Up-regulation of endothelial cyclooxygenase-2 and prostanoid synthesis by platelets. Role of thromboxane A2

Platelet-vascular endothelial cell interactions are central to the maintenance of vascular homeostasis. Thromboxane A2 (TXA2) and prostacyclin (prostaglandin (PG)I2) are the major products of cyclooxygenase (COX) metabolism by platelets and the vascular endothelium, respectively. Here we report the effects of platelet-endothelial interactions on human umbilical vein endothelial cells (HUVECs) COX-2 expression and prostanoid synthesis. Co-incubation of platelets with HUVECs resulted in a dose-dependent induction in COX-2 expression. This was accompanied by a relatively small increase in thromboxane B2 synthesis (2 ng) by comparison to the production of 6-keto-PGF1alpha and PGE2, which increased by approximately 14 and 12 ng, respectively. Abrogation of platelet-HUVEC interactions excluded direct cell-cell contact as a required event. Preincubation of HUVECs with SQ29548, a TXA2 receptor antagonist, dose-dependently inhibited platelet-induced COX-2 expression and prostanoid synthesis. Similarly, if platelet TXA2 synthesis was inhibited no induction of COX-2 was observed. Furthermore, a TXA2 analog, carbocyclic TXA2, induced HUVEC COX-2 expression and the synthesis of 6-keto-PGF1alpha and PGE2. This was also associated with an increase in the expression and activity of PGI synthase and PGE synthase but not TX synthase. Platelet co-incubation (or TXA2) also selectively activated the p44/42 mitogen-activated protein kinase pathway to regulate HUVEC COX-2 expression. Thus it seems that platelet-derived TXA2 can act in a paracrine manner to up-regulate endothelial COX-2 expression and PGI2 synthesis. These observations are of particular importance given the recent observations regarding selective COX-2 inhibitors and the suppression of PGI2 synthesis.     

Role of thromboxane A2 in early BDL-induced portal hypertension

Although the mechanisms of cirrhosis-induced portal hypertension have been studied extensively, the role of thromboxane A(2) (TXA(2)) in the development of portal hypertension has never been explicitly explored. In the present study, we sought to determine the role of TXA(2) in bile duct ligation (BDL)-induced portal hypertension in Sprague-Dawley rats. After 1 wk of BDL or sham operation, the liver was isolated and perfused with Krebs-Henseleit bicarbonate buffer at a constant flow rate. After 30 min of nonrecirculating perfusion, the buffer was recirculated in a total volume of 100 ml. The perfusate was sampled for the enzyme immunoassay of thromboxane B(2) (TXB(2)), the stable metabolite of TXA(2). Although recirculation of the buffer caused no significant change in sham-operated rats, it resulted in a marked increase in portal pressure in BDL rats. The increase in portal pressure was found concomitantly with a significant increase of TXB(2) in the perfusate (sham vs. BDL after 30 min of recirculating perfusion: 1,420 +/- 803 vs. 10,210 +/- 2,950 pg/ml; P < 0.05). Perfusion with a buffer containing indomethacin or gadolinium chloride for inhibition of cyclooxygenase (COX) or Kupffer cells, respectively, substantially blocked the recirculation-induced increases in both portal pressure and TXB(2) release in BDL group. Hepatic detection of COX gene expression by RT-PCR revealed that COX-2 but not COX-1 was upregulated following BDL, and this upregulation was confirmed at the protein level by Western blot analysis. In conclusion, these results clearly demonstrate that increased hepatic TXA(2) release into the portal circulation contributes to the increased portal resistance in BDL-induced liver injury, suggesting a role of TXA(2) in liver fibrosis-induced portal hypertension. Furthermore, the Kupffer cell is likely the source of increased TXA(2), which is associated with upregulation of the COX-2 enzyme.     

