Background-The neurotrophin nerve growth factor (NGF) regulates neuron survival and differentiation. Implication in neovascularization is supported by statement of NGF and its high-affinity receptor at vascular level and by NGF property of stimulating vascular endothelial cell proliferation. The present study investigated the involvement of endogenous NGF in spontaneous reparative response to ischemia. Mechanisms and therapeutic potential of NGFinduced neovascularization were examined. Methods and Results-Unilateral limb ischemia was produced in CD1 mice by femoral artery resection. By ELISA and immunohistochemistry, we documented that statement of NGF and its high-affinity receptor is upregulated in ischemic muscles. The functional relevance of this phenomenon was assessed by means of NGF-neutralizing antibody. Chronic NGF blockade abrogated the spontaneous capillarization response to ischemia and augmented myocyte apoptosis. Then we tested whether NGF administration may exert curative effects. Repeated NGF injection into ischemic adductors increased capillary and arteriole density, reduced endothelial cell and myofiber apoptosis, and accelerated perfusion recovery, without altering systemic hemodynamics. In normoperfused muscles, NFG-induced capillarization was blocked by vascular endothelial growth factor-neutralizing antibodies, dominant-negative Akt, or NO synthase inhibition. Conclusions-These results indicate that NGF plays a functional role in reparative neovascularization. Furthermore, supplementation of the growth factor promotes angiogenesis through a vascular endothelial growth factor-Akt-NOmediated mechanism. In the setting of ischemia, potentiation of NGF pathway stimulates angiogenesis and arteriogenesis, thereby accelerating hemodynamic recovery. NGF might be envisaged as a utilitarian target for the treatment of ischemic vascular disease.

Aims/hypothesis. The neurotrophin nerve growth factor (NGF) is pro-angiogenic and facilitates wound repair. The present study was conducted to (i) assess the statement of NGF system components in diabetic wounds and (ii) evaluate whether NGF supplementation could prevent impairment of wound neoangiogenesis by diabetes. Methods. Skin wounds were produced in the interscapular region of streptozotocin-induced diabetic mice. NGF (1 µg per day in PBS) or vehicle was applied onto the ulcers for 3 days after punching. Nondiabetic mice were used as controls.Results. In wounds of untreated diabetic mice, endogenous levels of immunoreactive NGF were lower than those in wounds of non-diabetic mice (p<0.01). Immunohistochemical analysis showed down-regulation of tyrosine kinase receptor-A (TrkA) and up-regulation of p75 receptor in granulation tissue microvasculature. Local NFG administration prevented diabetesinduced expressional alterations, enhanced reparative capillarisation (p<0.01), and accelerated wound closure (p<0.01). This was associated with a three-fold increase in endothelial cell proliferation (p<0.01), while apoptosis was reduced by 50% (p<0.05). Quantitative RT-PCR documented a 5.5-fold increase in the expression of vascular endothelial growth factor-A (VEGF-A) by exogenous NGF in diabetic tissues (p<0.01). In in vitro preparations of human endothelial cells from derma, NGF increased the release of immunoreactive VEGF-A, and reduced high-glucose-induced apoptosis (p<0.05), the latter effect being inhibited by a VEGF-A receptor-2 antagonist. Conclusions/interpretation. Diabetic ulcers display distinct alterations in reparative angiogenesis and in the expression of NGF and its receptors. NGF supplementation corrects endogenous liabilities, facilitates vascular regeneration, and suppresses endothelial apoptosis seemingly via VEGF-A. Our findings unravel new mechanisms responsible for NGF reparative action.

Background-Kinins are modulators of cardiovascular function. After ischemic injury, enhanced kinin generation may contribute in processes responsible for tissue healing. Methods and Results-Using pharmacological and genetic approaches, we investigated the role of kinin B 1 receptor in reparative angiogenesis in a murine model of limb ischemia. The effect of B 1 pharmacological manipulation on human endothelial cell proliferation and apoptosis was also studied in vitro. Abrogation of B 1 signaling dramatically inhibited the native angiogenic response to ischemia, severely compromising blood perfusion recovery. Outcome was especially impaired in B 1 knockouts that showed a very high incidence of limb necrosis, eventually leading to spontaneous auto-amputation. Conversely, local delivery of a long-acting B 1 receptor agonist enhanced collateral vascular growth in ischemic skeletal muscle, accelerated the rate of perfusion recovery, and improved limb salvage. In vitro, B 1 activation stimulated endothelial cell proliferation and survival, whereas B 1 antagonism induced apoptosis. Conclusions-Our results indicate that the B 1 plays an essential role in the host defense response to ischemic injury. B 1 signaling potentiation might be envisaged as a utilitarian target for the treatment of ischemic vascular disease.

