ABSTRACT
BACKGROUND: High prevalence of atherosclerotic cardiovascular events accounts for much of the mortality among patients suffering from end-stage renal disease (ESRD). Endothelial dysfunction as a pathogenic mechanism might contribute to increasing the cardiovascular risk of ESRD. Reduced endothelium-dependent vasodilation has consistently been observed in chronic renal failure patients. Since nitric oxide (NO) is the principal endothelium-derived vasodilator, a reduction in the NO bioavailability may be envisaged in ESRD patients. METHODS: To clarify whether exposure to erythrocytes from ESRD patients might modulate NO release by the endothelium, we evaluated endothelial NO synthase (eNOS) protein levels (Western blot), eNOS mRNA quantity (real-time PCR), and NOS activity (conversion of L-[3H] arginine in L-[3H] citruline) in endothelial cultures stimulated by erythrocytes from healthy subjects and ESRD patients. RESULTS: A time-dependent decrease in eNOS protein levels was evident in cultures treated with erythrocytes from ESRD patients. This observation was consistent with the decreased eNOS mRNA quantities induced by erythrocytes from such patients. Moreover, compared to controls, NOS activity exhibited a significant reduction after incubation with erythrocytes from ESRD patients. The observed eNOS reduction induced by erytrocytes from ESRD patients was totally abolished by annexin V, able to mask red blood cell (RBC) surface-exposed phosphatidylserine. CONCLUSION: These findings suggest that adhesion of erythrocytes from ESRD patients to vascular endothelium may cause a decrease in the levels of eNOS mRNA and protein, and inhibition of NOS activity. This might contribute to endothelial dysfunction, and may play a role in the pathogenesis of cardiovascular disease in ESRD patients.
Subject(s)
Cell Adhesion/physiology , Endothelium, Vascular/enzymology , Erythrocytes/physiology , Nitric Oxide Synthase/metabolism , Uremia/blood , Uremia/enzymology , Aged , Cardiovascular Diseases/etiology , Case-Control Studies , Cells, Cultured , Endothelium, Vascular/cytology , Erythrocytes/pathology , Female , Gene Expression , Humans , In Vitro Techniques , Kidney Failure, Chronic/blood , Kidney Failure, Chronic/complications , Kidney Failure, Chronic/enzymology , Male , Middle Aged , Nitric Oxide Synthase/genetics , Nitric Oxide Synthase Type III , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
Hypoxia is a potent regulator of various biological process. Mammalian cells respond to hypoxia by increased expression of several genes. The aim of this study was to evaluate the effects of chronic exposure to low oxygen tension on the induction of inducible nitric oxide synthase (iNOS) and heme oxygenase-1 (HO-1) in rat heart. Male Wistar rats were assigned randomly to 4 groups: (A) control rats maintained in normoxic conditions for 7 and 14 days; (B) rats maintained in hypoxic conditions for 7 and 14 days; (C) rats maintained in normoxic conditions for 7 days and then transferred to hypoxic conditions for 7 days; and (D) rats maintained in hypoxic conditions for 7 days and then transferred to normoxic conditions for 7 days. In Group A, iNOS and HO-1 immunoreactivities were not evident; in Group B these immunoreactivities increased from day 7 to 14; in Group C the immunoreactivities decreased on day 7, compared to day 14; and in Group D, the immunoreactivities increased on day 7, compared to day 14. These findings were confirmed by Western blot analyses of the respective proteins and by rt-PCR assays of the corresponding mRNAs. The results indicate that the adaptive response to hypoxia involves up-regulation of HO-1 through iNOS activation in cardiac cells. HO-1 helps to regulate vascular tone via CO and thereby participates in an important cardiac defense mechanism.
Subject(s)
Heme Oxygenase (Decyclizing)/biosynthesis , Hypoxia/enzymology , Myocardium/enzymology , Nitric Oxide Synthase/biosynthesis , Animals , Blotting, Western , DNA Primers/chemistry , Enzyme Induction , Fluorescent Antibody Technique, Indirect , Gene Expression , Heme Oxygenase (Decyclizing)/genetics , Heme Oxygenase-1 , Immunoenzyme Techniques , Myocardium/pathology , Nitric Oxide Synthase/genetics , Nitric Oxide Synthase Type II , RNA, Messenger/metabolism , Rats , Rats, Wistar , Reverse Transcriptase Polymerase Chain Reaction , Time FactorsABSTRACT
BACKGROUND: The exposure of phosphatidylserine (PS) on the outer leaflet of erythrocyte membrane may have several pathophysiological consequences including increased erythrocyte adherence to endothelial cells, a finding that seems relevant in pathologies with reported vascular injury. METHODS: Because PS externalization increases in erythrocytes from patients suffering from chronic uremia, which is frequently associated with vascular damage, the adherence of uremic erythrocytes to human umbilical vein endothelial cell (HUVEC) monolayers and the role of PS exposure on such cell-cell interaction were studied. RESULTS: The number of uremic erythrocytes adhering to HUVEC was markedly greater than with normal erythrocytes and significantly correlated (r = 0.88) with the percentage of PS-exposing erythrocytes in the population. Adhesion to the monolayers was significantly decreased when uremic erythrocytes were preincubated with either annexin V or PS-containing liposomes, and was strongly greater for PS-positive than PS-negative fluorescence-activated cell sorter (FACS)-sorted uremic erythrocytes. Binding occurred preferentially in the gaps of HUVEC monolayers and was enhanced by matrix exposure. Uremic erythrocytes adhered to immobilized thrombospondin, and binding to endothelial cells was significantly reduced when monolayers were incubated with antibodies to thrombospondin. CONCLUSIONS: These findings suggest that PS externalization may promote increased uremic erythrocyte adhesion to endothelium, possibly via a direct interaction with matrix thrombospondin.