Sinusoidal endothelial COX-1-derived prostanoids modulate the hepatic vascular tone of cirrhotic rat livers

CCl(4) cirrhotic rat liver exhibits a hyperresponse to the alpha(1)-adrenergic agonist methoxamine (Mtx) that is associated with enhanced thromboxane A(2) (TXA(2)) production and is abrogated by indomethacin. To further elucidate the molecular mechanisms involved in the hyperresponse to vasoconstrictors, portal perfusion pressure dose-response curves to Mtx were performed in CCl(4) cirrhotic rats livers after preincubation with vehicle, the cyclooxygenase (COX)-1 selective inhibitor SC-560, and the COX-2 selective inhibitor SC-236. TXA(2) production was determined in samples of the perfusate. COX-1 expression was analyzed and quantified in hepatocytes, Kupffer cells, sinusoidal endothelial cells (SEC), and hepatic stellate cells (HSC) isolated from control and cirrhotic rat livers by double-immunofluorescence staining, with specific markers for each population using flow cytometry or Western blot analysis. COX-1 protein levels were not significantly increased in cirrhotic livers, but COX-2 protein expression was increased. COX-1 inhibition, but not COX-2, significantly attenuated the response to Mtx and prevented the increased production of TXA(2). Cirrhotic livers showed an increased expression of COX-1 in SEC and reduced expression in HSC compared with control livers, whereas COX-1 was similarly distributed in Kupffer cells. Despite abundant hepatic COX-2 expression, the increased response to Mtx of cirrhotic livers is mainly dependent of COX-1. Upregulation of COX-1 in cirrhotic SEC may be responsible for the hyperesponse to Mtx.     

iNOS expression in vascular resident macrophages contributes to circulatory dysfunction of splanchnic vascular smooth muscle contractions in portal hypertensive rats

Portal hypertension, a major complication of cirrhosis, is caused by both increased portal blood flow due to arterial vasodilation and augmented intrahepatic vascular resistance due to sinusoidal constriction. In this study, we examined the possible involvement of resident macrophages in the tone regulation of splanchnic blood vessels using bile duct ligated (BDL) portal hypertensive rats and an in vitro organ culture method. In BDL cirrhosis, the number of ED2-positive resident macrophages increased by two- to fourfold in the vascular walls of the mesenteric artery and extrahepatic portal vein compared with those in sham-operated rats. Many ED1-positive monocytes were also recruited into this area. The expression of inducible nitric oxide (NO) synthase (iNOS) mRNA was increased in the vascular tissues isolated from BDL rats, and accordingly, nitrate/nitrite production was increased. Immunohistochemistry revealed that iNOS was largely expressed in ED1-positive and ED2-positive cells. We further analyzed the effect of iNOS expression on vascular smooth muscle contraction using an in vitro organ culture system. iNOS mRNA expression and nitrate production significantly increased in vascular tissues (without endothelium) incubated with 1 μg/ml lipopolysaccharide (LPS) for 6 h. Immunohistochemistry indicated that iNOS was largely expressed in ED2-positive resident macrophages. α-Adrenergic-stimulated contractility of the mesenteric artery was greatly suppressed by LPS treatment and was restored by N(G)-nitro-L-arginine methyl ester (NO synthase inhibitor); in contrast, portal vein contractility was largely unaffected by LPS. Sodium nitroprusside (NO donor) and 8-bromo-cGMP showed greater contractile inhibition in the mesenteric artery than in the portal vein with decreasing myosin light chain phosphorylation. In the presence of an α-adrenergic agonist, the mesenteric artery cytosolic Ca(2+) level was greatly reduced by sodium nitroprusside; however, the portal vein Ca(2+) level was largely unaffected. These results suggest that the induction of iNOS in monocytes/macrophages contributes to a hypercirculatory state in the cirrhosis model rat in which the imbalance of the responsiveness of visceral vascular walls to NO (mesenteric artery > portal vein) may account for the increased portal venous flow in portal hypertension.     

