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Ex vivo generation of a highly potent population of circulating angiogenic cells using a collagen matrix

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Abstract

Biomaterials that have the ability to augment angiogenesis are highly sought-after for applications in regenerative medicine, particularly for revascularization of ischemic and infarcted tissue. We evaluated the culture of human circulating angiogenic cells (CAC) on collagen type I-based matrices, and compared this to traditional selective-adhesion cultures on fibronectin. Culture on a collagen matrix supported the proliferation of CD133(+) and CD34(+)CD133(+) CACs. When subjected to serum starvation, the matrix conferred a resistance to cell death for CD34(+) and CD133(+) progenitors and increased phosphorylation of Akt. After 4days of culture, phenotypically enriched populations of endothelial cells (CD31(+)CD144(+)) and progenitor cells (CD34(+)CD133(+)) emerged. Culture on matrix upregulated the phosphorylation and activation of ERK1/2 pathway members, and matrix-cultured cells also had an enhanced functional capacity for adhesion and invasion. These functional improvements were abrogated when cultured in the presence of ERK inhibitors. The formation of vessel-like structures in an angiogenesis assay was augmented with matrix-cultured cells, which were also more likely to physically associate with such structures compared to CACs taken from culture on fibronectin. In vivo, treatment with matrix-cultured cells increased the size and density of arterioles, and was superior at restoring perfusion in a mouse model of hindlimb ischemia, compared to fibronectin-cultured cell treatment. This work suggests that a collagen-based matrix, as a novel substrate for CAC culture, possesses the ability to enrich endothelial and angiogenic populations and lead to clinically relevant functional enhancements.

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... EPCs can be generated from the culture of peripheral blood mononuclear cells (PBMCs) isolated from blood by density gradient centrifugation. PBMCs are cultured for 4–7 days in endothelial-promoting media on fibronectin and the subsequently generated therapeutic population is referred to as 'circulating angiogenic cells' (CACs), or early EPCs [2,14]. As cardiovascular disease is the number one leading cause of death in the Western world [15], there is a potential for CAC therapy to improve the quality of life for patients of this disease by aiding in the restoration of blood flow to the heart. ...
... Hindlimb ischemia mouse model. Nude BALB/C mice (7–8 weeks old) underwent ligation of proximal femoral arteries under 2% isoflurane, as previously described [14]. Subsequently, the ischemic hindlimb was injected downstream of the ligation site with 2 equivolumetric injections (50 ml total, suspended in PBS) containing a total of 5610 6 CACs that were derived from: i) fresh PBMCs; ii) day 1 cryopreserved PBMCs; or, iii) day 28 cryopreserved PBMCs, prepared as described above. ...
... Hindlimb perfusion was measured using laser Doppler analysis pre-operatively, post-operatively, and at days 4, 7, 10 and 14, as described previously [14]. Briefly, while mice were anaesthetized with 2% isoflurane, single point measurements were recorded (moorLD12; Moor Instruments, Axminster, UK) in both hindlimbs and used to evaluate perfusion. ...
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Cell transplantation for regenerative medicine has become an appealing therapeutic method; however, stem and progenitor cells are not always freshly available. Cryopreservation offers a way to freeze cells as they are generated, for storage and transport until required for therapy. This study was performed to assess the feasibility of cryopreserving peripheral blood mononuclear cells (PBMCs) for the subsequent in vitro generation of their derived therapeutic population, circulating angiogenic cells (CACs). PBMCs were isolated from healthy human donors. Freshly isolated cells were either analyzed immediately or cryopreserved in media containing 6% plasma serum and 5% dimethyl sulfoxide. PBMCs were thawed after being frozen for 1 (early thaw) or 28 (late thaw) days and analyzed, or cultured for 4 days to generate CACs. Analysis of the cells consisted of flow cytometry for viability and phenotype, as well as functional assays for their adhesion and migration potential, cytokine secretion, and in vivo angiogenic potential. The viability of PBMCs and CACs as well as their adhesion and migration properties did not differ greatly after cryopreservation. Phenotypic changes did occur in PBMCs and to a lesser extent in CACs after freezing; however the potent CD34(+)VEGFR2(+)CD133(+) population remained unaffected. The derived CACs, while exhibiting changes in inflammatory cytokine secretion, showed no changes in the secretion of important regenerative and chemotactic cytokines, nor in their ability to restore perfusion in ischemic muscle. Overall, it appears that changes do occur in cryopreserved PBMCs and their generated CACs; however, the CD34(+)VEGFR2(+)CD133(+) progenitor population, the secretion of pro-vasculogenic factors, and the in vivo angiogenic potential of CACs remain unaffected by cryopreservation.
... Procedures for isolating human peripheral blood mononuclear cells (PBMCs) were approved by the Human Research Ethics Board of the University of Ottawa Heart Institute. With informed consent, total PBMCs were isolated from the blood of healthy volunteers, and cultured for 4 days on fibronectin, as previously described [25]. PBMCs were then cultured on MG-modified or non-modified collagen gels in endothelial growth medium-2 (EGM-2) with fetal bovine serum, vascular endothelial growth factor, R 3 -insulin growth factor and endothelial growth factor supplements (Lonza) at 37°C, after which cells and/ or conditioned media were collected. ...
... For viability, PBMCs were cultured on collagen gels (±MG modification) for 2 days under serum deprivation and hypoxic conditions as described [25]. Cells were then stained for CD133, CD34, CD31, CD144 and 7-aminoactinomycin (7-AAD) and analyzed by flow cytometry. ...
... For adhesion, 5 9 10 5 DAPI-stained PBMCs were seeded on MG-modified or non-modified collagen gels for 60 min, fixed with 4% paraformaldehyde and counted per field-of-view in a blinded fashion (209 magnification). For chemotaxis, PBMCs on the two collagen gels were serum starved overnight, then lifted and placed in the upper chamber of Transwell Ò Permeable Supports (Corning), with 0.05 lg/mL of vascular endothelial growth factor (Cedarlane) in EBM in the bottom chamber as the migratory stimulus, as previously described [25]. After overnight culture, PBMCs were fixed with paraformaldehyde and the number of migrating cells was quantified. ...
Article
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Advanced glycation end-products (AGEs) have been associated with poorer outcomes after myocardial infarction (MI), and linked with heart failure. Methylglyoxal (MG) is considered the most important AGE precursor, but its role in MI is unknown. In this study, we investigated the involvement of MG-derived AGEs (MG-AGEs) in MI using transgenic mice that over-express the MG-metabolizing enzyme glyoxalase-1 (GLO1). MI was induced in GLO1 mice and wild-type (WT) littermates. At 6 h post-MI, mass spectrometry revealed that MG-H1 (a principal MG-AGE) was increased in the hearts of WT mice, and immunohistochemistry demonstrated that this persisted for 4 weeks. GLO1 over-expression reduced MG-AGE levels at 6 h and 4 weeks, and GLO1 mice exhibited superior cardiac function at 4 weeks post-MI compared to WT mice. Immunohistochemistry revealed greater vascular density and reduced cardiomyocyte apoptosis in GLO1 vs. WT mice. The recruitment of c-kit⁺ cells and their incorporation into the vasculature (c-kit⁺CD31⁺ cells) was higher in the infarcted myocardium of GLO1 mice. MG-AGEs appeared to accumulate in type I collagen surrounding arterioles, prompting investigation in vitro. In culture, the interaction of angiogenic bone marrow cells with MG-modified collagen resulted in reduced cell adhesion, increased susceptibility to apoptosis, fewer progenitor cells, and reduced angiogenic potential. This study reveals that MG-AGEs are produced post-MI and identifies a causative role for their accumulation in the cellular changes, adverse remodeling and functional loss of the heart after MI. MG may represent a novel target for preventing damage and improving function of the infarcted heart.
... It is well understood that response to cell therapy is dose-dependent due to poor retention and viability within affected areas and therefore limit their regenerative potential. The enhancement of EPC delivery is currently being developed by numerous groups and is of great interest (Bahlmann et al. 2005;Kuraitis et al. 2011). ...
... Cell homing and retention of transplanted cells within the target tissue relies on the adhesion of donor and host cells, and its survival rate which has been shown to be less than 15% (Muller-Ehmsen et al. 2002). Previous studies have shown that collagen-based matrices can support the differentiation of endothelial progenitor cells into mature endothelial cells, can improve the survival and function of EPCs in a serum deprivation environment, and can also produce a population of progenitor cells with enhanced functional capacities, such as cell migration, adhesion, and angiogenesis (Kuraitis et al. 2011). There are several advantages in using collagen-based matrices as they offer a large surface area for cell seeding, are very stable for mechanical support, its' porosity for vessel ingrowth, biodegradable, have minimal immunogenicity, and overall have had many successful years of use (Kofidis et al. 2003;Gonen-Wadmany et al. 2004;Boccafoschi et al. 2005). ...
... After 24 h, cells that had penetrated the basement membrane and adhered to the lower insert membrane were fixed with 4% paraformaldehyde, and stained with 4′,6-diamidino-2-phenylindol-(DAPI) containing mounting medium (Vector Laboratories). Six random fields-of-view were imaged using an Olympus BX60 fluorescent microscope, and DAPI+ cells were counted (Kuraitis et al., 2011). ...
... Collagen-based matrices were prepared on ice by blending type I rat tail collagen (0.4% wt/vol; BD Bioscience, Oakville, Canada) with chondroitin sulfate C (40% wt/vol; Wako Chemicals, Osaka, Japan), as described previously. 15 Briefly, components were mixed on ice and cross-linked with glutaraldehyde (1.5%). Residual aldehyde groups were inactivated by the addition of glycine (20%). ...
... 14 We also previously showed that CACs exposed to the matrix demonstrated higher levels of phosphorylated Akt (PI3K/Akt pathway) and increased survival under hypoxia. 15 Based on the ability of the collagen matrix to modulate the therapeutic phenotype and function of CACs, it may have induced similar effects in the pig model of this study. The increased retention and viability of cells within the matrix may also have provided more prolonged paracrine effects because transplanted cells secrete proangiogenic factors 37 and upregulate host-derived cytokine secretion. ...
