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Background Type 2 diabetes mellitus (T2DM) is commonly associated with both microvascular and macrovascular complications and a strong correlation exists between glycaemic control and the incidence and progression of vascular complications. Pioglitazone, a Peroxisome proliferator-activated receptor-γ (PPARγ) ligand indicated for therapy of type T2DM, induces vascular effects that seem to occur independently of glucose lowering. Methods By using a hindlimb ischemia murine model, in this study we have found that pioglitazone restores the blood flow recovery and capillary density in ischemic muscle of diabetic mice and that this process is associated with increased expression of Vascular Endothelial Growth Factor (VEGF). Importantly, these beneficial effects are abrogated when endogenous Akt is inhibited; furthermore, the direct activation of PPARγ, with its selective agonist GW1929, does not restore blood flow recovery and capillary density. Finally, an important collateral vessel growth is obtained with combined treatment with pioglitazone and selective PPARγ inhibitor GW9662. Conclusion These data demonstrate that Akt-VEGF pathway is essential for ischemia-induced angiogenic effect of pioglitazone and that pioglitazone exerts this effect via a PPARγ independent manner.

Local gene transfer of the human LIM Mineralization Protein (LMP), a novel intracellular positive regulator of the osteoblast differentiation program, can induce efficient bone formation in rodents. In order to develop a clinically relevant gene therapy approach to facilitate bone healing, we have used primary dermal fibroblasts transduced ex vivo with Ad.LMP3 and seeded on an hydroxyapatite/collagen matrix prior to autologous implantation. Here we demonstrate that genetically modified autologous dermal fibroblasts expressing Ad.LMP-3 are able to induce ectopic bone formation following implantation of the matrix into the mouse triceps and paravertebral muscles. Moreover, implantation of the Ad.LMP-3-modified dermal fibroblasts into a rat mandibular bone critical size defect model results in efficient healing as determined by X-ray, histology and three dimensional micro computed tomography (3DμCT). These results demonstrate the effectiveness of the non-secreted intracellular osteogenic factor LMP-3, in inducing bone formation in vivo. Moreover, the utilization of autologous dermal fibroblasts implanted on a biomaterial represents a promising approach for possible future clinical applications aimed at inducing new bone formation.

We have previously demonstrated that sonic hedgehog (Shh) gene transfer improves angiogenesis in the setting of ischemia by upregulating the expression of multiple growth factors and enhancing the incorporation of endogenous bone marrow (BM)-derived endothelial progenitor cells (EPCs). In this study, we hypothesized that combined therapy with Shh gene transfer and BM-derived EPCs is more effective than Shh gene therapy alone in an experimental model of peripheral limb ischemia. We used old mice, which have a significantly reduced angiogenic response to ischemia, and compared the ability of Shh gene transfer, exogenous EPCs, or both to improve regeneration after ischemia. We found a significantly higher capillary density in the Shh + EPC-treated muscles compared to the other experimental groups. We also found that Shh gene transfer increases the incorporation and survival of transplanted EPCs. Finally, we found a significantly higher number of regenerating myofibers in the ischemic muscles of mice receiving combined treatment with Shh and BM-derived EPCs. In summary, the combination of Shh gene transfer and BM-derived EPCs more effectively promotes angiogenesis and muscle regeneration than each treatment individually and merits further investigation for its potential beneficial effects in ischemic diseases.