Celecoxib reduces hepatic vascular resistance in portal hypertension by amelioration of endothelial oxidative stress

The balance between endothelial nitric oxide (NO) synthase (eNOS) activation and production of reactive oxygen species (ROS) is very important for NO homeostasis in liver sinusoidal endothelial cells (LSECs). Overexpression of cyclooxygenase-2 (COX-2), a major intravascular source of ROS production, has been observed in LSECs of cirrhotic liver. However, the links between low NO bioavailability and COX-2 overexpression in LSECs are unknown. This study has confirmed the link between low NO bioavailability and COX-2 overexpression by COX-2-dependent PGE2-EP2-ERK1/2-NOX1/NOX4 signalling pathway in LSECs in vivo and in vitro. In addition, the regulation of COX-2-independent LKB1-AMPK-NRF2-HO-1 signalling pathway on NO homeostasis in LSECs was also elucidated. The combinative effects of celecoxib on diminishment of ROS via COX-2-dependent and COX-2-independent signalling pathways greatly decreased NO scavenging. As a result, LSECs capillarisation was reduced, and endothelial dysfunction was corrected. Furthermore, portal hypertension of cirrhotic liver was ameliorated with substantial decreasing hepatic vascular resistance and great increase of portal blood flow. With the advance understanding of the mechanisms of LSECs protection, celecoxib may serve as a potential therapeutic candidate for patients with cirrhotic portal hypertension.     

Endothelial COX-1 and -2 differentially affect reactivity of MVB in portal hypertensive rats

Expression of constitutive and inducible cyclooxygenase (COX-1 and COX-2, respectively) and the role of prostanoids were investigated in the aorta and mesenteric vascular bed (MVB) from the portal vein-ligated rat (PVL) as a model of portal hypertension. Functional experiments were carried out in MVB from PVL and sham-operated rats in the absence or presence of the nonselective COX inhibitor indomethacin or the selective inhibitors of COX-1 (SC-560) or COX-2 (NS-398). Western blots of COX-1 and COX-2 proteins were evaluated in aorta and MVB, and PGI(2) production by enzyme immunoassay of 6-keto-PGF(1alpha) was evaluated in the aorta. In the presence of functional endothelium, decreased contraction to norepinephrine (NE) and increased vasodilatation to ACh were observed in MVB from PVL. Exposure of MVB to indomethacin, SC-560, or NS-398 reversed the hyporeactivity to NE and the increased endothelial vasodilatation to ACh in PVL, with NS-398 being more potent than the other two inhibitors. Upregulation of COX-1 and COX-2 expressions was detected in aorta and MVB from PVL portal hypertensive rats, and increased production of 6-keto-PGF(1alpha) was observed in aorta from portal hypertensive rats. These results suggest that generation of endothelial vasodilator prostanoids, from COX-1 and COX-2 isoforms, accounts for the increased mesenteric blood flow in portal hypertension.     

Posttranslational regulation of cyclooxygenase by tyrosine phosphorylation in cerebral endothelial cells

Endothelium-derived cyclooxygenase (COX) products regulatecerebral vascular tone in newborn pigs. Both COX-1 and COX-2 are constitutively expressed in endothelial cells from newborn pig cerebralmicrovessels. We investigated the role of protein phosphorylation inthe regulation of COX activity. The protein tyrosine phosphatase (PTP)inhibitors phenylarsine oxide, vanadate, and benzylphosphonic acidrapidly stimulated COX activity, whereas the protein tyrosine kinaseinhibitors, genistein and tyrphostins, inhibited it. Protein synthesisinhibitors did not reverse the stimulation of COX activity evoked byPTP inhibitors. Similar changes were observed in other vascular cellsfrom newborn pigs that also express COX-1 and COX-2 (cerebralmicrovascular smooth muscle cells and aortic endothelial cells) but notin human umbilical vein endothelial cells or Swiss 3T3 fibroblasts thatexpress COX-1 only. Tyrosine-phosphorylated proteins wereimmunodetected in endothelial cell lysates. COX-2 immunoprecipitatedfrom ³²P-loaded endothelial cellsincorporated ³²P that wasincreased by PTP inhibitors. COX-2, but not COX-1, was detected inendothelial fractions immunoprecipitated with anti-phosphotyrosine.These data indicate that tyrosine phosphorylation posttranslationallyregulates COX activity in newborn pig vascular cells and that COX-2 isa substrate for phosphorylation.

