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.

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.

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Background-Bone marrow-derived mononuclear cells (BM-MNCs) enhance postischemic neovascularization, and their therapeutic use is currently under clinical investigation. We evaluated the safety of BM-MNC-based therapy in the setting of atherosclerosis. Methods and Results-Apolipoprotein E (apoE)-knockout (KO) mice were divided into 4 groups: 20 nonischemic mice receiving intravenous injection of either saline (nϭ10) or 10 6 BM-MNCs from wild-type animals (nϭ10) and 20 mice with arterial femoral ligature receiving intravenous injection of either saline (nϭ10) or 10 6 BM-MNCs from wild-type animals (nϭ10) at the time of ischemia induction. Animals were monitored for 4 additional weeks. Atherosclerosis was evaluated in the aortic sinus. BM-MNC transplantation improved tissue neovascularization in ischemic hind limbs, as revealed by the 210% increase in angiography score (PϽ0.0001), the 33% increase in capillary density (Pϭ0.01), and the 65% increase in tissue Doppler perfusion score (Pϭ0.0002). Hindlimb ischemia without BM-MNC transplantation or BM-MNC transplantation without ischemia did not affect atherosclerotic plaque size. However, transplantation of 10

Objective-We analyzed the beneficial therapeutic effect of angiotensin converting enzyme inhibitor (ACEI) on both retinal and hind limb neovascularization in diabetic mice. Methods and Results-Diabetic mice (streptozotocin, 40 mg/kg) were treated with or without ACEI (Perindopril, 3 mg/kg per day) or AT1 receptor blocker (Candesartan, 20 mg/kg) for 4 months. Hind limb ischemia was then induced by right femoral artery ligature for 1 additional month. In the ischemic leg, angiographic score, capillary density, and foot perfusion were increased by 2.7, 2.0-fold, and 1.6-fold, respectively, in ACEI-treated diabetic mice compared with untreated diabetic animals (PϽ0.01). ACEI also raised vascular endothelial growth factor (VEGF) protein level by 1.4-fold in ischemic diabetic leg. This ACEI pro-angiogenic effect was totally blunted in diabetic bradykinin B2 receptor-deficient animals, suggesting that it was mediated by the bradykinin pathway. In the diabetic retina, angiotensinogen and ACE mRNA levels were increased by 2.8-fold and 4.1-fold, respectively (PϽ0.01 versus nondiabetic mice), highlighting a local activation of renin-angiotensin system. Diabetes also raised VEGF protein level by 1.5-fold (PϽ0.05 versus nondiabetic mice). Treatments with ACEI and AT1 receptor blocker hampered diabetes-induced VEGF upregulation and retinal neovascularization. Conclusion-ACE inhibition improved neovascularization in the diabetic ischemic leg through activation of bradykinin signaling, whereas it reduced vessel growth in the diabetic retina through inhibition of overacting Ang II pathway.

Abstract-Identification of factors that may stimulate ischemia-induced neovascularization without increasing atherosclerotic plaque progression is of major therapeutic importance. We hypothesized that interleukin-18 binding protein (IL-18BP), a major antiinflammatory protein with plaque-stabilizing activities, may affect the neovascularization in mice ischemic hindlimb. Ischemia was produced by artery femoral occlusion in mice that were subjected to in vivo intramuscular electrotransfer of either an empty plasmid or a murine IL-18BP plasmid. Angiographic score, capillary density (CD31 staining), and laser Doppler perfusion data at day 28 showed significant improvement in ischemic/ nonischemic leg ratio by respectively 1.6-, 1.4-, and 1.5-fold in IL-18BP-treated mice compared with controls (PϽ0.01). This was associated with a significant 2-fold increase in both vascular endothelial growth factor (VEGF) and phospho-Akt protein content in the ischemic hindlimb of IL-18BP-treated mice (PϽ0.05). Similar results were obtained in IL-18 -deficient mice. Because bone marrow-derived endothelial progenitor cells (BM-EPCs) are involved in postnatal vasculogenesis, EPCs were isolated and cultivated from bone marrow mononuclear cells. IL-18BP treatment led to a significant 1. Key Words: angiogenesis Ⅲ ischemia Ⅲ inflammation Ⅲ interleukin-18 Ⅲ endothelial progenitor cells M odulation of the angiogenic process is considered as an important therapeutic goal in numerous diseases including diabetic retinopathy, tumor growth, and ischemic diseases. In the setting of ischemia, the formation of new capillary blood vessels is under the control of both hypoxia and inflammation. 1 Macrophages and T lymphocytes promote angiogenesis through the release of proinflammatory cytokines that heighten the production of matrix metalloproteinases (MMPs), leading to matrix degradation, and through the expression of angiogenic factors, including vascular endothelial growth factor (VEGF) and fibroblast growth factor-2. 2,3 On the other hand, macrophages and subsets of T cells are also known to produce antiinflammatory cytokines, including interleukin (IL)-10, which we have recently shown to be a major negative regulator of ischemia-induced angiogenesis. 4,5 Therefore, the inflammatory balance seems to play a critical role in determining the extent of angiogenesis after ischemic injury.Vascular ischemic diseases are mainly the result of atherosclerotic plaque progression and rupture, in which inflammation plays a major role. 6,7 The benefit expected from the actors of the inflammatory response that stimulate tissue neovascularization in this setting, including inflammatory cytokines, chemokines, and VEGF, 1 may be outweighed by their potential to promote atherosclerotic plaque progression and complications. 6,8,9 Conversely, antiinflammatory cytokines like IL-10, although reducing plaque progression and complications, 10,11 may greatly limit the angiogenic process with its expected deleterious effects on tissue perfusion and functional recovery. 4,5 ...