Article
-Vasculogenic cell-based therapy combined with tissue engineering is a promising revascularization approach destined at patients with advanced coronary artery disease, many of whom exhibit myocardial hibernation. However, so far no experimental data have been available in this context; we therefore examined the biopolymer-supported delivery of circulating angiogenic cells (CACs) using a clinically relevant swine model of hibernating myocardium. -Twenty-five swine underwent placement of an ameroid constrictor on the left circumflex artery (LCx). After 2 weeks, animals underwent echocardiography, rest and stress NH3-PET perfusion, and FDG-PET viability scans. The following week, swine were randomized to receive intramyocardial injections of PBS-control (n=10), CACs (n=8), or CACs + collagen-based matrix (n=7). The imaging protocol was repeated after 7 weeks. Baseline PET myocardial blood flow (MBF) and myocardial flow reserve (MFR) were reduced in the LCx territory (both p<0.001), and hibernation (mismatch) was observed. At follow-up, stress MBF had increased (p≤0.01) and hibernation decreased (p<0.01) in the cells+matrix group only. Microsphere-measured MBF validated the perfusion results. Arteriole density and wall motion abnormalities improved in the cells+matrix group. There was also a strong trend towards an improvement in ejection fraction (p=0.07). -In this preclinical swine model of ischemic and hibernating myocardium, the combined delivery of CACs and a collagen-based matrix restored perfusion, reduced hibernation, and improved myocardial wall motion.
... Since they can be isolated from the peripheral blood of adult patients, CACs are the one of most accessible cell types for autologous cell therapy. Furthermore, it has been shown that their regenerative potential can be enhanced by the use of biomaterials [3][4][5]. Using direct cell radiolabeling methods with positron emission tomography (PET), we have reported that collagen-based matrices can significantly improve the early retention of CACs transplanted into ischemic tissue [6]. However, this technique can only monitor the cells for several hours due to the short half-life of PET radioisotopes. ...
... Collagen-based matrices (pH ~7.2-7.5) were prepared on ice, as previously described [5]. Briefly, matrices consisted of a mixture of 0.4% blended neutralized type I rat tail collagen (Becton Dickinson), and chondroitin 6-sulfate (Sigma), cross-linked with 0.02% glutaraldehyde, followed by glycine termination of unreacted aldehyde groups. ...
Article
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The use of biomaterials and tracking the long-term fate of the transplanted cells is expected to help improve the clinical translation of cell therapies for cardiac regeneration. To this end, reporter gene strategies are promising for monitoring the fate of cells transplanted with or without a delivery biomaterial; however, their application with primary adult progenitor cells (such as human circulating angiogenic cells (CACs)) has not been extensively evaluated. In this study, human CACs were transduced with reporter genes via one of two lentiviral (LV) vectors: LV-GFP-iresTK or LV-Fluc-RFP-tTK. The mean transduction efficiency was 15% (LV-GFP-iresTK) and 13% (LV-Fluc-RFP-tTK) at multiplicities of infection (MOI) of 10 and 50, respectively. Western blot analysis confirmed HSV1-tk protein expression in transduced CACs. There was no significant difference in viability between the transduced CACs and the untreated controls at a MOI of 50 or below. However, a reduction was observed in cell viability of CACs transduced with LV-Fluc-RFP-tTK at an MOI of 100. Cell migration and angiogenic potential were not affected by transduction protocol. After 4 weeks, 80.3 ± 8.4% of the labeled cells continued to express the reporters and could be visualized when embedded within a collagen matrix scaffold. Therefore, quiescent human CACs can be stably transduced to express reporter genes without affecting their function. This reporter gene technique is a promising approach to be further tested for tracking transplanted CACs (±delivery matrix) non-invasively and longitudinally.
... In turn, cells contribute to the complex cell-matrix feedback loop, with their overall functionalities resulting in proteolytic turnover and ECM structural integrity [276]. The design of new ingenious biomaterials must consider these functions of the native ECM, at least to a certain degree, to mimic the natural environment to regulate stem cell fate decisions. of human-derived biomaterials for therapeutics have now moved from the direct use of autogenic tissue grafts, allogenic tissues/organs from donors and a wide variety of allografts from cadavers toward the recreation of humanderived extracellular influences in simplified forms, the decellularization of tissues and organs for scaffolding use and the incorporation of short functional domains derived from human tissue into ECM-mimicking biomaterials to manipulate cell fate commitment [52,289,290]. ...
... This combination has also been successful in cell delivery and implantation within target ischemic tissue using a collagen I/CS tissue-engineered matrix [427]. Other such biomaterials with well-defined chemical, topographical, and mechanical cues and even gradients of these physicochemical cues may also enhance endogenous progenitor cell homing and engrafting to sites of ischemia [428] and may serve as novel substrates for human circulating angiogenic cells to augment angiogenesis for the revascularization of ischemic and infarcted tissue [290]. However, native GAGs derived from human tissue are heterogeneous and structurally complex, and specific GAG moieties have been demonstrated to trigger specific cellular responses during cell division, motility and migration. ...
Article
Biomaterials have played an increasingly prominent role in the success of biomedical devices and in the development of tissue engineering, which seeks to unlock the regenerative potential innate to human tissues/organs in a state of deterioration and to restore or reestablish normal bodily function. Advances in our understanding of regenerative biomaterials and their roles in new tissue formation can potentially open a new frontier in the fast-growing field of regenerative medicine. Taking inspiration from the role and multi-component construction of native extracellular matrices (ECMs) for cell accommodation, the synthetic biomaterials produced today routinely incorporate biologically active components to define an artificial in vivo milieu with complex and dynamic interactions that foster and regulate stem cells, similar to the events occurring in a natural cellular microenvironment. The range and degree of biomaterial sophistication have also dramatically increased as more knowledge has accumulated through materials science, matrix biology and tissue engineering. However, achieving clinical translation and commercial success requires regenerative biomaterials to be not only efficacious and safe but also cost-effective and convenient for use and production. Utilizing biomaterials of human origin as building blocks for therapeutic purposes has provided a facilitated approach that closely mimics the critical aspects of natural tissue with regard to its physical and chemical properties for the orchestration of wound healing and tissue regeneration. In addition to directly using tissue transfers and transplants for repair, new applications of human-derived biomaterials are now focusing on the use of naturally occurring biomacromolecules, decellularized ECM scaffolds and autologous preparations rich in growth factors/non-expanded stem cells to either target acceleration/magnification of the body's own repair capacity or use nature's paradigms to create new tissues for restoration. In particular, there is increasing interest in separating ECMs into simplified functional domains and/or biopolymeric assemblies so that these components/constituents can be discretely exploited and manipulated for the production of bioscaffolds and new biomimetic biomaterials. Here, following an overview of tissue auto-/allo-transplantation, we discuss the recent trends and advances as well as the challenges and future directions in the evolution and application of human-derived biomaterials for reconstructive surgery and tissue engineering. In particular, we focus on an exploration of the structural, mechanical, biochemical and biological information present in native human tissue for bioengineering applications and to provide inspiration for the design of future biomaterials.
... Animals receiving FGF-2 treatment demonstrated higher levels of therapeutic cell-mobilizing cytokines G-CSF, MCP-1 and VEGF. Other studies have correlated enhanced angiogenesis in animal models with increased levels of G-CSF [30], MCP-1 [19,31] and VEGF [30]. Based on the serum cytokine profile observed, it may prove to be that another effect of our FGF-2 delivery system is to induce a systemic environment that is supportive of angiogenesis. ...
... Animals receiving FGF-2 treatment demonstrated higher levels of therapeutic cell-mobilizing cytokines G-CSF, MCP-1 and VEGF. Other studies have correlated enhanced angiogenesis in animal models with increased levels of G-CSF [30], MCP-1 [19,31] and VEGF [30]. Based on the serum cytokine profile observed, it may prove to be that another effect of our FGF-2 delivery system is to induce a systemic environment that is supportive of angiogenesis. ...
... Dissolution products from a bioactive tissue engineering scaffold should stimulate genes in the regenerating tissue to promote efficient cell differentiation and proliferation. As type I collagen has been shown to promote osteogenesis of MSCs (28) and angiogenesis of EPCs (29), it is unknown whether intrafibrillar biosilicification confers additional benefits to the osteoinductivity or angiogenic potential of CSs. Thus, mRNA expressions of biomarkers associated with osteogenesis and angiogenesis were examined, using qRT-PCR. ...
Article
Traditional bone regeneration strategies relied on supplementation of biomaterials constructs with stem or progenitor cells or growth factors. By contrast, cell homing strategies employ chemokines to mobilize stem or progenitor cells from host bone marrow and tissue niches to injured sites. Although silica-based biomaterials exhibit osteogenic and angiogenic potentials, they lack cell homing capability. Stromal cell-derived factor-1 (SDF-1) plays a pivotal role in mobilization and homing of stem cells to injured tissues. In this work, we demonstrated that 3-dimensional collagen scaffolds infiltrated with intrafibrillar silica are biodegradable and highly biocompatible. They exhibit improved compressive stress-strain responses and toughness over nonsilicified collagen scaffolds. They are osteoconductive and up-regulate expressions of osteogenesis- and angiogenesis-related genes more significantly than nonsilicified collagen scaffolds. In addition, these scaffolds reversibly bind SDF-1α for sustained release of this chemokine, which exhibits in vitro cell homing characteristics. When implanted subcutaneously in an in vivo mouse model, SDF-1α-loaded silicified collagen scaffolds stimulate the formation of ectopic bone and blood capillaries within the scaffold and abrogate the need for cell seeding or supplementation of osteogenic and angiogenic growth factors. Intrafibrillar-silicified collagen scaffolds with sustained SDF-1α release represent a less costly and complex alternative to contemporary cell seeding approaches and provide new therapeutic options for in situ hard tissue regeneration.-Niu, L.-N., Jiao, K., Qi, Y.-P., Nikonov, S., Yiu, C. K. Y., Arola, D. D., Gong, S.-Q., El-Marakby, A., Carrilho, M. R. O., Hamrick, M. W., Hargreaves, K. M., Diogenes, A., Chen, J.-H., Pashley, D. H., Tay, F. R. Intrafibrillar silicification of collagen scaffolds for sustained release of stem cell homing chemokine in hard tissue regeneration.
