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.

H-kininogen (HK), a major factor involved in contact-phase activation, was recently immunolocalized on the external surface of human neutrophils. Experiments were, therefore, designed to consider the question of whether the complete assembly of contact factors occurs on the outer surface of the neutrophil membrane. By immunolocalization techniques, and using specific antibodies directed against the various contact factors, we now demonstrate that plasma prekallikrein (PK), factor XI (FXI), and factor XII (FXII) are present on the exterior face of the human neutrophil. Failure to localize HK, PK, or FXI by monoclonal antibodies directed to their reciprocal binding sites, and displacement of PK/FXI by peptide HK31, which mimics the relevant binding site(s) of HK, suggested that prekallikrein and FXI are anchored to the neutrophil membrane through attachment to the kininogen molecule. Probing of the kinin moiety by a specific antibody showed that kininogen molecules bound to the neutrophil cell membrane contain the kinin sequence, which can be released by plasma kallikrein or by tissue kallikrein. Our results led us to the novel conclusion that neutrophils provide a circulating platform for the components of the contact-phase system.

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An immunocytochemical study was performed to examine the cellular localization and the subcellular distribution of kininogens in human blood cells. Kininogens were visualised using the immunogold-silver staining method and confocal scanning laser microscopy. We confirmed the existence of high molecular weight kininogen in human neutrophils and describe for the first time the presence of low molecular weight kininogen on these cells. Both high and low molecular weight kininogens were restricted to the neutrophils where they localized as clusters of immunogold particles on the cell membrane. No labeling was observed intracellularly in organelles such as mitochondria, endoplasmic reticulum, and azurophilic or specific granules after permeabilization of the neutrophils with Triton X-100, a procedure that permitted the visualization of elastase in the azurophilic granules. Clusters of high molecular weight kininogen molecules attached to the neutrophil surface could serve as receptors for plasma kallikrein and/or be the source of substrate for a discrete and circumscribed formation of kinins that may in turn facilitate the local diapedesis of neutrophils and the transudation of plasma constituents during acute inflammation.

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