Abstract-Prostacyclin (prostaglandin I 2 , PGI 2 ) has a variety of functions, including inhibition of smooth muscle cell proliferation, vasodilation, and antiplatelet aggregation. PGI 2 production in endothelial cells has been reported to increase biphasically after shear loading, but the underlying mechanism is not well understood. To clarify the mechanism for the second phase of PGI 2 upregulation, we examined the gene expression of the enzymes involved in PGI 2 production in human umbilical vein endothelial cells (HUVECs) after shear-stress (24 dyne/cm 2 ) loading. The production of 6-keto-PGF 1␣ , a stable metabolite of PGI 2 , increased time-dependently under shear stress. The arachidonic acid liberation from membrane phospholipids in HUVECs after 12 hours of shear loading was increased significantly compared with the static condition. No change was observed for cytosolic phospholipase A 2 expression, as detected by reverse transcription-polymerase chain reaction and Western blotting. Cyclooxygenase (COX)-1 mRNA increased after 1 hour of shear loading, and the increase lasted for 12 hours, the longest time tested, whereas COX-2 mRNA increased after 1 hour of shear loading and peaked at 6 hours. An increase of COX-1 expression was detected at 12 hours of shear loading by Western blotting. No expression of COX-2 was detected in the static control, but induced expression was observed at 6 hours after shear loading. PGI 2 synthase was also found to be upregulated. These results suggest that the elevated PGI 2 production by shear stress is mediated by increased arachidonic acid release and a combination of increased expression of COXs and PGI 2 synthase. (Arterioscler Thromb Vasc Biol. 1998;18:1922-1926 Key Words: shear stress Ⅲ prostacyclin Ⅲ gene regulation Ⅲ endothelial cells V ascular endothelial cells were once considered to be a passive, antithrombogenic barrier; however, they are now known to have a variety of functions, such as regulation of vascular tone, atherogenic progression, and smooth muscle cell proliferation. Endothelial functions are regulated not only by humoral factors, such as growth factors and cytokines, but also by mechanical forces, such as shear stress, pressure, and stretch. Shear stress in particular plays an important role in endothelial functions. Shear stress can mediate a change in morphology and the release of various vasoactive substances from endothelial cells.1,2 Decreased shear stress has been suggested to be involved in the pathogenesis of atherosclerosis. It has been reported that areas of low shear stress are more likely to become sites of atherosclerosis 3 and that low shear stress promotes the formation of intimal hyperplasia. 4 Previous studies have shown that the release of NO, C-type natriuretic peptide, and prostaglandin I 2 (PGI 2 ) is augmented by shear stress through elevated intracellular Ca 2ϩ and increased transcription of endothelial NO synthase mRNA and C-type natriuretic peptide mRNA. [5][6][7] Frangos et al 7 reported that shear stress increases PGI 2 prod...

To elucidate the effect of blood flow on gene transcription of C-type natriuretic peptide (CNP), human umbilical vein endothelial cells were exposed to shear stress in a cone-plate viscometer. Expression of CNP mRNA, evaluated by reverse transcriptiou-polymerase chain reaction, was markedly increased by exposure to shear stress of 24 dyne/cm 2 at 3 h. The CNP mRNA level was maintained until 12 h. Thus, the present study demonstrated for the first time that shear stress induces expression of CNP gene in human endothelial cells.

ADP is an important physiological platelet agonist. The molecular identity of the ADP receptor(s) in human platelets, however, is still unclear. Although P2T purinoceptor was believed to be the ligand-gated cation channel for ADP in human platelets, recent patch clamp studies now suggest it is P2X 1 type. In the present study, we have cloned a cDNA encoding a P2X 1 purinoceptor from human platelets using degenerate reverse transcription and polymerase chain reaction. Northern blotting with a P2X 1 -specific probe revealed a band of 1.8 kilobases in human platelets as well as in several megakaryoblastic cell lines. 1321N1 human astrocytoma cells expressing the cloned P2X 1 cDNA exhibited both ATP-and ADP-stimulated Ca 2؉ influx that could be blocked by the purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2,4-disulfonic acid and suramin. Additionally, a polyclonal antibody raised against glutathione-S-transferase-P2X 1 fusion peptide reacted with a 70-kDa band on Western blot of human platelets. It is therefore concluded that functional P2X 1 purinoceptors are present in human platelets.Platelet activation is known to be involved in the development of atherosclerosis and restenosis after angioplasty. ADP is an important endogenous platelet agonist. Stimulation of platelets with ADP has been shown to mediate platelet shape change, aggregation, and further release of ADP and ATP from activated platelets. The ADP receptor in platelets is reported to possess a unique, pharmacologically characteristic P2T-type purinoceptor (1). Activation of the ADP receptor causes immediate activation of a non-selective cation channel that mediates calcium influx and mobilization of calcium from intracellular stores (2, 3). Membrane binding experiments have indicated the presence of both high and low affinity binding sites for ADP in platelets (4, 5). Furthermore, pharmacological and clinical studies have also suggested the presence of two types of ADP receptors (6, 7). Several platelet membrane ADP-binding proteins have been proposed as putative ADP receptor (8 -10), but no peptide sequence is available for the cloning purposes. Recently, it has been shown by patch clamp techniques and intracellular calcium measurements that both ADP and ATP can activate a channel and mediate an increase in intracellular calcium concentration in human platelets. It has also been suggested that the ADP/ATP-gated non-selective cation channel resembles a P2X 1 purinoceptor (11).Extracellular ATP and ADP interact with two subgroups of P2 purinoceptors, P2X and P2Y (12, 13). Molecular cloning has identified seven members of P2X, a non-selective cation channel with two transmembrane domains; P2Y, which belongs to the seven-transmembrane domain G-protein-coupled receptor family, consists of more than seven members identified to date. Based on the effects of ADP on adenylyl cyclase, activation of phospholipase C, and intracellular calcium mobilization, we hypothesize that the P2T receptor in human platelets may consist of one P2X-like and one or more P...

Abstract-Vascular smooth muscle cell (VSMC) proliferation still remains a poorly understood process, although it is believed to play a critical role in pathological states, including atherosclerosis and hypertension. Several reports have suggested that proteases may be directly involved in this process; however, it was still unclear which protease is responsible for VSMC proliferation. In this study, by use of a cell-permeable calpain inhibitor (calpeptin; benzyloxycarbonyl-Leu-nLeu-H), its analogue (benzyloxycarbonyl-Leu-Met-H), the cell-impermeable serine protease inhibitor leupeptin, and antisense oligonucleotide against m-calpain to inhibit proliferation of primarily cultured human VSMCs, we investigated whether calcium-activated neutral protease (calpain) is involved in VSMC proliferation. Calpeptin and its analogue, more specific for m-calpain, equally inhibited the proliferation of VSMCs in a dose-related manner, whereas a more limited antiproliferative effect was observed in leupeptin-treated VSMCs. Antisense oligonucleotide against m-calpain, but not scrambled antisense, dose-dependently inhibited m-calpain expression and proliferation of VSMCs. Maximal inhibition was an Ϸ50% reduction of cell number and m-calpain antigen observed at 50 mol/L of antisense oligonucleotide. Calpeptin or antisense oligonucleotide against m-calpain increased the expression of the endogenous calpain substrate pp125FAK (focal adhesion kinase), whereas the expression of the endogenous calpain inhibitor calpastatin was not affected. These results suggest that the proliferation of VSMCs requires protease activity, some of which is due to m-calpain.(Arterioscler Thromb Vasc Biol. 1998;18:493-498.)