... Approximately 100ml of blood was procured from healthy human volunteers and peripheral blood mononuclear cells (PBMCs) were isolated using Histopaque and density centrifugation. PBMCs were plated for 4d on fibronectin-coated plates to generate the heterogeneous population of CACs, as previously described [15]. ...
Article
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Islet transplantation to treat type 1 diabetes (T1D) has shown varied long-term success, due in part to insufficient blood supply to maintain the islets. In the current study, collagen and collagen:chitosan (10:1) hydrogels, +/- circulating angiogenic cells (CACs), were compared for their ability to produce a pro-angiogenic environment in a streptozotocin-induced mouse model of T1D. Initial characterization showed that collagen-chitosan gels were mechanically stronger than the collagen gels (0.7kPa vs. 0.4kPa elastic modulus, respectively), had more cross-links (9.2 vs. 7.4/µm(2)), and were degraded more slowly by collagenase. After gelation with CACs, live/dead staining showed greater CAC viability in the collagen-chitosan gels after 18h compared to collagen (79% vs. 69%). In vivo, collagen-chitosan gels, subcutaneously implanted for up to 6 weeks in a T1D mouse, showed increased levels of pro-angiogenic cytokines over time. By 6 weeks, anti-islet cytokine levels were decreased in all matrix formulations ± CACs. The 6-week implants demonstrated increased expression of VCAM-1 in collagen-chitosan implants. Despite this, infiltrating vWF(+) and CXCR4(+) angiogenic cell numbers were not different between the implant types, which may be due to a delayed and reduced cytokine response in a T1D versus non-diabetic setting. The mechanical, degradation and cytokine data all suggest that the collagen-chitosan gel may be a suitable candidate for use as a pro-angiogenic ectopic islet transplant site.
... The human CAC isolation protocol was approved by the Human Research Ethics Board of the University of Ottawa Heart Institute. As previously described (Kuraitis et al., 2011), peripheral blood mononuclear cells (PBMCs) were isolated by Histopaque 1077 (Sigma) density-gradient centrifugation from the blood of healthy male and female volunteers aged 20-35 years. Cells were seeded on fibronectin-coated plates (20 μg/plate) in EBM-2 (Clonetics) supplemented with EGM-2-MV SingleQuots (Clonetics). ...
Article
Islet transplantation is an emerging strategy for treating patients with type 1 diabetes mellitus. Although the proof of concept for cellular replacement therapy in diabetes has been firmly established, vascularity of the transplant site and the long-term survival and function of transplanted islets remains suboptimal. In the present study, human circulating angiogenic cells (CACs) and porcine islet cells embedded in collagen-chitosan hydrogels, with and without laminin, were investigated as potential engineered biomaterials for the treatment of type 1 diabetes. Hydrogels were evaluated in vitro for their physical properties (compression, degradation, porosity and wettability) and cell compatibility. Increasing the chitosan content in the collagen-based hydrogel resulted in increased stiffness (p ≤ 0.04) and time to gelation (p < 0.001), but reduced porosity (from 22-28% to 16-19%). The material design formulations (10:1 vs 20:1 collagen:chitosan ratio) directly affected the cell properties. The viability of both human CACs and porcine islets embedded in the 20:1 collagen-chitosan matrix was higher at 24 h compared to the 10:1 formulation. For islet function, glucose stimulation indices for the 20:1 formulation at 24 h compared favourably with values reported in the literature, more so than the 10:1 formulations. While laminin improved the short-term viability of CACs, its presence did not confer any benefit to islet viability or function. Overall, the design features outlined in this study provided the degree of control required to establish viable tissue with potential for islet transplantation and neovascularization. Copyright © 2013 John Wiley & Sons, Ltd.
... Our group has used various injectable collagen-based matrices to generate enhanced cells for therapy and/or to facilitate the delivery of progenitor cells into ischemic tissue (Fig. 5.2 ) (Suuronen et al. 2006a , b ;Deng et al. 2010 ;Kuraitis et al. 2011a ;Giordano et al. 2013b ;Ahmadi et al. 2014 ). By using the collagen-based matrix, cellular retention was enhanced while nonspecifi c distribution of EPCs to other tissues was limited Zhang et al. 2008d ). ...
Chapter
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Cardiac disease is one of the most common causes of death in the world. Currently, surgically invasive therapies, such as implantable devices and, ultimately, heart transplantation, are the only remedies for end-stage congestive heart disease, which constitutes the final common pathway for all cardiac disorders. However, these surgical treatments are all mechanical and do not repair the damaged heart tissue. On the other hand, regenerative therapy promises to revolutionize the treatment of these patients and provides a biological and natural solution for cardiac repair. For example, biomaterials for the delivery of cells, growth factors and/or signaling molecules have been developed to treat the diseased heart. Biomaterials can provide suitable microenvironments to promote angiogenesis, deliver several key signals needed for repair processes, enhance engraftment and differentiation of cells, and modify cell function in different ways. Biomaterials can mimic the natural extracellular matrix, which supports the structure and functions of cells. Over the last decade, many biomaterials have been developed and used. In this chapter, we provide an overview of the biomaterials most commonly used for cardiac tissue engineering in preclinical studies and discuss their roles in cardiac repair processes.
... Although injecting collagen post-MI may seem counterintuitive given the presence of the collagenous scar, the composition and mechanical properties of the scar are vastly different from the normal myocardium 10,15 . Supporting their use as a therapy post-MI, (animal-derived) collagen materials have been shown to improve angiogenesis and tissue integration, reduce inflammation and apoptosis, and limit negative remodeling and the loss of cardiac function 24,[26][27][28][29][30][31][47][48][49][50] . Furthermore, the safety and efficacy of a porcine myocardial ECM hydrogel, composed primarily of collagens, was demonstrated in a pre-clinical pig MI model 20 . ...
Article
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Despite the success of current therapies for acute myocardial infarction (MI), many patients still develop adverse cardiac remodeling and heart failure. With the growing prevalence of heart failure, a new therapy is needed that can prevent remodeling and support tissue repair. Herein, we report on injectable recombinant human collagen type I (rHCI) and type III (rHCIII) matrices for treating MI. Injecting rHCI or rHCIII matrices in mice during the late proliferative phase post-MI restores the myocardium’s mechanical properties and reduces scar size, but only the rHCI matrix maintains remote wall thickness and prevents heart enlargement. rHCI treatment increases cardiomyocyte and capillary numbers in the border zone and the presence of pro-wound healing macrophages in the ischemic area, while reducing the overall recruitment of bone marrow monocytes. Our findings show functional recovery post-MI using rHCI by promoting a healing environment, cardiomyocyte survival, and less pathological remodeling of the myocardium.
... Furthermore, the collagen scaffolds also provide structural and mechanical support for 3D cell culture in vitro and can be employed as a bioactive cue for the delivery of human cells in vivo. Collagen is one of the major ECM proteins that interacts with various growth factors and cells, and has a chemotactic activity to endothelial cells [21] in the wound healing process [22]. Theoretically, collagen scaffolds can be employed as cell carriers and as reservoirs for growth factors without compromising their activity which would be of great value to promote angiogenesis and vascular stabilization for tissue engineering [23,24]. ...
Article
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Here, we describe a porous 3-dimensional collagen scaffold material that supports capillary formation in vitro, and promotes vascularization when implanted in vivo. Collagen scaffolds were synthesized from type I bovine collagen and have a uniform pore size of 80 μm. In vitro, scaffolds seeded with primary human microvascular endothelial cells suspended in human fibrin gel formed CD31 positive capillary-like structures with clear lumens. In vivo, after subcutaneous implantation in mice, cell-free collagen scaffolds were vascularized by host neovessels, whilst a gradual degradation of the scaffold material occurred over 8 weeks. Collagen scaffolds, impregnated with human fibrinogen gel, were implanted subcutaneously inside a chamber enclosing the femoral vessels in rats. Angiogenic sprouts from the femoral vessels invaded throughout the scaffolds and these degraded completely after 4 weeks. Vascular volume of the resulting constructs was greater than the vascular volume of constructs from chambers implanted with fibrinogen gel alone (42.7±5.0 μL in collagen scaffold vs 22.5±2.3 μL in fibrinogen gel alone; p<0.05, n = 7). In the same model, collagen scaffolds seeded with human adipose-derived stem cells (ASCs) produced greater increases in vascular volume than did cell-free collagen scaffolds (42.9±4.0 μL in collagen scaffold with human ASCs vs 25.7±1.9 μL in collagen scaffold alone; p<0.05, n = 4). In summary, these collagen scaffolds are biocompatible and could be used to grow more robust vascularized tissue engineering grafts with improved the survival of implanted cells. Such scaffolds could also be used as an assay model for studies on angiogenesis, 3-dimensional cell culture, and delivery of growth factors and cells in vivo.
... Procedures were performed on 8-or 9-week-old athymic nude CD1 female mice as previously described (Kuraitis et al., 2011) with the approval of the University of Ottawa Animal Care Committee and in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. Unilateral left hindlimb ischemia in mice was induced by ligating the proximal end of the femoral artery using 4-0 silk surgical suture as described in Limbourg et al. (2009). ...
Article
A major goal of cell therapy for vascular diseases is to promote revascularization through the injection of endothelial stem/progenitor cells. The gene regulatory mechanisms that underlie endothelial progenitor-mediated vascular repair, however, remain elusive. Here, we identify the transcription factor TAL1/SCL as a key mediator of the vascular repair function of primary human endothelial colony-forming cells (ECFCs). Genome-wide analyses in ECFCs demonstrate that TAL1 activates a transcriptional program that promotes cell adhesion and migration. At the mechanistic level, we show that TAL1 upregulates the expression of migratory and adhesion genes through recruitment of the histone acetyltransferase p300. Based on these findings, we establish a strategy that enhances the revascularization efficiency of ECFCs after ischemia through ex vivo priming with the histone deacetylase inhibitor TSA. Thus, small molecule epigenetics drugs are effective tools for modifying the epigenome of stem/progenitor cells prior to transplantation as a means to enhance their therapeutic potential.
