Background— Adipose tissue development and remodeling are closely associated with the growth of vascular network. We hypothesized that adipose tissue may contain progenitor cells with angiogenic potential and that therapy based on adipose tissue-derived progenitor cells administration may constitute a promising cell therapy in patients with ischemic disease. Methods and Results— In mice, cultured stromal-vascular fraction (SVF) cells from adipose tissue have a great proangiogenic potential, comparable to that of bone marrow mononuclear cells in the mouse ischemic hindlimb model. Similarly, cultured human SVF cells differentiate into endothelial cells, incorporate into vessels, and promote both postischemic neovascularization in nude mice and vessel-like structure formation in Matrigel plug. In vitro, these cells represent a homogeneous population of CD34- and CD13-positive cells, which can spontaneously express the endothelial cell markers CD31 and von Willebrand factor when cultured in semisolid medium. Interestingly, dedifferentiated mature human adipocytes have the potential to rapidly acquire the endothelial phenotype in vitro and to promote neovascularization in ischemic tissue and vessel-like structure formation in Matrigel plug, suggesting that cells of endothelial and adipocyte phenotypes may have a common precursor. Conclusions— This study demonstrates, for the first time, that adipocytes and endothelial cells have a common progenitor. Such adipose lineage cells participate in vascular-like structure formation in Matrigel plug and enhance the neovascularization reaction in ischemic tissue. These results also highlight the concept that adipose lineage cells represent a suitable new cell source for therapeutic angiogenesis in ischemic disease.

Abstract-The potential role of anti-inflammatory cytokines in the modulation of the atherosclerotic process remains unknown. Interleukin (IL)-10 has potent deactivating properties in macrophages and T cells and modulates many cellular processes that may interfere with the development and stability of the atherosclerotic plaque. IL-10 is expressed in human atherosclerosis and is associated with decreased signs of inflammation. In the present study, we show that IL-10 -deficient C57BL/6J mice fed an atherogenic diet and raised under specific pathogen-free conditions exhibit a significant 3-fold increase in lipid accumulation compared with wild-type mice. Interestingly, the susceptibility of IL-10 -deficient mice to atherosclerosis was exceedingly high (30-fold increase) when the mice were housed under conventional conditions. Atherosclerotic lesions of IL-10 -deficient mice showed increased T-cell infiltration, abundant interferon-␥ expression, and decreased collagen content. In vivo, transfer of murine IL-10 achieved 60% reduction in lesion size. These results underscore the critical roles of IL-10 in both atherosclerotic lesion formation and stability. Moreover, IL-10 appears to be crucial as a protective factor against the effect of environmental pathogens on atherosclerosis. The full text of this article is available at http://www.circresaha.org. (Circ Res. 1999;85:e17-e24.)

Vascular endothelial growth factor (VEGF)-induced blood vessel growth is involved in both physiological and pathological angiogenesis and requires integrin-mediated signaling. We now show that an integrin-binding protein initially described in milk-fat globule, MFG-E8 (also known as lactadherin), is expressed in and around blood vessels and has a crucial role in VEGF-dependent neovascularization in the adult mouse. Using neutralizing antibodies and lactadherin-deficient animals, we show that lactadherin interacts with alphavbeta3 and alphavbeta5 integrins and alters both VEGF-dependent Akt phosphorylation and neovascularization. In the absence of VEGF, lactadherin administration induced alphavbeta3- and alphavbeta5-dependent Akt phosphorylation in endothelial cells in vitro and strongly improved postischemic neovascularization in vivo. These results show a crucial role for lactadherin in VEGF-dependent neovascularization and identify lactadherin as an important target for the modulation of neovascularization.

Ischemia induces both hypoxia and inflammation that trigger angiogenesis. The inflammatory reaction is modulated by production of anti-inflammatory cytokines. This study examined the potential role of a major anti-inflammatory cytokine, interleukin (IL)-10, on angiogenesis in a model of surgically induced hindlimb ischemia. Ischemia was produced by artery femoral occlusion in both C57BL/6J IL-10(+/+) and IL-10(-/-) mice. After 28 days, angiogenesis was quantified by microangiography, capillary, and arteriole density measurement and laser Doppler perfusion imaging. The protein levels of IL-10 and vascular endothelial growth factor (VEGF) were determined by Western blot analysis in hindlimbs. IL-10 was markedly expressed in the ischemic hindlimb of IL-10(+/+) mice. Angiogenesis in the ischemic hindlimb was significantly increased in IL-10(-/-) compared with IL-10(+/+) mice. Indeed, angiographic data showed that vessel density in the ischemic leg was 10.2+/-0.1% and 5.7+/-0.4% in IL-10(-/-) and IL-10(+/+) mice, respectively (P:<0.01). This corresponded to improved ischemic/nonischemic leg perfusion ratio by 1.4-fold in IL-10(-/-) mice compared with IL-10(+/+) mice (0.87+/-0. 05 versus 0.63+/-0.01, respectively; P:<0.01). Revascularization was associated with a 1.8-fold increase in tissue VEGF protein level in IL-10(-/-) mice compared with IL-10(+/+) mice (P:<0.01). In vivo electrotransfer of murine IL-10 cDNA in IL-10(-/-) mice significantly inhibited both the angiogenic process and the rise in VEGF protein level observed in IL-10(-/-) mice. No changes in vessel density or VEGF content were observed in the nonischemic hindlimb. These findings underscore the antiangiogenic effect of IL-10 associated with the downregulation of VEGF expression and suggest a role for the inflammatory balance in the modulation of ischemia-induced angiogenesis.

