The beta-specific protein kinase C inhibitor ruboxistaurin (LY333531) suppresses glucose-induced adhesion of human monocytes to endothelial cells in vitro.

AIMS: Strong evidence shows that late diabetic complications in diabetes mellitus are substantially related to an increased synthesis of diacylglycerol with a subsequent activation of protein kinase C (PKC) beta. Several studies have shown that specific inhibition of the PKC isoform beta by ruboxistaurin is able to attenuate the development of microvascular complications under diabetic conditions. The aim of this in vitro study was to investigate the effect of ruboxistaurin on glucose-induced adhesion of monocytes to endothelial cells, representing one of the first pivotal steps in the course of atherogenesis. METHODS: Human umbilical venous endothelial cells were isolated and cultured to confluence in microtiter plates. After coincubation with monocytes in the presence of 0, 10, or 400 ng ruboxistaurin to achieve PKC beta-specific and -unspecific PKC inhibition, cells were fixed and monocyte adhesion was determined by means of a standardized chemiluminescence assay. Expression of adhesion molecules (intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin) was also measured by chemiluminescence methods. RESULTS: Adhesion of monocytes to endothelial cells cultured under hyperglycemic conditions (27.7 mM glucose) was increased by 30.9 +/- 5.1% (p < 0.001) versus endothelial cells cultured under normoglycemic (NG) conditions (5.5 mM). Pretreatment of endothelial cells with 10 nM (PKC beta-specific concentration) and 400 nM (PKC beta-unspecific concentration) led to a significant reduction of glucose-induced adhesion of monocytes to endothelial cells that was statistically not different from endothelial adhesion under NG conditions (-7.2 +/- 3.1 and -8.1 +/- 2.6%, respectively; not significant vs NG). A nonsignificant tendency to lower the expression of adhesion molecules was seen with 10 ng of ruboxistaurin. CONCLUSIONS: We conclude that monocyte adhesion to endothelial cells under hyperglycemic conditions is at least mediated by PKC beta activation. Ruboxistaurin is able to suppress this monocyte adhesion even in a PKC beta-specific concentration. Further studies should evaluate these potential effects of ruboxistaurin in vivo.

Solid-phase synthesis of tetrahydro-beta-carbolines and tetrahydroisoquinolines by stereoselective intramolecular N-carbamyliminium Pictet-Spengler reactions.

A solid-phase method for the synthesis of tri-, tetra-, and pentacyclic compounds containing tetrahydro-beta-carboline, tetrahydroisoquinoline or analogous scaffolds is presented. The reaction proceeds with high stereoselectivity through an intermediate N-carbamyliminium ion that exclusively converts into Pictet-Spengler-type products with a variety of C-nucleophiles. Amino aldehydes masked with 3-Boc-(1,3)-oxazinane (Box) have been synthesized from amino acids, amino alcohols, or 2-nitro benzaldehydes. The amino moiety of these masked aldehydes has been converted into pentafluorophenyl carbamate to serve as a urea precursor. The building blocks were incorporated at the N-terminal of a resin-supported dipeptide through urea formation. Subsequent treatment with acid liberated the aldehyde quantitatively. A penultimate tryptophan residue gave rise, under the acetic conditions, to a spontaneous intramolecular Pictet-Spengler reaction with the liberated aldehyde. The reaction proceeded with a high degree of stereoselectivity affording tetrahydro-beta-carbolines with a de (de=diastereomeric excess) above 95 % and purity in the range of 90-99 %. This reaction has been extended to a range of other aromatic C-nucleophiles, including substituted indoles, benzenes, pyrene, furan, thiophenes, and benzothiophene with comparable stereoselectivity and purity. Prolonged exposure of the benzaldehyde-derived Pictet-Spengler products to strong acid and air lead to quantitative auto-oxidation which yielded compounds with a 3,4-dihydro-beta-carboline, a 3,4-dihydroisoquinoline, or a similar core structure.

Stimulation of endothelial nitric oxide synthase by proinsulin C-peptide.