... (3) Biomaterials act as a protective shell against the harsh inflammatory environment [173]. (4) Provide instructive cues for cells to proliferate, mobilize, and differentiate [171,[174][175][176]. (5) Modulates the secretion of trophic factors to enhance indirect myocardial repair [175,177]. ...
Article
Introduction: Over the past decade, it has become clear that long-term engraftment of any ex vivo expanded cell product transplanted into injured myocardium is modest and all therapeutic regeneration is mediated by stimulation of endogenous repair rather than differentiation of transplanted cells into working myocardium. Given that increasing the retention of transplanted cells boosts myocardial function, focus on the fundamental mechanisms limiting retention and survival of transplanted cells may enable strategies to help to restore normal cardiac function. Areas covered: This review outlines the challenges confronting cardiac engraftment of ex vivo expanded cells and explores means of enhancing cell-mediated repair of injured myocardium. Expert opinion: Stem cell therapy has already come a long way in terms of regenerating damaged hearts though the poor retention of transplanted cells limits the full potential of truly cardiotrophic cell products. Multifaceted strategies directed towards fundamental mechanisms limiting the long-term survival of transplanted cells will be needed to enhance transplanted cell retention and cell-mediated repair of damaged myocardium for cardiac cell therapy to reach its full potential.
... A more recent study described the differentiation of progenitor cells into ECs in a collagen matrix. This study showed that collagen protects progenitor cells from apoptosis and increases adhesion and invasion in an extracellular signal-regulated kinase pathway-dependent way [116]. Fibrin is also involved in the regulation of EPC biology . ...
Article
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In the last decade there have been multiple studies concerning the contribution of endothelial progenitor cells (EPCs) to new vessel formation in different physiological and pathological settings. The process by which EPCs contribute to new vessel formation in adults is termed postnatal vasculogenesis and occurs via four inter-related steps. They must respond to chemoattractant signals and mobilize from the bone marrow to the peripheral blood; home in on sites of new vessel formation; invade and migrate at the same sites; and differentiate into mature endothelial cells (ECs) and/or regulate pre-existing ECs via paracrine or juxtacrine signals. During these four steps, EPCs interact with different physiological compartments, namely bone marrow, peripheral blood, blood vessels and homing tissues. The success of each step depends on the ability of EPCs to interact, adapt and respond to multiple molecular cues. The present review summarizes the interactions between integrins expressed by EPCs and their ligands: extracellular matrix components and cell surface proteins present at sites of postnatal vasculogenesis. The data summarized here indicate that integrins represent a major molecular determinant of EPC function, with different integrin subunits regulating different steps of EPC biology. Specifically, integrin α4β1 is a key regulator of EPC retention and/or mobilization from the bone marrow, while integrins α5β1, α6β1, αvβ3 and αvβ5 are major determinants of EPC homing, invasion, differentiation and paracrine factor production. β2 integrins are the major regulators of EPC transendothelial migration. The relevance of integrins in EPC biology is also demonstrated by many studies that use extracellular matrix-based scaffolds as a clinical tool to improve the vasculogenic functions of EPCs. We propose that targeted and tissue-specific manipulation of EPC integrin-mediated interactions may be crucial to further improve the usage of this cell population as a relevant clinical agent.
... CS-CG scaffolds not only offer a mechanical support structure but also provide bioactive cues to induce vascularization of the implants. Collagen has a chemotactic activity to endothelial cells and macrophages during the healing response [28]. Research shows that macrophages are beneficial for scaffold vascularization in vivo [29], and three activated macrophage phenotypes (M1, M2a, and M2c) support angiogenesis in different ways [26]. ...
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... Procedures were performed on 8-or 9-week-old athymic nude CD1 female mice as previously described (Kuraitis et al., 2011) with the approval of the University of Ottawa Animal Care Committee and in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. Unilateral left hindlimb ischemia in mice was induced by ligating the proximal end of the femoral artery using 4-0 silk surgical suture as described in Limbourg et al. (2009). ...
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... The collagen matrix was prepared following previous methods [7]. Briefly, 3.3 ml of type I rat tail collagen (3.75 mg/ml; Becton Dickinson) was mixed on ice in a cold glass tube with 500 ml of buffer (29 ml buffer made from: 9 ml of FBS, 9 ml of 10 Â DMEM, 10 ml of 200 mM HEPES, 3e5 drops gentamycin) on ice. ...
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Myopathies of skeletal muscle are prevalent diseases worldwide. To address this, regenerative therapies are being developed to restore perfusion to ischemic muscle and to reverse muscle wasting. There are adult stem cell populations that inherently possess these therapeutic properties; however, cell transplantation trials in the clinic have shown modest results at best, being limited by poor cell persistence and viability post-transplantation, and by cell relocation to non-target sites. Many materials exist that can elicit and enhance beneficial cell responses - these materials can be applied directly, or used as stem cell delivery vehicles, for regenerative therapies. In particular, components of the body's extracellular matrices may be advantageous for therapeutic application because cells already have a pre-disposition for recognizing them, and also because their usage carries a low probability of inducing negative immune responses. This review will survey the major components of the extracellular matrix and their interactions with relevant stem cell populations for the regeneration of muscle. Future material-based therapies will benefit from a more precise control over therapeutic cell populations implicated in the regenerative response.
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Despite advances in treatment, heart failure remains one of the top killers in Canada. This recognition motivates a new research focus to harness the fundamental repair properties of the human heart. Since then, cardiac stem cells (CSCs) have emerged as a promising cell candidate to regenerate damaged hearts. The rationale of this approach is simple with ex vivo amplification of CSCs from clinical grade biopsies, followed by delivery to areas of injury, where they engraft and regenerate the heart. This review will summarize recent advances and future developments in CSC-mediated cardiac repair to treat the growing number of Canadians living and dying with heart failure.
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This study investigated the interaction of human circulating angiogenic cells (CACs) with a degradable polar hydrophobic ionic polyurethane (D-PHI) which has been previously shown to exhibit anti-inflammatory character and favorable interactions with human endothelial cells (ECs). Given the implication of the CACs in microvessel development it was of intrinsic interest to expand our knowledge of D-PHI biocompatibility with this relevant primary cell involved in angiogenesis. The findings will be compared to a well-established benchmark substrate for CACs, fibronectin-coated tissue culture polystyrene (TCPS). Immunoblotting analysis showed that CACs were a heterogeneous population of cells composed mostly of monocytic cells expressing the CD14 marker. Assessment of the cytokine release profile, using ELISA, showed that D-PHI supported a higher concentration of interleukin-10 (IL-10) when compared to the concentration of tumor necrosis factor alpha, which is indicative of an anti-inflammatory phenotype, and was different from the response with TCPS. It was found that the CACs were attached to D-PHI and remained viable and functional (nitric oxide production) during the seven days of culture. However, there did not appear to be any significant proliferation on D-PHI, contrary to the CAC growth on fibronectin-coated TCPS. It was concluded that D-PHI displayed some of the qualities suitable to enable the retention of CACs onto this substrate, as well as maintaining an anti-inflammatory phenotype, characteristics which have been reported to be important for angiogenesis in vivo.
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Endothelial progenitor cell (EPC) therapy has emerged as a promising treatment in cardiac regeneration. EPCs are capable of promoting neovascularization within ischemic tissues, but past studies have had limited success in vivo due to the poor retention and survival of cells within the injured area. In addition, the environment into which the cells are being transplanted may negatively influence the therapeutic potential of the transplanted cells. To overcome these hurdles, various biomaterials are being engineered to include a variety of proteins and peptides to increase cellular adhesion, survival and angiogenesis. This chapter will describe how such strategies are expected to enhance EPCs, and their ability to promote cardiac repair and regeneration.
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Recent advances in stem cell research have generated excitement for the use of cardiac stem cells (CSCs) as a viable cell therapy option for myocardial repair. This chapter reviews evidence for resident CSCs and examines the capacity of transplanted ex vivo proliferated CSCs to improve heart function after cardiac damage. Reflection upon recent preclinical and phase I studies of current first-generation CSC products will prompt a deeper understanding of challenges (and opportunities) confronting the next generation of this remarkable therapy.
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Immunocompromised hind-limb ischemia (HLI) murine models are essential for pre-clinical evaluation of human cell-based therapy or biomaterial based interventions. These models are used to generate proof of principle that the approach is effective and also regulatory pre-clinical data required for translation to the clinic. However, surgical variations in creation of hind-limb ischemia models reported in the literature introduce variability in the pathological manifestation of the model in consequence affecting therapeutic endpoints. This study aims to compare the extent of vascular regeneration in HLI induced immunocompromised murine models to obtain a stable and more reproducible injury model for testing. Athymic and Balb/C nude mice underwent hind-limb ischemia surgery with single and double ligation of femoral artery. The recovery from surgery was observed over a period of two weeks with respect to ischemia reperfusion using laser doppler and clinical signs of necrosis and ambulatory impairment. Double ligation of the femoral artery results in a more severe response to ischemia in Balb/C with endogenous perfusion recovery up to 50±10% compared to 75±20% in Athymic nude mice. Single iliac artery (IA) and femoral artery (FA) leads to creation of mild ischemia compared to femoral artery-vein (FAV) pair ligation in Balb/C. Microcirculatory parameters indicate significantly lower capillary numbers (26 ±3/mm2) and functional capillary density (203 ±5 cm/cm2) in the FAV group. Here in our study, we demonstrate a reproducible, arterial double ligation in immunocompromised Balb/C nude mouse model that exhibit characteristic pathological signs of ischemia with impaired endogenous recovery.
