Nerve growth factor supplementation reverses the impairment, induced by Type 1 diabetes, of hindlimb post-ischaemic recovery in mice.

AIMS/HYPOTHESIS: Type 1 diabetes increases the risk of peripheral ischaemia and impairs recovery once ischaemia occurs, probably because the healing process is hampered by diabetes-induced endothelial dysfunction. In normoglycaemic mice subjected to limb ischaemia, blockade of nerve growth factor (NGF) compromises reparative angiogenesis. In the present study, we evaluated if expressional alterations of endogenous NGF system components are associated with diabetes-related impairment in neovascularisation. In addition, we tested whether the correction of NGF liabilities benefits post-ischaemic healing of Type 1 diabetic animals. METHODS: Unilateral hindlimb ischaemia was produced in streptozotocin-induced Type 1 diabetic mice. Purified murine NGF (20 microg daily for 14 days) or PBS were injected into ischaemic adductors. Non-diabetic mice given PBS served as controls. Hindlimb blood flow was analysed sequentially for up to 14 days. At necroscopy, adductors were removed for quantification of microvessel density, endothelial cell apoptosis and NGF receptor expression. NGF content was determined by ELISA three days after ischaemia. In vitro, we tested whether NGF protects endothelial cells from apoptosis induced by high glucose and whether vascular endothelial growth factor-A (VEGF-A) is involved in this beneficial effect. RESULTS: Muscles removed from Type 1 diabetic mice showed reduced NGF content and up-regulation of the NGF p75 receptor. NGF supplementation promoted capillarisation and arteriogenesis, reduced apoptosis, and accelerated blood flow recovery. NGF stimulated VEGF-A production by human endothelial cells incubated in high-glucose medium and conferred resistance against high-glucose-induced apoptosis via a VEGF-A-mediated mechanism. CONCLUSIONS/INTERPRETATION: NGF protects endothelial cells from apoptosis induced by Type 1 diabetes and facilitates reparative neovascularisation. The findings may open up new therapeutic options for the treatment of diabetic complications.

Nerve growth factor promotes reparative angiogenesis and inhibits endothelial apoptosis in cutaneous wounds of Type 1 diabetic mice.

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 microg per day in PBS) or vehicle was applied onto the ulcers for 3 days after punching. Non-diabetic 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 diabetes-induced 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.