types. Pharmacological elevation of cAMP blocked collagen-induced actin polymerization and formation of cords by endothelial cells; conversely, pharmacological suppression of either cAMP or protein kinase A induced actin polymerization. Collectively, these studies identify a previously unrecognized and critical mechanism, involving suppression of cAMP-dependent protein kinase A and induction of actin polymerization, through which collagen I drives endothelial cell organization into multicellular pre-capillary cords.
Little is known about how the initial endothelial plexus is remodelled into a mature and functioning vascular network. Studying postnatal remodelling of the retina vasculature, we show that a critical step in vascular maturation, namely pericyte recruitment, proceeds by outmigration of cells positive for α-smooth muscle actin from arterioles and that coverage of primary and smaller branches lags many days behind formation of the endothelial plexus. The transient existence of a pericyte-free endothelial plexus coincides temporally and spatially with the process of hyperoxia-induced vascular pruning, which is a mechanism for fine tuning of vascular density according to available oxygen. Acquisition of a pericyte coating marks the end of this plasticity window.
To substantiate that association with pericytes stabilizes the vasculature, endothelial-pericyte associations were disrupted by intraocular injection of PDGF-BB. Ectopic PDGF-BB caused the detachment of PDGF-β receptor-positive pericytes from newly coated vessels, presumably through interference with endogenous cues, but had no effect on mature vessels. Disruption of endothelial-pericyte associations resulted in excessive regression of vascular loops and abnormal remodelling. Conversely, intraocular injection of VEGF accelerated pericyte coverage of the preformed endothelial plexus, thereby revealing a novel function of this pleiotropic angiogenic growth factor. These findings also provide a cellular basis for clinical observations that vascular regression in premature neonates subjected to oxygen therapy [i.e. in retinopathy of prematurity] drops precipitously upon maturation of retina vessels and a mechanistic explanation to our previous findings that VEGF can rescue immature vessels from hyperoxia-induced regression.
We studied the early pattern of neural tube (NT) vascularization in quail embryos and chick-quail chimeras. Angioblasts appeared first in the dorsal third at Hamburger and Hamilton (HH) stage 19 as single, migrating cells. Their distribution did not correspond to a segmental pattern. After this initial dorsal immigration, endothelial sprouts invaded the NT on either side of the floor plate (HH stage 21). These cells remained continuous with their arterial vascular sources, connected to the venous perineural vascular plexus at HH-stage 22, and formed the first perfused vessels of the NT at HH-stage 23. The same pattern of angiotrophic vascularization was observed in a craniocaudal sequence starting caudal to the rhombencephalic NT. Extremely long filopodia were observed on sprouting cells, extending toward the central canal and the mantle layer. The exclusively extraneuroectodermal origin of angioblastic cells was demonstrated with chick-quail chimeras. Following replacement of quail NT by chick NT graft, angioblast and sprout distribution in chimeras was the same as in controls. We conclude that the NT receives its first blood vessels by a combination of two different processes, dorsal immigration of isolated migrating angioblastic cells and ventral sprouting of endothelial cells, which derive from perfused vessels. The dorsal invasive angioblasts contribute to the developing intraneural vascular plexus after having traversed the neural tube. The initial distribution of blood vessels within the neuroepithelium corresponds to intrinsic random motility of angioblastic cells; a more regular pattern is seen later. The floor plate apparently prohibits connections between sprouts in both NT sides, whereas in the dorsal NT, such a separating effect on the migrating angioblasts does not exist.
The dramatic increase in sequence information in the form of expressed sequence tags (ESTs) and genomic sequence has created a 'gene function gap' with the identification of new genes far outpacing the rate at which their function can be identified. The ability to create mutations in embryonic stem (ES) cells on a large scale by tagged random mutagenesis provides a powerful approach for determining gene function in a mammalian system; this approach is well established in lower organisms. Here we describe a high-throughput mutagenesis method based on gene trapping that allows the automated identification of sequence tags from the mutated genes. This method traps and mutates genes regardless of their expression status in ES cells. To facilitate the study of gene function on a large scale, we are using these techniques to create a library of ES cells called Omnibank, from which sequence-tagged mutations in 2,000 genes are described.