Sonic hedgehog (Shh) is a morphogen regulating crucial epithelial-mesenchymal interactions during embryonic development, but its signaling pathway is considered generally silent in post-natal life. In this study, we demonstrate that Shh is de novo expressed after injury and during regeneration of the adult skeletal muscle. Shh expression is followed by significant upregulation of its receptor and target gene Ptc1 in injured and regenerating muscles. The reactivation of the Shh signaling pathway has an important regulatory role on injury-induced angiogenesis, as inhibition of Shh function results in impaired upregulation of prototypical angiogenic agents, such as vascular endothelial growth factor (VEGF) and stromal-derived factor (SDF)-1alpha, decreased muscle blood flow, and reduced capillary density after injury. In addition, Shh reactivation plays a regulatory role on myogenesis, as its inhibition impairs the activation of the myogenic regulatory factors Myf-5 and MyoD, decreases the upregulation of insulin-like growth factor (IGF)-1, and reduces the number of myogenic satellite cells at injured site. Finally, Shh inhibition results in muscle fibrosis, increased inflammatory reaction, and compromised motor functional recovery after injury. These data demonstrate that the Shh pathway is functionally important for adult skeletal muscle regeneration and displays pleiotropic angiogenic and myogenic potentials in post-natal life. These findings might constitute the foundation for new therapeutic approaches for muscular diseases in humans. Sonic hedgehog (Shh) is a morphogen-regulating crucial epithelial-mesenchymal interactions during embryonic development, but its signalling pathway is considered generally silent in post-natal life. In this study, we demonstrate that Shh is de novo expressed after injury and during regeneration of the adult skeletal muscle. Shh expression is followed by significant up-regulation of its receptor and target gene Ptc1 in injured and regenerating muscles. The reactivation of the Shh signalling pathway has an important regulatory role on injury-induced angiogenesis, as inhibition of Shh function results in impaired up-regulation of prototypical angiogenic agents, such as vascular endothelial growth factor (VEGF) and stromal-derived factor (SDF)-1alpha, decreased muscle blood flow and reduced capillary density after injury. In addition, Shh reactivation plays a regulatory role on myogenesis, as its inhibition impairs the activation of the myogenic regulatory factors Myf-5 and MyoD, decreases the up-regulation of insulin-like growth factor (IGF)-1 and reduces the number of myogenic satellite cells at injured site. Finally, Shh inhibition results in muscle fibrosis, increased inflammatory reaction and compromised motor functional recovery after injury. These data demonstrate that the Shh pathway is functionally important for adult skeletal muscle regeneration and displays pleiotropic angiogenic and myogenic potentials in post-natal life. These findings might constitute the foundation for new therapeutic approaches for muscular diseases in humans.

BACKGROUND & AIMS: Vascular endothelial growth factor A (VEGF-A) mediates angiogenesis and might also have a role in inflammation and immunity. We examined whether VEGF-A signaling has a role in inflammatory bowel disease (IBD). METHODS: Expression levels of VEGF-A, and its receptors VEGFR-1 and VEGFR-2, were examined in samples from patients with IBD and compared with those of controls. The capacity of VEGF-A to induce angiogenesis was tested in human intestinal microvascular endothelial cells using cell-migration and matrigel tubule-formation assays. Levels of vascular cellular adhesion molecule-1 and intercellular adhesion molecule were measured by flow cytometry to determine induction of inflammation; neutrophil adhesion was also assayed. Expression patterns were determined in tissues from mice with dextran sulfate sodium (DSS)-induced colitis; the effects of VEGF-A overexpression and blockade were assessed in these mice by adenoviral transfer of VEGF-A and soluble VEGFR-1. Intestinal angiogenesis was measured by quantitative CD31 staining and leukocyte adhesion in vivo by intravital microscopy. RESULTS: Levels of VEGF-A and VEGFR-2 increased in samples from patients with IBD and colitic mice. VEGF-A induced angiogenesis of human intestinal microvascular endothelial cells in vitro as well as an inflammatory phenotype and adherence of neutrophils to intestinal endothelium. Overexpression of VEGF-A in mice with DSS-induced colitis worsened their condition, whereas overexpression of soluble VEGFR-1 had the opposite effect. Furthermore, overexpression of VEGF-A increased mucosal angiogenesis and stimulated leukocyte adhesion in vivo. CONCLUSIONS: VEGF-A appears to be a novel mediator of IBD by promoting intestinal angiogenesis and inflammation. Agents that block VEGF-A signaling might reduce intestinal inflammation in patients with IBD.