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Antagonist of microRNA-21 improves balloon injury-induced rat iliac artery remodeling by regulating proliferation and apoptosis of adventitial fibroblasts and myofibroblasts
Abstract
Molecular pathways involved in adventitial fibroblasts (AFs) and myofibroblasts (MFs) proliferation and apoptosis contribute to vascular remodeling. MicroRNA-21 (miR-21) plays an important role in regulating cellular proliferation and apoptosis of many cell types; however, the effect of miR-21 on AFs and MFs is still unknown. In this study, we found that miR-21 was expressed in AFs and overexpressed in MFs. Inhibition of miR-21 decreased proliferation and increased apoptosis of AFs and MFs, and overexpression of miR-21 with pre-miR-21 had the reverse effect. Programmed cell death 4 (PDCD4), related to cell proliferation and apoptosis, was validated as a direct target of miR-21 by dual-luciferase reporter assay and gain and loss of function of miR-21 in AFs and MFs. PDCD4 knockdown with siRNA partly rescued the reduced proliferation with miR-21 inhibition and alleviated the increased apoptosis induced by miR-21 inhibition in AFs and MFs. Moreover, increasing PDCD4 expression by miR-21 inhibition significantly decreased JNK/c-Jun activity. In contrast, decreasing PDCD4 expression by pre-miR-21 treatment increased JNK/c-Jun activity, while the effect of miR-21 inhibition on JNK/c-Jun activity could be rescued by PDCD4 siRNA. Moreover, miR-21 inhibition could regulate proliferation and apoptosis of vascular AFs and MFs in vivo. Furthermore, miR-21 inhibition reversed vascular remodeling induced by balloon injury. In summary, our findings demonstrate that miR-21 may have a critical role in regulating proliferation and apoptosis of AFs and MFs, and PDCD4 is a functional target gene involved in the miR-21-mediated cellular effects in vascular remodeling by a miR-21/PDCD4/JNK/c-Jun pathway.
... So far, many microRNAs related to vascular development have been reported, and many are related to vascular smooth muscle cells. For example, miR-21 has an important regulatory role in the proliferation and migration of vascular smooth muscle cells [64], and There are also higher levels of expression in endothelial cells [65]. Down-regulation of miR-21 expression increases apoptosis and inhibits the proliferation of adventitial fibroblasts and myofibroblasts [66]. ...
... In vitro experiments in serum-depleted conditions, human or mouse aortic smooth muscle cell differentiation reduces miR-21 expression [65]. In vitro experiments in mouse aortic smooth muscle cells and injured mouse carotid arteries, silencing of miR-21 will inhibit cell proliferation and increase apoptosis [65]. ...
... In vitro experiments in serum-depleted conditions, human or mouse aortic smooth muscle cell differentiation reduces miR-21 expression [65]. In vitro experiments in mouse aortic smooth muscle cells and injured mouse carotid arteries, silencing of miR-21 will inhibit cell proliferation and increase apoptosis [65]. In mice, knockdown of miR-21 inhibits vascular remodeling in carotid injury [65]. ...
The development of the vertebrate vascular system is an extremely important and complex process. The circulatory system is the first organ system to develop during embryogenesis. The development of the vasculature into highly branched canals must occur clearly in many places in order to supply oxygen and nutrients to the rapidly developing embryo. This process is mediated by a coordinated response of vascular endothelial and parietal cells to heterogeneous angiogenic signals provided by tissues and organs. MicroRNAs regulate gene expression at the transcriptional and post-transcriptional levels and participate in many important physiological and pathological processes. MicroRNAs mainly play an important role in the developmental regulation of vascular smooth muscle cells and vascular endothelial cells. This article summarizes the research progress of microRNAs in vascular development in recent years, focusing on the regulatory mechanism of miR-126 and miR-17/92 families in vascular endothelial cells, as well as the miR-143/145 family, miR-21 in vascular smooth muscle cell's regulation. The research prospects of the role of microRNAs in vascular development are also presented in this article.
... Based on these data, therefore, we propose that the infiltrating macrophage may contribute to transplantation-induced neointima formation by stimulating miRNA expressions in vascular mural cells. In particular, we selected miR-21 as a target to prove this concept, because miR-21 has been shown to have crucial effects in the pathogenesis of neointima formation in angioplastic and vein graft models [19,[27][28][29][30]. ...
... Oncotarget 30106 www.impactjournals.com/oncotarget arterial neointima formation in various animal models [19,27,29,30]. Using this established molecule as a test target, here we provided evidence suggesting that macrophages could promote transplantation-induced neointima formation by promoting miRNA expression in mural cells. ...
... An interesting finding in the present study is that macrophage can stimulate miRNA expressions in vascular mural cells via a paracrine signaling mechanism. Specifically, using miR-21 as an example of vascular enriched miRNA [19,27,29,30], we have identified that macrophages may promote miR-21 expression in VSMCs and adventitial fibroblasts via the liberation of TNF-α. We suggest that this process may represent a novel mechanism by which macrophages contribute to the development of pathological neointimal remodeling. ...
In this study, we tested the possibility that macrophages might contribute to neointima formation by stimulating microRNA expressions in mural cells. Thoracic aortas from F344 rats were transplanted into recipient Lewis rats. Clodronate liposome was used for in vivo macrophage depletion. Using miR-21 as a prototypic example of vascular enriched microRNA, we showed that macrophage depletion reduced the expression level of miR-21, which was upregulated in the allograft. This effect of macrophage depletion was accompanied by attenuations in neointimal hyperplasia and transplantation-induced vascular inflammation. Using in vitro assays, we identified that macrophages might stimulate miR-21 expression in smooth muscle cells and adventitial fibroblasts via the release of tumor necrosis factor-α. We also showed that silencing of miR-21 suppressed tumor necrosis factor-induced proliferation, migration, and inflammatory responses in mural cells. Our results suggest that macrophage may promote transplantation-induced neointima formation by stimulating miR-21 expression in vascular mural cells, which promotes mural cell proliferation, migration and/or inflammation. Moreover, we have established that tumor necrosis factor-α has a major role in mediating this paracrine process.
... Based on these data, therefore, we propose that the infiltrating macrophage may contribute to transplantation-induced neointima formation by stimulating miRNA expressions in vascular mural cells. In particular, we selected miR-21 as a target to prove this concept, because miR-21 has been shown to have crucial effects in the pathogenesis of neointima formation in angioplastic and vein graft models [19,[27][28][29][30]. ...
... MiR-21 has been implicated in angioplasty-induced arterial neointima formation in various animal models [19,27,29,30]. Using this established molecule as a test target, here we provided evidence suggesting that macrophages could promote transplantation-induced neointima formation by promoting miRNA expression in mural cells. ...
... An interesting finding in the present study is that macrophage can stimulate miRNA expressions in vascular mural cells via a paracrine signaling mechanism. Specifically, using miR-21 as an example of vascular enriched miRNA [19,27,29,30], we have identified that macrophages may promote miR-21 expression in VSMCs and adventitial fibroblasts via the liberation of TNF-α. We suggest that this process may represent a novel mechanism by which macrophages contribute to the development of pathological neointimal remodeling. ...
In this study, we tested the possibility that macrophages might contribute to neointima formation by stimulating microRNA expressions in mural cells. Thoracic aortas from F344 rats were transplanted into recipient Lewis rats. Clodronate liposome was used for in vivo macrophage depletion. Using miR-21 as a prototypic example of vascular enriched microRNA, we showed that macrophage depletion reduced the expression level of miR-21, which was upregulated in the allograft. This effect of macrophage depletion was accompanied by attenuations in neointimal hyperplasia and transplantation-induced vascular inflammation. Using in vitro assays, we identified that macrophages might stimulate miR-21 expression in smooth muscle cells and adventitial fibroblasts via the release of tumor necrosis factor-α. We also showed that silencing of miR-21 suppressed tumor necrosis factor-induced proliferation, migration, and inflammatory responses in mural cells. Our results suggest that macrophage may promote transplantation-induced neointima formation by stimulating miR-21 expression in vascular mural cells, which promotes mural cell proliferation, migration and/or inflammation. Moreover, we have established that tumor necrosis factor-α has a major role in mediating this paracrine process.
... PDCD4 also plays a role in cardiovascular cell biology by inhibiting proliferation and inducing apoptosis of most cardiovascular cells, including vascular smooth muscle cells [9,10], cardiac myocytes [11] and fibroblasts [12], and repressing contractile gene expression in vascular smooth muscle cells [13]. However, the action of PDCD4 in endothelial cells is unclear. ...
... The ubiquitin-proteasome pathway mediates the degradation of PDCD4 under various conditions [18,19], and miR-21 negatively regulates PDCD4 expression translationally in various tumor cells [20] and other cardiovascular cells [9,11,12]. We investigated whether these pathways were involved in decreased PDCD4 level with pulsatile shear stress. ...
... As a tumor suppressor, PDCD4 inhibits the oncogenesis and progression of cancers and improves the sensitivity of tumor cells to chemotherapy [1]. In the cardiovascular system, PDCD4 was found mainly as an inducer of apoptosis and a suppressor of proliferation [9][10][11][12]. Here in HUVECs, PDCD4 is shown to be an inducer of both proliferation and apoptosis. ...
We recently found low level of tumor suppressor programmed cell death 4 (PDCD4) associated with reduced atherosclerotic plaque area (unpublished). We investigated whether atheroprotective unidirectional pulsatile shear stress affects the expression of PDCD4 in endothelial cells.
En face co-immunostaining of the mouse aortic arch revealed a low level of PDCD4 in endothelial cells undergoing pulsatile shear stress. Application of unidirectional pulsatile shear stress to human umbilical vein endothelial cells (HUVECs) decreased PDCD4 protein but not mRNA level. Immunoprecipitation revealed that pulsatile shear stress induced the coupling of ubiquitin with PDCD4 expression. The phosphatidyl inositol 3-kinase (PI3K)/Akt pathway was involved in this ubiquitin-proteasome-mediated degradation of PDCD4. Gain of function and loss of function experiments showed that PDCD4 induced turnover (proliferation and apoptosis) of HUVECs. Low PDCD4 level was associated with reduced proliferation but not apoptosis or phosphorylation of endothelial nitric oxide synthase caused by pulsatile shear stress to help maintain the homeostasis of endothelial cells.
Pulsatile shear stress induces ubiquitin-proteasome-mediated degradation of PDCD4 via a PI3K/Akt pathway in HUVECs. PDCD4 induces turnover (proliferation and apoptosis) of HUVECs. Low PDCD4 level is associated with reduced proliferation for maintenance of HUVEC homeostasis under pulsatile shear stress.
... PTEN is an established target known to be expressed in fibroblasts, EC, and SMCs, where it regulates cell survival/apoptosis and is thought to have a key role in many cardiovascular diseases.32 In agreement with previous studies,14,26,33 we demonstrated an increase in PTEN expression in treated vessels. We also found significant de-repression of STAT3 and BMPR2, which have been shown to be direct targets of miR-21 in mesenchymal stem cells31 and pulmonary vascular SMCs,34 respectively. ...
... Global miRNA profiling analysis revealed that 21 out of 377 miRNAs were up-regulated following engraftment (see Supplementary material online, Figure S1 and Table S1), 6 of which were up-regulated more than 4-fold (FDR < 0.1) at 7 and 28 days post-engraftment (Figure 1B). From this data set, we focused on miR-21 due to the substantial up-regulation observed in pig grafts compared with controls (Figure 1C) and since it has previously been implicated in SMC and fibroblast proliferation following acute vascular injury.25,26 ...
... This is consistent with previous reports showing that knockdown of miR-21 levels in arterial SMCs and fibroblasts reduced rates of proliferation, migration, and neointimal formation in a rat balloon injury model, which was at least in part caused by de-repression of PTEN and BCL-2.14,25,26 A subsequent study in a mouse model of abdominal aneurysm demonstrated that lentiviral-mediated over-expression of miR-21 reduced PTEN expression and increased SMC proliferation.33 ...
AimsThe long-term failure of autologous saphenous vein bypass grafts due to neointimal thickening is a major clinical burden. Identifying novel strategies to prevent neointimal thickening is important. Thus, this study aimed to identify microRNAs (miRNAs) that are dysregulated during neointimal formation and determine their pathophysiological relevance following miRNA manipulation.Methods and resultsWe undertook a microarray approach to identify dysregulated miRNAs following engraftment in an interpositional porcine graft model. These profiling experiments identified a number of miRNAs which were dysregulated following engraftment. miR-21 levels were substantially elevated following engraftment and these results were confirmed by quantitative real-time PCR in mouse, pig, and human models of vein graft neointimal formation. Genetic ablation of miR-21 in mice or grafted veins dramatically reduced neointimal formation in a mouse model of vein grafting. Furthermore, pharmacological knockdown of miR-21 in human veins resulted in target gene de-repression and a significant reduction in neointimal formation.Conclusion
This is the first report demonstrating that miR-21 plays a pathological role in vein graft failure. Furthermore, we also provided evidence that knockdown of miR-21 has therapeutic potential for the prevention of pathological vein graft remodelling.
... We further explored the functional molecules that mediate the pro-calcification effect of AFs HPi -Exos. We first selected eight miRNAs that have been reported to be highly expressed in fibroblasts and associated with osteogenic differentiation [44][45][46][47][48][49][50][51][52][53][54][55][56][57][58][59][60]. qRT-PCR analysis was used to detect the expression levels of 8 miRNAs in AFs NPi -Exos and AFs HPi -Exos. ...