A growing number of studies have established reciprocal linkages between extracellular matrix (ECM)-integrins, growth factor signaling and cell-cell adhesion molecules. ECM-dependent tissue-specific gene expression has also been linked to chromatin remodeling. With respect to tissue morphogenesis and differentiation, crosstalk has been established between the ECM and the homeobox morphoregulatory genes. Each of these linkages is profoundly influenced by the cell's microenvironment and the resulting tissue form. Thus for a cell to achieve a differentiated phenotype, the ECM molecules and their receptors must integrate both form and function. In contrast, mutated genes and aberrant interactions with the microenvironment conspire to undermine this integration, often resulting in malignant transformation.
The vasculature is one of the most important and complex organs in the mammalian body. The first functional organ to form during embryonic development, the intricately branched network of endothelial and supporting periendothelial cells is essential for the transportation of oxygen and nutrients to and the removal of waste products from the tissues. Serious disruptions in the formation of the vascular network are lethal early in post-implantation development, while the maintenance of vessel integrity and the control of vessel physiology and hemodynamics have important consequences throughout embryonic and adult life. A full understanding of the signaling pathways of vascular development is important not just for understanding normal development but because of the importance of reactivation of angiogenic pathways in disease states. Clinically there is a need to develop therapies to promote new blood vessel formation in situations of severe tissue ischemia, such as coronary heart disease. In addition, there is considerable interest in developing angiogenic inhibitors to block the new vessel growth that solid tumors promote in host tissue to enhance their own growth. Already studies on the signaling pathways of normal vascular development have provided new targets for therapeutic intervention in both situations. Further understanding of the complexities of the pathways should help refine such strategies.
The vascular system matures during embryonic development to form a stable, well-organized tubular network. In vivo data have established that the extracellular matrix (ECM) is crucial in providing structural support to the vascular system. In vitro studies are defining the involvement of ECM-smooth-muscle cell signaling in establishing and maintaining the mature tubular structure. However, correlating cell signaling with established structural functions for the ECM and determining the relative importance of these two roles in vivo is often difficult. Here, we examine human genetics, murine gene targeting and cell biology to better understand the relationship between structural and signaling roles for the ECM in vascular morphogenesis and disease.
The protein tyrosine kinase focal adhesion kinase (FAK) plays a prominent role in integrin signaling. FAK activation, demonstrated by an increase in phosphorylation of Tyr397 as well as other sites in the protein, is best understood in the context of the engagement of integrins at the cell surface. Activation of FAK results in recruitment of a number of SH2-domain- and SH3-domain-containing proteins, which mediate signaling to several downstream pathways. FAK-dependent activation of these pathways has been implicated in a diverse array of cellular processes, including cell migration, growth factor signaling, cell cycle progression and cell survival.
The zebrafish has recently emerged as an important model for the study of vascular embryogenesis. Its genetic accessibility, external development, and optically clear embryo are just a few of the features that set the zebrafish apart as a particularly well-suited model for studying vascular development. However, there is little precedent for its use as a tool for the experimental study of therapeutic angiogenesis. Here, we review the use of the zebrafish for studying vascular development and patterning, and discuss how the zebrafish might be used more directly as a model for developing and testing effective therapeutic angiogenesis approaches.
British Journal of Pharmacology (2003) 140, 585–594. doi:10.1038/sj.bjp.0705496
Vascular development is a complex but orderly process that is tightly regulated. A number of secreted factors produced by surrounding cells regulate endothelial cell (EC) differentiation, proliferation, migration and coalescence into cord-like structures. Vascular cords then undergo tubulogenesis to form vessels with a central lumen. But little is known about how tubulogenesis is regulated in vivo. Here we report the identification and characterization of a new EC-derived secreted factor, EGF-like domain 7 (Egfl7). Egfl7 is expressed at high levels in the vasculature associated with tissue proliferation, and is downregulated in most of the mature vessels in normal adult tissues. Loss of Egfl7 function in zebrafish embryos specifically blocks vascular tubulogenesis. We uncover a dynamic process during which gradual separation and proper spatial arrangement of the angioblasts allow subsequent assembly of vascular tubes. This process fails to take place in Egfl7 knockdown embryos, leading to the failure of vascular tube formation. Our study defines a regulator that controls a specific and important step in vasculogenesis.