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Introduction: The feasibility and safety of bone marrow cell (BMC) therapy for cardiac repair following myocardial infarction has been demonstrated in clinical studies, albeit with relatively modest structural and functional benefits. In response to the shortcomings of BMC therapy, the use of biomaterials to enhance cell transplantation is being investigated. Areas covered: The authors first review what has been learned from BMC therapies for the treatment of myocardial infarction in animal models and in clinical trials. Some issues that may be limiting the efficacy of BMC therapy are then described. Lastly, they summarize several biomaterial approaches that have been reported to improve transplanted cell retention and functional outcome, and then focus on how a material can enhance cell function such as proliferation, viability, endothelial differentiation and angiogenic potential. Expert opinion: Improvements are needed if BMC therapy is to become a viable treatment in the clinic. There is optimism that a biomaterial strategy will lead to superior results compared to the cell therapy alone. Through the identification of underlying cell-biomaterial mechanisms, the establishment of comparative standards, and an awareness of the lessons learned from cell therapy trials, biomaterial-enhanced BMC therapy may become an option for the treatment of heart disease patients.
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Human endothelial colony-forming cells (ECFCs) represent a promising source of adult stem cells for vascular repair, yet their regenerative capacity is limited. Here, we set out to understand the molecular mechanism restricting the repair function of ECFCs. We found that key pro-angiogenic pathways are repressed in ECFCs due to the presence of bivalent (H3K27me3/H3K4me3) epigenetic marks, which decreases the cells' regenerative potential. Importantly, ex vivo treatment with a combination of epigenetic drugs that resolves bivalent marks toward the transcriptionally active H3K4me3 state leads to the simultaneous activation of multiple pro-angiogenic signaling pathways (VEGFR, CXCR4, WNT, NOTCH, SHH). This in turn results in improved capacity of ECFCs to form capillary-like networks in vitro and in vivo. Furthermore, restoration of perfusion is accelerated upon transplantation of drug-treated ECFCs in a model of hindlimb ischemia. Thus, ex vivo treatment with epigenetic drugs increases the vascular repair properties of ECFCs through transient activation of pro-angiogenic signaling pathways.
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CD34+ cells are promising for revascularization therapy, but their clinical use is limited by low cell counts, poor engraftment, and reduced function after transplantation. In this study, a collagen type I biomaterial was used to expand and enhance the function of human peripheral blood CD34+ cells, and potential underlying mechanisms were examined. Compared to the fibronectin control substrate, biomaterial-cultured CD34+ cells from healthy donors had enhanced proliferation, migration toward VEGF, angiogenic potential, and increased secretion of CD63+CD81+ extracellular vesicles (EVs). In the biomaterial-derived EVs, greater levels of the angiogenic microRNAs (miRs), miR-21 and -210, were detected. Notably, biomaterial-cultured CD34+ cells had reduced mRNA and protein levels of Sprouty (Spry)1, which is an miR-21 target and negative regulator of endothelial cell proliferation and angiogenesis. Similar to the results of healthy donor cells, biomaterial culture increased miR-21 and -210 expression in CD34+ cells from patients who underwent coronary artery bypass surgery, which also exhibited improved VEGF-mediated migration and angiogenic capacity. Therefore, collagen biomaterial culture may be useful for expanding the number and enhancing the function of CD34+ cells in patients, possibly mediated through suppression of Spry1 activity by EV-derived miR-21. These results may provide a strategy to enhance the therapeutic potency of CD34+ cells for vascular regeneration.-McNeill, B., Ostojic, A., Rayner, K. J., Ruel, M., Suuronen, E. J. Collagen biomaterial stimulates the production of extracellular vesicles containing microRNA-21 and enhances the proangiogenic function of CD34+ cells.
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Injectable hydrogels are increasingly being developed for biomedical applications due to their ability to be delivered in a minimally invasive manner. One potential use for such materials is in cell delivery for cardiac regeneration. While the materials' properties are often characterized, how these properties (and in particular gelation) are affected by the addition of the therapeutic agent(s) they are designed to deliver is often overlooked. The aim of this study was to examine the interactive effects between collagen-based hydrogels and different additives (cells and microspheres). The results demonstrated that the incorporation of either cells or microspheres to a collagen hydrogel decreased its gelation time and increased its viscosity. Increased concentrations of the EDC/NHS cross-linker resulted in greater loss of cell viability. However, it was found that this cell loss could be minimized by delivering cells with the cross-linker scavenger glycine. A better understanding of how materials and cells (and other additives) respond to each other will help towards the goal of improving scaffolds being developed for regenerative therapy.
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Endothelial precursor cells (EPCs) have been identified in adult peripheral blood. We examined whether EPCs could be isolated from umbilical cord blood, a rich source for hematopoietic progenitors, and whether in vivo transplantation of EPCs could modulate postnatal neovascularization. Numerous cell clusters, spindle-shaped and attaching (AT) cells, and cord-like structures developed from culture of cord blood mononuclear cells (MNCs). Fluorescence-trace experiments revealed that cell clusters, AT cells, and cord-like structures predominantly were derived from CD34-positive MNCs (MNC(CD34+)). AT cells and cell clusters could be generated more efficiently from cord blood MNCs than from adult peripheral blood MNCs. AT cells incorporated acetylated-LDL, released nitric oxide, and expressed KDR, VE-cadherin, CD31, and von Willebrand factor but not CD45. Locally transplanted AT cells survived and participated in capillary networks in the ischemic tissues of immunodeficient nude rats in vivo. AT cells thus had multiple endothelial phenotypes and were defined as a major population of EPCs. Furthermore, laser Doppler and immunohistochemical analyses revealed that EPC transplantation quantitatively augmented neovascularization and blood flow in the ischemic hindlimb. In conclusion, umbilical cord blood is a valuable source of EPCs, and transplantation of cord blood-derived EPCs represents a promising strategy for modulating postnatal neovascularization.
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The aim was to investigate that a bio-degradable alginate and poly lactide-co-glycolide (PLG) system capable of delivering growth factors sequentially would be superior to single growth factor delivery in promoting neovascularization and improving perfusion. Three groups of apoE null mice underwent unilateral hindlimb ischemia surgery and received ischemic limb intramuscular injections of alginate (Blank), alginate containing VEGF(165) (VEGF), or alginate containing VEGF(165) combined with PLG microspheres containing PDGF-BB (VEGF/PDGF). Vascularity in the ischemic hindlimb was assessed by morphologic and immunohistochemical end-points, while changes in blood flow were assessed by Laser Doppler Perfusion Index. Muscle VEGF and PDGF content was assessed at multiple time points. In the VEGF/PDGF group, local tissue VEGF and PDGF levels peaked at week 2 and 4, respectively, with detectable PDGF levels at week 6. At week 6, mean vessel mean diameter was significantly greater in the VEGF/PDGF group compared to the VEGF or Blank groups with evidence of well-formed smooth muscle-lined arterioles. Sequential delivery of VEGF and PDGF using an injectable, biodegradable platform resulted in stable and sustained improvements in perfusion. This sustained, control-released, injectable alginate polymer system is a promising approach for multiple growth factor delivery in clinical application.
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Proper tissue function and regeneration rely on robust spatial and temporal control of biophysical and biochemical microenvironmental cues through mechanisms that remain poorly understood. Biomaterials are rapidly being developed to display and deliver stem-cell-regulatory signals in a precise and near-physiological fashion, and serve as powerful artificial microenvironments in which to study and instruct stem-cell fate both in culture and in vivo. Further synergism of cell biological and biomaterials technologies promises to have a profound impact on stem-cell biology and provide insights that will advance stem-cell-based clinical approaches to tissue regeneration.
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Current evidence suggests that endothelial progenitor cells (EPC) contribute to ischemic tissue repair by both secretion of paracrine factors and incorporation into developing vessels. We tested the hypothesis that cell-free administration of paracrine factors secreted by cultured EPC may achieve an angiogenic effect equivalent to cell therapy. EPC-derived conditioned medium (EPC-CM) was obtained from culture expanded EPC subjected to 72 hours of hypoxia. In vitro, EPC-CM significantly inhibited apoptosis of mature endothelial cells and promoted angiogenesis in a rat aortic ring assay. The therapeutic potential of EPC-CM as compared to EPC transplantation was evaluated in a rat model of chronic hindlimb ischemia. Serial intramuscular injections of EPC-CM and EPC both significantly increased hindlimb blood flow assessed by laser Doppler (81.2+/-2.9% and 83.7+/-3.0% vs. 53.5+/-2.4% of normal, P<0.01) and improved muscle performance. A significantly increased capillary density (1.62+/-0.03 and 1.68+/-0.05/muscle fiber, P<0.05), enhanced vascular maturation (8.6+/-0.3 and 8.1+/-0.4/HPF, P<0.05) and muscle viability corroborated the findings of improved hindlimb perfusion and muscle function. Furthermore, EPC-CM transplantation stimulated the mobilization of bone marrow (BM)-derived EPC compared to control (678.7+/-44.1 vs. 340.0+/-29.1 CD34(+)/CD45(-) cells/1x10(5) mononuclear cells, P<0.05) and their recruitment to the ischemic muscles (5.9+/-0.7 vs. 2.6+/-0.4 CD34(+) cells/HPF, P<0.001) 3 days after the last injection. Intramuscular injection of EPC-CM is as effective as cell transplantation for promoting tissue revascularization and functional recovery. Owing to the technical and practical limitations of cell therapy, cell free conditioned media may represent a potent alternative for therapeutic angiogenesis in ischemic cardiovascular diseases.
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The interferon-inducible, double-stranded (ds) RNA-dependent serine/threonine protein kinase (PKR) plays a role in viral pathogenesis, cell growth, and differentiation and is implicated as a tumor suppressor gene. Expression of atrans-dominant negative, catalytically inactive mutant PKR protected NIH3T3 cells from apoptosis in response to either treatment with tumor necrosis factor α (TNFα), serum deprivation. In cells expressing mutant PKR, TNFα, but not dsRNA induced transcription from a nuclear factor κ B-dependent promoter, demonstrating specificity for dsRNA in signaling through the PKR pathway. Serum or platelet-derived growth factor addition to serum-deprived mutant PKR-expressing cells induced transcription of the early response genes c-fos and c-jun, indicating that the immediate early response signaling was intact. Overexpression of wild-type PKR in a transient DNA transfection system was sufficient to induce apoptosis. TNFα-induced apoptosis correlated with increased phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF-2α), the primary physiological substrate of the PKR. Furthermore, forced expression of a nonphosphorylatable S51A mutant eIF-2α partially protected cells from TNFα-induced apoptosis, and expression of a S51D mutant eIF-2α, a mutant that mimics phosphorylated eIF-2α, was sufficient to induce apoptosis. Taken together, these studies identify a novel requirement for PKR in stress-induced apoptosis that is mediated through eIF-2α phosphorylation.