... The 5' seed region (bases 2-8) of miRNAs can bind to the 3'-UTR region of mRNAs to inhibit the expression of target genes by inducing mRNA degradation or translational repression [78][79][80][81][82][83]. MiR-21-5p was found to be expressed in rat AFs, with higher expression in myofibroblasts (MFs), furthermore, down-expression of miR-21 enhanced the expression of PDCD4 and inhibited the JNK/c-Jun pathway, thereby decreasing the proliferation and increasing the apoptosis of AFs and MFs, while overexpression of miR-21 had the opposite effects [60]. Macrophages may stimulate the expression of miR-21 in VSMCs and AFs by releasing TNF-α, thereby promoting the proliferation and migration of VSMCs and AFs [57]. ...
Vascular calcification often occurs in patients with chronic renal failure (CRF), which significantly increases the incidence of cardiovascular events in CRF patients. Our previous studies identified the crosstalk between the endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), and the paracrine effect of VSMCs, which regulate the calcification of VSMCs. Herein, we aim to investigate the effects of exosomes secreted by high phosphorus (HPi) -induced adventitial fibroblasts (AFs) on the calcification of VSMCs and the underlying mechanism, which will further elucidate the important role of AFs in high phosphorus vascular wall microenvironment. The conditioned medium of HPi-induced AFs promotes the calcification of VSMCs, which is partially abrogated by GW4869, a blocker of exosomes biogenesis or release. Exosomes secreted by high phosphorus-induced AFs (AFsHPi-Exos) show similar effects on VSMCs. miR-21-5p is enriched in AFsHPi-Exos, and miR-21-5p enhances osteoblast-like differentiation of VSMCs by downregulating cysteine-rich motor neuron 1 (Crim1) expression. AFsHPi-Exos and exosomes secreted by AFs with overexpression of miR-21-5p (AFsmiR21M-Exos) significantly accelerate vascular calcification in CRF mice. In general, AFsHPi-Exos promote the calcification of VSMCs and vascular calcification by delivering miR-21-5p to VSMCs and subsequently inhibiting the expression of Crim1. Combined with our previous studies, the present experiment supports the theory of vascular wall microenvironment.
Graphical Abstract
... 13 They also demonstrated that PDCD4 functioned as a negative regulator of various phenotypic genes of myofibroblasts, 13 which was consistent with our data showing overexpression of PDCD4 downregulated the myofibroblast features. Moreover, one of the recent studies has shown that knockdown of PDCD4 partly rescued the decreased proliferation of myofibroblasts in response to miR-21 inhibition, 14 which was also in line with our finding. Besides, Wang et al. demonstrated that elevation of PDCD4 expression markedly decreased JNK/c-Jun activity, 14 a known pathway that may antagonize the TGF-b1/Smad2 signaling. ...
... Moreover, one of the recent studies has shown that knockdown of PDCD4 partly rescued the decreased proliferation of myofibroblasts in response to miR-21 inhibition, 14 which was also in line with our finding. Besides, Wang et al. demonstrated that elevation of PDCD4 expression markedly decreased JNK/c-Jun activity, 14 a known pathway that may antagonize the TGF-b1/Smad2 signaling. 15 PDCD4 has been shown to involve in the miR-21-regulated cardiac epithelial-to-mesenchymal transition (EMT), 16 a possible source of myofibroblast. ...
Background/purpose
Oral submucous fibrosis (OSF) has been regarded as a premalignant disorder of oral cancer, and myofibroblasts are the main cells that are responsible for pathological fibrosis. Hence, elucidation of the molecular mechanism underlying myofibroblast activation is important to treat OSF. MicroRNA-21 (miR-21) is a well-known fibrosis non-coding RNA, and its role in the development of OSF remains largely unclear.
Materials and methods
Luciferase reporter assay was used to confirm the direct interaction between miR-21 and its target programmed cell death 4 (PDCD4). The expression level of PDCD4 in OSF was examined by qRT-PCR. Myofibroblast activities were assessed by collagen gel contraction and transwell migration assays.
Results
Our result validated the direct binding of miR-21 to PDCD4. We showed the expression of PDCD4 was downregulated in OSF specimens and negatively correlated with miR-21. Our results suggested that overexpression of PDCD4 in fibrotic buccal mucosal fibroblasts (fBMFs) mitigated the myofibroblast activities, including collagen gel contractility and migration capacity. Moreover, we showed miR-21 contributed to myofibroblast activation of BMFs through repression of PDCD4.
Conclusion
Our results suggest that the miR-21/PDCD4 axis mediates the myofibroblast activation of BMFs, and targeting this axis may exert an anti-fibrosis effect.
... 23 miRNA-21 was the first miRNA demonstrated to be involved in the regulation of vascular smooth muscle cell phenotype; it is highly expressed in both vascular smooth muscle and endothelial cells and it promotes their differentiation, proliferation, and apoptosis. 24 Inhibition of miRNA-21 decreases the proliferation of smooth muscle cells and increases their apoptosis. 24 miRNA-21 is upregulated in proliferative vessels, as was confirmed in a mouse ligation model. ...
... 24 Inhibition of miRNA-21 decreases the proliferation of smooth muscle cells and increases their apoptosis. 24 miRNA-21 is upregulated in proliferative vessels, as was confirmed in a mouse ligation model. 25 Moreover, increased shear stress, as in arterial hypertension, induces the expression of miRNA-21 at the transcriptional level in cultured human umbilical vein endothelial cells via an increased binding of c-Jun to the promoter region of miRNA-21. ...
MicroRNAs (miRNAs) play a crucial role in myocardial and vascular remodeling and have emerged as potential diagnostic and prognostic biomarkers or as therapeutic targets. The authors aimed to investigate the expression profile of selected miRNAs in the peripheral blood of patients with well-controlled essential hypertension in relation to arterial stiffness. Expression levels of miRNAs miRNA-1, miRNA-133a, miRNA-26b, miRNA-208b, miRNA-499, and miRNA-21 in peripheral blood mononuclear cells were quantified by real-time reverse transcription polymerase chain reaction. Carotid-femoral pulse wave velocity (cfPWV) and carotid radial pulse wave velocity (crPWV) were evaluated at baseline and after 1 year of effective antihypertensive therapy. A total of 95 patients (50 men, mean age 62±9 years) with well-controlled essential hypertension were included in the analysis. Only miRNA-21 was independently correlated with changes in both cfPWV and crPWV, independently of blood pressure levels (r=−0.56 and r=−0.46, respectively; P<.001 for both). Low levels of miRNA-21 are strongly associated with an improvement in arterial stiffness in patients with well-controlled essential hypertension, independently of their blood pressure levels. These data highlight the significance of miRNA-21 in vascular remodeling and its role as a potential prognostic marker and future therapeutic target.
... 16 miR-21 is also implicated in vascular remodelling. 17 This effect is partly mediated via silencing of programmed cell death 4 (PDCD4). Inhibition of miR-21 reversed vascular remodelling induced by balloon injury. ...
... Inhibition of miR-21 reversed vascular remodelling induced by balloon injury. 17 In the skeletal muscle, fibrosis is directly implicated in the death of patients suffering from Duchenne muscular dystrophy. Recently, it was demonstrated that extracellular plasminogen activator inhibitor-1 (PAI-1)/urokinase-type plasminogen activator balance regulates miR-21 biogenesis. ...
This editorial refers to an article by S. Roy et al .[1][1] published in Cardiovascular Research in 2009. It is accompanied by an editorial by J. Bauersachs (doi:10.1093/cvr/cvs200) as part of this Spotlight on Landmark Papers in Cardiovascular Research.
This editorial celebrates the extraordinary citation of our recent publication1 in Cardiovascular Research highlighting the emerging significance of miR-21 in the heart, particularly in the context of tissue injury, inflammation, and remodelling. The work was first submitted to a leading cardiovascular journal in January 2008. The submission was summarily rejected, citing ‘paucity of novel mechanistic insight’. The work went on to be published in Cardiovascular Research and has now been cited 150 times in 3 years, being recognized as one of the most highly cited articles in the journal. Had the work been published in the original journal it would have been published almost a year in advance and would have today ranked third in citation score among all of nearly 300 articles published in that journal in 2009. Here, we briefly review the progress in the field since our original work was published.⇓
Box 1
Title page including abstract from the original 2009 publication in Cardiovascular Research .1 Used with permission of Oxford University Press on behalf of the European Society of Cardiology.
Identified as one of the first mammalian microRNAs (miRs), the miR-21 sequence is strongly conserved throughout evolution. During the early phase of development of miR-21 biology, most of the target coding genes reported were tumour suppressors. Thus, miR-21 represents one of the first ‘oncomirs’ named. Two concurrent works1,2 published by the Engelhardt laboratory2 and us1 markedly changed the landscape by introducing the cardiac fibroblast as a major hub for miR-21 action. Taken together, these studies drew attention to the role of miR-21 …
[1]: #ref-1
... In addition to the role of PDCD4 in tumor progression, PDCD4 was also shown to be involved in glucose and lipid metabolism disorders [8], oxidative stress [9], inflammatory responses [10], and intestinal microflora imbalance [11]. PDCD4 also plays a role in regulating cardiovascular diseases by inhibiting proliferation and inducing apoptosis of most cardiovascular cells, including VSMCs [12], cardiac myocytes [13], and fibroblasts [14]. PDCD4 expression is suppressed by cardiac progenitor cell-derived exosomal miR-21, which protects myocardial cells against oxidative stress-related apoptosis [15]. ...
WWP2 is a HECT-type E3 ubiquitin ligase that regulates various physiological and pathological activities by binding to different substrates, but its role in atherosclerosis (AS) remains largely unknown. The objective of the present study is to investigate the role and underlying molecular mechanisms of WWP2 in endothelial injury. We found that WWP2 expression is significantly decreased in Apolipoprotein E (ApoE) mice. Overexpression of WWP2 attenuates oxidative stress and inflammation in AS mice, while knockdown of WWP2 has opposite effects. WWP2 overexpression alleviates oxidized low-density lipoprotein (ox-LDL)-induced human umbilical vein endothelial cells (HUVECs) injury, evidenced by the decreased oxidative stress levels and the secretion of inflammatory cytokines. Programmed cell death 4 (PDCD4) is identified as a potential substrate of WWP2. Co-immunoprecipitation (Co-IP) further demonstrates that WWP2 interacts with PDCD4, which is enhanced by ox-LDL treatment. Furthermore, the level of PDCD4 ubiquitination is significantly increased by WWP2 overexpression under the condition of MG132 treatment, while WWP2 knockdown shows opposite results. Subsequently, rescue experiments demonstrate that WWP2 knockdown further aggravates oxidative stress and inflammation in ox-LDL-treated HUVECs, while knockdown of PDCD4 alleviates this effect. Moreover, the use of sn-protoporphyrin (SnPP), an inhibitor of HO-1 pathway, confirms that PDCD4 enhances endothelial injury induced by ox-LDL through inhibiting HO-1 pathway. In conclusion, our results suggest that WWP2 protects against atherosclerosis progression via the PDCD4/HO-1 pathway, which may provide a novel treatment strategy for atherosclerosis.
... miR-21 is overexpressed in response to high glucose and protects endothelial cells from apoptosis ( Zeng et al., 2013). Antagonists of miRNA-21 can regulate the proliferation and apoptosis of adventitial fibroblasts and myofibroblasts ( Wang et al., 2012). miR-21 is the only post-transcriptional regulator that has a highly conserved effect on apoptosis in species. ...
Aim:
The target molecule regulatory function of microRNA-21 (miR-21) in multiple signalling pathways has become a main focus of genetic and pharmacological regulatory studies of various diseases. The identification of target genes for miRNA-21 in the development of hair follicles can provide new research pathways for the regulation of cell development.
Methods:
In the present study, eight six-month-old ewes from Super Merino (SM) and Small Tailed Han (STH) sheep breeds were selected. Target prediction and dual-luciferase wild-type and mutant vectors were used to identify the target genes of miR-21. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) and bioinformatics analysis were conducted to analyze the effects of miR-21.
Results:
The results show that the expressions of CNKSR2, KLF3 and TNPO1 were downregulated by miRNA-21 at rates of 36%, 26% and 48%, respectively. Moreover, there was a significant negative correlation between the expression of miR-21 and the three target genes in sheep with two extreme phenotypes. The expression of microRNA-21in October was significantly lower than that in January and February; while the expression of CNKSR2, KLF3 and TNPO1 in October was higher than that in January and February. Conclusions: These results suggest that CNKSR2, KLF3 and TNPO1 are three newly discovered target genes of miR-21 and might be involved in the effects of miR-21 on hair follicle development.
... These findings demonstrate that PDCD4 gene silencing may play a protective role in vascular injury. Wang et al. have confirmed in their studies that PDCD4 gene participates in vascular remodeling through regulation of the JNK signaling pathway [22]. ...
Background
We aimed to investigate the role of PDCD4-mediated Akt signaling pathway in vascular endothelial cell injury caused by ischemia-reperfusion in the lower extremities.
Material/Methods
Ten rats were used as control, while 50 rats were used for creating disease models and were assigned to 5 groups: model group (no injection), NC group (injected with vectors containing PDCD negative control sequence), sh-PDCD4 group (injected with vectors containing sh-PDCD4 sequence), IGF-1 group (injected with IGF-1), and sh-PDCD4+IGF-1 group (injected with IGF-1 and vectors containing sh-PDCD4 sequence).
