Histamine covalently bound to glutaraldehyde-activated ferritin was prepared as either monomers or as small aggregates of -0 .05 to 0.15 p,m Diam, suitable for electron microscopic detection of histamine cellular binding sites. The histamine-ferritin conjugates (MF) maintain the histamine capability to induce the opening of endothelial junctions in venules. To investigate the distribution of histamine receptors in the vascular endothelium, monomers or aggregates of MF were perfused in situ (mice), and various vascular beds, particularly that of the diaphragm, were fixed and processed for electron microscopy . The conjugate was preferentially bound on restricted areas of luminal endothelial cell plasmalemma especially in regions rich in filaments, and near the junctions between endothelial cells. The density of histamine binding sites was characteristically high in venules; it occurred to a much lesser extent in arterioles, veins, and muscular arteries whereas capillaries and aorta showed the lowest values . A similar distribution was obtained after perfusion of H, or H 2 receptor agonists coupled to ferritin (2-pyridylethylamine-ferritin [PF], or 4-methylhistamine-ferritin [MF], respectively) . The binding specificity was assessed through control experiments with either native or activated ferritin or by competition with histamine. The findings suggest that histamine receptors are largely represented in the cell membrane of the vascular endothelium, particularly in venules. Experiments using specific H, and H2 receptor agonists (PF and MF) and antagonists (mepyramine and cimetidine) indicate that the venular endothelium contains mainly H 2 receptors.

Emerging evidence demonstrates the involvement of endothelin-1 (ET-1) in the pathophysiology of cardiovascular disorders associated with diabetes mellitus. The molecular mechanisms accountable for the increased production of ET-1 are not completely defined. The Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway is an essential pathogenic mechanism leading to endothelial cell dysfunction. Our aim has been to investigate the role of JAK/STAT in the regulation of ET-1 synthesis in human endothelial cells (EAhy926 cells line). EAhy926 cells were exposed to normal (5 mM) or high (25 mM) glucose concentrations in the presence/absence of various JAK/STAT inhibitors. Using real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and gene reporter assay, we found that JAK/STAT inhibitors (STAT1 decoy oligodeoxynucleotides, AG490, S3I201, WP1066) significantly diminished the high-glucose-dependent up-regulation of ET-1 mRNA, peptide synthesis, and promoter activity. In silico analysis of the human ET-1 promoter revealed the presence of typical STAT1-gamma-activated sequence (STAT1-GAS) elements. Transient overexpression of STAT1 indicated an up-regulation of ET-1 promoter activity. Chromatin immunoprecipitation demonstrated the physical interaction of STAT1 proteins with the predicted GAS sites. Regulation of ET-1 synthesis by the JAK/STAT pathway thus represents a novel mechanism by which high glucose induces endothelial cell dysfunction in diabetes. Since the JAK/STAT system is an important regulator of the response of endothelial cells to injury, the modulation of this system and the subsequent decrease in ET-1 level may represent a key pharmacological target in diabetes-associated cardiovascular disorders.

The endothelial cell (EC) dysfunction is a common characteristic of various pathologies that include atherosclerosis, hypertension, and Fabry's disease. Aware of the role of eNO and ACE in EC dysfunction, we questioned whether polymorphism of eNOS and/or ACE gene may be a common denominator in these pathologies. Patients with CHD (108), HT (109), Fabry's disease (37) and healthy subjects (control, 141) were genotyped for the eNOSG894T by RFLP‐PCR technique and for eNOS4b/a, and ACEI/D polymorphisms by PCR amplification. The results of these studies were statistically evaluated. Compared to controls, the frequency of the eNOSG894T (T allele) was higher in CHD (P=0.03) and Fabry (P=0.01), while the eNOS4b/a (a allele) in CHD (P=0.01) and HT patients (P=0.01). The proportion of the ACEI/D was similar in all subjects. In CHD patients at “low risk” of atherogenic factors, the frequency of the T and a alleles of eNOS gene was high (P=0.03 and 0.02, respectively). Carriers of the T allele of eNOSG894T were over‐represented (P=0.04) in Fabry subgroup with renal failure. Compared to women, the eNOS894T alleles were more frequent (P=0.03) in men with CHD and HT, whereas ACE I/D in men (P=0.03) with HT. These findings suggest: (i) the frequency of eNOSG894T and/or eNOS4b/a is significantly associated with coronary dysfunction; (ii) eNOS4b/a confers a relatively high risk of hypertension in subjects with atherogenic risk factors; (iii) the frequency of eNOSG894T is high in Fabry hemizygotes with renal complications. Therefore, eNOS gene polymorphism represent a frequent risk factor for vascular abnormalities in CHD, HT and Fabry's disease, afflictions which have in common, the endothelial dysfunction.

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