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 endothelial 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 +/- 0.3- and 2.8 +/- 0.3-fold elevation of cNOS protein levels in BAEC (n = 3, P < 0.01) and HUVEC (n = 3, P < 0.01), respectively, in the presence or absence of 1 microM 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 microM of NG-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 microM) 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.

Arterial levels of shear stress (25 dynes/cm2) can elevate constitutive endothelial nitric oxide synthase (eNOS) gene expression in cultured endothelial cells (Ranjan et al., 1995). By PhosphorImaging of Northern blots, we report that the eNOS/glyceraldehyde 3-phosphate dehydrogenase (GAPDH) messenger RNA (mRNA) ratio in bovine aortic endothelial cells (BAEC) increased by 4.8- and 7.95-fold after 6-hr shear stress exposure of 4 and 25 dynes/cm2, respectively. Incubation of BAEC with dexamethasone (1 microM) had no effect on shear stress induction of eNOS mRNA. Buffering of intracellular calcium in BAEC with bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl)-ester (BAPTA/AM) reduced shear stress induction of eNOS mRNA by 70%. Yet, stimulation of BAEC with ionomycin (0.1-1.0 microM) for 6-24 hr to elevate intracellular calcium had no effect on eNOS mRNA. These studies indicated that the shear stress induction of eNOS mRNA was a calcium-dependent, but not calcium-activated, process. Shear stress was a very potent and rapid inducer of the eNOS mRNA, which could not be mimicked with phorbol myristrate acetate or endotoxin. Inhibition of tyrosine kinases with genistein (10 microM) or tyrphostin B46 (10 microM) or inhibition of G-protein signaling with guanosine 5'-O-(2-thiodiphosphate) (GDP-betaS) (600 microM, 6-hr preincubation) did not block the shear stress elevation of eNOS mRNA.

Arterial levels of shear stress (25 dynes/cm2) can elevate constitutive endothelial nitric oxide synthase (eNOS) gene expression in cultured endothelial cells (Ranjan et al., 1995). By PhosphorImaging of Northern blots, we report that the eNOS/glyceraldehyde 3-phosphate dehydrogenase (GAPDH) messenger RNA (mRNA) ratio in bovine aortic endothelial cells (BAEC) increased by 4.8- and 7.95-fold after 6-hr shear stress exposure of 4 and 25 dynes/cm2, respectively. Incubation of BAEC with dexamethasone (1 microM) had no effect on shear stress induction of eNOS mRNA. Buffering of intracellular calcium in BAEC with bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl)-ester (BAPTA/AM) reduced shear stress induction of eNOS mRNA by 70%. Yet, stimulation of BAEC with ionomycin (0.1-1.0 microM) for 6-24 hr to elevate intracellular calcium had no effect on eNOS mRNA. These studies indicated that the shear stress induction of eNOS mRNA was a calcium-dependent, but not calcium-activated, process. Shear stress was a very potent and rapid inducer of the eNOS mRNA, which could not be mimicked with phorbol myristrate acetate or endotoxin. Inhibition of tyrosine kinases with genistein (10 microM) or tyrphostin B46 (10 microM) or inhibition of G-protein signaling with guanosine 5'-O-(2-thiodiphosphate) (GDP-betaS) (600 microM, 6-hr preincubation) did not block the shear stress elevation of eNOS mRNA.

C-type natriuretic peptide (CNP) is secreted by endothelial cells and has vasodilatory and antiproliferative activity against smooth muscle cells. Using defined laminar shear stress exposures of cultured bovine aortic endothelial cells, we investigated the regulation of CNP gene by PhosphorImaging the ratio of CNP mRNA to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA. A 6 h exposure to arterial shear stress of 25 dyn/cm2 caused a marked elevation (10.5 +/- 6.2-fold: n=10, p<0.001) of CNP/GAPDH mRNA ratio compared to stationary controls. Arterial shear stress was 2.6 times more potent than a venous level of shear stress of 4 dyn/cm2 in elevating the CNP/GAPDH mRNA ratio. After 6 h, CNP secretion by shear stressed BAEC was elevated over stationary controls by 3.1-fold (n=5, p<0.001) to a level of 34 +/- 7.5 pg/cm2 BAEC. Shear stress elevated CNP mRNA in the presence of L-NAME (400 microM) indicating that autocrine signaling through shear-induced NO production or guanylate cyclase activation was not involved. Similarly, the tyrosine kinase inhibitor genistein (10 microM), which can also block shear-induced NO production, had no effect on CNP mRNA induction by shear stress in BAEC. The intracellular calcium chelator BAPTA/AM (5 microM) attenuated the shear stress-induced CNP mRNA expression by 71%. Interestingly, dexamethasone (1 microM) potentiated by 2-fold the shear stress enhancement of CNP mRNA. Shear stress was a more potent inducer of CNP than either phorbol myristrate acetate or lipopolysaccharide. Hemodynamic shear stress may be an important physiological regulator of CNP expression with consequent effects on vasodilation and regulation of intimal hyperplasia.

The c-fos protein belongs to a family of transcriptional cofactors that can complex with proteins of the Jun family and activate mRNA transcription from gene promoters containing an activator protein 1 (AP-1) binding element. The shear stress inducibility of the c-fos protein was studied in human and animal cell lines of vastly different origins. Primary human umbilical vein endothelial cells (HUVEC), bovine aortic endothelial cells (BAEC, passage 2-14), HeLa cells, and Chinese hamster ovary (CHO) cells were subjected to steady laminar shear stress using a parallel plate flow apparatus. After 1 h of flow exposure at 25 dyn/cm2, the c-fos levels in nuclei of shear stress HUVEC, BAEC, HeLa, and CHO were 5.4 2 2.0 ( n = 3). 2.25 2 1.38 ( n = 61, 2.14 2 0.07 ( n = 81, 1.92 ? 0.58 ( n = 2) times higher, respectively, than in matched stationary controls. Flow exposure at 4 dyn/cm2 caused no enhancement of c-fos levels in any of the cell lines tested, but caused significant reduction in c-fos expression in the HeLa cells. The c-fos induction by shear stress could be blocked by pharmacological agents. For example, the flow induction of the c-fos protein levels was blocked by 50% with the preincubation of HUVEC with a protein kinase C inhibitor, H7 (10 pM) and blocked completely in HeLa cells preincubated with the phospholipase C inhibitor, neomycin (5 mM). The minimum time of shear stress exposure required t o induce the c-fos protein expression in HeLa cells was found to be as low as 1 min. By Northern analysis, the c-fos mRNA levels were found to be elevated in BAEC, CHO, and HeLa cells exposed t o 25 dyn/cm2 for 30 min. These studies indicate that c-fos induction is a consistent genetic response in a variety of mammalian cells that may alter cellular phenotype in mechanical environments.