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.

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Diabetes macro-and microvascular disease causes tissue hypoperfusion. This deficit, together with a failure to mount an adequate angiogenic response, might explain why vascular occlusion evolves more severely among diabetic patients. The present study investigated whether prophylactic gene therapy with human tissue kallikrein (hTK) may protect diabetic limbs from the consequences of supervening ischemia. Vehicle (saline) or an adenovirus carrying the gene for either hTK (Ad.hTK) or luciferase (Ad.Luc) was injected into left adductor muscles of streptozotocin-induced type 1 diabetic mice 2 weeks before operative occlusion of the ipsilateral femoral artery. Saline-injected nondiabetic mice served as controls. Hindlimb blood flow recovery was analyzed sequentially over the 2 weeks after ischemia induction. At necroscopy, microvessel density and endothelial cell proliferation and apoptosis were quantified in skeletal muscles. We found that limb perfusion recovery of saline-injected type 1 diabetic mice is delayed because of insufficient reparative neovascularization and excessive activation of endothelial cell apoptosis. By contrast, prophylactic Ad.hTK renewed the ability to mount an appropriate neovascularization response to ischemia, suppressed apoptosis, and upregulated endothelial nitric oxide synthase expression. Ultimately, correction of diabetic endotheliopathy by Ad.hTK allowed proper perfusion recovery as seen in nondiabetic mice. These discoveries disclose new therapeutic options for the treatment of diabetic complications. Diabetes 53

Background— Angiogenesis gene therapy with human tissue kallikrein (hTK) has shown promise for ischemic disease. The present study was undertaken to (1) assess an optimal gene transfer modality, (2) clarify hTK angiogenic pathways, and (3) discount possible side effects. Methods and Results— The hTK gene was transferred to murine adductors by increasing doses of an adenovirus (Ad.hTK). Heterologous protein production was evaluated by ELISA and immunohistochemistry. Structural and functional characteristics of hTK-induced neovascularization were assessed. Muscular endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF)-A mRNA and protein content were evaluated by real-time polymerase chain reaction and Western blotting. The ability of hTK to phosphorylate-activate Akt/protein kinase B (Akt-B) and VEGF receptor 2 (VEGF-R2) was also determined. Implication of the aforementioned mechanisms in Ad.hTK-induced neovascularization was challenged by blocking Akt-B with a dominant-negative Akt construct; NOS with N G -nitro- l -arginine methyl ester; and VEGF-A with neutralizing antibody, VEGF-R2 antagonist, or Ad carrying soluble VEGF-R1 gene. We found that 10 7 PFU Ad.hTK led to peak increases in capillary and arteriole density. Newly developed arterioles persisted for up to 8 weeks. Ad.hTK did not change microvascular permeability. Ad.hTK upregulated eNOS mRNA and protein and activated Akt-B through Ser-473 phosphorylation. Inhibitory studies documented that these biochemical events were instrumental to Ad.hTK-induced neovascularization. In contrast, Ad.hTK neither affected VEGF-A and VEGF-R2 levels nor increased VEGF-R2 phosphorylation. Consistently, Ad.hTK-induced neovascularization was not disturbed by any of the different approaches used to block VEGF-A. Conclusions— Our findings provide new information on the pathway involved in hTK-induced neoangiogenesis and represent an advancement toward clinical applications with Ad.hTK.

Background— Vascular growth factors are upregulated in stroke patients, but it remains unknown if they correlate with carotid atherosclerosis. Methods and Results— A case-control study was conducted to determine: (1) possible association between biomarkers of angiogenesis or inflammation and carotid stenosis; and (2) the impact of revascularization on the same biomarkers. Circulating vascular endothelial growth factor (VEGF), basic fibroblast GF (bFGF), tissue kallikrein (tK), and high-sensitivity C-reactive protein (hs-CRP) were measured in 89 patients with carotid obstruction and 45 age-matched controls. Patients were stratified as <50% carotid stenosis (CAS; n=16); 50% to 69% CAS (n=12); 70% to 99% CAS (n=43); and carotid occlusion (CAO; n=18). No association was found between VEGF, bFGF, or hs-CRP and obstruction grading. TK augmented from 360±30 in <50% CAS ( P =NS versus controls) to 509±72 in moderate CAS ( P <0.05), 1159±178 in high-grade CAS ( P <0.02), and 1616±403 pg/mL in CAO ( P <0.01). A threshold of 508 pg/mL provided the maximized predictive value of high-grade obstruction. After revascularization, tK decreased from 1410±352 to 782±86 pg/mL ( P <0.01), whereas no change was detected in nonoperated cases. Hs-CRP was unaffected by revascularization. Conclusions— Angiogenic factors are heterogeneously expressed in patients with carotid atherosclerosis. The tK measurement may be useful for the diagnosis and monitoring of atherosclerotic disease.