Results
Compared with the control group, the expression levels of PDCD4 mRNA and protein, as well as levels of circulating endothelial cells, von Willebrand factor, thrombomodulin, and malondialdehyde, increased in the other 5 groups, while the mRNA and protein expression levels of Akt and eNOS, the protein expression levels of p-Akt and p-eNOS, and superoxide dismutase content decreased in these groups (all P<0.05). Compared with the model group, the sh-PDCD4 and sh-PDCD4+1GF-1 groups had lower mRNA and protein expressions of PDCD4 (all P<0.05), whereas the IGF-1 group had similar levels (all P>0.05). These 3 groups had lower levels of circulating endothelial cells, von Willebrand factor, thrombomodulin, and malondialdehyde, and higher mRNA and protein expressions of Akt and eNOS, protein expressions of p-Akt and p-eNOS, and superoxide dismutase content (all P<0.05). The NC group did not differ from the model group (all P>0.05).
Conclusions
PDCD4 gene silencing can activate the Akt signaling pathway and attenuate vascular endothelial cell injury caused by ischemia-reperfusion in the lower extremities in rats.
... Overexpression of miR-21 by using pre-miR-21 treatment increases the proliferation and decreases apoptosis of AFs and MFs. Furthermore, miR-21 inhibition can reverse the vascular remodeling induced by balloon injury [71]. In cardiac remodeling, abnormal up-regulation of miR-21 targets SPRY1 and hence augments the ERK-MAPK signaling in cardiac fibroblast, which leads to fibroblast survival and its differentiation into myocyte [72]. ...
MicroRNA-21 (miR-21), probably one of the most studied miRNAs to date, is found pleiotropic in various biological events. Its emerging role in pulmonary remodeling has attracted extensive attention. This review summarizes the genomic information of its primary transcript and various transcriptional regulations on its promoter. In addition, the role of miR-21 in pulmonary remodeling related signaling such as transforming growth factor β (TGF-β), bone morphogenetic protein (BMP), epidermal growth factor receptor (EGFR) and Notch signaling is discussed. Various validated miR-21 target genes participate in controlling of the overactive cell accumulation, smooth muscle contraction, inflammatory stress (trigger for lung epithelium damage), extracellular matrix deposition and hypoxia-induced disorders. Moreover, we focus on its particular implication in events including inflammatory stress-driven epithelium damage, epithelial-to-mesenchymal transition (EMT), transdifferentiation of fibroblasts into myofibroblasts, hypoxia stimuli and ROS response, as well as some other pulmonary remodeling related events such as overactive fibroblast (myofibroblast) accumulation, extracellular matrix deposition, and angiogenesis. Here, we summarize the strong potential of miR-21 in pulmonary remodeling and provide novel clues for further research in this area.
... miR-21 has been highlighted due to its importance in tumor progression and metastasis, specifically in the process of cell proliferation and differentiation, which is vital to chondrogenesis and cartilage remodeling [13][14][15]. However, little is known about the function of miR-21 in OA. ...
Objective
Osteoarthritis (OA) is a prevalent degenerative disease caused by various factors. MicroRNAs are important regulators in OA. MiR-21-5p expression is decreased in OA cartilage, but the effects of modulating miR-21-5p on cartilage regeneration are unknown. Therefore, our aim was to investigate the effects of miR-21-5p on cartilage metabolism of OA chondrocytes.
Design
We used IL-1β (10 ng/ml) to mimic OA chondrocytes. OA chondrocytes were transfected with miR-21-5p, the gene expression of COL2A1, MMP13, and ADAMTS5 was detected by qPCR. At the same time, COL2A1, MMP13, and ADAMTS5 were analyzed at the protein level by Western blot. CCK8 measured the cell’s viability and SA-β-gal detected the cell’s senescence.
Results
Upregulation of miR-21-5p had increased COL2A1 expression and decreased MM P13 and ADAMTS5 expression, which were in accord with Western blot data. SA-β-gal activity significantly increased, the viability was decreased in OA chondrocytes, and upregulation of miR-21-5p can decrease the SA-β-gal activity and increase cell viability.
Conclusion
MiR-21-5p might be a potential disease-modifying compound in OA, as it promotes hyaline cartilage production. These results provided that novel insights into the important function in OA pathological development.
... Programmed cell death 4 (PDCD4) was first identified as a tumor inhibitor and its expression is significantly increased by apoptosis [6]. In addition to tumor cells, cardiac muscle and endothelial cells also express PDCD4 [7] which is known to play a critical function in cardiovascular cell biology by inhibiting the expression of contractile genes in vascular smooth muscle cells and by triggering apoptosis and suppressing proliferation of many types of cardiovascular cells, such as fibroblasts, cardiac myocytes and vascular smooth muscle cells (endothelial cells) [7][8][9][10]. Low PDCD4 level was related to lower proliferation of ECs [11]. ...
Background/aims:
LncRNA is a growth arrest-specific transcript 5 (GAS5) with tumor suppressor activities in some cancers, but its role in atherosclerosis is unclear.
Methods:
Bioinformatics algorithm analysis was utilized to search the target of GAS5 and miR-21, followed by luciferase assay to confirm these targets. Real-time PCR and western-blot were utilized to verify the connection among GAS5, miR-21 and Programmed cell death 4 (PDCD4). MTT assay and flow cytometry analysis were performed to explore the mechanism of GAS5 in the regulation of atherosclerosis.
Results:
GAS5 directly targets miR-21 and functions as a competing endogenous RNA to suppress miR-21 expression. We also observed that rs145204276 polymorphism, including INS/INS and DEL/DEL, on GAS5 promoter increased transcription activity of GAS5, but the presence of rs145204276 DEL/DEL allele significantly promoted the transcription of GAS5 promoter compared with rs145204276 INS/INS allele. PDCD4 was predicted as a direct target gene of miR-21 with a binding site on PDCD4 3'UTR. It was further confirmed by luciferase assay that miR-21 significantly reduced the luciferase activity of wild-type PDCD4 3'UTR but not that of mutant PDCD4 3'UTR. In addition, high glucose significantly inhibited the growth rate of EC genotyped as DEL/DEL or INS/ INS, and apparently promoted the apoptotic rate of either DEL/DEL or INS/INS genotype ECs. Furthermore, the effect of high glucose was stronger in the INS/INS group, while the expression of GAS5 was dramatically upregulated with the presence of GAS5 DEL/DEL, while GAS5 positively regulated PDCD4 expression via inhibiting miR-21 expression. GAS5 siRNA and miR-21 mimics significantly decreased GAS5 and PDCD4 expressions, and the inhibitory effects of GAS5 siRNA or miR-21 mimics on GAS5 and PDCD4 expressions in the INS/INS group was stronger. Moreover, GAS5 siRNA and miR-21 mimics remarkably triggered cells proliferation and suppressed cell apoptosis, and the inhibition effects of GAS5 siRNA or miR-21 mimics on either cell viability and apoptosis in the INS/INS group was stronger. In this study, we enrolled 1,306 subjects with or without atherosclerosis and found that the INS/DEL or DEL/DEL genotypes significantly decreased the risk of atherosclerosis compared with the ins/ins genotype (adjusted odds ratio: 0.74 and 0.40, respectively).
Conclusion:
In summary, rs145204276 was associated with the risk of atherosclerosis by affecting the proliferation and apoptosis of endothelial cells via regulating the GAS5/miR-21/PDCD4 signaling pathway.
... For the CCK-8 assay, cells were seeded into a 96-well plate andCCK-8 solution added. For the EdU incorporation assay, 5×10 3 cells/well were seeded into 96-well plates, transfected for 24 h, and incubated with EdU for 2 h [13]. The EdU incorporation rate was expressed as the ratio of EdU-positive to total Hoechst 33342-positive cells (blue cells). ...
Background/aims:
Keloids are fibrous overgrowths induced by cutaneous injury. MicroRNAs (miRNAs) have recently emerged as post-transcriptional gene repressors and participants in a diverse array of pathophysiological processes leading to skin disease. The purpose of the current study was to explore the precise functions of miR-181a in human keloid development and the underlying mechanisms.
Methods:
A miRNA microarray analysis was performed to compare expression profiles between keloid and normal skin tissues. Quantitative real-time PCR was conducted to estimate miR-181a expression. Cell proliferation was determined using the cell counting kit-8 (CCK-8) and 5-ethynyl-2-deoxyuridine (EdU) assays, and cell cycle and apoptosis were detected with flow cytometry. Direct targets of miR-181a were identified using the luciferase reporter assay.
Results:
miR-181a was significantly upregulated in human keloid tissues and fibroblasts, compared with their control counterparts. Overexpression of miR-181a enhanced keloid fibroblast DNA synthesis and proliferation and inhibited apoptosis, whereas miR-181a suppression triggered the opposite effects. Moreover, miR-181a suppressed the expression of PH domain leucine-rich repeat protein phosphatase 2 (PHLPP2) through direct interactions with its 3'UTR region and subsequently enhanced AKT activation. Overexpression of PHLPP2 without its 3'UTR attenuated the effects of miR-181a on cell proliferation and apoptosis in keloid fibroblast cells. Furthermore, miR-181a mimics increased normal skin fibroblast proliferation.
Conclusions:
Our results highlight a novel pathway mediated by miR-181a, which may be effectively used as a therapeutic target for treatment of keloids.
... 55 Interestingly, similar effects were observed in adventitial fibroblast and myofibroblast through the miR-21/PDCD4/JNK/c-Jun pathway. 57 In addition, miR-21 modulates VSMCs proliferation also in human pulmonary arteries. In this latter setting, miR-21 targets several molecules, including bone morphogenetic protein receptor type II, WWP1, YOD1, and SATB1, finally leading to the chronic hypoxia-induced vascular remodeling. ...
Percutaneous revascularization revolutionized the therapy of patients with coronary artery disease. Despite continuous technical advances that substantially improved patients' outcome after percutaneous revascularization, some issues are still open. In particular, restenosis still represents a challenge, even though it was dramatically reduced with the advent of drug-eluting stents. At the same time, drug-eluting stent thrombosis emerged as a major concern because of incomplete or delayed re-endothelialization after vascular injury. The discovery of microRNAs revealed a previously unknown layer of regulation for several biological processes, increasing our knowledge on the biological mechanisms underlying restenosis and stent thrombosis, revealing novel promising targets for more efficient and selective therapies. The present review summarizes recent experimental and clinical evidence on the role of microRNAs after arterial injury, focusing on practical aspects of their potential therapeutic application for selective inhibition of smooth muscle cell proliferation, enhancement of endothelial regeneration, and inhibition of platelet activation after coronary interventions. Application of circulating microRNAs as potential biomarkers is also discussed.
... The assay was performed in triplicate and repeated three times. The proliferation rate of the RASMCs was calculated as the percentage of EDU-positive nuclei to total nuclei in five high-power fields per well [20]. ...
Recent studies have shown that nicotine, a major component of cigarette smoke, can stimulate the proliferation of non-neuronal cells. Cigarette smoking can promote a variety of pulmonary and cardiovascular diseases, such as chronic obstructive pulmonary disease (COPD), atherosclerosis, and cancer. A predominant feature of COPD is airway remodeling, which includes increased airway smooth muscle (ASM) mass. The mechanisms underlying ASM remodeling in COPD have not yet been fully elucidated. Here, we show that nicotine induces a profound and time-dependent increase in DNA synthesis in rat airway smooth muscle cells (RASMCs) in vitro. Nicotine also significantly increased the number of RASMCs, which was associated with the increased expression of Cyclin D1, phosphorylation of the retinoblastoma protein (RB) and was dependent on the activation of Akt. The activation of Akt by nicotine occurred within minutes and depended upon the nicotinic acetylcholine receptors (nAchRs). Activated Akt increased the phosphorylation of downstream substrates such as GSK3β. Our data suggest that the binding of nicotine to the nAchRs on RASMCs can regulate cellular proliferation by activating the Akt pathway.
... 8 MiR-21 has been highlighted due to its importance in tumor progression and metastasis, specifically in the process of cell proliferation and differentiation, which is vital to chondrogenesis and cartilage remodeling. [11][12][13] However, the role of miR-21 in the progression of osteoarthritis and its underlying mechanism has remained unclear. GDF-5 acts as a signal for chondrogenesis and promotes chondrocyte differentiation. ...
Osteoarthritis is a common cause of functional deterioration in older adults and is an immense burden on the aging population. Altered chondrogenesis is the most important pathophysiological process involved in the development of osteoarthritis. However, the molecular mechanism underlying the regulation of chondrogenesis in patients with osteoarthritis requires further elucidation, particularly with respect to the role of microRNAs. MiR-21 expression in cartilage specimens was examined in 10 patients with knee osteoarthritis and 10 traumatic amputees. The effect of miR-21 on chondrogenesis was also investigated in a chondrocyte cell line. The effect of miR-21 on the expression of growth differentiation factor 5 (GDF-5) was further assessed by luciferase reporter assay and western blot. We found that endogenous miR-21 is upregulated in osteoarthritis patients, and overexpression of miR-21 could attenuate the process of chondrogenesis. Furthermore, we identified GDF-5 as the direct target of miR-21 during the regulation of chondrogenesis. Our data suggest that miR-21 has an important role in the pathogenesis of osteoarthritis and is a potential therapeutic target.
