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.

SUMMARY:Although accumulating lines of evidence indicate the proangiogenic role of angiotensin II (Ang II), little is known about the molecular mechanisms associated with such an effect. This study aimed to identify molecular events involved in Ang II-induced angiogenesis in the Matrigel model in mice. C57Bl/6 female mice received a subcutaneous injection of either Matrigel or Matrigel with Ang II (10 Ϫ7 M) alone, with Ang II and an AT1 receptor antagonist (candesartan, 10 Ϫ6 M), or with Ang II and AT2 receptor antagonist (PD123319, 10 Ϫ6 M). After 14 days, angiogenesis was assessed in the Matrigel-plug by histological evaluation and cellular counting. Ang II increased by 1.9-fold the number of cells within the Matrigel (p Ͻ 0.01 versus control). Immunohistological analysis revealed the presence of macrophages, endothelial and smooth muscle cells, and the development of vascular-like structure. Such an angiogenic effect was associated with an increase in vascular endothelial growth factor (VEGF) (1.5-fold, p Ͻ 0.01), endothelial nitric oxide (eNOS) (1.7-fold, p Ͻ 0.01), and cyclooxygenase-2 (1.4-fold, p Ͻ 0.05) protein levels measured by Western blotting. Conversely, Ang II treatment did not affect MMP-9 and MMP-2 activity, assessed by zymography. Blockade of AT1 receptor completely prevented the Ang II-induced angiogenesis and protein regulations, whereas that of AT2 was ineffective. Administration of VEGF neutralizing antibody (2.5 g ip twice a week) and cyclooxygenase-2 selective inhibitor (nimesulide, 30 mg/L) also hampered Ang II proangiogenic effect. In addition, Ang II-induced cell ingrowth was impaired by treatment with nitric oxide synthase inhibitor (L-NAME, 10 mg/kg/day) and in eNOS-deficient mice. Therefore, in an in vivo model, Ang II induced angiogenesis through AT1 receptor, which involved activation of VEGF/eNOS-related pathway and of the inflammatory process. (Lab Invest 2002, 82:747-756).

Abstract-Recent studies have suggested a proangiogenic effect of angiotensin-converting enzyme (ACE) inhibition. We hypothesized that such a proangiogenic effect of ACE inhibition may be mediated, in part, by bradykinin (BK) B 2 -receptor pathway. This study therefore examined the neovascularization induced by ACE inhibitor treatment in B 2 receptor-deficient mice (B 2 Ϫ/Ϫ ) in a model of surgically induced hindlimb ischemia. After artery femoral occlusion, wild-type and B 2 Ϫ/Ϫ mice were treated with or without ACE inhibitor (perindopril, 3 mg/kg/d) for 28 days. Angiogenesis was then quantitated by microangiography, capillary density measurement, and laser Doppler perfusion imaging. The protein levels of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) were determined by Western blot. In wild-type animals, vessel density and capillary number in the ischemic leg were raised by 1.8-and 1.4-fold, respectively, in mice treated with ACE inhibitor when compared with the nontreated animals (PϽ0.01). This corresponded to an improved ischemic/nonischemic leg perfusion ratio by 1.5-fold in ACE inhibitor-treated animals when compared with the untreated ones (0.87Ϯ0.07 versus 0.59Ϯ0.05, respectively, PϽ0.01). Activation of the angiogenic process was also associated with a 1.7-fold increase in tissue eNOS protein level in mice treated with ACE inhibitor (PϽ0.05 versus control) but not with changes in VEGF protein level. Conversely, ACE inhibition did not affect vessel density, blood flow, and eNOS protein level in ischemic hindlimb of B 2 Ϫ/Ϫ mice. Therefore, proangiogenic effect of ACE inhibition is mediated by B 2 -receptor signaling and was associated with upregulation of eNOS content, independently of VEGF expression. Key Words: angiogenesis Ⅲ ischemia Ⅲ kinins Ⅲ angiotensin-converting enzyme Ⅲ B 2 receptor I n ischemic diseases, both hypoxia and inflammation play a major role in the control of new vessel growth. 1 The main mechanism of hypoxia-induced angiogenesis involves the rise in hypoxia-inducible factor-1␣ protein, resulting in increased expression of vascular endothelial growth factor (VEGF), a specific angiogenic factor. 1 The angiogenic effect of VEGF might be mediated by activation of endothelial nitric oxide synthase (eNOS) and subsequently, by production of nitric oxide, as previously described in ischemia-induced angiogenesis. 2 Neovascularization appears also to be controlled by monocyte/macrophage accumulation that occurs within the ischemic area. 3 The presence of these inflammatory cells is associated with local secretion of several angiogenic factors, including cytokines and growth factors. 3,4 Numerous factors are involved in the regulation of new vessel growth from preexisting ones. Among these factors, the angiogenic role of the renin-angiotensin system remains to be defined. Angiotensin II increases vessel density in rat cremaster muscle 5 and the chorioallantoic membrane of the chick embryo 6 and stimulates angiogenesis in the rat subcutaneous sponge granulom...

