Proteases and receptors in the recruitment of endothelial progenitor cells in neovascularization.

Since the initial discovery of endothelial progenitor cells (EPC), and their promise in increasing angiogenesis and vasculogenesis, a myriad of papers have highlighted their potential application in experimental and clinical neovascularization and in tissue engineering. However, promising reports are contrasted by other studies that could not find a role for EPC in neovascularization. Presently, two types of endothelial progenitor cell populations are recognized. The first population provides early-outgrowth CD34+/VEGFR-2+ cells, or colony-forming unit endothelial cells (CFU-EC), which represent myeloid cells with some endothelial properties, but no ability to form endothelial colonies. They can stimulate neovascularization by paracrine means, but are not incorporated in the endothelial lining themselves. The second population generates the late-outgrowth endothelial colony-forming cells (ECFC) from a very scant blood-derived cell population. ECFC have a very high proliferative potential, can insert into the endothelial lining of new blood vessels, and can also form endothelial tubes by themselves after stimulation with the proper angiogenic stimulus. This review surveys the mobilization of progenitor cells from the bone marrow, the homing of EPC (CFU-EC) to areas of neovascularization, and the participation of EPC (ECFC) in the endothelial lining of newly formed blood vessels. Specific emphasis has been placed on the role of proteases, which include serine proteases, including urokinase, L-cathepsin, and several ADAM- and matrix metalloproteinases. The specific properties of ECFC make them a potential source of cells for tissue engineering applications, but much has to be learned about their nature, origin and properties.

Differential gene expression analysis of tubule forming and non-tubule forming endothelial cells: CDC42GAP as a counter-regulator in tubule formation.

The formation of new tubular structures from a quiescent endothelial lining is one of the hallmarks of sprouting angiogenesis. This process can be mimicked in vitro by inducing capillary-like tubular structures in a three-dimensional (3D) fibrin matrix. We aimed to analyze the differential mRNA expression in two phenotypically distinct cell populations from the same culture, namely in tubule-forming endothelial cells and monolayer endothelial cells not participating in tubule formation. A fibrin-rich 3D matrix derived from human plasma was used to facilitate tubule formation by human foreskin microvascular endothelial cells (hMVEC). After 7 days of stimulation with VEGF, bFGF, and TNF-alpha, the culture consisted of a monolayer and capillary-like sprouts that had grown into the fibrinous matrix. A method was developed to separate the monolayer and tubule-forming populations of hMVEC, keeping their cellular integrity intact to ensure mRNA extraction and cDNA production. Subsequent array analysis resulted in an inventory of differentially expressed genes that were associated with either tube-forming (angiogenic) or non-angiogenic capacity. Differential gene expression was verified by real-time PCR on the original RNA samples as well as on RNA obtained from laser-capture microdissected cross sections of monolayers and capillary structures in the 3D fibrinous matrix. The expression of CDC42GAP, an inhibitor of active-state small Rho GTPases, was reduced in tubular hMVEC. Overexpression of CDC42GAP in hMVEC attenuated endothelial tubule formation, while its suppression by siRNA slightly enhanced this process. Thus, CDC42GAP was identified as a counter-regulatory mediator for tubule formation.

Many novel mammalian microRNA candidates identified by extensive cloning and RAKE analysis.

MicroRNAs are 20- to 23-nucleotide RNA molecules that can regulate gene expression. Currently > 400 microRNAs have been experimentally identified in mammalian genomes, whereas estimates go up to 1000 and beyond. Here we show that many more mammalian microRNAs exist. We discovered novel microRNA candidates using two approaches: testing of computationally predicted microRNAs by a modified microarray-based detection system, and cloning and sequencing of large numbers of small RNAs from different human and mouse tissues. Together these efforts experimentally identified 348 novel mouse and 81 novel human microRNA candidate genes. Most novel microRNAs candidates are not conserved beyond mammals, and ~10% are taxon-specific. Our analyses indicate that the entire microRNA repertoire is not remotely exhausted.

Hypercholesterolemia reduces collateral artery growth more dominantly than hyperglycemia or insulin resistance in mice.