     

Human neutrophil peptides upregulate expression of COX-2 and endothelin-1 by inducing oxidative stress

Polymorphonuclear leukocytes (PMNs) play an important role during inflammation in cardiovascular diseases. Human neutrophil peptides (HNPs) are released from PMN granules upon activation and are conventionally involved in microbial killing. Recent studies suggested that HNPs may be involved in the pathogenesis of vascular abnormality by modulating inflammatory responses and vascular tone. Since HNPs directly interact with endothelium upon release from PMNs in the circulation, we tested the hypothesis that the stimulation with HNPs of endothelial cells modulates the expression of vasoactive by-products through altering cyclooxygenase (COX) activity. When human umbilical vein endothelial cells were stimulated with purified HNPs, we observed a time- and dose-dependent increase in the expression of COX-2, whereas COX-1 levels remained unchanged. Despite an increased expression of COX-2 at the protein level, HNPs did not significantly enhance the COX-2 activity, thus the production of the prostaglandin PGI2. HNPs significantly induced the release of endothelin-1 (ET-1) as well as the formation of nitrotyrosine. The HNP-induced COX-2 and ET-1 production was attenuated by the treatment with the oxygen free radical scavenger N-acetyl-L-cysteine and the inhibitors of p38 MAPK and NF-kappaB, respectively. The angiontensin II pathway did not seem to be involved in the HNP-induced upregulation of COX-2 and ET-1 since the use of the angiotensin-converting enzyme inhibitor enalapril had no effect in this context. In conclusion, HNP may play an important role in the pathogenesis of inflammatory cardiovascular diseases by activating endothelial cells to produce vasoactive by-products as a result of oxidative stress.     

Apelin/APJ：血管功能调节因子和药物治疗新靶点

Apelin(APJendogenousligand)是血管紧张素Ⅱ1型受体相关蛋白(angiotensin receptor-like 1,APJ)的内源性配体.Apelin/APJ系统在机体内广泛分布,在众多血管系统表达水平较高,如心血管系统、肺血管系统等.研究发现,apelin可调节血管张力,促进血管平滑肌细胞增殖、视网膜血管新生以及单核细胞向内皮细胞黏附,促进肝门静脉和冠状动脉侧枝形成等.本文就apelin调节血管功能及其相关疾病(高血压、肺动脉高压、动脉粥样硬化、胶质瘤、肺癌、门静脉高压、糖尿病血管并发症等)进行综述,揭示了apelin与血管及其相关疾病的内在联系,表明apelin/APJ可作为血管疾病的治疗靶点.     

Celecoxib Ameliorates Portal Hypertension of the Cirrhotic Rats through the Dual Inhibitory Effects on the Intrahepatic Fibrosis and Angiogenesis

Background

Increased intra-hepatic resistance to portal blood flow is the primary factor leading to portal hypertension in cirrhosis. Up-regulated expression of cyclooxygenase-2 (COX-2) in the cirrhotic liver might be a potential target to ameliorate portal hypertension.

Objective

To verify the effect of celecoxib, a selective inhibitor of COX-2, on portal hypertension and the mechanisms behind it.

Methods

Cirrhotic liver model of rat was established by peritoneal injection of thiacetamide (TAA). 36 rats were randomly assigned to control, TAA and TAA+celecoxib groups. Portal pressures were measured by introduction of catheters into portal vein. Hepatic fibrosis was assessed by the visible hepatic fibrotic areas and mRNAs for collagen III and α-SMA. The neovasculature was determined by hepatic vascular areas, vascular casts and CD31 expression. Expressions of COX-2, vascular endothelial growth factor (VEGF), VEGF receptor-2 (VEGFR-2) and related signal molecules were quantitated.

