Drew Kuraitis

A Collagen–Chitosan Hydrogel for Endothelial Differentiation and Angiogenesis

Cell therapy for the treatment of cardiovascular disease has been hindered by low cell engraftment, poor survival, and inadequate phenotype and function. In this study, we added chitosan to a previously developed injectable collagen matrix, with the aim of improving its properties for cell therapy and neovascularization. Different ratios of collagen and chitosan were mixed and chemically crosslinked to produce hydrogels. Swell and degradation assays showed that chitosan improved the stability of the collagen hydrogel. In culture, endothelial cells formed significantly more vascular-like structures on collagen–chitosan than collagen-only matrix. While the differentiation of circulating progenitor cells to CD31+ cells was equal on all matrices, vascular endothelial-cadherin expression was increased on the collagen–chitosan matrix, suggesting greater maturation of the endothelial cells. In addition, the collagen–chitosan matrix supported a significantly greater number of CD133+ progenitor cells than the collagen-only matrix. In vivo, subcutaneously implanted collagen–chitosan matrices stimulated greater vascular growth and recruited more von Willebrand factor (vWF+) and CXCR4+ endothelial/angiogenic cells than the collagen-only matrix. These results indicate that the addition of chitosan can improve the physical properties of collagen matrices, and enhance their ability to support endothelial cells and angiogenesis for use in cardiovascular tissue engineering applications.

Exploiting extracellular matrix-stem cell interactions: A review of natural materials for therapeutic muscle regeneration

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 bodys 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.

An acellular matrix-bound ligand enhances the mobilization, recruitment and therapeutic effects of circulating progenitor cells in a hindlimb ischemia model

Circulating progenitor cells home to and engraft to sites of ischemia, mediated in part by the adhesion molecule Lrselectin; however, accumulation in tissues such as the heart is low. In this study, an acellular collagen‐based matrix containing sialyl LewisX (sLeX), 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 sLeX– collagen matrix recruited more CD133+CD34+L‐selectm+ 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 sLeX‐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 sLeX‐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 sLeX‐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.— Suuronen, E. J., Zhang, P., Kuraitis, D., Cao, X., Melhuish, A., McKee, D., Li, F., Mesana, T. G., Veinot, J. P., Ruel, M. An acellular matrix‐bound ligand enhances the mobilization, recruitment and therapeutic effects of circulating progenitor cells in a hindlimb ischemia model. FASEBJ. 23, 1447–1458 (2009)

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

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.

Human Blood and Cardiac Stem Cells Synergize to Enhance Cardiac Repair When Cotransplanted Into Ischemic Myocardium

Background— Blood-derived circulatory angiogenic cells (CACs) and resident cardiac stem cells (CSCs) have both been shown to improve cardiac function after myocardial infarction. The superiority of either cell type has long been an area of speculation with no definitive head-to-head trial. In this study, we compared the effect of human CACs and CSCs, alone or in combination, on myocardial function in an immunodeficient mouse model of myocardial infarction. Methods and Results— CACs and CSCs were cultured from left atrial appendages and blood samples obtained from patients undergoing clinically indicated heart surgery. CACs expressed a broader cytokine profile than CSCs, with 3 cytokines in common. Coculture of CACs and CSCs further enhanced the production of stromal cell–derived factor-1&agr; and vascular endothelial growth factor (P⩽0.05). Conditioned media promoted equivalent vascular networks and CAC recruitment with superior effects using cocultured conditioned media. Intramyocardial injection of CACs or CSCs alone improved myocardial function and reduced scar burdens when injected 1 week after myocardial infarction (P⩽0.05 versus negative controls). Cotransplantation of CACs and CSCs together improved myocardial function and reduced scar burdens to a greater extent than either stem cell therapy alone (P⩽0.05 versus CAC or CSC injection alone). Conclusions— CACs and CSCs provide unique paracrine repertoires with equivalent effects on angiogenesis, stem cell migration, and myocardial repair. Combination therapy with both cell types synergistically improves postinfarct myocardial function greater than either therapy alone. This synergy is likely mediated by the complimentary paracrine signatures that promote revascularization and the growth of new myocardium.