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Animal studies and preliminary results in humans suggest that lower extremity and myocardial ischemia can be attenuated by treatment with angiogenic cytokines. The resident population of endothelial cells that is competent to respond to an available level of angiogenic growth factors, however, may potentially limit the extent to which cytokine supplementation enhances tissue neovascularization. Accordingly, we transplanted human endothelial progenitor cells (hEPCs) to athymic nude mice with hindlimb ischemia. Blood flow recovery and capillary density in the ischemic hindlimb were markedly improved, and the rate of limb loss was significantly reduced. Ex vivo expanded hEPCs may thus have utility as a "supply-side" strategy for therapeutic neovascularization.
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Biomaterials not only serve as scaffolds for bone regeneration, but may also exhibit inductive capability for bone growth. The goal of this study was to identify the best extracellular matrix protein for enhancing osteogenesis by hMSCs (human mesenchymal stem cells) and to investigate the underlying mechanism. Coating with collagen I, but not fibronectin, laminin, gelatin, and poly-L-lysine, enhanced late cell proliferation and promoted osteogenesis by hMSCs, as evidenced by an increase in Alizarin Red S staining, alkaline phosphatase activity and mRNA levels of Runx2 and osteocalcin. Coating with collagen I induced activation of ERK and Akt but not FAK, and treatment with PD98059 and LY294002 blocked the activation of ERK and Akt, respectively. Interestingly, LY294002 also blocked ERK activation, indicating the activation of PI3K/ERK pathway upon contact with collagen I. Furthermore, PD98059 or LY294002 abolished collagen I-induced promotion of osteogenesis by hMSCs. However, blocking antibodies against alpha2beta1 integrins did not inhibit collagen I-induced osteogenesis by hMSCs. These data demonstrate that collagen I promotes proliferation and osteogenesis of hMSCs via activation of ERK and Akt pathways.
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Gene and stem-cell therapies hold promise for the treatment of ischemic cardiovascular disease. Combined stem cell, chemokine, and angiogenic growth factor gene therapy could augment angiogenesis, and better improve heart function in the infarcted myocardium. In order to prove this action, we established the animal model of myocardial infarction (MI) was by occlusion of the left anterior descending artery in rats. Seven days after surgery, 5.0 x 10(6) Ad-EGFP-MSC, 5.0 x 10(6) Ad-SDF-1-MSC, 5.0 x 10(6) Ad-VEGF-MSC, or 5.0 x 10(6) Ad-SDF-VEGF-MSC (Ad-SDF-1-VEGF-MSC) suspension in 0.2 ml of serum-free medium was injected into four sites in the infarcted hearts. Results showed that MSCs transfected with Ad-VEGF and Ad-SDF-1 produced more SDF-1 and VEGF protein than MSCs alone, the increased protein levels of VEGF and SDF-1 activated Akt in MSCs transfected with Ad-VEGF and Ad-SDF-1, and improved the survival capability of the MSCs in vitro and in vivo. These transplanted cells showed that the characteristic phenotype of cardiomyocyte (e.g., cTnt) and endothelial cells (e.g., CD31). Four weeks after transplantation, reduced infarct size and fibrosis, greater vascular density, and a thicker left ventricle wall were observed in Ad-SDF-VEGF-MSC group. Measurement of hemodynamic parameters showed an improvement in left ventricular performance in Ad-SDF-VEGF-MSC group compared with other groups. These results demonstrated that combination of chemokine and angiogenic factor gene and stem cells could enhance angiogenesis and improves cardiac function after acute myocardial infarction in rats.
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Blood vessel growth in adult organisms involves the following two fundamental processes: angiogenesis, the proliferation and extension of capillary networks; and arteriogenesis, the growth of functional arteries. We provide a protocol for the evaluation of postnatal arteriogenesis and angiogenesis in a mouse model of hind-limb ischemia. Surgical ligation of the femoral artery at a specific site triggers arteriogenesis of small, pre-existing collateral arteries into functional conduit vessels proximally and ischemic angiogenesis distally. The vascular response to hind-limb ischemia can be readily evaluated by laser Doppler-based perfusion measurements, histological quantification of arteriogenesis and angiogenesis or whole-mount visualization of arteries in limb muscles. Depending on the experimental design, the protocol takes between 4 and 29 d to complete; however, the net working time is about 2 d per mouse. The concurrent and specific analysis of postnatal angiogenesis and arteriogenesis in the same animal is a unique feature of the protocol.
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Prorenin is an enzymatically inactive precursor of renin, and its biological function in endothelial cells (ECs) is unknown despite its relevance with the incidence of diabetic microvascular complications. Recently, (pro)renin receptor was identified, and the receptor-associated prorenin system has been discovered, whereas its expression as well as function in ECs remain unclear. In the present study, we found that ECs express the (pro)renin receptor, and that prorenin provoked ERK activation through (pro)renin receptor independently of the renin-angiotensin system (RAS). Prorenin stimulated the proliferation, migration and tube-formation of ECs, while it inhibited endothelial apoptosis induced by serum and growth factor depletion. MEK inhibitor abrogated these proangiogenic effects of prorenin, while AT1 receptor antagonist or angiotensin-converting enzyme inhibitor failed to block them. In vivo neovascularization in the Matrigel-plugs implanted into mouse flanks was significantly enhanced by prorenin, in which significant ERK activation was detected in ECs. Furthermore, tumor xenografts stably transfected with prorenin demonstrated the significantly accelerated growth rate concomitantly with enhanced intratumoral neovascularization. Our data demonstrated that the RAS-independent (pro)renin receptor-mediated signal transduction plays a pivotal role in the regulation of ECs function as well as in the neovascularization, and thus prorenin is potentially involved in the pathophysiology of diabetic microvascular complications as well as cancers.
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Collagen delivery matrices have been reported to improve the results of cell therapy, but knowledge of their mechanisms of action is limited. To evaluate whether a collagen matrix improves early engraftment posttransplantation, 2-[(18)F]fluoro-2-deoxy-d-glucose ((18)F-FDG) was used to label transplanted circulating progenitor cells (CPCs) and track them in vivo with positron-emission tomography. Efficiency of (18)F-FDG cell labeling was CPC-concentration dependent (r=0.61, P<0.001) but not (18)F-FDG-dose dependent. Labeled human CPCs (2x10(6)) were injected with or without a collagen-based matrix in the ischemic hind limb of rats (n=12 per group) 2 weeks after femoral artery ligation. Imaging of labeled cells, acquired by small animal positron-emission tomography at 150 minutes postinjection, revealed greater CPC retention in the ischemic hind limb and less nonspecific leakage to other tissues (retention ratio, 0.44+/-0.08) when CPCs were delivered within the matrix, compared with cells injected alone (0.22+/-0.13, P=0.040) and with (18)F-FDG injected with or without the matrix (0.10+/-0.05 and 0.11+/-0.05, respectively, P<0.005). Tissue radionuclide biodistribution was performed after completion of positron-emission tomography imaging. When (18)F-FDG-labeled cells were injected with the collagen matrix, accumulation was significantly increased (by 69.6%, P=0.021) in the target ischemic hind limb muscle and significantly reduced (by 14.8% to 31.4%, P<0.05) in nonspecific tissues, compared with cells injected alone. Histology confirmed the increased retention in target tissue associated with the matrix. Early posttransplantation, a collagen matrix enhances progenitor cell retention and limits distribution to nonspecific tissues, as measured by the use of (18)F-FDG labeled cells and positron-emission tomography imaging and confirmed by biodistribution and histology.
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Circulating progenitor cells home to and engraft to sites of ischemia, mediated in part by the adhesion molecule L-selectin; however, accumulation in tissues such as the heart is low. In this study, an acellular collagen-based matrix containing sialyl Lewis(X) (sLe(X)), which binds L-selectin, was developed in order to enhance the endogenous progenitor cell therapeutic response. Its effect on progenitor cells and angiogenesis were assessed in vitro and using a hindlimb ischemia model with rats. In culture, the sLe(X)-collagen matrix recruited more CD133(+)CD34(+)L-selectin(+) cells than collagen-only matrix, with adhesion mediated by L-selectin binding. Increased angiogenic/chemotactic cytokine production and improved resistance to apoptosis appeared in cells cultured on sLe(X)-collagen matrix. In vivo, mobilization of endogenous circulating progenitor cells was increased, and greater recruitment of these and systemically injected human peripheral blood CXCR4(+)L-selectin(+) cells to sLe(X)-collagen treated limbs was observed compared to collagen-only. This condition was associated with differences in angiogenic/chemotactic cytokine levels, with greater arteriole density and increased perfusion in sLe(X)-collagen treated hindlimbs. With these factors taken together, we demonstrated that an acellular matrix-bound ligand approach can enhance the mobilization, recruitment, and therapeutic effects of endogenous and/or transplanted progenitor cells, possibly through paracrine and antiapoptotic mechanisms, and could be used to improve cell-based regenerative therapies.