Results

Compared with TAA group, the portal pressure in TAA+celecoxib group was significantly decreased by 17.8%, p<0.01. Celecoxib treatment greatly reduced the tortuous hepatic portal venules. The data of fibrotic areas, CD31expression, mRNA levels of α-SMA and collagen III in TAA+celecoxib group were much lower than those in TAA group, p<0.01. Furthermore, the up-regulation of hepatic mRNA and protein levels of VEGF, VEGFR-2 and COX-2 induced by TAA was significantly inhibited after celecoxib treatment. The expressions of prostaglandin E2 (PGE2), phosphorylated extracellular signal-regulated kinase (p-ERK), hypoxia-inducible factor-1α (HIF-1α), and c-fos were also down-regulated after celecoxib treatment.

Conclusions

Long term administration of celecoxib can efficiently ameliorate portal hypertension in TAA rat model by its dual inhibitory effects on the intrahepatic fibrosis and angiogenesis. The anti-angiogenesis effect afforded by celecoxib may attribute to its modulation on VEGF/VEGFR-2 through the down-regulation of integrated signal pathways involving PGE2- HIF-1α- VEGF and p-ERK- c-fos- VEGFR-2.     

Transgenic mice over-expressing ET-1 in the endothelial cells develop systemic hypertension with altered vascular reactivity

Endothelin-1 (ET-1) is a potent vasoconstrictor involved in the regulation of vascular tone and implicated in hypertension. However, the role of small blood vessels endothelial ET-1 in hypertension remains unclear. The present study investigated the effect of chronic over-expression of endothelial ET-1 on arterial blood pressure and vascular reactivity using transgenic mice approach. Transgenic mice (TET-1) with endothelial ET-1 over-expression showed increased in ET-1 level in the endothelial cells of small pulmonary blood vessels. Although TET-1 mice appeared normal, they developed mild hypertension which was normalized by the ET(A) receptor (BQ123) but not by ET(B) receptor (BQ788) antagonist. Tail-cuff measurements showed a significant elevation of systolic and mean blood pressure in conscious TET-1 mice. The mice also exhibited left ventricular hypertrophy and left axis deviation in electrocardiogram, suggesting an increased peripheral resistance. The ionic concentrations in the urine and serum were normal in 8-week old TET-1 mice, indicating that the systemic hypertension was independent of renal function, although, higher serum urea levels suggested the occurrence of kidney dysfunction. The vascular reactivity of the aorta and the mesenteric artery was altered in the TET-1 mice indicating that chronic endothelial ET-1 up-regulation leads to vascular tone imbalance in both conduit and resistance arteries. These findings provide evidence for the role of spatial expression of ET-1 in the endothelium contributing to mild hypertension was mediated by ET(A) receptors. The results also suggest that chronic endothelial ET-1 over-expression affects both cardiac and vascular functions, which, at least in part, causes blood pressure elevation.     

High-density lipoprotein induces cyclooxygenase-2 expression and prostaglandin I-2 release in endothelial cells through sphingosine kinase-2

High-density lipoprotein (HDL) has a significant cardioprotective effects. HDL induces cyclooxygenase-2 (COX-2) expression and prostacyclin I-2 (PGI-2) release in vascular endothelial cells, which contributes to its anti-atherogenic effects. However, the underlying mechanisms are not fully understood. In the present study, we observed that HDL-stimulated COX-2 expression and PGI-2 production in human umbilical vein endothelial cells (HUVECs) in a time- and dose-dependent manner. These effects triggered by HDL were inhibited by pertussis toxin (PTX), protein kinase C (PKC) inhibitor GF109203X, and ERK inhibitor PD98059, suggesting that Gαi/Gαo-coupled GPCR, PKC, and ERK pathways are involved in HDL-induced COX-2/PGI-2 activation. More importantly, we found that silencing of sphingosine kinase 2 (SphK-2) also blocked HDL-induced COX-2/PGI-2 activation. In addition, HDL-activated SphK-2 phosphorylation accompanied by increased S1P level in the nucleus. Our ChIP data demonstrated that SphK-2 is associated with CREB at the COX-2 promoter region. Collectively, these results indicate that HDL induces COX-2 expression and PGI-2 release in endothelial cells through activation of PKC, ERK1/2, and SphK-2 pathways. These findings implicate a novel mechanism underlying anti-atherothrombotic effects of HDL.     

