Constitutive NOS expression in cultured endothelial cells is elevated by fluid shear stress VIBHU RANJAN, ZESHUAI XIAO, AND SCOTT L. DIAMOND Bioengineering Laboratory, Department of Chemical Engineering, The State University of New York, Buffalo, New York 14260 Ranjan, Vibhu, Zeshuai Xiao, and Scott L. Diamond. Constitutive NOS expression in cultured endothelial cells is elevated by fluid shear stress. Am. J. PhysioZ. 269 (Heart Circ. Physiol. 38): H550-H555, 1995.-The role of chronic fluid shear stress on endothelial constitutive nitric oxide synthase (cNOS) levels may have an important role in vessel diameter control. We subjected primary human umbilical vein endothe- lial cells (HUVEC) or bovine aortic endothelial cells (BAEC, passages 2-14) to steady laminar shear stress. In both cell types, the intracellular level of cNOS was elevated within 3 h of flow exposure at 25 dyn/cm2 and remained elevated at 6 and 12 h of flow exposure, compared with stationary controls, as indicated by digital immunofluorescence microscopy. Shear stress exposure for 6 h caused a 2.2 t 0.3- and 2.8 t 0.3-fold elevation of cNOS protein levels in BAEC (n = 3, P < 0.01) and HUVEC (n = 3, P < O.Ol), respectively, in the presence or absence of 1 FM dexamethasone. Dexamethasone suppresses induction of the inducible NOS gene, indicating that cNOS was elevated by fluid shear stress. Flow exposure at 4 dyn/cm2 caused no enhancement of cNOS levels in either cell type. The flow induction of the cNOS protein levels was not blocked by preincubation of BAEC with 100-400 PM of hTG-nitro-L- arginine methyl ester, indicating that flow-induced NO (or guanosine 3’,5’-cyclic monophosphate) was not involved in the elevation of cNOS levels. Protein kinase C inhibitor H-7 (10 FM) had no effect on induction of NOS protein in BAEC exposed to 25 dyn/cm2. The cNOS mRNA levels were found to be elevated by two- to threefold in BAEC after 6 or 12 h of flow exposure at either 4 or 25 dyn/cm2, and this induction of NOS mRNA occurred in the presence of dexamethasone. The elevation of cNOS levels by chronic flow exposure may provide a mechanism for chronic regulation of vessel diameter by endothelial response to prevailing blood flow. nitric oxide synthase; endothelium; hemodynamics NITRIC OXIDE from the vascular endothelium mediates vasodilation and can be elicited by endothelium-depen- dent vasodilators such as acetylcholine, bradykinin, and calcium ionophores (9) as well as fluid shear stress (5, 24). NO also inhibits the adhesion of monocytes and platelets to endothelial cells and inhibits smooth muscle cell proliferation. In endothelial cells, NO synthase (NOS) activity is predominantly membrane-bound (9), and two distinct gene products are responsible for the biosynthesis of NO. The activity of constitutive NOS (cNOS, also called endothelial NOS or eNOS) is Ca2+/ calmodulin and NADPH dependent (3). The inducible NOS (iNOS) protein is found in macrophages and endothelial cells (29), and its level is elevated by endo- toxin and some cytokines. Furthermore, the activity of iNOS is Ca2+ independent and requires NADPH and tetrahydrobiopterin. Glucocorticoids such as dexametha- sone inhibit the induced expression of iNOS but not the expression of cNOS in vascular endothelial cells, with- out direct effect on the activity of either enzyme (29). The flow-induced release of NO by endothelial cells has been demonstrated in vitro, in vivo, and in perfused vessels (51516). Fewer studies have been conducted to study the effect of hemodynamic forces on endothelial NOS gene expression. Recently, it was shown that chronic exercise in dogs increased coronary vascular NO production and endothelial cell NOS gene expression (33). Cultured bovine endothelial cells exposed to shear stress are reported to have elevated levels of NOS mRNA levels (27>, and cyclic stretching of cultured endothelial cells may also enhance NOS levels (1). Several endothelial genes have been shown to be enhanced by shear stress either transiently, such as fos andjun (12,301, platelet-derived growth factor (PDGF)- B (11, 21, 31), basic fibroblast growth factor (21), and monocyte chemotactic protein 1 (35), or in a sustained manner, such as tissue plasminogen activator (tPA)(7, 8) and intracellular adhesion molecule (ICAM)(26). Other endothelial genes are downregulated by arterial levels of fluid shear stress, such as endothelin (15, 20, 22, 34) or fibronectin (10). Chronic shear stress exposure appears to have little effect on plasminogen activator-inhibitor type 1 or glyceraldehyde 3-phosphate dehydrogenase expression levels (7, 8). Using an in vitro perfusion system to expose cultured endothelial cells to steady laminar flow, we have investigated the regulation of the cNOS gene in response to fluid shear stress exposures. We show that cNOS gene expression can be elevated in a sustained manner by fluid shear stress in human and bovine endothelial cells of venous and arterial origin, respectively. The induction was independent of flow- induced NO production or protein kinase C (PKC) activation. MATERIALS AND METHODS Cell cuZture. Bovine aortic endothelial cells (BAEC) were grown to confluence in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% heat-inactivated newborn calf se- rum, 0.30 mg/ml glutamine, 150 U/ml penicillin, and 0.15 mg/ml streptomycin (GIBCO, Grand Island, NY) (10% C- DMEM). Primary human umbilical vein endothelial cells (HUVEC) were extracted and cultured as previously described (7) and grown in DMEM with 20% serum (20% C-DMEM). Exposure of endotheZiaZ cells to shear stress. Confluent monolayers of BAEC (passages 2-14) and primary, confluent monolayers of HUVEC were treated with 1 FM dexametha- sone for 24 h to inhibit the expression of iNOS during the experiment (29). The cells were then exposed to a steady laminar shear stress of 4 or 25 dyn/cm2 in individual, sterile, H550 0363-6135/95 $3.00 Copyright 0 1995 the American Physiological Society