... Twentyfour hours after transfection, cells were incubated with EdU at 37°C and 5% CO 2 for 2 hours. 26 The cells were fixed using 4% paraformaldehyde and incubated with 2 mg/mL aminoacetic acid for 5 minutes. After washing with phosphate-buffered saline (PBS), the cells were incubated with penetrant for 10 minutes and then Apollo reaction cocktail (RiboBio Corporation, Guangzhou, China) was added for 30 minutes in the dark. ...
Background:
Micro-ribonucleic acids (miR) are small, noncoding RNA molecules 19 to 25 nucleotides in length that typically function as negative regulators of expression for many target genes involved in cell proliferation, differentiation, and apoptosis. However, the effects of miR-21 on keloid fibroblasts are currently unknown.
Objectives:
The authors investigate whether miR-21, a specific miR implicated in multiple aspects of keloid fibroblasts, affects the expression of Fas ligand (FasL) in the presence of transforming growth factor (TGF)-β1.
Methods:
The relationship between TGF-β1 and miR-21 expression was investigated by TaqMan quantitative real-time polymerase chain reaction (Life Technologies, Grand Island, New York). FasL protein was determined by Western blotting, and regulation of cell proliferation/migration/apoptosis ability by TGF-β1 inhibitor or plasmid was evaluated respectively by EdU incorporation, Transwell assay, and flow cytometry analysis.
Results:
Fibroblasts from keloid tissue were confirmed to express high levels of TGF-β1 and miR-21 compared with normal skin fibroblasts. Expression of TGF-β1 and miR-21 was positively correlated in fibroblasts. In addition, cells transfected with TGF-β1 inhibitor or miR-21 inhibitor showed significant increases in FasL protein levels and number of apoptotic cells compared with control cells, whereas cell growth and migration significantly decreased. The opposite results could also be confirmed when TGF-β1 was upregulated in normal skin fibroblasts.
Conclusions:
TGF-β1 could effectively influence cell proliferation, apoptosis, and migration via its control of miR-21. These findings also identify a novel mechanism of interaction between TGF-β1 and miR-21 in the regulation of FasL protein, which is involved in keloid formation.
... The stimulus for miR-21 upregulation is at least partly mediated via NF-κB upregulation, and as newer data suggest, also via cyclic stretch [64]. These results are in line with some previous publications [65][66][67] which have identified miR-21 as a proliferative, anti-apoptotic messenger in fibroblasts and SMCs. However, Davis et al. provide evidence [68] that miR-21 induction via TGF-ß may be involved in SMC differentiation, not proliferation, which could have the opposite effect. ...
Abdominal aortic aneurysms (AAA) are an important source of morbidity and mortality in the U.S. and worldwide. Treatment options are limited, with open surgery or endovascular repair remaining the only curative treatments. Classical cardiovascular medications have generally failed to prevent or significantly alter AAA formation or progression. Therefore, there is a tremendous need for better therapeutic approaches. With increasing knowledge of microRNA (miR) regulation in the context of cardiovascular disease, and with improving technical options permitting alteration of miR-expression levels in vitro and in vivo, we are offered a glimpse into the diagnostic and therapeutic possibilities of using miRs to treat vascular pathobiology. This review focuses on the role of miRs in aneurysmal disease of the abdominal aorta, summarizing recent publications regarding this topic, and outlining known effects of relevant miRs in AAA formation, including miR-21 and miR-29b. Despite there being only limited studies available, several other miRs also display clear potential for alteration of the disease process including miR-26a, the miR-17-92-cluster, miRs-221/222, miR-133 and miR-146a. While studies have shown that miRs can regulate the activity and interplay of vascular inflammatory cells, endothelial cells, smooth muscle cells and fibroblasts, all key elements leading to AAA formation, much work remains to be done.
... [ 67 , 76 ] Fibroblast proliferation is also activated by miR-21 through programmed cell death 4/JNK pathway. [ 76 ] In vascular smooth muscle cells, the proliferative actions of miR-21 are mediated through PTEN and Bcl-2. [ 67 , 77 ] In patients with thromboangiitis obliterans, a non-atherosclerotic peripheral vascular disease, arterial miR-21 levels were increased and shown to modulate smooth muscle proliferation by targeting tropomyosin 1. [ 78 ] The number of samples in the clinical study was relatively small precluding any adjustment for potential confounders; thus a larger study will be necessary to replicate these findings. ...
Peripheral artery disease (PAD) produces significant disability attributable to lower extremity ischemia. Limited treatment modalities exist to ameliorate clinical symptoms in patients with PAD. Growing evidence links microRNAs to key processes that govern disease expression in PAD including angiogenesis, endothelial function, inflammation, vascular regeneration, vascular smooth muscle cell function, restenosis, and mitochondrial function. MicroRNAs have been identified in circulation and may serve as novel biomarkers in PAD. This article reviews the potential contribution of microRNA to key pathways of disease development in PAD that may lead to microRNA-based diagnostic and therapeutic approaches.
... Additionally, anti-apoptosis approaches signi cantly improved CVB3-induced heart injury [32,33], further con rming the pathological role of apoptosis in the acute myocarditis. Our results were in line with previous studies that show miR-21 targeting PDCD4 to produce antiapoptotic e ect; however, these studies are mainly focused on the elds of tumor [34][35][36] and vascular diseases [37,38]. Only a few studies report that miR-21 exerts an anti-apoptotic e ect by targeting PDCD4 in heart diseases. ...
Purpose:
The participation of microRNAs (miRNAs) in cardiovascular diseases suggests them as potential targets for novel preventive and therapeutic strategies. In this study, the key myocardial miRNA, miR-21, was identified in the murine coxsackievirus B3 (CVB3)-induced myocarditis model and its contribution to disease progression was explored.
Methods:
Myocardial microRNA expression changes in CVB3-infected mice were analyzed by real-time PCR and miR-21 was found to be the miRNA whose expression was significantly reduced. Mice were injected with plasmid encoding miR-21 (pMDH-miR-21) at day 1 post CVB3 infection and myocarditis severity was evaluated 7 days post-infection. The underlying mechanism of miR-21 in viral myocarditis was also investigated.
Results:
Myocardial miR-21 expression was negatively related to viral myocarditis severity. Recovery of miR-21 expression, by injecting with pMDH-miR-21, significantly relieved CVB3-induced myocarditis as shown by increased body weight, reduced myocardial injury, lowered myocarditis score and increased survival rate. Further study showed that miR-21 could protect myocardial apoptosis by specifically inhibiting its target programmed cell death 4 (PDCD4) expression.
Conclusion:
miR-21 administration efficiently alleviated CVB3-induced myocarditis by repressing PDCD4-mediated apoptosis. Our study not only helps to better understand the pathogenesis of viral myocarditis, but also proves the potential of miR-21 as a novel therapeutic target for treatment of CVB3-induced myocarditis and other apoptosis-mediated cardiovascular diseases.
... This effect is mediated via down-regulation of expression of programmed cell death 4 (PDCD4), itself a negative regulator of myofibroblast-specific proteins such as α-SMA [58]. MiR-21 is overexpressed in myofibroblasts following treatment of primary rat adventitial fibroblasts with TGF-β1, and the resultant decreased PDCD4 expression also leads to up-regulated JNK/c-Jun activity [59]. ...
MicroRNAs are short noncoding RNA regulators that repress synthesis of their targets post-transcriptionally. On average, each microRNA is estimated to regulate several hundred protein-coding genes, and about 60% of proteins are thought to be regulated by microRNAs in total. A subset of these genes, including the key profibrotic cytokine Transforming Growth Factor Beta-1 (TGF-β1), exhibit particularly strong levels of post-transcriptional control of protein synthesis, involving microRNAs and other mechanisms. Changes in microRNA expression pattern are linked to profound effects on cell phenotype, and microRNAs have an emerging role in diverse physiological and pathological processes. In this review, we provide an overview of microRNA biology with a focus on their emerging role in diseases typified by organ fibrosis. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Vascular calcification often occurs in patients with chronic renal failure (CRF), which significantly increases the incidence of cardiovascular events in CRF patients. Our previous studies identified the crosstalk between the endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), and the paracrine effect of VSMCs, which regulates the calcification of VSMCs. Herein, we aim to investigate the effects of exosomes secreted by high phosphorus (HPi) -induced adventitial fibroblasts (AFs) on the calcification of VSMCs, which will construct the novel theory of “Vascular Wall Microenvironment”. The conditioned medium of HPi-induced AFs promotes the calcification of VSMCs, which is partially abrogated by GW4869, a blocker of exosomes biogenesis or release. Exosomes secreted by high phosphorus-induced AFs (AFs HPi -Exos) show similar effects on VSMCs. miR-21-5p is enriched in AFs HPi -Exos, and miR-21-5p enhances osteoblast-like differentiation of VSMCs by downregulating Crim1 expression. AFs HPi -Exos and exosomes secreted by AFs with overexpression of miR-21-5p (AFs miR21M -Exos) significantly accelerate vascular calcification in CRF mice. In general, the enriched miR-21-5p in AFs HPi -Exos promotes the calcification of VSMCs and vascular calcification by inhibiting the expression of cysteine-rich Crim1 protein (Crim1). Combined with our previous studies, the present experiment supports the theory of vascular wall microenvironment.
Myofibroblasts escape apoptosis and proliferate abnormally under pathological conditions, especially fibrosis; they synthesize and secrete a large amount of extracellular matrix (ECM), such as α-SMA and collagen, which leads to the distortion of organ parenchyma structure, an imbalance in collagen deposition and degradation, and the replacement of parenchymal cells by fibrous connective tissues. Fibroblast to myofibroblast transition (FMT) is considered to be the main source of myofibroblasts. Therefore, it is crucial to explore the influencing factors regulating the process of FMT for the prevention, treatment, and diagnosis of FMT-related diseases. In recent years, non-coding RNAs, including microRNA, long non-coding RNAs, and circular RNAs, have attracted extensive attention from scientists due to their powerful regulatory functions, and they have been found to play a vital role in regulating FMT. In this review, we summarized ncRNAs which regulate FMT during fibrosis and found that they mainly regulated signaling pathways, including TGF-β/Smad, MAPK/P38/ERK/JNK, PI3K/AKT, and WNT/β-catenin. Furthermore, the expression of downstream transcription factors can be promoted or inhibited, indicating that ncRNAs have the potential to be a new therapeutic target for FMT-related diseases.
Long-term noise exposure is reported to damage cardiovascular system, but the relationship between occupational noise exposure and arterial stiffness (AS) and the underlying mechanism is still unclear. We aimed to investigate the association of occupational noise exposure with arterial stiffness (AS), and further explore the mediation roles of microRNAs (miRNAs). A total of 838 workers were recruited from two companies in Wuhan, Hubei, China. Cumulative occupational noise exposure (CNE) was assessed through noise level of job title and work years in occupational noise. The AS for the participants were evaluated using brachial-ankle pulse wave velocity (baPWV) measured by an oscillometric device. Each 1-unit increase in CNE levels was significantly associated with a 0.002 (95% confidence interval (CI) = 0.001–0.003) unit increase in ln-transformed values of baPWV. In the sex-specific analysis, the association was significant in males (β = 0.002, 95%CI = 0.001–0.003). Meanwhile, the risk of bilateral hearing loss at high frequency was significantly higher in the high-exposed group than non-exposed group (OR = 1.895, 95%CI = 1.024–3.508), and participants with bilateral hearing loss at high frequency had a significantly higher level of ln-transformed baPWV (β = 0.032, 95%CI = 0.003–0.061). Occupational noise exposure and AS were both negatively associated with plasma miR-92a-3p and miR-21–5p, and the two miRNAs mediated 15.0% and 16.8% of the association of occupational noise with AS (P < 0.05). Our findings suggest that occupational noise exposure is positively associated with AS, and plasma miR-92a-3p and miR-21–5p may partly mediate such association.
Myocarditis is a common heart disease which lacks effective treatment till now. Baicalin possesses plenty of activities, including anti‐inflammation. In this investigation, we attempted to investigate the influences of Baicalin on Lipopolysaccharide (LPS)‐evoked H9c2 cells.Cells viability, apoptosis, and expressions of apoptosis‐associated proteins were, respectively, measured utilizing CCK‐8 assay, flow cytometry and western blot. The levels of IL‐6 and TNF‐α were detected through enzyme‐linked immunosorbent assay, western blot and qRT‐PCR. miR‐21 expression was detected through qRT‐PCR and was silenced using cell transfection. The expressions of NF‐κB and PDCD4/JNK pathways related proteins were measured through western blot. We found that LPS stimulation induced cell apoptosis and upregulation of IL‐6 and TNF‐α. Baicalin treatment effectively suppressed LPS‐induced inflammation and apoptosis. The NF‐κB and PDCD4/JNK pathways were blocked by Baicalin. Additionally, the enhanced expression of miR‐21 triggered by LPS was further elevated by Baicalin. Further study revealed that the inhibiting effects of Baicalin on LPS‐evoked injury were largely attenuated by knockdown of miR‐21. Moreover, the associated NF‐κB and JNK pathways, which were suppressed by Baicalin treatment, were then activated by knockdown of miR‐21. Our present study revealed that Baicalin alleviated LPS‐evoked inflammatory injury via suppressing the NF‐κB and PDCD4/JNK pathways through regulating miR‐21 expression.
