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Abstract-Hypercholesterolemia is associated with impaired neovascularization in response to ischemia. Potential mechanisms include defective NO bioactivity and a reduction in the number/function of endothelial progenitor cells (EPCs). Here we tested the hypothesis that sildenafil, a phosphodiesterase 5 inhibitor that increases NO-driven cGMP levels, could stimulate EPC function and improve ischemia-induced neovascularization in hypercholesterolemic conditions. Apolipoprotein E-deficient (ApoE Ϫ/Ϫ ) mice were treated (or not treated) with sildenafil (40 mg/kg per day in water), and hindlimb ischemia was surgically induced by femoral artery removal. Sildenafil treatment led to an improved blood flow recovery, an increased capillary density, and a reduction of oxidative stress levels in ischemic muscles at day 7 after surgery. Sildenafil therapy is associated with an increased activation of angiogenic transduction pathways, including Akt, p44/42 mitogen-activated protein kinase, and p38. In vitro, sildenafil increases cellular migration and tubule formation of mature endothelial cells (human umbilical vascular endothelial cells) in a cGMP-dependent manner. In vivo, ApoE Ϫ/Ϫ mice treated with sildenafil exhibit a significant increase in the number of bone marrow-derived EPCs. Moreover, the angiogenic activities of EPCs (migration and adhesion) are significantly improved in ApoE Ϫ/Ϫ mice treated with sildenafil. In summary, this study demonstrates that sildenafil treatment is associated with improved ischemia-induced neovascularization in hypercholesterolemic ApoE Ϫ/Ϫ mice. The mechanisms involve beneficial effects on angiogenic transduction pathways together with an increase in the number and the functional activity of EPCs. Sildenafil could constitute a novel therapeutic strategy to reduce tissue ischemia in atherosclerotic diseases. Key Words: sildenafil Ⅲ endothelial Ⅲ progenitor Ⅲ cells Ⅲ angiogenesis hypercholesterolemia T he ability of the organism to develop new blood vessels (neovascularization) constitutes an important adaptive response to vascular occlusive diseases. 1 Postnatal neovascularization necessitates the activation, migration, and proliferation of mature endothelial cells (angiogenesis). 2 In response to ischemia, hypoxia-inducible factor 1 (HIF-1) and vascular endothelial growth factor (VEGF) have been shown to be critical limiting factors for the induction of angiogenesis. 3,4 The importance of NO for endothelial cell migration and VEGF-induced angiogenesis was also demonstrated recently. 5,6 However, increasing evidence suggests that postnatal neovascularization relies not exclusively on the sprouting of preexisting vessels, but also the contribution of bone marrowderived circulating endothelial progenitor cells (EPCs). 7 In young patients and in animal models with young and healthy animals, the neovascularization process is very effective so that blood flow restoration after ischemia is almost complete. Neovascularization, however, is impaired in several clinical situations, which leads to i...

Objective-Because Nox2-containing NADPH oxidase is a major source of ROS in the vasculature, we investigated its potential role for the modulation of ischemia-induced neovascularization in conditions of increased oxidative stress. Methods and Results-To mimic a clinical situation of increased oxidative stress, mice were exposed to cigarette smoke before and after the surgical induction of hindlimb ischemia. Nox2 expression and oxidative stress in ischemic tissues were significantly increased in wild-type mice, but not in mice deficient for the Nox2-containing NADPH oxidase (Nox2 Ϫ/Ϫ ). Nox2 Ϫ/Ϫ mice demonstrated faster blood flow recovery, increased capillary density in ischemic muscles, and improved endothelial progenitor cell functional activities compared to Nox2 ϩ/ϩ mice. In addition, Nox2 deficiency was associated with increased antioxidant and nitrite concentrations in plasma, together with a preserved expression of eNOS in ischemic tissues. In vitro, Nox2Ϫ/Ϫ endothelial cells exhibit resistance against superoxide induction and improved VEGF-dependent angiogenic activities compared to Nox2 ϩ/ϩ endothelial cells. Importantly, the beneficial effects of Nox2 deficiency on neovascularization in vitro and in vivo were lost after treatment with the NO inhibitor L-NAME. Conclusions-Nox2-containing NADPH oxidase deficiency protects against ischemia in conditions of increased oxidative stress. The mechanism involves improved neovascularization through a reduction of ROS formation, preserved activation of the VEGF/NO angiogenic pathway, and improved functional activities of endothelial progenitor cells.

Objective Long bones develop through the strictly regulated process of endochondral ossification within the growth plate, resulting in the replacement of cartilage by bone. Defects in this process result in skeletal abnormalities and can predispose to disease such as osteoarthritis (OA). Studies suggest that activation of the transcription factor peroxisome proliferator activated receptor gamma (PPARγ) is a therapeutic target for OA. In order to devise PPARγ-related therapies in OA and related diseases, it is critical to identify its role in cartilage biology. Therefore, we determined the in vivo role of PPARγ in endochondral ossification and cartilage development using cartilage-specific PPARγ knockout (KO) mice. Methods Cartilage-specific PPARγ KO mice were generated using LoxP/Cre system. Histomorphometric and immunohistochemical analysis was performed to account for ossification patterns, chondrocyte proliferation, differentiation, hypertrophy, skeletal organization, bone density and calcium deposition. Real-Time PCR and western blotting was performed to determine the expression of key markers involved in endochondral ossification. Results PPARγ KO mice exhibited reduced body length, weight, length of long bones, skeletal growth, cellularity, bone density, calcium deposition and trabecular bone thickness, abnormal growth plate organization, loss of columnar organization, shorter hypertrophic zones, and delayed primary and secondary ossification. Immunohistochemistry for Sox9, BrdU, p57, collagen X and PECAM revealed reduction in chondrocyte differentiation and proliferation, and hypertrophy and vascularisation in growth plates of mutant mice. Isolated chondrocytes and cartilage explants from mutant mice showed aberrant expression of ECM markers including aggrecan, collagen II and MMP-13. Conclusion PPARγ is required for normal endochondral ossification and cartilage development in vivo.

Patients with systemic sclerosis (SSc) display altered intestinal microbiota. However, the influence of intestinal dysbiosis on the development of experimental SSc remains unknown. Topoisomerase I peptide-loaded dendritic cell immunization induces SSc-like disease, with progressive skin and lung fibrosis. Breeders were given streptomycin and pups continued to receive antibiotic (ATB) until endpoint (lifelongATB). Alternately, ATB was withdrawn (earlyATB) or initiated (adultATB) during adulthood. Topoisomerase I peptide-loaded dendritic cell (no ATB) immunization induced pronounced skin fibrosis, with increased matrix (Col1a1), profibrotic (Il13, Tweakr), and vascular function (Serpine1) gene expression. Remarkably, earlyATB exposure was sufficient to augment skin Col5a1 and Il13 expression, and inflammatory cell infiltration, which included IL-13 cells, mononuclear phagocytes, and mast cells. Moreover, skin pathology exacerbation was also observed in lifelongATB and adultATB groups. Oral streptomycin administration induced intestinal dysbiosis, with exposure limited to early life (earlyATB) being sufficient to cause long-term modification of the microbiota and a shift toward increased Bacteroidetes/Firmicutes ratio. Finally, aggravated lung fibrosis and dysregulated pulmonary T-cell responses were observed in earlyATB and lifelongATB but not adultATB-exposed mice. Collectively, intestinal microbiota manipulation with streptomycin exacerbated pathology in two distinct sites, skin and lungs, with early life being a critical window to affect the course of SSc-like disease.