IRF2BP2 is a skeletal and cardiac muscle-enriched ischemia-inducible activator of VEGFA expression

We sought to identify an essential component of the TEAD4/VGLL4 transcription factor complex that controls vascular endothelial growth factor A (VEGFA) expression in muscle. A yeast 2-hybrid screen was used to clone a novel component of the TEAD4 complex from a human heart cDNA library. We identified interferon response factor 2 binding protein 2 (IRF2BP2) and confirmed its presence in the TEAD4/VGLL4 complex in vivo by coimmunoprecipitation and mammalian 2-hybrid assays. Coexpression of IRF2BP2 with TEAD4/VGLL4 or TEAD1 alone potently activated, whereas knockdown of IRF2BP2 reduced, VEGFA expression in C(2)C(12) muscle cells. Thus, IRF2BP2 is required to activate VEGFA expression. In mouse embryos, IRF2BP2 was ubiquitously expressed but became progressively enriched in the fetal heart, skeletal muscles, and lung. Northern blot analysis revealed high levels of IRF2BP2 mRNA in adult human heart and skeletal muscles, but immunoblot analysis showed low levels of IRF2BP2 protein in skeletal muscle, indicating post-transcriptional regulation of IRF2BP2 expression. IRF2BP2 protein levels are markedly increased by ischemia in skeletal and cardiac muscle compared to normoxic controls. IRF2BP2 is a novel ischemia-induced coactivator of VEGFA expression that may contribute to revascularization of ischemic cardiac and skeletal muscles.

Improving Cell Engraftment with Tissue Engineering

Cardiac cell therapy has not yet resulted in long-term clinical benefits or major recovery of myocardial function in humans. To date, most of the cardiac effects of cell-based therapy are believed to be mediated by a local angiogenic response rather than by the formation of neosyncytial contractile units such as had initially been hoped for. Therefore, repopulation of the ischemic or infarcted heart with progenitor cells that have vasculogenic potential may be an important mechanism to improve contractile function, both in the presence of viable and nonviable myocardium. This constitutes a focus within scientific reach; however, the low engraftment and viability of progenitor cells after transplantation necessitate the exploration of novel delivery techniques. Because biomaterials have the capacity to improve cell retention, survival, and differentiation, tissue engineering is now being explored as an approach to support cell-based therapies and enhance their efficacy. In this article, we address current progress made in tissue engineering to support cell therapy for the heart, and summarize our work in the development of biomaterials toward improving cell delivery and vascularization of ischemic tissue.

Mesenchymal Stem Cells for Cardiovascular Regeneration

Despite recent studies suggesting that the heart has instrinsic mechanisms of self-regeneration following myocardial infarction, it cannot regenerate itself to an optimal level. Mesenchymal stem cells (MSCs) are currently being investigated for regeneration of mesenchyme-derived tissues, such as bone, cartilage and tendon. In vitro evidence suggests that MSCs can also differentiate into cardiomyogenic and vasculogenic lineages, offering another cell source for cardiovascular regeneration. In vivo, MSCs may contribute to the re-growth and protection of vasculature and cardiomyocytes, mediated by paracrine actions, and/or persist within the myocardium in a differentiated state; although proof of cardiomyocytic phenotype and functional integration remains elusive. Herein, we review the evidence of MSCs as a cell source for cardiovascular regeneration, as well as their limitations that may prevent them from being effectively used in the clinic.

Glyoxalase-1 overexpression in bone marrow cells reverses defective neovascularization in STZ-induced diabetic mice