The intermediate filament vimentin might play a key role in vascular resistance to mechanical stress. We investigated the responses to pressure (tensile stress) and flow (shear stress) of mesenteric resistance arteries perfused in vitro from vimentin knockout mice. Arteries were isolated from homozygous (Vim-/-, n = 14) or heterozygous vimentin-null mice (Vim+/-, n = 5) and from wild-type littermates (Vim+/+, n = 9). Passive arterial diameter (175+/-15 micron in Vim+/+ at 100 mmHg) and myogenic tone were not affected by the absence of vimentin. Flow-induced (0-150 microl/min) dilation (e. g., 19+/-3 micron dilation at 150 mmHg in Vim+/+) was significantly attenuated in Vim-/- mice (13+/-2 micron dilation, P < 0.01). Acute blockade of nitric oxide synthesis (NG-nitro- L-arginine, 10 microM) significantly decreased flow-induced dilation in both groups, whereas acute blockade of prostaglandin synthesis (indomethacin, 10 microM) had no significant effect. Mean blood pressure, in vivo mesenteric blood flow and diameter, and mesenteric artery media thickness or media to lumen ratio were not affected by the absence of vimentin. Thus, the absence of vimentin decreased selectively the response of resistance arteries to flow, suggesting a role for vimentin in the mechanotransduction of shear stress.

Background-Dystrophin has a key role in striated muscle mechanotransduction of physical forces. Although cytoskeletal elements play a major role in the mechanotransduction of pressure and flow in vascular cells, the role of dystrophin in vascular function has not yet been investigated. Thus, we studied endothelial and muscular responses of arteries isolated from mice lacking dystrophin (mdx mice). Methods and Results-Carotid and mesenteric resistance arteries 120 m in diameter were isolated and mounted in vitro in an arteriograph to control intraluminal pressure and flow. Blood pressure was not affected by the absence of dystrophin. Pressure-induced (myogenic), phenylephrine-induced, and KCl-induced forms of tone were unchanged. Flow (shear stress)-induced dilation in arteries isolated from mdx mice was decreased by 50% to 60%, whereas dilation to acetylcholine or sodium nitroprusside was unaffected. NG-nitro-L-arginine methyl ester-sensitive flow dilation was also decreased in arteries from mdx mice. Thus, the absence of dystrophin was associated with a defect in signal transduction of shear stress. Dystrophin was present in vascular endothelial and smooth muscle cells, as shown by immunolocalization, and localized at the level of the plasma membrane, as seen by confocal microscopy of perfused isolated arteries. Conclusions-This

We hypothesized that diabetes-induced oxidative stress may affect postischemic neovascularization. The response to unilateral femoral artery ligation was studied in wild-type or gp91 phox -deficient control or type 1 diabetic mice or in animals treated with the anti-oxidant N-acetyl-L-cysteine (NAC) or with in vivo electrotransfer of a plasmid encoding dominant-negative Rac1 (50 g) for 21 days. Postischemic neovascularization was reduced in diabetic mice in association with down-regulated vascular endothelial growth factor-A protein levels. In diabetic animals vascular endothelial growth factor levels and postischemic neovascularization were restored to nondiabetic levels by the scavenging of reactive oxygen species (ROS) by NAC administration or the inhibition of ROS generation by gp91 phox deficiency or by administration of dominant-negative Rac1. Finally, diabetes reduced the ability of adherent bone marrow-derived mononuclear cells (BM-MNCs) to differentiate into endothelial progenitor cells. Treatment with NAC (3 mmol/L), apocynin (200 mol/L), or the p38MAPK inhibitor LY333351 (10 mol/L) up-regulated the number of endothelial progenitor cell colonies derived from diabetic BM-MNCs by 1.5-, 1.6-, and 1.5-fold, respectively (P < 0.05). In the ischemic hindlimb model, injection of diabetic BM-MNCs isolated from NACtreated or gp91 phox -deficient diabetic mice increased neovascularization by ϳ1.5-fold greater than untreated diabetic BM-MNCs (P < 0.05). Thus, inhibition of NADPH oxidase-derived ROS overproduction improves the angiogenic and vasculogenic processes and restores postischemic neovascularization in type 1 diabetic mice.

These results demonstrate that MPs isolated from human atherosclerotic lesions express CD40L, stimulate endothelial cell proliferation after CD40 ligation, and promote in vivo angiogenesis. Therefore, MPs could represent a major determinant of intraplaque neovascularization and plaque vulnerability.