Objective
The aim was to prepare a new calcium-decorated titanium surface and investigate whether this new modified titanium surface could enhance bone formation and osseointegration.
Materials and methods
In this study, we prepared a calcium-decorated titanium (Ti) surface through hydrothermal treatment with a mixed solution of phytic acid (PA) and calcium hydroxide [Ca (OH)2]. Pure titanium with a sandblasted/acid-etched (SLA) surface was used as the control group. The physical properties of different titanium surfaces such as the surface morphology, elemental composition, wettability, and surface roughness were investigated. The effect of biomineralization was studied by soaking samples in simulated body fluid (SBF). The cell adhesion, proliferation, and osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) were evaluated as well.
Results
Calcium-decorated titanium surfaces with superior hydrophilicity were successfully prepared. X-ray photoelectron spectroscopy (XPS) and energy-dispersive X-ray spectroscopy (EDX) confirmed that the calcium ions were decorated onto the titanium surface and were continuously released with time. After immersion in 10SBF for 1 day, a hydroxyapatite layer formed on both modified surfaces. In vitro tests showed that the modified surface enhanced the cell adhesion, proliferation, and osteogenic differentiation of hBMSCs. The expression levels of the osteogenic-related genes were significantly upregulated compared to the control samples.
Conclusions
The calcium-decorated titanium surface enhanced the bioactivity and osteogenesis ability of the titanium surface, which suggests that calcium-decorated materials would have great potential for not only dental implants but also various other biomaterial applications.
Background:
In active inflammatory bowel disease (IBD), microRNA expression profiling consistently features disease-specific signatures, and microRNA-21 (miR-21) has been shown to be upregulated in the inflamed colon of patients with active ulcerative colitis (UC). However, the cellular sources of miR-21 expression in IBD tissues have not yet been identified. We sought to determine the expression levels of miR-21 and one of its downstream target genes, programmed cell death 4 (PDCD4), in CD3 T cells isolated from the colonic mucosa of patients with active IBD, inactive IBD, and non-IBD controls.
Methods:
Colonic biopsies were treated with collagenase V. CD3 T cells were isolated using MACS CD3 positive selection. Total RNA was converted to cDNA. Real-time PCR reactions were performed with PCR primers for miR-21, SNORD95, PDCD4, and GAPDH.
Results:
The expression of miR-21 was statistically significantly downregulated in CD3 T cells from patients with UC in remission as compared to active disease (P = 0.0193). miR-21 negatively regulates PDCD4 expression. As predicted, the mRNA level of PCDC4 in CD3 T cells was upregulated in UC and Crohn's disease in remission as compared to active disease (UC active versus UC remission: P = 0.0008, Crohn's disease active versus Crohn's disease remission: P = 0.0215) and in patients with UC in remission as compared to healthy controls (P = 0.0226).
Conclusions:
Although miR-21 expression is downregulated, PDCD4 is upregulated in CD3 T cells during the remission phase of UC. Our results indicate that miR-21 and related pathways in colonic T cells may play a role in limiting pathogenic T-cell responses and may constitute future target candidates to induce remission in UC.
Recent studies suggest that peripheral nerve injury converts resting spinal cord astroglial cells into an activated state, which is required for the development and maintenance of neuropathic pain. However, the underlying mechanisms of how resting astrocytes are activated after nerve injury remain largely unknown. Astroglial cell proliferation and activation could be affected by endogenous factors including chemokines, growth factors, and neurotropic factor. Chemokine (C-C motif) ligand 7 (Ccl7) is essential in facilitating the development of neuropathic pain; however, the mechanism is unknown. In the present study, we found that Ccl7 promoted astrocyte proliferation and thus contributed toward neuropathic pain. Spinal nerve ligation increased the expression in the spinal cord of neuronal Ccl7. Behavioral analyses showed that knockdown of Ccl7 alleviated spinal nerve ligation-induced neuropathic pain. Further in-vitro study showed that neuronal-derived Ccl7 was sufficient for the proliferation and activation of astroglial cells. We found a novel mechanism of Ccl7 stimulating the proliferation and activation of spinal cord astrocytes that contributes toward neuropathic pain.
There is increasing evidence that microRNAs (miRNAs) are deregulated in autoimmune and cardiovascular diseases. The present study aimed to identify if miRNAs are deregulated in giant cell arteritis (GCA), a vasculitis affecting large-sized and medium-sized arteries, and to determine if miRNA levels might allow to discriminate between patients with GCA and those without.
58 patients who had temporal artery biopsy (TAB) for suspected GCA were included in the study and divided into three groups: patients with TAB-positive GCA showing a transmural inflammation (n=27), patients with TAB-negative GCA (n=8) and TAB-negative non-GCA patients with a final diagnosis different from GCA (n=23). To identify candidate miRNAs deregulated in GCA, we profiled the expression of 1209 miRNAs in inflamed TABs and normal TABs. Selected miRNAs were then validated by real-time PCRs and in situ hybridisation (ISH).
MiR-146b-5p, -146a, -155, -150, -21 and -299-5p were significantly more expressed in inflamed TABs from patients with GCA. miRNAs were mainly deregulated at the tissue level because peripheral blood mononuclear cells and polymorphonuclear cells from the three groups of patients and age-matched healthy controls had similar levels of miRNAs. ISH showed that miR-21 was mainly expressed by cells in the medial and intimal layers of inflamed TABs. Patients with TAB-negative GCA had a miRNA profile similar to TAB-negative non-GCA patients.
MiR-146b-5p, -146a, -21, -150, -155, -299-5p are overexpressed in the presence of inflammation in TABs from patients with GCA.
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Background:
MicroRNAs (miRNAs) are small noncoding RNA molecules that regulate gene expression in the progress of proliferation, differentiation, and apoptosis. A keloid is considered to be a type of benign tumor. The exact contribution of miRNAs in keloid fibroblasts remains largely unknown.
Methods:
Loss and gain of function was used by transfecting the keloid fibroblast cells with chemically synthesized oligonucleotides complementary to microRNA-21 (miR-21). Expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) in keloid tissues and adjacent normal skin tissues were investigated by quantitative real-time polymerase chain reaction and Western blot assay. Moreover, cell apoptosis and proliferation were checked in keloid cells, and related proteins were determined by Western blot assay.
Results:
MiR-21 inhibitor and mimic transfection changed the apoptosis and DNA synthesis. Of the 23 paired samples analyzed, expression of PTEN was low in keloid tissues relative to adjacent normal skin tissues. Cells showed an inverse correlation between miR-21 and PTEN protein after transfection. In addition, cell proliferation was increased when normal skin fibroblasts were transfected with miR-21 mimics. It is worth noting that the expression of phosphorylated AKT decreased to relatively low levels after miR-21 inhibitor treatment.
Conclusions:
This in vitro study demonstrates important interactions among miR-21 expression, keloid fibroblast apoptosis, cell proliferation, and some related proteins. These findings may provide some hints toward effective applications of miR-21 as a therapy target for keloids.
A number of cardiovascular diseases, such as restenosis, aneurysm and atherosclerosis, lead to vascular remodeling associated with complex adaptive reactions of different cell populations. These reactions include growth of smooth muscle cells (SMCs), proliferation of endothelial cells (ECs) and the inflammatory response of macrophages. MicroRNAs (miRNAs), a class of short RNAs, play key roles in various biological processes and in the development of human disease by post-transcriptional regulation of gene expression. Here, we review the molecular mechanisms of a subset of miRNAs involved in vascular remodeling, including miR-143/145, miR-221/222, miR-126, miR-21 and miR-155. Some of these miRNAs, such as miR-143/145 and miR-126, have been shown to be protective during vascular remodeling, whereas others, such as miR-21, may promote the cellular response that leads to neointima formation. The increasing knowledge regarding the roles of miRNAs in vascular remodeling opens novel avenues for the treatment of various cardiovascular diseases. However, more in vivo studies on the functional roles of these miRNAs are required in the future.
Cardiac fibrosis is characterized by aberrant proliferation of cardiac fibroblasts and exaggerated deposition of extracellular matrix (ECM) in the myocardial interstitial, and ultimately impairs cardiac function. It is still controversial whether microRNA-21 (miR-21) participates in the process of cardiac fibrosis. Our previous study confirmed that transforming growth factor beta receptor III (TGFβRIII) is a negative regulator of TGF-β pathway. Here, we aimed to decipher the relationship between miR-21 and TGFβRIII in the pathogenic process of myocardial fibrosis. We found that TGF-β1 and miR-21 were up-regulated, whereas TGFβRIII was down-regulated in the border zone of mouse hearts in response to myocardial infarction. After transfection of miR-21 into cardiac fibroblasts, TGFβRIII expression was markedly reduced and collagen content was increased. And, luciferase results confirmed that TGFβRIII was a target of miR-21. It suggests that up-regulation of miR-21 could increase the collagen content and at least in part through inhibiting TGFβRIII. Conversely, we also confirmed that overexpression of TGFβRIII could inhibit the expression of miR-21 and reduce collagen production in fibroblasts. Further studies showed that overexpression of TGFβRIII could also deactivate TGF-β1 pathway by decreasing the expression of TGF-β1 and phosphorylated-Smad3 (p-Smad3). TGF-β1 has been proven as a positive regulator of miR-21. Taken together, we found a novel reciprocal loop between miR-21 and TGFβRIII in cardiac fibrosis caused by myocardial infarction in mice, and targeting this pathway could be a new strategy for the prevention and treatment of myocardial remodeling.
MicroRNAs (miRNAs) are a recently discovered class of endogenous, small, noncoding RNAs that regulate about 30% of the encoding genes of the human genome. However, the role of miRNAs in vascular disease is currently completely unknown. Using microarray analysis, we demonstrated for the first time that miRNAs are aberrantly expressed in the vascular walls after balloon injury. The aberrantly expressed miRNAs were further confirmed by Northern blot and quantitative real-time polymerase chain reaction. Modulating an aberrantly overexpressed miRNA, miR-21, via antisense-mediated depletion (knock-down) had a significant negative effect on neointimal lesion formation. In vitro, the expression level of miR-21 in dedifferentiated vascular smooth muscle cells was significantly higher than that in fresh isolated differentiated cells. Depletion of miR-21 resulted in decreased cell proliferation and increased cell apoptosis in a dose-dependent manner. MiR-21-mediated cellular effects were further confirmed in vivo in balloon-injured rat carotid arteries. Western blot analysis demonstrated that PTEN and Bcl-2 were involved in miR-21-mediated cellular effects. The results suggest that miRNAs are novel regulatory RNAs for neointimal lesion formation. MiRNAs may be a new therapeutic target for proliferative vascular diseases such as atherosclerosis, postangioplasty restenosis, transplantation arteriopathy, and stroke.
MicroRNAs (miRNAs) have been shown to have an important role in various cellular processes, such as apoptosis, differentiation and development. Recent studies have shown that miRNAs are mis-expressed in human cancers where they can exert their effect as oncogenes or tumor suppressors. Here, we review the potential for using miRNAs as biomarkers for diagnosis, prognosis and cancer therapies.
Arteries are composed of 3 concentric tissue layers which exhibit different structures and properties. Because arterial injury is generally initiated at the interface with circulating blood, most studies performed to unravel the mechanisms involved in injury-induced arterial responses have focused on the innermost layer (intima) rather than on the outermost adventitial layer. In the present review, we focus on the involvement of the adventitia in response to various types of arterial injury leading to vascular remodeling. Physiologically, soluble vascular mediators are centrifugally conveyed by mass transport toward the adventitia. Moreover, in pathological conditions, neomediators and antigens can be generated within the arterial wall, whose outward conveyance triggers different patterns of local adventitial response. Adventitial angiogenesis, immunoinflammation, and fibrosis sequentially interact and their net balance defines the participation of the adventitial response in arterial pathology. In the present review we discuss 4 pathological entities in which the adventitial response to arterial wall injury participates in arterial wall remodeling. Hence, the adventitial adaptive immune response predominates in chronic rejection. Inflammatory phagocytic cell recruitment and initiation of a shift from innate to adaptive immunity characterize the adventitial response to products of proteolysis in abdominal aortic aneurysm. Adventitial sprouting of neovessels, leading to intraplaque hemorrhages, predominates in atherothrombosis. Adventitial fibrosis characterizes the response to mechanical stress and is responsible for the constrictive remodeling of arterial segments and initiating interstitial fibrosis in perivascular tissues. These adventitial events, therefore, have an impact not only on the vessel wall biology but also on the surrounding tissue.
MicroRNA-21 (miR-21) is highly up-regulated during hypertrophic and cancerous cell growth. In contrast, we found that it declines
in cardiac myocytes upon exposure to hypoxia. Thus, the objective was to explore its role during hypoxia. We show that miR-21
not only regulates phosphatase and tensin homologue deleted on chromosome 10 (PTEN), but also targets Fas ligand (FasL). During
prolonged hypoxia, down-regulation of miR-21 proved necessary and sufficient for enhancing expression of both proteins. We
demonstrate here for the first time that miR-21 is positively regulated via an AKT-dependent pathway, which is depressed during
prolonged hypoxia. Accordingly, hypoxia-induced down-regulation of miR-21 and up-regulation of FasL and PTEN were reversed
by activated AKT and reproduced by a dominant negative mutant, wortmannin, or PTEN. Moreover, the antiapoptotic function of
AKT partly required miR-21, which was sufficient for inhibition of caspase-8 activity and mitochondrial damage. In consensus,
overexpression of miR-21 in a transgenic mouse heart resulted in suppression of ischemia-induced up-regulation of PTEN and
FasL expression, an increase in phospho-AKT, a smaller infarct size, and ameliorated heart failure. Thus, we have identified
a unique aspect of the function of AKT by which it inhibits apoptosis through miR-21-dependent suppression of FasL.