Abstract-We have previously shown that deficiency in the anti-inflammatory cytokine interleukin-10 (IL-10) is responsible for enhanced angiogenesis after hindlimb ischemia. This study examined the putative involvement of matrix metalloproteinase (MMP) activation in this process. Ischemia was produced by artery femoral occlusion in both C57BL6 IL-10

To determine the events leading to cardiac fibrosis in aldosterone-salt hypertensive rats, we studied protein and mRNA accumulation of procollagens I and III for 60 days. After 3 and 7 days of treatment systolic pressure was normal, and no histological or biochemical changes were seen in rat hearts. At day 15 arterial pressure was raised (+40%) and left ventricular hypertrophy was +15%. Cardiac examination after hemalun-eosin staining and immunolabeling with anticollagen I and III antibodies showed no structural alterations, but an 83% increase in right ventricular type III procollagen mRNA levels was found. At 30 and 60 days we found progressive cardiac fibrosis, with inflammatory cells, myocyte necrosis, and elevation of both types I and III procollagen mRNA levels in both ventricles. To determine whether aldosterone had effects on Na,K-ATPase that might lead to ionic disturbances and induce myocyte necrosis, we studied the major cardiac Na,K-ATPase isoform genes. Although Na,K-ATPase alpha 1- and beta 1-subunit mRNA levels were elevated in kidney at day 1, neither of these cardiac transcripts nor the specific alpha 2 isoform was altered between 1 and 15 days. These results show that accumulation of procollagen mRNAs occurs before collagen deposition. Cardiac alterations are late and not preceded by changes in Na,K-ATPase cardiac gene expression, precluding a direct modulation of cardiac collagen synthesis and Na,K-ATPase by aldosterone.

Extracellular vesicles secreted by iPSC-Pg are effective in the treatment of CHF, possibly, in part, through their specific miRNA signature and the associated stimulation of distinct cardioprotective pathways. The processing and regulatory advantages of EV could make them effective substitutes for cell transplantation.

Abstract-This study tests the hypothesis that aldosterone induces cardiac fibrosis through an increase of cardiac angiotensin II (Ang II) AT 1 receptor levels, thereby potentiating the fibrotic effect of Ang II by determining the effects of spironolactone and losartan on cardiac fibrosis, AT 1 density, and gene expression in aldosterone-salttreated rats. Fibrosis was quantified by slot blots of collagen I and III mRNA levels and videomorphometry of Sirius red-stained collagen. AT 1 receptor density was determined by ( 125 I-Sar 1 -Ile 8 )-Ang II competition binding, and AT 1 mRNA levels were analyzed by quantitative reverse transcriptase polymerase chain reaction. One month of aldosterone-salt treatment induced a decrease in plasma Ang II and an increase in blood pressure, left ventricular hypertrophy, and ventricular fibrosis. Spironolactone (20 mg/kg per day) and losartan spironolactone (10 mg/kg per day) had no effect on the first 3 parameters. Losartan was as effective as spironolactone in preventing ventricular collagen mRNA increase and fibrosis. Ventricular density of AT 1 receptors increased 2-fold and was accompanied by a 3-fold increase in the corresponding mRNA in aldosterone-salt compared with sham-operated rats. Both spironolactone and losartan prevented the elevation of ventricular AT 1 density and that of right ventricular AT 1 mRNA levels. These results demonstrate that the mechanism by which aldosterone-salt induces cardiac fibrosis involves Ang II acting through AT 1 receptors. They also suggest that the cardiac AT 1 receptor is a target for aldosterone. (Hypertension. 1999;33:981-986.)

Acute myocardial infarction is a common condition responsible for heart failure and sudden death. Here, we show that following acute myocardial infarction in mice, CD8+ T lymphocytes are recruited and activated in the ischemic heart tissue and release Granzyme B, leading to cardiomyocyte apoptosis, adverse ventricular remodeling and deterioration of myocardial function. Depletion of CD8+ T lymphocytes decreases apoptosis within the ischemic myocardium, hampers inflammatory response, limits myocardial injury and improves heart function. These effects are recapitulated in mice with Granzyme B-deficient CD8+ T cells. The protective effect of CD8 depletion on heart function is confirmed by using a model of ischemia/reperfusion in pigs. Finally, we reveal that elevated circulating levels of GRANZYME B in patients with acute myocardial infarction predict increased risk of death at 1-year follow-up. Our work unravels a deleterious role of CD8+ T lymphocytes following acute ischemia, and suggests potential therapeutic strategies targeting pathogenic CD8+ T lymphocytes in the setting of acute myocardial infarction.