Gene expression changes of prostanoid synthases in endothelial cells and prostanoid receptors in vascular smooth muscle cells caused by aging and hypertension.

The present study was designed to assess whether or not changes in genomic expression of cyclooxygenases (COX-1, COX-2), endothelial nitric oxide synthase (eNOS), and prostanoid synthases in the endothelium and of prostanoid receptors in vascular smooth muscle contribute to the occurrence of endothelium-dependent contractions during aging and hypertension. Gene expression was quantified by real-time PCR using isolated endothelial cells and smooth muscle cells (SMC) from the aorta of Wistar-Kyoto and spontaneously hypertensive rats. Genes for all known prostanoid synthases and receptors were present in endothelial cells and SMC, respectively. Aging caused overexpression of eNOS, COX-1, COX-2, thromboxane synthase, hematopoietic-type prostaglandin D synthase, membrane prostaglandin E synthase-2, and prostaglandin F synthase in endothelial cells and COX-1 and prostaglandin E(2) (EP)(4) receptors in SMC. Hypertension augmented the expression of COX-1, prostacyclin synthase, thromboxane synthase, and hematopoietic-type prostaglandin D synthase in endothelial cells and prostaglandin D(2) (DP), EP(3), and EP(4) receptors in SMC. The increase in genomic expression of endothelial COX-1 explains why in aging and hypertension the endothelium has greater propensity to release cyclooxygenase-derived vasoconstrictive prostanoids. The expression of prostacyclin synthase was by far the most abundant, explaining why the majority of the COX-1-derived endoperoxides are transformed into prostacyclin, substantiating the role of prostacyclin as an endothelium-derived contracting factor. The expression of thromboxane synthase was increased in the cells of aging or hypertensive rats, explaining why the prostanoid can contribute to endothelium-dependent contractions. It is uncertain whether the gene modifications caused by aging and hypertension directly contribute to endothelium-dependent contractions or rather to vascular aging and the vascular complications of the hypertensive process.     

Role of caveolin-1 in EGCG-mediated protection against linoleic-acid-induced endothelial cell activation

Flavonoids can protect against inflammatory diseases such as atherosclerosis by decreasing vascular endothelial cell activation. Plasma microdomains called caveolae may be critical in regulating endothelial activation. Caveolae are particularly abundant in endothelial cells and play a major role in endothelial trafficking and the regulation of signaling pathways associated with the pathology of vascular diseases. We hypothesize that flavonoids can down-regulate endothelial inflammatory parameters by modulating caveolae-regulated cell signaling. We focused on the role of caveolae and its major protein, caveolin-1, in mechanisms of linoleic-acid-induced endothelial cell activation and protection by the catechin epigallocatechin-3-gallate (EGCG). Exposure to linoleic acid for 6 h induced expression of both caveolin-1 and cyclooxygenase (COX)-2. Pretreatment with EGCG blocked fatty-acid-induced caveolin-1 and COX-2 expression in a time- and concentration-dependent manner. Similar results were observed with nuclear factor-kappa B DNA binding activity, which was also reduced by caveolin-1 silencing. Exposure to linoleic acid rapidly increased phosphorylation of several kinases, including p38 MAPK, extracellular signal regulated kinase 1/2 (ERK1/2) and amino kinase terminal (Akt), with maximal induction at about 10 min. Inhibitors of ERK1/2 and Akt down-regulated the linoleic-acid-induced increase in COX-2 protein, which also occurred after pretreatment with EGCG. Caveolin-1 silencing blocked linoleic-acid-induced phosphorylation of ERK1/2 and protein expression of COX-2, suggesting that specific MAPK signaling is caveolae dependent. Our data provide evidence that caveolae may play a critical role in regulating vascular endothelial cell activation and protection by flavonoids such as EGCG.     