Left ventricular hypertrophy (LVH) was produced in guinea pigs after aortic stenosis (AS). The percentage of LVH in AS was determined by normalizing left ventricular (LY) weight by the mean LV weight of sham-operated controls (n = 12). After 3 weeks of cardiac overload, a mild LVH (30 ± 3%) was induced in 17 animals and a relatively severe LVH (56 ±3%) was induced in 7 animals. LV papillary muscles were rapidly excised for mechanical studies. No significant differences were observed between control and mild hypertrophy groups. In contrast, a marked decrease in myocardial performance was seen in the more severe cardiac hypertrophy group and was expressed as a percentage of sham-operated levels (Vmax, 22%; active isometric force/mm 2 ,23%; + dF/dt max/mm 2 ,26%). Relaxation in this group was still more impaired than contraction (peak lengthening velocity, 14%; -dF/dt max/mm 2 , 19%). Moreover, the load sensitivity of relaxation was present in both shamoperated controls and mild hypertrophy but almost disappeared in more severe hypertrophy. Isometric relaxation was delayed in the latter group, as shown by the 15% increase of the half-time of the decline of isometric relaxation (t w ). On the other hand, acute hypoxia (95% N 2 -5% CO 2 for 20 minutes) also induced a fall in contractility and the disappearance of the load sensitivity of relaxation but with a 67% decrease of ti.. Thus, the mechanical analysis of relaxation allows the effects of chronic overload in relatively severe cardiac hypertrophy to be separated from those of acute hypoxia. Moreover, in severe cardiac hypertrophy, the impairment of the load sensitivity of relaxation with increased ti/, strongly suggests alterations of the sarcoplasmic reticulum. especially since the moderate decrease in the myofibrillar ATPase activity, which has been observed previously in guinea pig pressure overload, cannot account completely for the marked fall in myocardial performance. (Circulation Research 1987;61:I07-116) C hronic cardiac overload appears to modify the myocyte homeostasis in a complex manner, as shown by numerous biochemical, metabolic, or mechanical alterations described in the literature, These changes observed during cardiac hypertrophy are, in part, species specific. Thus, the rat myocardium adapts to chronic overload by changing the fast V, isomyosin to the slow V,, 1 and this isomyosin shift is related linearly to alterations in contractility 21 Also, it has been shown that relaxation in the hypertrophied rat myocardium remains sensitive to the loading conditions, a mechanical property that generally seems to be present when the sarcoplasmic reticulum is normally functional and that, in the rat, is never impaired, whatever the degree or type of cardiac overload.

Inflammatory cell infiltration is a feature of postischemic neovascularization. However, mechanisms leading to leukocyte attraction to the site of neovascularization are still undefined. We hypothesized that the CXC chemokine receptor 3 (CXCR3) may contribute to leukocyte accumulation and subsequently to blood vessel growth in the ischemic area. Ischemia induced by femoral artery ligature improved the number of CXCR3-expressing cells and the level of its ligand, CXCL10. Angiographic score, blood flow recovery measurement, and capillary density analysis showed a significant decrease of ischemic/nonischemic leg ratio in CXCR3-deficient mice when compared with controls (P<0.05), at day 21 after ischemia. Interestingly, this impairment was as important as that observed in mice deficient for the well known CC-chemokine monocyte chemoattractant protein-1 (MCP-1). At day 7 of ischemic injury, the number of CD3-positive T cells and Mac-3-positive monocytes/macrophages was 38% and 45% lower, respectively, in the ischemic leg of CXCR3-deficient mice compared with the control group (P<0.05), suggesting an important role for CXCR3 in leukocyte recruitment into the ischemic area. VEGF protein content, a classical proangiogenic factor, was also markedly reduced (80% reduction) in ischemic leg of CXCR3-deficient mice (P<0.01). Injection of bone marrow-derived mononuclear cells (BM-MNCs) isolated from wild-type animals restored the neovascularization reaction in CXCR3-deficient mice whereas BM-MNCs from CXCR3-deficient mice was ineffective. In conclusion, CXCR3 plays a key role in neovascularization and provides novel information on the mechanisms leading to leukocyte infiltration in the vessel growth area.