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The recruitment of endothelial progenitor cells (EPC) to ischemia has recently been suggested as an important mechanism of tissue repair. Although tissue ischemia can facilitate EPC mobilization, recruitment, and retention at the hypoxic site, the effects of hypoxia on EPC survival are not well known. In the present study, we examined whether hypoxia (2% O2) would suppress apoptosis induced by serum withdrawal and whether survival signals, such as the phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated protein kinase (ERK) pathways, were involved in this process. After being serum-starved for 24 h, EPC were cultured under normoxic or hypoxic conditions (2% O2) for 24 h. Cell survival was assessed by 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, annexin V-propidium iodide dual-color flow cytometry, and terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling assay. The expressions of signaling proteins were evaluated by Western blot analysis. Under hypoxic conditions, EPC were resistant to apoptosis induced by serum starvation. The inhibition of the PI3K/Akt pathway using the LY294002 inhibitor prevented hypoxia-inhibited apoptosis in EPC and altered the phosphorylation state of glycogen synthase kinase-3beta, an effector protein involved in regulation of EPC apoptosis. However, ERK inhibitor PD98059 had no significant effect on cell survival. Our data demonstrated that hypoxia inhibited serum withdrawal-induced apoptosis in EPC, which might be associated with the activation of the PI3K/Akt pathway.
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Nearly all cell surface receptors utilize one or more of the mitogen-activated protein kinase cascades in their repertoire of signal transduction mechanisms. Recent advances in the study of such cascades include the cloning of genes encoding novel members of the cascades, further definition of the roles of the cascades in responses to extracellular signals, and examination of cross-talk between different cascades.
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Circulating endothelial progenitor cells (EPCs) have been isolated in peripheral blood of adult species. To determine the origin and role of EPCs contributing to postnatal vasculogenesis, transgenic mice constitutively expressing beta-galactosidase under the transcriptional regulation of an endothelial cell-specific promoter (Flk-1/LZ or Tie-2/LZ) were used as transplant donors. Localization of EPCs, indicated by flk-1 or tie-2/lacZ fusion transcripts, were identified in corpus luteal and endometrial neovasculature after inductive ovulation. Mouse syngeneic colon cancer cells (MCA38) were implanted subcutaneously into Flk-1/LZ/BMT (bone marrow transplantation) and Tie-2/LZ/BMT mice; tumor samples harvested at 1 week disclosed abundant flk-1/lacZ and tie-2/lacZ fusion transcripts, and sections stained with X-gal demonstrated that the neovasculature of the developing tumor frequently comprised Flk-1- or Tie-2-expressing EPCs. Cutaneous wounds examined at 4 days and 7 days after skin removal by punch biopsy disclosed EPCs incorporated into foci of neovascularization at high frequency. One week after the onset of hindlimb ischemia, lacZ-positive EPCs were identified incorporated into capillaries among skeletal myocytes. After permanent ligation of the left anterior descending coronary artery, histological samples from sites of myocardial infarction demonstrated incorporation of EPCs into foci of neovascularization at the border of the infarct. These findings indicate that postnatal neovascularization does not rely exclusively on sprouting from preexisting blood vessels (angiogenesis); instead, EPCs circulate from bone marrow to incorporate into and thus contribute to postnatal physiological and pathological neovascularization, which is consistent with postnatal vasculogenesis.
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Vascular endothelial growth factor (VEGF) induces endothelial cell proliferation, migration, and actin reorganization, all necessary components of an angiogenic response. However, the distinct signal transduction mechanisms leading to each angiogenic phenotype are not known. In this study, we examined the ability of VEGF to stimulate cell migration and actin rearrangement in microvascular endothelial cells infected with adenoviruses encoding beta-galactosidase (beta-gal), activation-deficient Akt (AA-Akt), or constitutively active Akt (myr-Akt). VEGF increased cell migration in cells transduced with beta-gal, whereas AA-Akt blocked VEGF-induced cell locomotion. Interestingly, myr-Akt transduction of bovine lung microvascular endothelial cells stimulated cytokinesis in the absence of VEGF, suggesting that constitutively active Akt, per se, can initiate the process of cell migration. Treatment of beta-gal-infected endothelial cells with an inhibitor of NO synthesis blocked VEGF-induced migration but did not influence migration initiated by myr-Akt. In addition, VEGF stimulated remodeling of the actin cytoskeleton into stress fibers, a response abrogated by infection with dominant-negative Akt, whereas transduction with myr-Akt alone caused profound reorganization of F-actin. Collectively, these data demonstrate that Akt is critically involved in endothelial cell signal transduction mechanisms leading to migration and that the Akt/endothelial NO synthase pathway is necessary for VEGF-stimulated cell migration.
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We investigated the therapeutic potential of ex vivo expanded endothelial progenitor cells (EPCs) for myocardial neovascularization. Peripheral blood mononuclear cells obtained from healthy human adults were cultured in EPC medium and harvested 7 days later. Myocardial ischemia was induced by ligating the left anterior descending coronary artery in male Hsd:RH-rnu (athymic nude) rats. A total of 10(6) EPCs labeled with 1,1'-dioctadecyl-1 to 3,3,3',3'-tetramethylindocarbocyanine perchlorate were injected intravenously 3 hours after the induction of myocardial ischemia. Seven days later, fluorescence-conjugated Bandeiraea simplicifolia lectin I was administered intravenously, and the rats were immediately killed. Fluorescence microscopy revealed that transplanted EPCs accumulated in the ischemic area and incorporated into foci of myocardial neovascularization. To determine the impact on left ventricular function, 5 rats (EPC group) were injected intravenously with 10(6) EPCs 3 hours after ischemia; 5 other rats (control group) received culture media. Echocardiography, performed just before and 28 days after ischemia, disclosed ventricular dimensions that were significantly smaller and fractional shortening that was significantly greater in the EPC group than in the control group by day 28. Regional wall motion was better preserved in the EPC group. After euthanization on day 28, necropsy examination disclosed that capillary density was significantly greater in the EPC group than in the control group. Moreover, the extent of left ventricular scarring was significantly less in rats receiving EPCs than in controls. Immunohistochemistry revealed capillaries that were positive for human-specific endothelial cells. Ex vivo expanded EPCs incorporate into foci of myocardial neovascularization and have a favorable impact on the preservation of left ventricular function.
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We sought to optimize vascular endothelial growth factor (VEGF) treatment for therapeutic angiogenesis in myocardial ischemia, we explored the efficacy of five different regimens. Although VEGF165 is one of the most potent pro-angiogenic growth factors, VEGF165 treatment for myocardial ischemia has been hampered by low efficacy and dose-limiting hypotension after systemic or intracoronary delivery. This study evaluated the effect of intravenous or intracoronary rhVEGF165 in the presence or absence of nitric oxide (NO) synthase inhibition in a porcine model of chronic myocardial ischemia. Forty-two Yorkshire pigs with chronically occluded left circumflex coronary arteries were randomly assigned to receive 10 microg/kg of VEGF165: 1) rapid (40 min) intravenous VEGF165 0.25 microg/kg/min, 2) slow (200 min) intravenous VEGF165 0.05 microg/kg/min, 3) rapid intracoronary VEGF165 0.25 microg/kg/min, 4) rapid intracoronary VEGF165 0.25 microg/kg/min + nitro-L-arginine methyl ester hydrochloride (L-NAME) or 5) rapid vehicle infusion. Intracoronary and intravenous VEGF165 induced hypotension. Intracoronary VEGF-induced hypotension was blocked by L-NAME. Coronary angiography three weeks after treatment showed improvement in collateral index in both intracoronary groups but not the intravenous VEGF165 groups. Likewise, myocardial blood flow and microvascular function in the ischemic territory improved in both intracoronary groups but not in the intravenous groups. Global and regional myocardial function showed no significant improvements in any groups. Intracoronary infusion of VEGF165 significantly improves blood flow to the ischemic myocardium. Concomitant administration of L-NAME inhibits VEGF-induced hypotension while most likely preserving VEGF-induced angiogenesis. Intravenous infusion of VEGF165 was not effective in augmenting either myocardial flow or function in this model.
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Angiogenesis is a complex process, involving functional cooperativity between cytokines and endothelial cell (EC) surface integrins. In this study, we investigated the mechanisms through which the alpha(1)beta(1) and alpha(2)beta(1) integrins support angiogenesis driven by vascular endothelial growth factor (VEGF). Dermal microvascular EC attachment through either alpha(1)beta(1) or alpha(2)beta(1) supported robust VEGF activation of the Erk1/Erk2 (p44/42) mitogen-activated protein kinase signal transduction pathway that drives EC proliferation. Haptotactic EC migration toward collagen I was dependent on alpha(1)beta(1) and alpha(2)beta(1) as was VEGF-stimulated chemotaxis of ECs in a uniform collagen matrix. Consistent with the functions of alpha(1)beta(1) and alpha(2)beta(1) in supporting signal transduction and EC migration, antibody antagonism of either integrin resulted in potent inhibition of VEGF-driven angiogenesis in mouse skin. Moreover, combined antagonism of alpha(1)beta(1) and alpha(2)beta(1) substantially reduced tumor growth and angiogenesis of human squamous cell carcinoma xenografts. Collectively, these studies identify critical collaborative functions for the alpha(1)beta(1) and alpha(2)beta(1) integrins in supporting VEGF signal transduction, EC migration, and tumor angiogenesis.
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An in vitro angiogenesis system was designed for screening angiogenic agonists and antagonists. In order to obtain large quantities of cells and reproducibility, human endothelial cells with extended life spans were developed by retroviral transfection. The resulting cells grown in a serum-free medium containing endothelial cell growth supplement (ECGS) have a telomerase activity, extended life spans of at least 21 passages, and an endothelial cell phenotype (diI-acetylated-LDL upake, factor VIII-related antigen, VEGFR-1 and R-2, and tissue-type plasminogen activator (tPA)) that resembled that of unaltered primary endothelial cells. Exceptions were (i) a higher expression of tPA, and (ii) a non-significant growth response to FGF-2 or VEGF stimulation. Within three-dimensional fibrin gels, specific cell clones rapidly formed tubular structures in a more reproducible manner than those observed with low-passage primary cells. Tube formation by primary endothelial cells and those with extended life spans was dependent upon FGF-2 and ECGS, respectively. Both cell types produced FGF-2 and VEGF cytokines. Increasing doses of suramin significantly decreased the size of microvessels formed by both cell lines. These functional results indicate that a vascular matrix system containing human cells with extended life spans can be successfully utilized as an in vitro assay for antiangiogenic compounds.