Cellular localization of the endothelin receptor subtypes ET(A) and ET(B) in the rat heart and their differential expression in coronary arteries,veins, and capillaries

In the heart, the endothelin (ET)/endothelin-receptor system is markedly involved in pathophysiological mechanisms underlying various cardiac diseases. Based upon pharmacological studies both ET-receptor subtypes take part in the regulation of coronary vascular tone, however, their detailed cellular distribution in the coronary vascular bed based upon direct mRNA and protein detection is unknown. This issue was addressed in the rat heart by means of non-radioactive in situ hybridization, RT-PCR, and immunohistochemistry. Expression of vascular ET(A)-receptors was detected in arterial smooth muscle and capillary endothelium while ET(B)-receptors were present in arterial, venous, and capillary endothelium, and in arterial and venous smooth muscle cells. This differential distribution of the ET-receptor subtypes supports the concept that ET(A)- as well as ET(B)-receptors mediate arterial vasoconstriction, while postcapillary vascular resistance is exclusively regulated by ET(B)-receptors. The observed capillary endothelial expression of the ET(A)-receptor correlates with the known ability of ET(A)-receptor antagonists to attenuate increases in cardiac microvascular permeability during endotoxin shock and ischemia/reperfusion injury.     

Histamine directly and synergistically with lipopolysaccharide stimulates cyclooxygenase-2 expression and prostaglandin I(2) and E(2) production in human coronary artery endothelial cells

Although histamine plays an essential role in inflammation, its influence on cyclooxygenases (COX) and prostanoid homeostasis is not well understood. In this study, we investigated the effects of histamine on the expression of COX-1 and COX-2 and determined their contribution to the production of PGE(2), prostacyclin (PGI(2)), and thromboxane A(2) in human coronary artery endothelial cells (HCAEC). Incubation of HCAEC monolayers with histamine resulted in marked increases in the expression of COX-2 and production of PGI(2) and PGE(2) with no significant change in the expression of COX-1. Histamine-induced increases in PGI(2) and PGE(2) production were due to increased expression and function of COX-2 because gene silencing by small interfering RNA or inhibition of the catalytic activity by a COX-2 inhibitor blocked prostanoid production. The effects of histamine on COX-2 expression and prostanoid production were mediated through H(1) receptors. In addition to the direct effect, histamine was found to amplify LPS-stimulated COX-2 expression and PGE(2) and PGI(2) production. In contrast, histamine did not stimulate thromboxane A(2) production in resting or LPS-activated HCAEC. Histamine-induced increases in the production of PGE(2) and PGI(2) were associated with increased expression of mRNA encoding PGE(2) and PGI(2) synthases. The physiological role of histamine on the regulation of COX-2 expression in the vasculature is indicated by the findings that the expression of COX-2 mRNA, but not COX-1 mRNA, was markedly reduced in the aortic tissues of histidine decarboxylase null mice. Thus, histamine plays an important role in the regulation of COX-2 expression and prostanoid homeostasis in vascular endothelium.     

Caveolae and caveolae constituents in mechanosensing

Studies in modeled microgravity or during orbital space flights have clearly demonstrated that endothelial cell physiology is strongly affected by the reduction of gravity. Nevertheless, the molecular mechanisms by which endothelial cells may sense gravity force remain unclear. We previously hypothesized that endothelial cell caveolae could be a mechanosensing system involved in hypergravity adaptation of human endothelial cells. In this study, we analyzed the effect on the physiology of human umbilical vein endothelial cell monolayers of short exposure to modeled microgravity (24–48h) obtained by clinorotation. For this purpose, we evaluated the levels of compounds, such as nitric oxide and prostacyclin, involved in vascular tone regulation and synthesized starting from caveolae-related enzymes. Furthermore, we examined posttranslational modifications of Caveolin (Cav)-1 induced by simulated microgravity. The results we collected clearly indicated that short microgravity exposure strongly affected endothelial nitrix oxide synthase activity associated with Cav-1 (Tyr 14) phosphorylation, without modifying the angiogenic response of human umbilical vein endothelial cells. We propose here that one of the early molecular mechanisms responsible for gravity sensing of endothelium involves endothelial cell caveolae and Cav-1 phosphorylation.