We report the cloning and characterization of a novel epidermal growth factor (EGF) domain gene that was identified in a retroviral gene entrapment screen and is expressed in endothelial cells. This gene encodes a protein of 278 amino acids with an amino-terminal signal peptide and two centrally located EGF-like domains. We have named this novel gene in accordance with the guidelines of the Mouse Genome Informatics group Egfl7, for EGF-like domain 7. Egfl7 mRNA is expressed in highly vascularized adult tissues such as the lung, heart, uterus, and ovary. In addition, Egfl7 is expressed early during mouse embryogenesis and in undifferentiated murine embryonic stem cells. The analysis of Egfl7 expression in embryonic day 9.5 embryos by in situ hybridization indicates that Egfl7 is expressed in vascular structures in both the embryo proper and the yolk sac and at sites of mesodermal precursors of angioblasts. Within the cell, EGFL7 protein is localized to the endoplasmic reticulum and Golgi apparatus, suggesting that the protein is targeted for secretion. Indeed, recombinant EGFL7 is readily detectable in the supernatant media of transiently transfected HEK293 cells. We also report the identification of an Egfl7 paralog, Egfl8, and show that EGFL8 protein shares similar domains and molecular weight with EGFL7.
The movement of cells that maintain cell-cell junctions yet protrude along or within tissues is an important mechanism for cell positioning in morphogenesis, tissue repair and cancer. Collective cell migration shares similarities but also important differences to individually migrating cells. Coherent groups of cells are arranged and held together by cell-cell adhesion molecules, including cadherins, integrins, ALCAM and NCAM. Integrins of the beta 1 and beta 3 families further provide polarized interactions with the extracellular tissue environment, while matrix-degrading proteases become focalized to substrate contacts to widen tissue space for the advancing cell mass. By generating one functional unit, in contrast to individual cell migration, collective migration provides the active and passive translocation of mobile and non-mobile cells, respectively. This review highlights cellular and molecular principles of collective migration in the context of morphogenic tissue patterning and tumor cell invasion.
In sprouting angiogenesis, endothelial cells must orientate in the tissue environment in order to effectively invade tissues and form vascular patterns according to the local needs. Here, we review recent data indicating that sprouting angiogenesis is a guided process resembling axonal guidance and insect trachea formation. Angiogenesis requires functional specialization of endothelial cells within the sprout. Cells situated at the tip of the sprouts sense and navigate the environment using long filopodia, whereas cells in the sprout stalks proliferate and form a vascular lumen. Migration of the tip cells depends on a graded distribution of VEGF-A and activation of VEGFR2 located on the tip-cell filopodia. Proliferation in the stalk is concomitantly regulated by the local VEGF-A levels. Thus, the shape of the VEGF-A gradient controls the balance between tip cell migration and stalk cell proliferation, which in turn determines the initial vascular pattern. An imbalance between the two processes may explain why abnormal vascular patterns develop in pathological angiogenesis.
The semaphorins are a large family of proteins involved in the patterning of both the vascular and the nervous systems. In
order to analyze the function of the membrane-bound semaphorin 5A (Sema5A), we generated mice homozygous for a null mutation
in the Sema5a gene. Homozygous null mutants die between embryonic development days 11.5 (E11.5) and E12.5, indicating an essential role
of Sema5A during embryonic development. Mutant embryos did not show any morphological defects that could account for the lethality
of the mutation. A detailed analysis of the vascular system uncovered a role of Sema5A in the remodeling of the cranial blood
vessels. In Sema5A null mutants, the complexity of the hierarchically organized branches of the cranial cardinal veins was
decreased. Our results represent the first genetic analysis of the function of a class 5 semaphorin during embryonic development
and identify a role of Sema5A in the regional patterning of the vasculature.