Background— Microvascular insufficiency represents a major cause of end-organ failure among diabetics. Methods and Results— In streptozotocin-induced diabetic mice, we evaluated the potential of human tissue kallikrein (hTK) gene as a sole therapy against peripheral microangiopathy. Local delivery of hTK gene halted the progression of microvascular rarefaction in hindlimb skeletal muscle by inhibiting apoptosis, thus ensuring an improved hemodynamic recovery in case of supervening vascular occlusion. The curative action of hTK did not necessitate insulin supplementation. Application of gene therapy at a stage of established microangiopathy stimulated vascular regeneration. Conclusions— Our studies indicate that hTK may represent a useful tool for the treatment of microvascular complications in diabetics.

Abstract-Proteinase-activated receptors (PAR-2) are expressed by the cardiovascular system and mediate vasodilation, plasma protein extravasation, and endothelial cell proliferation, all regarded as essential steps for neovascularization. We investigated the angiogenic action of PAR-2 signaling in vivo. The effect of the PAR-2 activating peptide (PAR-2AP, SLIGRL-NH 2 ) was assessed in the absence of ischemia, and the therapeutic potential of PAR-2AP and the PAR-2 agonist trypsin (at 300 and 1.5 nmol IM daily for 21 days, respectively) was also tested in mice subjected to unilateral limb ischemia. PAR-2AP increased capillarity in normoperfused adductor skeletal muscles, whereas neither the vehicle of the PAR2-AP nor the PAR-2 reverse peptide (PAR-2RP, LRGILS-NH 2 ) did produce any effect. In addition, both PAR-2AP and trypsin enhanced reparative angiogenic response to limb ischemia, an effect that was not produced by PAR-2RP or the vehicle of PAR-2 agonists. Potentiation of reparative angiogenesis by PAR-2AP or trypsin resulted in an accelerated hemodynamic recovery and enhanced limb salvage. In conclusions, our study is the first to demonstrate the angiogenic potential of PAR-2 stimulation in vivo.

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a b s t r a c tA challenge in using bioactive melt-derived glass in bone regeneration is to produce scaffolds with interconnected pores while maintaining the amorphous nature of the glass and its associated bioactivity. Here we introduce a method for creating porous melt-derived bioactive glass foam scaffolds with low silica content and report in vitro and preliminary in vivo data. The gel-cast foaming process was adapted, employing temperature controlled gelation of gelatin, rather than the in situ acrylic polymerisation used previously. To form a 3D construct from melt derived glasses, particles must be fused via thermal processing, termed sintering. The original Bioglass Ò 45S5 composition crystallises upon sintering, altering its bioactivity, due to the temperature difference between the glass transition temperature and the crystallisation onset being small. Here, we optimised and compared scaffolds from three glass compositions, ICIE16, PSrBG and 13-93, which were selected due to their widened sintering windows. Amorphous scaffolds with modal pore interconnect diameters between 100-150 mm and porosities of 75% had compressive strengths of 3.4 ± 0.3 MPa, 8.4 ± 0.8 MPa and 15.3 ± 1.8 MPa, for ICIE16, PSrBG and 13-93 respectively. These porosities and compressive strength values are within the range of cancellous bone, and greater than previously reported foamed scaffolds. Dental pulp stem cells attached to the scaffold surfaces during in vitro culture and were viable. In vivo, the scaffolds were found to regenerate bone in a rabbit model according to X-ray micro tomography imaging. Statement of SignificanceThis manuscript describes a new method for making scaffolds from bioactive glasses using highly bioactive glass compositions. The glass compositions have lower silica content that those that have been previously made into amorphous scaffolds and they have been designed to have similar network connectivity to that of the original (and commercially used) 45S5 Bioglass. The aim was to match Bioglass' bioactivity. The scaffolds retain the amorphous nature of bioactive glass while having an open pore structure and compressive strength similar to porous bone (the original 45S5 Bioglass crystallises during sintering, which can cause reduced bioactivity or instability).The new scaffolds showed unexpectedly rapid bone regeneration in a rabbit model.

To fully exploit the potential of self-assembly in a single step, we have designed an integrated process to obtain mesoporous graphene nanocomposite films. The synthesis allows incorporating graphene sheets with a small number of defects into highly ordered and transparent mesoporous titania films. The careful design of the porous matrix at the mesoscale ensures the highest diffusivity in the films. These exhibit an enhanced photocatalytic efficiency, while the high order of the mesoporosity is not affected by the insertion of the graphene sheets and is well-preserved after a controlled thermal treatment. In addition, we have proven that the nanocomposite films can be easily processed by deep X-ray lithography to produce functional arrays.