Abstract
Background
Balloon injury (BI) of the rat carotid artery (CCA) is widely used to study intimal hyperplasia (IH) and decrease in lumen diameter (LD), but CCA's small diameter impedes the evaluation of endovascular therapies. Therefore, we validated BI in the aorta (AA) and iliac artery (CIA) to compare it with CCA.
Methods
Rats underwent BI or a sham procedure (control). Light microscopic evaluation was performed either directly or at 1, 2, 3, 4 and 16 weeks follow-up. The area of IH and the change in LD (LD at 16 weeks minus LD post BI) were compared.
Results
In the BI-groups the area of IH increased to 0.14 ± 0.08 mm<sup>2</sup> (CCA), 0.14 ± 0.03 mm<sup>2</sup> (CIA) and 0.12 ± 0.04 mm<sup>2</sup> (AA) at 16 weeks (NS). The LD decreased with 0.49 ± 0.07 mm (CCA), compared to 0.22 ± 0.07 mm (CIA) and 0.07 ± 0.10 mm (AA) at 16 weeks (p < 0.05). The constrictive vascular remodelling (CVR = wall circumference loss combined with a decrease in LD) was -0.17 ± 0.05 mm in CIA but absent in CCA and AA. No IH, no decrease in LD and no CVR was seen in the control groups.
Conclusions
BI resulted in: (1.) a decrease in LD in CCA due to IH, (2.) a decrease in LD in CIA due to IH and CVR, (3.) no change in LD in AA, (4.) Comparable IH development in all arteries, (5.) CCA has no vasa vasorum compared to CIA and AA, (6.) The CIA model combines good access for 2 F endovascular catheters with a decrease in LD due to IH and CVR after BI.
Xeroderma pigmentosum (XP) is an autosomal recessive genetic disorder. Afflicted patients show extreme sun-sensitivity and
skin cancer predisposition. XP is in most cases associated with deficient nucleotide excision repair (NER), which is the process
responsible for removing photolesions from DNA. Measuring NER activity by nucleotide incorporation into repair patches, termed
‘unscheduled DNA synthesis (UDS)’, is one of the most commonly used assays for XP-diagnosis and NER research. We have established
a rapid and accurate procedure for measuring UDS by replacement of thymidine with 5-ethynyl-2'-deoxyuridine (EdU). EdU incorporated
into repair patches can be directly conjugated to fluorescent azide derivatives, thereby obviating the need for either radiolabeled
thymidine or denaturation and antibody detection of incorporated bromodeoxyuridine (BrdU). We demonstrate that the EdU incorporation
assay is compatible with conventional techniques such as immunofluorescent staining and labeling of cells with micro-latex
beads. Importantly, we can complete the entire UDS assay within half a day from preparation of the assay coverslips; this
technique may prove useful as a method for XP diagnosis.
MicroRNAs (miRNAs) comprise a novel class of endogenous, small, noncoding RNAs that negatively regulate approximately 30% of genes in a cell via degradation or translational inhibition of their target mRNAs. However, the effects of reactive oxygen species (ROS) on miRNA expression and the roles of miRNAs in ROS-mediated gene regulation and biological functions of vascular cells are unclear. Using microarray analysis, we demonstrated that miRNAs are aberrantly expressed in vascular smooth muscle cells (VSMCs) after treatment with hydrogen peroxide (H(2)O(2)). H(2)O(2)-mediated up-regulation of microRNA-21 (miR-21) was further confirmed by quantitative real-time PCR. To determine the potential roles of miRNAs in H(2)O(2)-mediated gene regulation and cellular effects, miR-21 expression was down-regulated by miR-21 inhibitor and up-regulated by pre-miR-21. H(2)O(2)-induced VSMC apoptosis and death were increased by miR-21 inhibitor and decreased by pre-miR-21. Programmed cell death 4(PDCD4) was a direct target of miR-21 that was involved in miR-21-mediated effects on VSMCs. Pre-miR-21-mediated protective effect on VSMC apoptosis and death was blocked via adenovirus-mediated overexpression of PDCD4 without the miR-21 binding site. Moreover, activator protein 1 was a downstream signaling molecule of PDCD4 in miR-21-modulated VSMCs. The results suggest that miRNAs in VSMCs are sensitive to H(2)O(2) stimulation. miRN-21 participates in H(2)O(2)-mediated gene regulation and cellular injury response through PDCD4 and the activator protein 1 pathway. miRNAs might play a role in vascular diseases related to ROS.
MicroRNAs comprise a broad class of small non-coding RNAs that control expression of complementary target messenger RNAs. Dysregulation of microRNAs by several mechanisms has been described in various disease states including cardiac disease. Whereas previous studies of cardiac disease have focused on microRNAs that are primarily expressed in cardiomyocytes, the role of microRNAs expressed in other cell types of the heart is unclear. Here we show that microRNA-21 (miR-21, also known as Mirn21) regulates the ERK-MAP kinase signalling pathway in cardiac fibroblasts, which has impacts on global cardiac structure and function. miR-21 levels are increased selectively in fibroblasts of the failing heart, augmenting ERK-MAP kinase activity through inhibition of sprouty homologue 1 (Spry1). This mechanism regulates fibroblast survival and growth factor secretion, apparently controlling the extent of interstitial fibrosis and cardiac hypertrophy. In vivo silencing of miR-21 by a specific antagomir in a mouse pressure-overload-induced disease model reduces cardiac ERK-MAP kinase activity, inhibits interstitial fibrosis and attenuates cardiac dysfunction. These findings reveal that microRNAs can contribute to myocardial disease by an effect in cardiac fibroblasts. Our results validate miR-21 as a disease target in heart failure and establish the therapeutic efficacy of microRNA therapeutic intervention in a cardiovascular disease setting.
Apoptotic and mitogenic stimuli activate c-Jun NH2-terminal kinases (JNKs) in T cells. Although T cells express both JNK1 and JNK2 isozymes, the absence of JNK2 alone can result in resistance to anti-CD3-induced thymocyte apoptosis and defective mature T cell proliferation. Similar defects in thymocyte apoptosis and mature T cell proliferation, the latter due to reduced interleukin 2 production, are also caused by JNK1 deficiency. Importantly, T cell function was compromised in Jnk1(+/-)Jnk2(+/-) double heterozygous mice, indicating that JNK1 and JNK2 play similar roles in regulating T cell function. The reduced JNK dose results in defective c-Jun NH2-terminal phosphorylation in thymocytes but not in peripheral T cells, in which nuclear factors of activated T cells (NK-ATs)-DNA binding activity is affected. Thus, JNK1 and JNK2 control similar functions during T cell maturation through differential targeting of distinct substrates.
The adventitial layer surrounding the blood vessels has long been exclusively considered a supporting tissue the main function of which is to provide adequate nourishment to the muscle layers of tunica media. Although functionally interconnected, the adventitial and medial layers are structurally interfaced at the external elastic lamina level, clearly distinguishable at the maturational phase of vascular morphogenesis. Over the last few years the "passive" role that the adventitia seemed to play in experimental and spontaneous vascular pathologies involving proliferation, migration, differentiation, and apoptosis of vascular smooth muscle cells (VSMCs) has been questioned. It has been demonstrated that fibroblasts from the adventitia display an important partnership with the resident medial VSMCs in terms of phenotypic conversion, proliferation, apoptotic, and migratory properties the result of which is neointima formation and vascular remodeling. This article is an attempt at reviewing the major themes and more recent findings dealing with the phenotypic conversion process that leads adventitial "passive" (static) fibroblasts to become "activated" (mobile) myofibroblasts. This event shows some facets in common with vascular morphogenesis, ie, the process of recruitment, incorporation, and phenotypic conversion of cells surrounding the primitive endothelial tube in the definitive vessel wall. We hypothesize that during the response to vascular injuries in the adult, "activation" of adventitial fibroblasts is, at least in part, reminiscent of a developmental program that also invests, although with distinct spatiotemporal features, medial VSMCs.
We previously reported that activator protein-1 (AP-1), containing c-Jun, is rapidly activated in balloon-injured artery. Therefore, we examined the role of c-Jun in vascular smooth muscle cell (SMC) proliferation, by using in vitro and in vivo gene transfer techniques. (1) Serum (2%) stimulation significantly increased AP-1 DNA binding activity in aortic SMCs, followed by the increase in both 3H-thymidine incorporation and cell number. Aortic SMCs were infected with recombinant adenovirus containing TAM67, a dominant negative c-Jun lacking transactivation domain of wild c-Jun (Ad-DN-c-Jun), to specifically inhibit AP-1. Ad-DN-c-Jun significantly inhibited serum-induced SMC proliferation, by inhibiting the entrance of SMC into S phase. (2) The effect of DN-c-Jun was examined on balloon injury-induced intimal hyperplasia in rats. Before balloon injury, DN-c-Jun was transfected into rat carotid artery using the hemagglutinating virus of Japan-liposome method. In vivo transfection of DN-c-Jun significantly inhibited vascular SMC proliferation in the intima and the media and subsequently prevented intimal thickening at 14 days after balloon injury. We obtained the first evidence that DN-c-Jun gene transfer prevented vascular SMC proliferation in vitro and in vivo, and c-Jun was involved in balloon injury-induced intimal hyperplasia. Thus, AP-1 seems to be the new therapeutic target for treatment of vascular diseases.
Balloon injury (BI) of the rat carotid artery (CCA) is widely used to study intimal hyperplasia (IH) and decrease in lumen diameter (LD), but CCA's small diameter impedes the evaluation of endovascular therapies. Therefore, we validated BI in the aorta (AA) and iliac artery (CIA) to compare it with CCA.
Rats underwent BI or a sham procedure (control). Light microscopic evaluation was performed either directly or at 1, 2, 3, 4 and 16 weeks follow-up. The area of IH and the change in LD (LD at 16 weeks minus LD post BI) were compared.
In the BI-groups the area of IH increased to 0.14 +/- 0.08 mm2 (CCA), 0.14 +/- 0.03 mm2 (CIA) and 0.12 +/- 0.04 mm2 (AA) at 16 weeks (NS). The LD decreased with 0.49 +/- 0.07 mm (CCA), compared to 0.22 +/- 0.07 mm (CIA) and 0.07 +/- 0.10 mm (AA) at 16 weeks (p < 0.05). The constrictive vascular remodelling (CVR = wall circumference loss combined with a decrease in LD) was -0.17 +/- 0.05 mm in CIA but absent in CCA and AA. No IH, no decrease in LD and no CVR was seen in the control groups.
BI resulted in: (1.) a decrease in LD in CCA due to IH, (2.) a decrease in LD in CIA due to IH and CVR, (3.) no change in LD in AA, (4.) Comparable IH development in all arteries, (5.) CCA has no vasa vasorum compared to CIA and AA, (6.) The CIA model combines good access for 2 F endovascular catheters with a decrease in LD due to IH and CVR after BI.
Myofibroblast differentiation and activation by transforming growth factor-beta1 (TGF-beta1) is a critical event in the pathogenesis of human fibrotic diseases, but regulatory mechanisms for this effect are unclear. In this report, we demonstrate that stable expression of the myofibroblast phenotype requires both TGF-beta1 and adhesion-dependent signals. TGF-beta1-induced myofibroblast differentiation of lung fibroblasts is blocked in non-adherent cells despite the preservation of TGF-beta receptor(s)-mediated signaling of Smad2 phosphorylation. TGF-beta1 induces tyrosine phosphorylation of focal adhesion kinase (FAK) including that of its autophosphorylation site, Tyr-397, an effect that is dependent on cell adhesion and is delayed relative to early Smad signaling. Pharmacologic inhibition of FAK or expression of kinase-deficient FAK, mutated by substituting Tyr-397 with Phe, inhibit TGF-beta1-induced alpha-smooth muscle actin expression, stress fiber formation, and cellular hypertrophy. Basal expression of alpha-smooth muscle actin is elevated in cells grown on fibronectin-coated dishes but is decreased on laminin and poly-d-lysine, a non-integrin binding polypeptide. TGF-beta1 up-regulates expression of integrins and fibronectin, an effect that is associated with autophosphorylation/activation of FAK. Thus, a safer and more effective therapeutic strategy for fibrotic diseases characterized by persistent myofibroblast activation may be to target this integrin/FAK pathway while not interfering with tumor-suppressive functions of TGF-beta1/Smad signaling.