The endothelium of the adult vasculature is normally quiescent, with the exception of the vasculature of the female reproductive system. However, in response to appropriate stimuli (ie, wound healing, atherosclerosis, tumor growth and metastasis, arthritis) the vasculature becomes activated and grows new capillaries through angiogenesis. We have recently identified a novel endothelial-restricted gene, Egfl7, that encodes a 41-kd secreted protein (Fitch MJ, Campagnolo L, Kuhnert F, Stuhlmann H: Egfl7, a novel epidermal growth factor-domain gene expressed in endothelial cells. Dev Dyn 2004, 230:316-324). Egfl7 is expressed at high levels early during mouse embryonic development and is strictly associated with the vascular bed. In this study, we investigated Egfl7 expression in the quiescent adult vasculature, in the pregnant uterus, and in two different models of arterial injury, namely ballooning and ferric chloride injury. By RNA in situ hybridization, Egfl7 expression in the vasculature was found to be restricted to the endothelium of the capillaries and mature vessels. In the pregnant uterus, increased vascularization was accompanied by up-regulation of Egfl7. On arterial injury, Egfl7 expression was up-regulated in the regenerating endothelium, but not in the neointima. Importantly, the EGFL7 protein acted as a chemoattractant for embryonic endothelial cells and fibroblasts in a cell migration assay. Together, these results suggest that Egfl7 functions in the formation and maintenance of endothelial integrity and that its up-regulation may be a critical component in the reorganization of the vascular bed in response to angiogenic stimuli.
Blood vessels and nerve fibres course throughout the body in an orderly pattern, often alongside one another. Although superficially distinct, the mechanisms involved in wiring neural and vascular networks seem to share some deep similarities. The discovery of key axon guidance molecules over the past decade has shown that axons are guided to their targets by finely tuned codes of attractive and repulsive cues, and recent studies reveal that these cues also help blood vessels to navigate to their targets. Parallels have also emerged between the actions of growth factors that direct angiogenic sprouting and those that regulate axon terminal arborization.
Blood vessels are constantly exposed to hemodynamic forces in the form of cyclic stretch and shear stress due to the pulsatile nature of blood pressure and flow. Endothelial cells (ECs) are subjected to the shear stress resulting from blood flow and are able to convert mechanical stimuli into intracellular signals that affect cellular functions, e.g., proliferation, apoptosis, migration, permeability, and remodeling, as well as gene expression. The ECs use multiple sensing mechanisms to detect changes in mechanical forces, leading to the activation of signaling networks. The cytoskeleton provides a structural framework for the EC to transmit mechanical forces between its luminal, abluminal and junctional surfaces and its interior, including the cytoplasm, the nucleus, and focal adhesion sites. Endothelial cells also respond differently to different modes of shear forces, e.g., laminar, disturbed, or oscillatory flows. In vitro studies on cultured ECs in flow channels have been conducted to investigate the molecular mechanisms by which cells convert the mechanical input into biochemical events, which eventually lead to functional responses. The knowledge gained on mechano-transduction, with verifications under in vivo conditions, will advance our understanding of the physiological and pathological processes in vascular remodeling and adaptation in health and disease.
The extracellular matrix (ECM) is critical for all aspects of vascular biology. In concert with supporting cells, endothelial cells (ECs) assemble a laminin-rich basement membrane matrix that provides structural and organizational stability. During the onset of angiogenesis, this basement membrane matrix is degraded by proteinases, among which membrane-type matrix metalloproteinases (MT-MMPs) are particularly significant. As angiogenesis proceeds, ECM serves essential functions in supporting key signaling events involved in regulating EC migration, invasion, proliferation, and survival. Moreover, the provisional ECM serves as a pliable scaffold wherein mechanical guidance forces are established among distal ECs, thereby providing organizational cues in the absence of cell-cell contact. Finally, through specific integrin-dependent signal transduction pathways, ECM controls the EC cytoskeleton to orchestrate the complex process of vascular morphogenesis by which proliferating ECs organize into multicellular tubes with functional lumens. Thus, the composition of ECM and therefore the regulation of ECM degradation and remodeling serves pivotally in the control of lumen and tube formation and, finally, neovessel stability and maturation.
The appropriate guidance and patterning of vessels during vascular development is critical for proper tissue function. The loss of these guidance mechanisms can lead to abnormal vascularization and a number of pathological conditions. The molecular basis of endothelial cell guidance and subsequent tissue specific vascular patterning remains largely unknown in spite of its clinical relevance and biological importance. In this regard, retinal vascular development offers many advantages for studying endothelial cell guidance and the mechanisms by which characteristic vascular patterns are formed. In this review, we will provide an overview of the known mechanisms that mediate vascular patterning during mouse retinal development, synthesizing these data to formulate a model of how growth factors, cellular adhesion molecules, and vascular-associated cells mediate directed endothelial cell migration and appropriate vascular remodeling. Finally, we will discuss the many aspects of retinal vascular development that remain unknown and cite evidence that many of these gaps may be addressed by further studying the guidance cues shared by vascular and neuronal elements in the retina and other parts of the central nervous system.