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Endothelial progenitor cells (EPC) in one study group is not the same as EPC in other investigators, suggesting that EPC is not a single type of cell population. In this study, we tried to demonstrate the heterogeneity of EPC. We cultured total mononuclear cells from human peripheral blood to get two types of EPC sequentially from the same donors. We called them early EPC and late EPC. Early EPC with spindle shape showed peak growth at 2 to 3 weeks and died at 4 weeks, whereas late EPC with cobblestone shape appeared late at 2 to 3 weeks, showed exponential growth at 4 to 8 weeks, and lived up to 12 weeks. Late EPC was different from early EPC in the expression of VE-cadherin, Flt-1, KDR, and CD45. Late EPC produced more nitric oxide, incorporated more readily into human umbilical vein endothelial cells monolayer, and formed capillary tube better than early EPC. Early EPC secreted angiogenic cytokines (vascular endothelial growth factor, interleukin 8) more so than late EPC during culture in vitro. Both types of EPC showed comparable in vivo vasculogenic capacity. We found two types of EPC from a source of adult peripheral blood that might have different roles in neovasculogenesis based on the identified differences.
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Bone marrow and peripheral blood of adults contain a special sub-type of progenitor cells which are able to differentiate into mature endothelial cells, thus contributing to re-endothelialization and neo-vascularization. These angiogenic cells have properties of embryonal angioblasts and were termed endothelial progenitor cells (EPCs). In general, three surface markers (CD133, CD34 and the vascular endothelial growth factor receptor-2) characterize the early functional angioblast, located predominantly in the bone marrow. Later, when migrating to the systemic circulation EPCs gradually lose their progenitor properties and start to express endothelial marker like VE-cadherin, endothelial nitric oxide synthase and von Willebrand factor. The number of circulating EPCs in healthy subjects is rather low and a variety of conditions or factors may further influence this number. In the context of possible therapeutic application of EPCs recent clinical studies employing these cells for neo-vascularization of ischemic organs have just been published. However, the specificity of the observed positive clinical effects, the mechanisms regulating the differentiation of EPCs and their homing to sites of injured tissue remain partially unknown at present.
Article
Two types of cells are cultured from the human peripheral blood, early endothelial progenitor cells (EPCs) and outgrowth endothelial cells (OECs), as previously reported. Here, we further characterize these cells, especially with respect to their different origins and functions both in vitro and in vivo. We also investigated whether the combination of these different cell types shows synergism during neovascularization. Early EPCs were heterogeneously made up of both CD14+ monocyte-derived cells, which secrete cytokines, and CD14(-)-derived cells, which contain high levels of (CD34+)KDR+ cells. OECs were cultured almost exclusively from CD14- cells, not CD14+ cells, and were distinct from mature endothelial cells in terms of proliferation potential, KDR+ expression level, and telomerase activity. A portion of cells from CD14- cells and early EPCs produced rapidly proliferating, capillary-forming cells in both the Matrigel plug and the ischemic hind limb similar to OECs. Early EPCs and OECs expressed receptors for vascular endothelial growth factor and interleukin-8, cytokines secreted by early EPCs. There was a differential increase in matrix metalloproteinases (MMPs): MMP-9 in early EPCs and MMP-2 in OECs. In vitro, the angiogenic capability of the 2 cell types was augmented by mutual interaction through cytokines and MMPs. Injection of a mixture of the 2 cells resulted in superior neovascularization in vivo to any single-cell-type transplantation. Distinct origins of the different types of EPCs exist that have different functions in neovascularization. Mixed transplantation of these cells results in synergistic neovascularization through cytokines and MMPs.
Article
Cardiac cell transplantation is limited by poor graft viability. We aimed to enhance the survival of transplanted cardiomyoblasts using growth factor-supplemented collagen matrices. H9c2 cardiomyoblasts were lentivirally transduced to express firefly luciferase and green fluorescent protein (GFP). Lewis rats underwent ligation of the left anterior descending artery (LAD) ligation to induce an anterior wall myocardial infarction. Hearts (n=9/group) were harvested and restored ex vivo with 1 x 10(6) genetically labeled H9c2 cells either in (1) saline-suspension, or seeded onto (2) collagen-matrix (Gelfoam [GF];), (3) GF/Matrigel (GF/MG), (4) GF/MG/VEGF (10 microg/mL), or (5) GF/MG/FGF (10 microg/mL). Hearts were then abdominally transplanted into syngeneic recipients (working heart model). Controls (n=6/group) underwent infarction followed by GF implantation or saline injection. Cell survival was evaluated using optical bioluminescence on days 1, 5, 8, 14, and 28 postoperatively. At 4 weeks, fractional shortening and ejection fraction were determined using echocardiography and magnetic resonance imaging, respectively. Graft characteristics were assessed by immunohistology. Bioluminescence signals on days 5, 8, and 14 were higher for GF-based grafts compared with plain H9c2 injections (P<0.03). Signals were higher for GF/MG grafts compared with GF alone (P<0.02). GFP-positive, spindle-shaped H9c2 cells were found integrated in the infarct border zones at day 28. Left ventricular (LV) function of hearts implanted with collagen-based grafts was better compared with controls (P<0.05). Vascular endothelial growth factor or fibroblast growth factor did not further improve graft survival or heart function. Collagen matrices enhance early survival of H9c2 cardiomyoblasts after transplantation into ischemic hearts and lead to improved LV function. Further optimization of the graft design should make restoration of large myocardial infarctions by tissue engineering approaches effective.
Article
The use of stem and/or progenitor cells to achieve potent vasculogenesis in humans has been hindered by low cell numbers, implant capacity, and survival. This study investigated the expansion of CD133+ cells and the use of an injectable collagen-based tissue engineered matrix to support cell delivery and implantation within target ischemic tissue. Adult human CD133+ progenitor cells from the peripheral blood were generated and expanded by successive removal and culture of CD133- cell fractions, and delivered within an injectable collagen-based matrix into the ischemic hindlimb of athymic rats. Controls received injections of phosphate-buffered saline, matrix, or CD133+ cells alone. Immunohistochemistry of hindlimb muscle 2 weeks after treatment revealed that the number of CD133+ cells retained within the target site was >2-fold greater when delivered by matrix than when delivered alone (P<0.01). The transplanted CD133+ cells incorporated into vascular structures, and the matrix itself also was vascularized. Rats that received matrix and CD133+ cells demonstrated greater intramuscular arteriole and capillary density than other treatment groups (P<0.05 and P<0.01, respectively). Compared with other experimental approaches, treatment of ischemic muscle tissue with generated CD133+ progenitor cells delivered in an injectable collagen-based matrix significantly improved the restoration of a vascular network. This work demonstrates a novel approach for the expansion and delivery of blood CD133+ cells with resultant improvement of their implantation and vasculogenic capacity.
Article
Multipotent stem cells in the body facilitate tissue regeneration, growth, and wound healing throughout life. The microenvironment in which they reside provides signals that direct these progenitors to proliferate, differentiate, or remain dormant; these factors include soluble molecules, the extracellular matrix, neighboring cells, and physical stimuli. Recent advances in the culture of embryonic stem cells and adult progenitors necessitate an increased understanding of these phenomena. Here, we summarize the interactions between stem cells and their local environment, drawing on in vivo observations and tissue culture studies. In addition, we describe novel methods of characterizing the effects of various environmental factors and review new techniques that enable scientists and engineers to more effectively direct stem cell fate.
Article
Therapeutic efficacy of bone marrow (BM) cell injection for treating ischemic chronic heart failure has not been established. In addition, experimental data are lacking on arrhythmia occurrence after BM cell injection. We hypothesized that therapeutic efficacy and arrhythmia occurrence induced by BM cell injection may be affected by the cell delivery route. Three weeks after left coronary artery ligation, wild-type female rats were injected with 1x10(7) mononuclear BM cells derived from green fluorescent protein-transgenic male rats through either a direct intramyocardial or a retrograde intracoronary route. Both intramyocardial and intracoronary injection of BM cells demonstrated similar improvement in left ventricular ejection fraction measured by echocardiography and a similar graft size analyzed by real-time polymerase chain reaction for the Y chromosome-specific Sry gene. Noticeably, intramyocardial injection of BM cells induced frequent ventricular premature contractions (108+/-73 per hour at 7 days after BM cell injection), including multiform, consecutive ventricular premature contractions and ventricular tachycardia for the initial 14 days; intracoronary injection of BM cells and intramyocardial injection of phosphate-buffered saline rarely induced arrhythmias. Immunohistochemistry demonstrated that intramyocardial BM cell injection formed distinct cell clusters containing donor-derived cells and accumulated host-derived inflammatory cells in the infarct border zone, whereas intracoronary BM cell injection provided more homogeneous donor cell dissemination with less inflammation and without disrupting the native myocardial structure. BM cell injection is able to improve cardiac function in ischemic chronic heart failure but has a risk of arrhythmia occurrence when the intramyocardial route is used. Such arrhythmias may be prevented by using the intracoronary route.
Article
Here we address the effect of Akt signaling on endothelial progenitor cells (EPCs). Human peripheral blood mononuclear cells (PBMCs) were cultured on fibronectin-coated dishes in EPC differentiation medium. PBMCs differentiated in a series of three steps: proliferation for foci formation, tight attachment to the dishes in the early stages of differentiation, and maturation in the late stages. In Western blot analysis, Akt expression was attenuated in the early stages of differentiation and was gradually upregulated during EPC maturation. Forkhead box-containing protein, class O 3a (FOXO3a), an Akt downstream target, was downregulated through phosphorylation in the late stages of EPC differentiation. Adenovirus-mediated overexpression of activated FOXO3a in PBMCs markedly increased the number of cell foci but reduced the number of DiI-acetyl LDL EPCs that appear at later time points. These data suggest that Akt/FOXO3a signaling is an important regulator of EPC maturation.
Mitogen-activated protein kinase pathways.
  • Robinson M.J.
  • Cobb M.H.
Collagen-based matrices improve the delivery of transplanted circulating progenitor cells.