The c-Jun NH(2)-terminal kinase (JNK) subgroup of mitogen-activated protein kinases has been implicated largely in stress responses, but an increasing body of evidence has suggested that JNK also plays a role in cell proliferation and survival. We examined the effect of JNK inhibition, using either SP600125 or specific antisense oligonucleotides, on cell proliferation and cell cycle progression. SP600125 was selective for JNK in vitro and in vivo versus other kinases tested including ERK, p38, cyclin-dependent protein kinase 1 (CDK1), and CDK2. SP600125 inhibited JNK activity and KB-3 cell proliferation with the same dose dependence, suggesting that inhibition of proliferation was a direct consequence of JNK inhibition. Inhibition of proliferation by SP600125 was associated with an increase in the G(2)-M and apoptotic fractions of cells but was not associated with p53 or p21 induction. Antisense oligonucleotides to JNK2 but not JNK1 caused highly significant inhibition of cell proliferation. Wild-type mouse fibroblasts responded similarly with proliferation inhibition and apoptosis induction, whereas c-jun(-/-) fibroblasts were refractory to the effects of SP600125, suggesting that JNK signaling to c-Jun is required for cell proliferation. Studies in synchronized KB-3 cells indicated that SP600125 delayed transit time through S and G(2)-M phases. Correspondingly, JNK activity increased in late S phase and peaked in late G(2) phase. During synchronous mitotic progression, cyclin B levels increased concomitant with phosphorylation of c-Jun, H1 histone, and Bcl-2. In the presence of SP600125, mitotic progression was prolonged, and c-Jun phosphorylation was inhibited, but neither H1 nor Bcl-2 phosphorylation was inhibited. However, the CDK inhibitor roscovitine inhibited mitotic Bcl-2 phosphorylation. These results indicate that JNK, and more specifically the JNK2 isoform, plays a key role in cell proliferation and cell cycle progression. In addition, conclusive evidence is presented that a kinase other than JNK, most likely CDK1 or a CDK1-regulated kinase, is responsible for mitotic Bcl-2 phosphorylation.
Oxidative stress activates various signal transduction pathways, including Jun N-terminal kinase (JNK) and its substrates, that induce apoptosis. We reported here the role of angiopoietin-1 (Ang1), which is a prosurvival factor in endothelial cells, during endothelial cell damage induced by oxidative stress. Hydrogen peroxide (H2O2) increased apoptosis of endothelial cells through JNK activation, whereas Ang1 inhibited H2O2-induced apoptosis and concomitant JNK phosphorylation. The inhibition of H2O2-induced JNK phosphorylation was reversed by inhibitors of phosphatidylinositol (PI) 3-kinase and dominant-negative Akt, and constitutively active-Akt attenuated JNK phosphorylation without Ang1. These data suggested that Ang1-dependent Akt phosphorylation through PI 3-kinase leads to the inhibition of JNK phosphorylation. H2O2-induced phosphorylation of SAPK/Erk kinase (SEK1) at Thr261, which is an upstream regulator of JNK, was also attenuated by Ang1-dependent activation of the PI 3-kinase/Akt pathway. In addition, Ang1 induced SEK1 phosphorylation at Ser80, suggesting the existence of an additional signal transduction pathway through which Ang1 attenuates JNK phosphorylation. These results demonstrated that Ang1 attenuates H2O2-induced SEK1/JNK phosphorylation through the PI 3-kinase/Akt pathway and inhibits the apoptosis of endothelial cells to oxidative stress.
Tumor-suppressor Pdcd4 inhibits transformation and invasion and is downregulated in cancers. So far, it has not been studied as to whether miRNAs, suppressing target expression by binding to the 3'-UTR, regulate Pdcd4 or invasion. The present study was conducted to investigate the regulation of Pdcd4, and invasion/intra-vasation, by miRNAs. A bioinformatics search revealed a conserved target-site for miR-21 within the Pdcd4-3'-UTR at 228-249 nt. In 10 colorectal cell lines, an inverse correlation of miR-21 and Pdcd4-protein was observed. Transfection of Colo206f-cells with miR-21 significantly suppressed a luciferase-reporter containing the Pdcd4-3'-UTR, whereas transfection of RKO with anti-miR-21 increased activity of this construct. This was abolished when a construct mutated at the miR-21/nt228-249 target site was used instead. Anti-miR-21-transfected RKO cells showed an increase of Pdcd4-protein and reduced invasion. Moreover, these cells showed reduced intra-vasation and lung metastasis in a chicken-embryo-metastasis assay. In contrast, overexpression of miR-21 in Colo206f significantly reduced Pdcd4-protein amounts and increased invasion, while Pdcd4-mRNA was unaltered. Resected normal/tumor tissues of 22 colorectal cancer patients demonstrated an inverse correlation between miR-21 and Pdcd4-protein. This is the first study to show that Pdcd4 is negatively regulated by miR-21. Furthermore, it is the first report to demonstrate that miR-21 induces invasion/intravasation/metastasis.
MicroRNAs are emerging as important regulators of cancer-related processes. The miR-21 microRNA is overexpressed in a wide variety of cancers and has been causally linked to cellular proliferation, apoptosis,
and migration. Inhibition of mir-21 in MCF-7 breast cancer cells causes reduced cell growth. Using array expression analysis of MCF-7 cells depleted of miR-21, we have identified mRNA targets of mir-21 and have shown a link between miR-21 and the p53 tumor suppressor protein. We furthermore found that the tumor suppressor protein Programmed Cell Death 4 (PDCD4)
is regulated by miR-21 and demonstrated that PDCD4 is a functionally important target for miR-21 in breast cancer cells.
Curcumin induces cancer cell growth arrest and apoptosis in vitro, but its poor bioavailability in vivo limits its antitumor efficacy. We have previously evaluated the bioavailability of novel analogues of curcumin compared with curcumin, and we found that the analogue CDF exhibited greater systemic and pancreatic tissue bioavailability. In this study, we evaluated the effects of CDF or curcumin alone or in combination with gemcitabine on cell viability and apoptosis in gemcitabine-sensitive and gemcitabine-resistant pancreatic cancer (PC) cell lines. Mechanistic investigations revealed a significant reduction in cell viability in CDF-treated cells compared with curcumin-treated cells, which were also associated with the induction of apoptosis, and these results were consistent with the downregulation of Akt, cyclooxygenase-2, prostaglandin E(2), vascular endothelial growth factor, and NF-kappaB DNA binding activity. We have also documented attenuated expression of miR-200 and increased expression of miR-21 (a signature of tumor aggressiveness) in gemcitabine-resistant cells relative to gemcitabine-sensitive cells. Interestingly, CDF treatment upregulated miR-200 expression and downregulated the expression of miR-21, and the downregulation of miR-21 resulted in the induction of PTEN. These results prompt further interest in CDF as a drug modality to improve treatment outcome of patients diagnosed with PC as a result of its greater bioavailability in pancreatic tissue.
We have recently reported that adventitial fibroblasts are able to express endothelin-1 (ET-1) in response to angiotensin II (Ang II) stimulation. However, the mechanism by which this occurs in the adventitia remains unclear. As Ang II has been reported to increase oxidant production by NADPH oxidase, we examined the role of this complex in Ang II stimulated ET-1 expression in vascular adventitial fibroblasts.
Adventitial fibroblasts were isolated and cultured from mouse aorta. Cells were treated with Ang II (100 nmol/L) in the presence or absence of NADPH oxidase inhibitors, apocynin (100 micromol/L) and diphenyleneiodonium (10 micromol/L), superoxide scavengers, SOD (350 units/mL), tempol (100 micromol/L), tiron (100 micromol/L), and ET-receptor antagonists (10 microM), BQ123 (for ET(A)-) and BQ788 (for ET(B)-). PreproET-1 mRNA and ET-1 level were determined by relative RT-PCR and ELISA, respectively. Type I procollagen-alpha-I (collagen) level was detected by Western blot. Superoxide anion (superoxide) production was determined by coelenterazine or lucigenin chemiluminescence. Ang II-induced collagen expression was inhibited by BQ123, suggesting that adventitial ET-1 plays a significant role in regulating the extracellular matrix. NADPH oxidase inhibitors and superoxide scavengers significantly decreased Ang II-induced ET-1 mRNA and peptide expression, superoxide production as well as collagen expression. Furthermore, deletion of gp91(phox) (a key subunit of NADPH oxidase) and overexpression of SOD1 attenuated Ang II-induced responses.
Ang II-evoked expression of ET-1 in adventitial fibroblasts appears to be mediated, at least in part, by NADPH oxidase. Functionally, this mechanism stimulates collagen expression thereby implicating the adventitia as a potential contributor to the vascular pathophysiology associated with oxidative stress and vascular remodeling.
Mitotic catastrophe is a phenomenon displayed by cells undergoing aberrant mitosis to eliminate cells that fail to repair the errors. Why and how mitotic catastrophe would lead to cell death remains to be resolved and the answer will prove valuable in design of better therapeutic agents that specifically target such cells in mitosis. The antibiotic actinomycin D has been shown to induce chromosomal lesions in lower order organisms as well as in human interphase cells. Relatively few studies have been conducted to elucidate molecular events in the context of mitotic DNA damage. We have previously established a model of mitotic catastrophe in human HeLa cells induced by actinomycin D. Here, we show that actinomycin D induce cellular stress via DNA damage during mitosis. The higher order packing of chromosomes during mitosis might impede efficient DNA repair. gammaH2AX serves as a marker for DNA repair and active JNK interacts with gammaH2AX in actinomycin D-treated mitotic extracts. We believe JNK might be in part, responsible for the phosphorylation of H2AX and thereby, facilitate the propagation of a positive signal for cell death, when repair is not achieved. The mitotic cell activates JNK-mediated cell death response that progresses through a caspase cascade downstream of the mitochondria. In the mean time, remaining checkpoint signals may be sufficient to put a restraining hand on entry into anaphase and the cell eventually dies in mitosis.
MicroRNAs (miRNAs) are a newly identified class of small regulatory non-coding endogenous RNAs that are ubiquitous from animals to plants and have pivotal functions in nearly all biological and metabolic processes. Increasing evidence shows that miRNAs are also new players regulating many protein-coding genes and specific pathways during the cell cycle. This review focuses on the functions of miRNAs in the cell cycle of differentiated cells and embryonic stem (ES) cells. Aberrant expression of these cell-cycle-related miRNAs may result in carcinogenesis, revealing the potential of miRNAs as therapeutic targets for clinical purposes.
MicroRNA-21 (miR-21) expression is activated in multiple types of cancers, such as breast, liver, brain, prostate, myometrial cancers but also in cardiovascular diseases. MiR-21 regulates a plethora of target proteins which are involved in cellular survival, apoptosis and cell invasiveness. MiR-21 regulation is complex due to an own promoter that is target for various transcription factors and hormones. The consistent miR-21 overexpression under pathophysiological conditions points to miR-21 as a valuable tool for new therapeutic strategies. In this review, we present and analyze current data about miR-21 expression in various pathologies ranging from cancer to cardiovascular disease. Further, miR-21 regulatory mechanisms and miR-21 downstream targets are discussed. Finally, we highlight the particular role of miR-21 as a therapeutic target in various diseases.
The aims of the present study are to determine the miRNA expression signature in rat hearts after ischaemic preconditioning (IP) and to identify an IP-regulated miRNA, miR-21, in IP-mediated cardiac protection, and the potential cellular and molecular mechanisms involved.
The miRNA expression signature was investigated in rat hearts. Among the 341 arrayed miRNAs, 40 miRNAs were differentially expressed (21 up and 19 down) in rat hearts with IP, compared with their controls. Some of these differentially expressed miRNAs were further verified by quantitative reverse transcriptase-polymerase chain reaction. Remarkably, miR-21 was one of most upregulated miRNAs in hearts after IP. In vivo, IP-mediated cardiac protection against ischaemia/reperfusion injury was inhibited by knockdown of cardiac miR-21. In cultured cardiac myocytes, we identified that miR-21 also had a protective effect on hypoxia/reoxygenation-induced cell apoptosis that was associated with its target gene, programmed cell death 4. The protective effect of miR-21 on cardiac cell apoptosis was further confirmed in rat hearts after ischaemia/reperfusion injury in vivo.
The results suggest that miRNAs are involved in IP-mediated cardiac protection. Identifying the roles of IP-regulated miRNAs in cardiac protection may provide novel therapeutic and preventive targets for ischaemic heart disease.
c-Jun N-terminal kinases (JNKs), first characterized as stress-activated members of the mitogen-activated protein kinase (MAPK) family, have become a focus of inhibitor screening strategies following studies that have shown their critical roles in the development of a number of diseases, such as diabetes, neurodegeneration and liver disease. We discuss recent advances in the discovery and development of ATP-competitive and ATP-noncompetitive JNK inhibitors. Because understanding the modes of actions of these inhibitors and improving their properties will rely on a better understanding of JNK structure, JNK catalytic mechanisms and substrates, recent advances in these areas of JNK biochemistry are also considered. In addition, the use of JNK gene knockout animals is continuing to reveal in vivo functions for these kinases, with tissue-specific roles now being dissected with tissue-specific knockouts. These latest advances highlight the many challenges now faced, particularly in the directed targeting of the JNK isoforms in specific tissues.