OBJECTIVE: Collateral artery development (arteriogenesis), a vital compensatory mechanism in patients with arterial obstructive disease, may be deregulated by vascular risk factors, eg, diabetes or hypercholesterolemia. Here, we compared the effects of either disturbed glucose metabolism or disturbed lipid metabolism on arteriogenesis. METHODS AND RESULTS: Femoral artery occlusion was performed in streptozotocin(STZ)-treated mice, nonobese diabetic (NOD) mice, and insulin-resistant Ob/Ob mice on regular diet, and APOE3*Leiden mice on different hypercholesterolemic diets. Angiography and laser Doppler perfusion analysis of hindlimbs were performed postoperatively. Surprisingly, angiographic arteriogenesis was not impaired in diabetic and insulin-resistant mice. Perfusion recovery in STZ-treated and Ob/Ob mice was only decreased by 19% and 16%, respectively (P<0.05). Furthermore, perfusion recovery was unchanged between high-glycemic and mild-glycemic NOD mice. Angiographic arteriogenesis in APOE3*Leiden mice, however, was markedly impaired at 7 days and 14 days (P< or =0.01). Correspondingly, perfusion recovery was 41% decreased in APOE3*Leiden mice (P<0.05). There was an inverse correlation of perfusion recovery with plasma cholesterol (P=0.02), but not with triglyceride, free fatty acid, glucose, or insulin levels. CONCLUSIONS: Hypercholesterolemia reduces arteriogenesis more dominantly than hyperglycemia or hyperinsulinemia in mice. This suggests that a disturbed lipid metabolism as observed in diabetic patients might be crucial for the impairment of collateral formation.

CD34+ cells home, proliferate, and participate in capillary formation, and in combination with CD34- cells enhance tube formation in a 3-dimensional matrix.

OBJECTIVE: Emerging evidence suggests that human blood contains bone marrow (BM)-derived endothelial progenitor cells that contribute to postnatal neovascularization. Clinical trials demonstrated that administration of BM-cells can enhance neovascularization. Most studies, however, used crude cell populations. Identifying the role of different cell populations is important for developing improved cellular therapies. METHODS AND RESULTS: Effects of the hematopoietic stem cell-containing CD34+ cell population on migration, proliferation, differentiation, stimulation of, and participation in capillary-like tubule formation were assessed in an in vitro 3-dimensional matrix model using human microvascular endothelial cells. During movement over the endothelial monolayer, CD34+ cells remained stuck at sites of capillary tube formation and time- and dose-dependently formed cell clusters. Immunohistochemistry confirmed homing and proliferation of CD34+ cells in and around capillary sprouts. CD34+ cells were transduced with the LNGFR marker gene to allow tracing. LNGFR gene-transduced CD34+ cells integrated in the tubular structures and stained positive for CD31 and UEA-1. CD34+ cells alone stimulated neovascularization by 17%. Coculture with CD34- cells led to 68% enhancement of neovascularization, whereas CD34- cells displayed a variable response by themselves. Cell-cell contact between CD34+ and CD34- cells facilitated endothelial differentiation of CD34+ cells. CONCLUSIONS: Our data suggest that administration of CD34+-enriched cell populations may significantly improve neovascularization and point at an important supportive role for (endogenous or exogenous) CD34- cells.

Involvement of furin-like proprotein convertases in the arterial response to injury.

BACKGROUND: Furin-like proprotein convertases (PCs) are proteolytic activators of proproteins, like membrane type 1-matrix metalloproteinase (MT1-MMP) and transforming growth factor beta (TGF-beta), that are described in the arterial response to injury. However, the involvement of furin-like PCs in the arterial response to injury has not been studied yet. We studied furin, MT1-MMP, MMP levels and TGF-beta signaling after arterial injury. We also investigated the effect of an inhibitor of furin-like PCs, alpha1-antitrypsin Portland (alpha1-PDX), on arterial injury following balloon dilation. METHODS AND RESULTS: NZW rabbit femoral and iliac arteries (N=42) were balloon dilated unilaterally and harvested after 2, 7, 14, 28 or 42 days. Furin mRNA levels were increased after 2 and 7 days. MMP-2 and MT1-MMP levels were increased after day 7 and TGF-beta signaling, by phosphorylating Smad 1/5 and 2/3, was increased at all time points. Inhibition of furin-like PCs, by adenoviral over-expression of alpha1-PDX, blocked proTGF-beta activation and Smad phosphorylation, and reduced MT1-MMP and MMP-2 activation (N=5). In vivo adventitial inhibition of furin-like PCs (N=9) resulted in a reduction of 13.1+/-5.2% in advential and 23.6+/-7.9% in intimal areas (P<0.05), but had no effect on lumen size due to decreased vessel areas. CONCLUSIONS: This study demonstrates that furin-like PCs are involved in the arterial response to injury possibly through activation of the TGF-beta-Smad signaling pathway and identifies furin-like PCs as a possible target to inhibit intimal hyperplasia.