Isolation of "side population" progenitor cells from healthy arteries of adult mice.

OBJECTIVE: Circulating progenitors and stem cells have been reported to contribute to angiogenesis and arterial repair after injury. In the present study, we investigated whether the arterial wall could host permanently residing progenitor cells under physiological context. METHODS AND RESULTS: Using the Hoechst-based flow cytometry method, we identified and isolated progenitor cells termed side population (SP) cells at a prevalence of 6.0+/-0.8% in the tunica media of adult mice aortas. Arterial SP cells expressed the ATP-binding cassette transporter subfamily G member 2, frequently present on SP cell surface, and displayed a Sca-1+ c-kit(-/low) Lin- CD34(-/low) profile. They did not form myeloid or lymphoid hematopoietic colonies after plating in methylcellulose-based medium. Importantly, cultured SP cells were able to acquire the phenotype of endothelial cells (CD31, VE-cadherin, and von Willebrand factor expression) or of smooth muscle cells (alpha-smooth muscle actin, calponin, and smooth muscle myosin heavy chain expression), in presence of either vascular endothelial growth factor or transforming growth factor (TGF)-beta1/PDGF-BB, respectively. Moreover, they generated vascular-like branching structures, composed of both VE-cadherin+ cells and alpha-smooth muscle actin+ cells on Matrigel. CONCLUSIONS: In this study, we provide the first evidence to our knowledge that in the adult mice, the normal arterial wall harbors SP cells with vascular progenitor properties.

Decorin overexpression reduces atherosclerosis development in apolipoprotein E-deficient mice.

Atherosclerosis results from accumulation of macrophages and extracellular matrix in the arterial wall. Decorin, a small matrix proteoglycan, is able to regulate cell proliferation, migration and growth factors' activity. We investigated the effect of decorin overexpression on atherosclerosis progression in apolipoprotein E-deficient (ApoE(-/-)) mice. Female ApoE(-/-) mice, 10 weeks old (early treatment, n = 20) and 20 weeks old (delayed treatment, n = 20) were administered intravenously with either an adenovirus (2.5 x 10(9) plaque-forming units/mouse) containing human decorin gene (Ad-Dcn) or beta-galactosidase (LacZ), or PBS. Transgenic decorin was mainly expressed in the liver, and was secreted in the plasma up to 4 weeks. Six weeks after treatment, no significant difference in aortic root lesion size was observed between LacZ- and PBS-control groups. In contrast, Ad-Dcn-treated mice showed significantly reduced atherosclerotic lesions as compared to controls in both early and delayed treatment groups (2.9 +/- 1.1% versus 5.5 +/- 0.4%; p = 0.004 and 13.4 +/- 1.3% versus 19.9 +/- 1.41%; p = 0.009, respectively). In parallel, macrophage, gelatinase activity and collagen plaque content were also reduced. Interestingly, plasma triglycerides were reduced and decorin formed complexes with transforming growth factor-beta1 (TGF-beta1) that resulted in reduced circulating free-TGF-beta1. In conclusion, systemic overexpression of decorin reduces inflammation, triglycerides and fibrosis in atherosclerotic plaques of ApoE(-/-) mice resulting in slowing down of disease progression.

Effect of local heating on restenosis and in-stent neointimal hyperplasia in the atherosclerotic rabbit model: a dose-ranging study.

AIMS: In-stent restenosis is related to neointimal hyperplasia. Heating reduces neointimal hyperplasia but promotes constrictive remodeling after balloon angioplasty. We aimed to assess the ability of local heating in inhibiting restenosis and in-stent neointimal hyperplasia and its potential side effects on arterial thrombosis. METHODS AND RESULTS: Atherosclerotic-like lesions were induced in iliac rabbit arteries. One month later, both iliac rabbit arteries were stented. In each animal, one artery was randomized to local heating at four temperatures (50, 60, 80, and 100 degrees C). The contra lateral artery was used as control. Angiographic and histomorphometric analysis were performed 42 days after angioplasty. Immunohistochemistry was performed 3, 15, and 42 days after angioplasty. Angiographic significant reduction of in-stent restenosis after moderate heating (50 degrees C) was related to in-stent neointimal hyperplasia trend to be lower after moderate local heating when compared with controls. In contrast, in-stent thrombosis was similar to controls. Higher temperatures (i.e. 80 and 100 degrees C) also reduced in-stent neointimal hyperplasia but were most frequently associated with severe in-stent thrombosis. Local heating was associated with decreased cell proliferation, collagen density, and increased smooth muscle cell apoptosis and heat shock protein expression. CONCLUSION: Moderate heating represents a promising approach to prevent in-stent restenosis via the limitation of the proliferative response without thrombosis induction.

The N-terminal domain of hepatocyte growth factor inhibits the angiogenic behavior of endothelial cells independently from binding to the c-met receptor.

Hepatocyte growth factor (HGF) is a pleiotropic factor that plays an important role in complex biological processes such as embryogenesis, tissue regeneration, cancerogenesis, and angiogenesis. HGF promotes cell proliferation, survival, motility, and morphogenesis through binding to its receptor, a transmembrane tyrosine kinase encoded by the MET proto-oncogene (c-met). Structurally speaking, HGF is a polypeptide related to the enzymes of the blood coagulation cascade. Thus, it comprises kringle domains that in some other proteins have been shown to be responsible for the anti-angiogenic activity. To check whether the isolated kringles of HGF were able to inhibit angiogenesis, we produced them as recombinant proteins and compared their biological activity with that of the recombinant HGF N-terminal domain (N). We showed that (i) none of the isolated HGF kringle exhibits an anti-angiogenic activity; (ii) N is a new anti-angiogenic polypeptide; (iii) the inhibitory action of N is not specific toward HGF, because it antagonized the angiogenic activity of other growth factors, such as fibroblast growth factor-2 and vascular endothelial growth factor; and (iv) in contrast with full-length HGF, N does not bind to the c-met receptor in vitro, but fully retains its heparin-binding capacity. Our results suggest that N inhibits angiogenesis not by disrupting the HGF/c-met interaction but rather by interfering with the endothelial glycosaminoglycans, which are the secondary binding sites of HGF.