Pdcd4 (programmed cell death 4) has been known as a tumour suppressor gene and potential target for anticancer therapies for several years. Initially, Pdcd4 was identified as a gene that is up-regulated during apoptosis, but its precise role still remains to be defined. However, there is increasing evidence that Pdcd4 levels influence transcription, as well as translation, modulate different signal transduction pathways and might act as a tumour suppressor. Interestingly, recent data suggest that Pdcd4 function may depend on cell type and/or genetic background. This review summarizes the current knowledge regarding the function and regulation of Pdcd4.
miR‐21 expression in cancer and other diseases
Mechanisms of miR‐21 elevation in cancer: multi‐level regulatory control
Transcriptional control
Post‐trancriptional regulation
miR‐21 functions in cancer
Identification of direct miR‐21 targets
miR‐21 in gliomas: targeting cell cycle, apoptosis and invasion
miR‐21 networking and feedback regulation
miR‐21 as a diagnostic and prognostic marker
Potential therapeutic target
Acknowledgements
Abstract
More than 1000 microRNAs (miRNAs) are expressed in human cells, some tissue or cell type specific, others considered as house‐keeping molecules. Functions and direct mRNA targets for some miRNAs have been relatively well studied over the last years. Every miRNA potentially regulates the expression of numerous protein‐coding genes (tens to hundreds), but it has become increasingly clear that not all miRNAs are equally important; diverse high‐throughput screenings of various systems have identified a limited number of key functional miRNAs over and over again. Particular miRNAs emerge as principal regulators that control major cell functions in various physiological and pathophysiological settings. Since its identification 3 years ago as the miRNA most commonly and strongly up‐regulated in human brain tumour glioblastoma [ 1 ], miR‐21 has attracted the attention of researchers in various fields, such as development, oncology, stem cell biology and aging, becoming one of the most studied miRNAs, along with let‐7, miR‐17–92 cluster (‘oncomir‐1’), miR‐155 and a few others. However, an miR‐21 knockout mouse has not yet been generated, and the data about miR‐21 functions in normal cells are still very limited. In this review, we summarise the current knowledge of miR‐21 functions in human disease, with an emphasis on its regulation, oncogenic role, targets in human cancers, potential as a disease biomarker and novel therapeutic target in oncology.
Intraluminal thrombus formation and medial smooth muscle (SM) cell proliferation are recognized responses of the arterial system to injury. In contrast to these well-characterized processes during vascular repair, changes involving the adventitia have been largely neglected in previous studies. Hence, the goal of this investigation was to assess the response of the adventitia to coronary arterial injury.
Adventitial changes in porcine coronary arteries subjected to medial injury were characterized by immunohistochemistry, histochemistry, and microscopic morphometry. The rapid development of a hypercellular response in the adventitia was evident 3 days after balloon-induced medial injury. Cell proliferation, as assessed by proliferating cell nuclear antigen immunostaining, reached the maximum level in the adventitia at 3 days, whereas at 14 and 28 days, the number of replicating cells reverted toward the baseline. The proliferating activity in the adventitia exceeded that seen in the media at all times after injury. To further define the changes in the phenotype of adventitial cells, the expression of three cytoskeletal proteins (vimentin, alpha-SM actin, and desmin) was characterized. Fibroblasts in normal adventitia expressed vimentin but no alpha-SM actin or desmin. After injury, these cells acquired characteristics of myofibroblasts expressing alpha-SM actin, which peaked at 7 and 14 days. Desmin expression was patchy in the adventitia, as opposed to its homogeneous distribution in medial SM cells. The modulation of fibroblast phenotype was transient, inasmuch as alpha-SM actin immunostaining declined at 28 days after injury, when dense, collagen-rich scar was evident within the adventitia. The above-described changes involving hypercellularity of the adventitia, myofibroblast formation, and fibrosis were associated with a significant focal adventitial thickening at 3, 7, 14, and 28 days after injury (P < .01 versus uninjured coronary arteries).
This study demonstrates the involvement of the adventitia in the vascular repair process after medial injury. The hypercellularity of the adventitial layer, proliferation of fibroblasts, and modulation of their phenotype to myofibroblasts are associated with the development of the thickened adventitia. It is postulated that these phenomena affect vascular remodeling and may provide an important insight into the mechanisms of vascular disorders.
It is postulated that vascular lesion formation and remodeling involves a balance between vascular cell death and cell proliferation. Transforming growth factor-beta1 (TGF-beta1) is a pleiotropic factor expressed within vascular cells that regulates cell growth in a tissue-specific manner. This study tested the hypothesis that the control of vascular cell apoptosis involves cell type-specific regulation by TGF-beta1.
In response to serum withdrawal, cultured endothelial cells and vascular smooth muscle cells exhibited apoptosis as evidenced by DNA laddering and quantitated by analysis of nuclear chromatin morphology. Addition of TGF-beta1 to endothelial cells in serum-free media further potentiated the induction of apoptosis in a dose-dependent fashion. Moreover, TGF-beta1 promoted endothelial cell death despite the presence of 10% serum. However, endothelial cells plated on collagen I were resistant to TGF-beta1-induced apoptosis. This antiapoptotic influence of the matrix was mimicked by integrin activation with anti-beta1 antibodies and associated with increased expression of the antiapoptotic factor bcl-2. In accord with the hypothesis that the modulation of antiapoptotic gene expression may mediate the effects of TGF-beta1 and beta1 integrins on cell fate, we observed that endothelial cells with constitutive upregulation of bcl-2 remained viable despite exposure to TGF-beta1 in serum-free conditions. In contrast to the proapoptotic effect of TGF-beta1 in endothelial cells, addition of TGF-beta1 to vascular smooth muscle cells in serum-free media inhibited apoptosis.
These findings suggest that the effect of cytokines such as TGF-beta1 on cell fate is contextual and is modulated by cell-matrix interactions in a cell type-specific manner.
A series of molecular pathways have in common a significant role in the pathogenesis and progression of atherosclerosis and cancer. Shared mechanisms implicated for both diseases include oxidative stress and the cellular damage that results from it, toxic metabolites produced by cigarette smoking, and increased dietary fat intake. Atherosclerosis may begin when an injury or infection mutates or transforms a single arterial smooth muscle cell in the progenitor of a proliferative clone, similar to the most widely held carcinogenesis theory. Cell proliferation regulatory pathways have been associated with plaque progression, stenosis, and restenosis after angioplasty and with cancer progression. Alterations in cell adhesion molecules have been linked to plaque formation and thrombosis and to tumor invasion and metastasis. Altered expression of proteases associated with thrombolysis has been implicated in atherosclerotic plaque expansion and hemorrhage and in the invasion and metastasis of malignant neoplasms. Ligand-growth factor receptor interactions have been associated with early atherosclerotic lesions and with cancer development and spread. Nuclear transcription factors have been associated with progression of both diseases. Angiogenesis modulators have been linked to plaque expansion and restenosis of atherosclerotic lesions and to local and metastatic tumor expansion.
All tissues in the body are subjected to physical forces originating either from tension, created by cells themselves, or from the environment. Particularly, the cardiovascular system is continuously subjected to haemodynamic forces created by blood flow and blood pressure. While biomechanical force at physiological levels is essential to develop and maintain organic structure and function, elevated mechanical stress may result in cell death leading to pathological conditions. In recent years, however, it has been widely recognized that cell death, namely apoptosis, is not just the response to an injury but a highly regulated and controlled process. Therefore, physical stimuli must be sensed by cells and transmitted through intracellular signal transduction pathways to the nucleus, resulting in cell apoptosis. Disturbances in the regulatory mechanisms of apoptosis often precede the development of a disease. Exploration of the molecular signalling mechanisms leading to mechanical stress-induced apoptosis in cardiovascular disorders revealed the crucial role of apoptosis in the pathogenesis of these diseases. For instance, heart failure, hypertension and atherosclerosis are believed to be related to sustained mechanical overloading or stress. In this review we summarize the recent data focusing on molecular mechanisms of mechanical stress-induced apoptosis and highlight the role of apoptosis in the development of cardiovascular disorders, which may lead to new therapeutic strategies for these diseases.
Migration of adventitial fibroblasts, in addition to smooth muscle cell proliferation, plays a role in neointima formation following vascular injury. Previous studies have not directly addressed whether endogenous adventitial cells migrate towards the intima following balloon injury in the absence of medial dissection. We have employed an in vivo gene transfer technique to the rat carotid artery to directly label adventitial fibroblasts prior to balloon injury.
An adenoviral vector coordinating expression of nuclear targeted beta-galactosidase (AdLacZ) suspended in pluronic gel was applied to the perivascular surface of left carotid arteries of male Sprague-Dawley rats. Balloon catheter mediated vascular injury was performed on these arteries 4 days later and animals killed at 3, 7 and 14 days after injury.
Expression of LacZ up to 14 days after application of the adenovirus was restricted only to the adventitia of uninjured arteries and absent from untransfected right carotid arteries. However, following balloon catheter injury, LacZ positive cells were observed within the medial layer of vessels by 3 days, and contributed to the population of cells within the neointima at 7-14 days. Adventitial cells in uninjured arteries did not express smooth muscle alpha-actin but after injury, LacZ positive cells migrating towards the lumen exhibited alpha-actin immunostaining, suggesting their change to a myofibroblastic phenotype.
These findings provide direct evidence that adventitial fibroblasts migrate and contribute to neointima formation after balloon injury and show that in vivo gene transfer to the adventitia results in sustained transgene expression capable of labelling migrating adventitial cells within the media and neointima of injured vessels.
Traditional concepts of vascular inflammation are considered "inside-out" responses centered on the monocyte adhesion and lipid oxidation hypotheses. These mechanisms likely operate in concert, holding the central tenet that the inflammatory response is initiated at the luminal surface. However, growing evidence supports a new paradigm of an "outside-in" hypothesis, in which vascular inflammation is initiated in the adventitia and progresses inward toward the intima. Hallmarks of the outside-in hypothesis include population of the adventitia with exogenous cell types, including monocytes, macrophages, and lymphocytes, the phenotypic switch of adventitial fibroblasts into migratory myofibroblasts, and increased vasa vasorum neovascularization. The resident and migrating cells deposit collagen and matrix components, respond to and upregulate inflammatory chemokines and/or antigens, and regulate the local redox state of the adventitia. B cells and T cells generate local humoral immune responses against local antigen presentation by foam cells and antigen presenting cells. These events result in increased local expression of cytokines and growth factors, evoking an inflammatory response that propagates inward toward the intima. Ultimately, it appears that the basic mechanisms of cellular activation and migration in vascular inflammation are highly conserved across a variety of cardiovascular disease states and that major inflammatory events begin in the adventitia.
microRNAs (miRNAs) are short noncoding RNAs that regulate gene expression negatively. Although a role for aberrant miRNA expression in cancer has been postulated, the pathophysiologic role and relevance of aberrantly expressed miRNA to tumor biology has not been established.
We evaluated the expression of miRNA in human hepatocellular cancer (HCC) by expression profiling, and defined a target gene and biologically functional effect of an up-regulated miRNA.
miR-21 was noted to be highly overexpressed in HCC tumors and cell lines in expression profiling studies using a miRNA microarray. Inhibition of miR-21 in cultured HCC cells increased expression of the phosphatase and tensin homolog (PTEN) tumor suppressor, and decreased tumor cell proliferation, migration, and invasion. In contrast-enhanced miR-21 expression by transfection with precursor miR-21 increased tumor cell proliferation, migration, and invasion. Moreover, an increase in cell migration was observed in normal human hepatocytes transfected with precursor miR-21. PTEN was shown to be a direct target of miR-21, and to contribute to miR-21 effects on cell invasion. Modulation of miR-21 altered focal adhesion kinase phosphorylation and expression of matrix metalloproteases 2 and 9, both downstream mediators of PTEN involved in cell migration and invasion.
Aberrant expression of miR-21 can contribute to HCC growth and spread by modulating PTEN expression and PTEN-dependent pathways involved in mediating phenotypic characteristics of cancer cells such as cell growth, migration, and invasion.
Vascular remodeling is the result of a close interplay of changes in vascular tone and structure. In this review, the role of angiotension-converting enzyme (ACE) and the impact of ACE inhibition on vascular remodeling processes during vascular injury and restenosis, hypertension, atherosclerosis, and aneurysm formation are discussed. The role of ACE and angiotensin II (Ang II) in neointimal thickening has been firmly established by animal studies and is mediated by Ang II type 1 (AT(1)) receptor signaling events via monocyte chemoattractant protein-1 and NAD(P)H oxidase. ACE and Ang II are involved in the remodeling of large and resistance arteries during hypertension; here, cell proliferation and matrix remodeling are also regulated by signaling events downstream of the AT(1) receptor. In atherosclerosis, Ang II is involved in the inflammatory and tissue response, mediated by various signaling pathways downstream of the AT(1) receptor. Although ACE inhibition has been shown to inhibit atherosclerotic processes in experimental animal models, results of large clinical trials with ACE inhibitors were not conclusive. Remodeling of vessel dimensions and structure during aneurysm formation is counteracted by ACE inhibition. Here, a direct effect of ACE inhibitors on matrix metalloproteinase activity has to be considered as part of the working mechanism. The role of ACE2 in vascular remodeling has yet to be established; however, ACE2 has been shown to be associated with vascular changes in hypertension and atherosclerosis.
We have developed a method to detect DNA synthesis in proliferating cells, based on the incorporation of 5-ethynyl-2′-deoxyuridine (EdU) and its subsequent detection by a fluorescent azide through a Cu(I)-catalyzed [3 + 2] cycloaddition reaction (“click” chemistry). Detection of the EdU label is highly sensitive and can be accomplished in minutes. The small size of the fluorescent azides used for detection results in a high degree of specimen penetration, allowing the staining of whole-mount preparations of large tissue and organ explants. In contrast to BrdU, the method does not require sample fixation or DNA denaturation and permits good structural preservation. We demonstrate the use of the method in cultured cells and in the intestine and brain of whole animals.
• BrdU
• click chemistry
• DNA replication
• EdU
• microscopy
Programmed cell death 4 (PDCD4) is an important functional target of the microRNA miR-21 in breast cancer cells
- Frankel
The role of the adventitia in vascular inflammation
- Maiellaro