Incubation of endothelial cells in vitro with high concentrations of glucose activates protein kinase C (PKC) and increases nitric oxide synthase (NOS III) gene expression as well as superoxide production. The underlying mechanisms remain unknown. To address this issue in an in vivo model, diabetes was induced with streptozotocin in rats. Streptozotocin treatment led to endothelial dysfunction and increased vascular superoxide production, as assessed by lucigenin-and coelenterazine-derived chemiluminescence. The bioavailability of vascular nitric oxide (as measured by electron spin resonance) was reduced in diabetic aortas, although expression of endothelial NOS III (mRNA and protein) was markedly increased. NOS inhibition with N G-nitro-L-arginine increased superoxide levels in control vessels but reduced them in diabetic vessels, identifying NOS as a superoxide source. Similarly, we found an activation of the NADPH oxidase and a 7-fold increase in gp91 phox mRNA in diabetic vessels. In vitro PKC inhibition with chelerythrine reduced vascular superoxide in diabetic vessels, whereas it had no effect on superoxide levels in normal vessels. In vivo PKC inhibition with N-benzoyl-staurosporine did not affect glucose levels in diabetic rats but prevented NOS III gene upregulation and NOS-mediated superoxide production, thereby restoring vascular nitric oxide bioavailability and endothelial function. The reduction of superoxide in vitro by chelerythrine and the normalization of NOS III gene expression and reduction of superoxide in vivo by N-benzoyl-staurosporine point to a decisive role of PKC in mediating these phenomena and suggest a therapeutic potential of PKC inhibitors in the prevention or treatment of vascular complications of diabetes mellitus. The full text of this article is available at http://www.circresaha.org. (Circ Res. 2001; 88:e14-e22.) Key Words: diabetes nitric oxide synthase protein kinase C uncoupling NADPH oxidase M yocardial infarction and stroke constitute major causes of death in patients with diabetes mellitus. Long-term diabetes is associated with macroangiopathy and microangi-opathy. Several hypotheses have been put forth to explain the adverse effects of hyperglycemia on the vasculature. These include the activation of the polyol pathway, nonenzymatic glycation, redox potential alterations, and stimulation of the diacylglycerol (DAG)-protein kinase C (PKC) pathway (for review, see Ishii et al 1). Although studies on the polyol pathway and nonenzymatic glycation remain inconclusive so far, more recent studies strongly point to a decisive role of the DAG-PKC pathway for the vascular complications associated with diabetes.

Our findings are the first in vitro and in vivo demonstration that leptin is a renal growth and profibrogenic factor. These results may be an important contribution to our understanding of how leptin can contribute to renal damage, characterized by endocapillary proliferation and subsequent development of glomerulosclerosis, in pathophysiological situations with high circulating levels such as in diabetics or obese individuals. Although the effects of leptin itself are moderate, growth-promoting and profibrogenic effects may be enhanced in concert with other factors such as angiotensin II.

Regulatory macrophages (M regs) were administered to two living-donor renal transplant recipients. Both patients were minimized to low-dose tacrolimus monotherapy within 24 wk of transplantation and subsequently maintained excellent graft function. After central venous administration, most M regs remained viable and were seen to traffic from the pulmonary vasculature via the blood to liver, spleen, and bone marrow. By 1 y posttransplantation, both patients displayed patterns of peripheral blood gene expression converging upon the IOT-RISET signature. Furthermore, both patients maintained levels of peripheral blood FOXP3 and TOAG-1 mRNA expression within the range consistent with nonrejection. It is concluded that M regs warrant further study as a potential immune-conditioning therapy for use in solid-organ transplantation. The results of this work are being used to inform the design of The ONE Study, a multinational clinical trial of immunomodulatory cell therapy in renal transplantation.