Tumours are known as wounds that do not heal - this implies that cells that are involved in angiogenesis and the response to injury, such as endothelial cells and fibroblasts, have a prominent role in the progression, growth and spread of cancers. Fibroblasts are associated with cancer cells at all stages of cancer progression, and their structural and functional contributions to this process are beginning to emerge. Their production of growth factors, chemokines and extracellular matrix facilitates the angiogenic recruitment of endothelial cells and pericytes. Fibroblasts are therefore a key determinant in the malignant progression of cancer and represent an important target for cancer therapies.
The shape of most complex organ systems arises from the directed migration of cohesive groups of cells. Here, we dissect the role of the chemokine guidance receptor Cxcr4b in regulating the collective migration of one such cohesive tissue, the zebrafish lateral line primordium. Using in vivo imaging, we show that the shape and organization of the primordium is surprisingly labile, and that internal cell movements are uncoordinated in embryos with reduced Cxcr4b signaling. Genetic mosaic experiments reveal that single cxcr4b mutant cells can migrate in a directional manner when placed in wild-type primordia, but that they are specifically excluded from the leading edge. Moreover, a remarkably small number of SDF1a-responsive cells are able to organize an entire cxcr4b mutant primordium to restore migration and organogenesis in the lateral line. These results reveal a role for chemokine signaling in mediating the self-organizing migration of tissues during morphogenesis.
Two distinct mechanisms, vasculogenesis and angiogenesis implement the formation of the vascular network in the embryo. Vasculogenesis gives rise to the heart and the first primitive vascular plexus inside the embryo and in its surrounding membranes, as the yolk sac circulation. Angiogenesis is responsible for the remodeling and expansion of this network. While vasculogenesis refers to in situ differentiation and growth of blood vessels from mesodermal derived hemangioblasts, angiogenesis comprises two different mechanisms: endothelial sprouting and intussusceptive microvascular growth (IMG). The sprouting process is based on endothelial cell migration, proliferation and tube formation. IMG divides existing vessel lumens by formation and insertion of tissue folds and columns of interstitial tissue into the vessel lumen. The latter are termed interstitial or intervascular tissue structures (ITSs) and tissue pillars or posts. Intussusception also includes the establishment of new vessels by in situ loop formation in the wall of large veins. The molecular regulation of these distinct mechanisms is discussed in respect to the most important positive regulators, VEGF and its receptors flk-1 (KDR) and flt-1, the Angiopoietin/tie system and the ephrin-B/EpH-B system. The cellular mechanisms and the molecular regulation of angiogenesis in the pathological state are summarized and the differences of physiological and pathological angiogenesis elaborated.
Wholemount immunohistochemistry (IHC) and immunofluorescence (IF) were performed as described
- Gerhardt
Wholemount immunohistochemistry (IHC) and immunofluorescence (IF) were performed as described (http://spot.colorado.edu/~klym/) (Gerhardt et al., 2003).
Collective cell migration in morphogenesis and cancer
- P Friedl
- Y Hegerfeldt
- M Tusch
Friedl, P., Hegerfeldt, Y. and Tusch, M. (2004). Collective cell migration in
morphogenesis and cancer. Int. J. Dev. Biol. 48, 441-449.
Spatially restricted patterning cues provided by heparin-binding VEGF-A control blood vessel branching morphogenesis
- C Ruhrberg
- H Gerhardt
- M Golding
- R Watson
- S Ioannidou
- H Fujisawa
- C Betsholtz
- D T Shima
Ruhrberg, C., Gerhardt, H., Golding, M., Watson, R., Ioannidou, S.,
Fujisawa, H., Betsholtz, C. and Shima, D. T. (2002). Spatially restricted
patterning cues provided by heparin-binding VEGF-A control blood vessel
branching morphogenesis. Genes Dev. 16, 2684-2698.
A model describing how EGFL7 regulates sprout morphology and motility. DEVELOPMENT biosynthesis, remodeling, and functions during vascular morphogenesis and neovessel stabilization
- Fig
Fig. 8. A model describing how EGFL7 regulates sprout morphology and motility.
DEVELOPMENT
biosynthesis, remodeling, and functions during vascular morphogenesis and
neovessel stabilization. Circ. Res. 97, 1093-1107.