There is increasing evidence for biological functions of human C-peptide. Recently, we have described that proinsulin C-peptide increases nutritive capillary blood flow and restores erythrocyte deformability in type 1 diabetic patients, whereas it has no such effect in non-diabetic subjects. The aim of the current study was to elucidate cellular mechanisms of this vasodilator effect in vitro by measuring the nitric oxide (NO)-mediated increase of cGMP production in a RFL-6 reporter cell assay and by demonstrating endothelial calcium influx with the Fluo-3 technique. C-peptide increased the release of NO from endothelial NO synthase (eNOS) in bovine aortic endothelial cells in a concentration- and time-dependent manner. At physiological concentrations of C-peptide, endothelial NO production was more than doubled (208+/-12% vs control; p<0.001). The NO release was abolished by the inhibitor of NO synthase N(G)-nitro-L-arginine or when Ca(2+) was removed from the medium superfusing the endothelial cells. C-peptide stimulated the influx of Ca(2+) into endothelial cells. No change in Ser-1179 phosphorylation of eNOS was detected after 6.6nM C-peptide. C-peptide did not change eNOS mRNA levels after 1, 6 or 24h. These data indicate that C-peptide is likely to stimulate the activity of the Ca(2+)-sensitive eNOS by increasing the influx of Ca(2+) into endothelial cells. We suggest that this effect may contribute to the increase in skin and muscle blood flow previously demonstrated in human in vivo.

Rheopheresis in patients with ischemic diabetic foot syndrome: results of an open label prospective pilot trial.

Rheopheresis is a specific application of membrane differential filtration, synonymous with double filtration plasmapheresis, for extracorporeal hemorheotherapy. Safety and efficacy of Rheopheresis for wound healing and skin oxygenation were investigated in patients with ischemic diabetic foot syndrome. Eight patients with type 2 diabetes mellitus and non-healing foot ulcers caused by severe ischemic diabetic foot syndrome were treated by a series of seven Rheopheresis sessions in a time span of 11 weeks. Wound healing had not been detectable under conditions of standardized wound care during at least 2 months. Wound status was classified by its morphology, severity and location, according to the criteria of Wagner. Transcutaneous oxygen pressure (tcPO2), laser Doppler flowmetry and vital capillary microscopy were repeatedly performed to monitor the effects of the Rheopheresis treatment series on microcirculation and skin blood flow. Laboratory parameters of blood rheology, endothelial function and inflammatory state were measured in addition to safety parameters. In four patients (baseline Wagner stage 2), Rheopheresis accelerated wound healing of foot ulcers and was associated with an improvement of Wagner stage and a pronounced increase in tcPO2. In two patients (baseline Wagner stage 2), wound healing was unchanged but mean tcPO2 increased, allowing successful minor amputation. Values of tcPO2 remained stable and enhanced for the 3 months follow-up period. In two patients (baseline Wagner stage 4 or 5), no improvements in foot lesions were observed within the treatment period. As an adjunct therapeutic option, Rheopheresis may preserve a functional lower extremity, delay amputation or reduce the extent of amputation.

[Interdisciplinary diagnosis and therapy of ischemic-osteomyelitic diabetic foot syndrome]. / Interdisziplinäre Diagnostik und Therapie des ischämisch-osteomyelitischen diabetischen Fusssyndroms.

Advanced stages of the diabetic foot syndrome complicated by ischemia and osteomyelitis frequently result in minor amputation, followed by impaired wound healing and higher-level amputation in the context of regular health structures. Even in specialized foot care centers, peripheral arterial occlusive disease and osteomyelitis still represent the greatest challenge in the strife for limb salvage. Whereas the treatment of nonischemic foot lesions has increasingly become a matter of conservative medicine within recent years, for advanced diabetic foot wounds a multidisciplinary treatment policy is essential. A well-coordinated treatment concept aiming at the elimination of the most relevant prognostic factors ischemia and osteomyelitis is required to achieve high limb salvage rates. Surgical revascularization by distal bypass is a crucially important element of this approach. Percutaneous transluminal angioplasty represents a complementary option for short-segment arterial occlusive disease. Foot-sparing minor surgery improves healing time and rates. A specialized diabetologic foot care clinic provides preclinical diagnosis, planning of inpatient procedures, and selection of patients requiring hospitalization for surgical intervention. In addition, it safeguards postinterventional care for wounds with secondary healing and measures of secondary prevention.