AIMS Methylglyoxal (MG) accumulates in diabetes and impairs neovascularization. This study assessed whether overexpressing the MG-metabolizing enzyme glyoxalase-1 (GLO1) in only bone marrow cells (BMCs) could restore neovascularization in ischaemic tissue of streptozotocin-induced diabetic mice. METHODS AND RESULTS After 24 h of hyperglycaemic and hypoxic culture, BMCs from GLO1 overexpressing and wild-type (WT) diabetic mice were compared for migratory potential, viability, and mRNA expression of anti-apoptotic genes (Bcl-2 and Bcl-XL). In vivo, BMCs from enhanced green fluorescent protein (eGFP) mice that overexpress GLO1 were used to reconstitute the BM of diabetic mice (GLO1-diabetics). Diabetic and non-diabetic recipients of WT GFP(+) BM served as controls (WT-diabetics and non-diabetics, respectively). Following hindlimb ischaemia, the mobilization of BMCs was measured by flow cytometry. In hindlimbs, the presence of BM-derived angiogenic (GFP(+)CXCR4(+)) and endothelial (GFP(+)vWF(+)) cells and also arteriole density were determined by immunohistochemistry. Hindlimb perfusion was measured using laser Doppler. GLO1-BMCs had superior migratory potential, increased viability, and greater Bcl-2 and Bcl-XL expression, compared with WT BMCs. In vivo, the mobilization of pro-angiogenic BMCs (CXCR4(+), c-kit(+), and Flk(+)) was enhanced post-ischaemia in GLO1-diabetics compared to WT-diabetics. A greater number of GFP(+)CXCR4(+) and GFP(+)vWF(+) BMCs incorporated into the hindlimb tissue of GLO1-diabetics and non-diabetics than in WT-diabetics. Arteriole and capillary density and perfusion were also greater in GLO1-diabetics and non-diabetics. CONCLUSION This study demonstrates that protection from MG uniquely in BM is sufficient to restore BMC function and neovascularization of ischaemic tissue in diabetes and identifies GLO1 as a potential therapeutic target.

18F-FDG Cell Labeling May Underestimate Transplanted Cell Homing: More Accurate, Efficient, and Stable Cell Labeling with Hexadecyl-4-[18F]Fluorobenzoate for in Vivo Tracking of Transplanted Human Progenitor Cells by Positron Emission Tomography:

Cell therapy is expected to restore perfusion and improve function in the ischemic/infarcted myocardium; however, the biological mechanisms and local effects of transplanted cells remain unclear. To assess cell fate in vivo, hexadecyl-4-[18F]fluorobenzoate (18F-HFB) cell labeling was evaluated for tracking human circulating progenitor cells (CPCs) with positron emission tomography (PET) and was compared to the commonly used 2-[18F]fluoro-2-deoxy-d-glucose (18F-FDG) labeling method in a rat myocardial infarction model. CPCs were labeled with 18F-HFB or 18F-FDG ex vivo under the same conditions. 18F-HFB cell-labeling efficiency (23.4 ± 7.5%) and stability (4 h, 88.4 ± 6.0%) were superior to 18F-FDG (7.6 ± 4.1% and 26.6 ± 6.1%, respectively; p < 0.05). Neither labeling approach significantly altered cell viability, phenotype or migration potential up to 24 h postlabeling. Two weeks after left anterior descending coronary artery ligation, rats received echo-guided intramyocardial injection in the infarct border zone with 18F-HFB-CPCs, 18F-FDG-CPCs, 18F-HFB, or 18F-FDG. Dynamic PET imaging of both 18F-HFB-CPCs and 18F-FDG-CPCs demonstrated that only 16–37% of the initial injection dose (ID) was retained in the injection site at 10 min postdelivery, and remaining activity fell significantly over the first 4 h posttransplantation. The 18F-HFB-CPC signal in the target area at 2 h (23.7 ± 14.7% ID/g) and 4 h (17.6 ± 13.3% ID/g) postinjection was greater than that of 18F-FDG-CPCs (5.4 ± 2.3% ID/g and 2.6 ± 0.7% ID/g, respectively; p < 0.05). Tissue biodistribution confirmed the higher radioactivity in the border zone of 18F-HFB-CPC rats. Immunostaining of heart tissue sections revealed no significant difference in cell retention between two labeled cell transplantation groups. Good correlation with biodistribution results was observed in the 18F-HFB-CPC rats (r = 0.81, p < 0.05). Compared to 18F-FDG, labeling human CPCs with 18F-HFB provides a more efficient, stable, and accurate way to quantify the distribution of transplanted cells. 18F-HFB cell labeling with PET imaging offers a better modality to enhance our understanding of early retention, homing, and engraftment with cardiac cell therapy.