Hypergravity impairs angiogenic response of in vitro cultured human primary endothelial cells

A variety of evidence suggest that cardiovascular system functions are impaired in altered gravity conditions. In this study we investigated the influence of hypergravity environment (3g) on endothelial cell proliferation, endothelial vasoactive compound production and on in vitro angiogenesis. We found that cultured primary human endothelial cells were very sensitive to mild hypergravity conditions. Even if we did not record changes in cell viability and apoptosis, we found significant differences in cell proliferation, prostacyclin (PGI2) synthesis, nitric oxide (NO) synthesis, and in angiogenic responses. Using western blotting technique we detected an increased expression of cycloxygenase-2 (COX-2) in primary endothelial cells exposed for 48 hours to hypergravity, in comparison to those exposed to normal gravity.     

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.     

Possible mechanisms underlying pregnancy-induced changes in uterine artery endothelial function

The last 10 years has seen a dramatic increase in our understanding of the mechanisms underlying the pregnancy-specific adaptation in cardiovascular function in general and the dramatic changes that occur in uterine artery endothelium in particular to support the growing fetus. The importance of these changes is clear from a number of studies linking restriction of uterine blood flow (UBF) and/or endothelial dysfunction and clinical conditions such as intrauterine growth retardation (IUGR) and/or preeclampsia in both humans and animal models; these topics are covered only briefly here. The recent developments that prompts this review are twofold. The first is advances in an understanding of the cell signaling processes that regulate endothelial nitric oxide synthase (eNOS) in particular (Govers R and Rabelink TJ. Am J Physiol Renal Physiol 280: F193-F206, 2001). The second is the emerging picture that uterine artery (UA) endothelial cell production of nitric oxide (NO) as well as prostacyclin (PGI2) may be as much a consequence of cellular reprogramming at the level of cell signaling as due to tonic stimuli inducing changes in the level of expression of eNOS or the enzymes of the PGI2 biosynthetic pathway (cPLA2, COX-1, PGIS). In reviewing just how we came to this conclusion and outlining the implications of such a finding, we draw mostly on data from ovine or human studies, with reference to other species only where directly relevant.     

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.     

Colocalization prostacyclin (PGI2) synthase--caveolin-1 in endothelial cells and new roles for PGI2 in angiogenesis

In vascular cells, prostacyclin (PGI2) synthase (PGI2s) has been localized in the endoplasmic reticulum of endothelial cells and in the nuclear and plasma membrane of smooth muscle cells. In human umbilical vein endothelial (HUVE) cells, we detected the enzyme in abundant cytoplasmic vesicles apparently originating from the plasma membrane and similar to those stained by gold-albumin, which interacts with a caveolar receptor. This prompted us to try a direct confocal microscopy approach aimed at colocalizing gold-albumin, caveolin-1, and PGI2 synthase. Moreover, the staining of HUVE cells with an anti-BiP7Grp78 antibody (a marker of endoplasmic reticulum) shows a perinuclear localization, sharply separated from PGI2 synthase localization. The results indicate that more than 80% of the enzyme resides in cellular sites costaining with caveolin-1 antibody and gold-albumin. This evidence was confirmed by the demonstration that PGI2 synthase and caveolin-1 coimmunoprecipitate in HUVE cell lysates and that they are associated to detergent-insoluble membrane domains in the same low-density fractions of a sucrose gradient. In addition, depletion of cellular cholesterol by mevalonate and methyl-beta-cyclodextrin leads to the shift of PGI2 synthase and caveolin-1 to higher density fractions of the gradient. Biochemical evidence about colocalization was supported by the use of a fusion protein glutathione S-transferase (GST)/caveolin-1, which retained either PGI2s purified from ram seminal vesicles or PGI2s present in HUVE cell lysates. Binding of PGI2s to caveolin "scaffolding domain" and to C-terminal region was deduced by using full-length GST--Cav-1, GST--Cav 61--101, and GST C- and N-terminal fusion proteins. A double approach based on the usage of filipin as a specific caveolae-disrupting agent and antisense oligonucleotides targeting PGI2 synthase mRNA suggests that the production of PGI2 in caveolae is likely to be connected to the regulation of angiogenesis, at least in vitro.     

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.     

Endostatin induces acute endothelial nitric oxide and prostacyclin release

Chronic exposure to endostatin (ES) blocks endothelial cell (EC) proliferation, and migration and induces EC apoptosis thereby inhibiting angiogenesis. Nitric oxide (NO) and prostacyclin (PGI(2)), in contrast, play important roles in promoting angiogenesis. In this study, we examined the acute effects of ES on endothelial NO and PGI(2) production. Unexpectedly, a cGMP reporter cell assay showed that ES-induced acute endothelial NO release in cultured bovine aortic endothelial cells (BAECs). Enzyme immunoassay showed that ES also induced an acute increase in PGI(2) production in BAECs. These results were confirmed by ex vivo vascular ring studies that showed vascular relaxation in response to ES. Immunoblot analysis showed that ES stimulated acute phosphorylation of endothelial nitric oxide synthase (eNOS) at Ser116, Ser617, Ser635, and Ser1179, and dephosphorylation at Thr497 in BAECs, events associated with eNOS activation. Short-term exposure of EC to ES, therefore, unlike long-term exposure which is anti-angiogenic, may be pro-angiogenic.     

Oxidized low density lipoprotein displaces endothelial nitric-oxide synthase (eNOS) from plasmalemmal caveolae and impairs eNOS activation.

Hypercholesterolemia-induced vascular disease and atherosclerosis are characterized by a decrease in the bioavailability of endothelium-derived nitric oxide. Endothelial nitric-oxide synthase (eNOS) associates with caveolae and is directly regulated by the caveola protein, caveolin. In the present study, we examined the effects of oxidized low density lipoprotein (oxLDL) on the subcellular location of eNOS, on eNOS activation, and on caveola cholesterol in endothelial cells. We found that treatment with 10 microgram/ml oxLDL for 60 min caused greater than 90% of eNOS and caveolin to leave caveolae. Treatment with oxLDL also inhibited acetylcholine-induced activation of eNOS but not prostacyclin production. oxLDL did not affect total cellular eNOS abundance. Oxidized LDL also did not affect the palmitoylation, myristoylation or phosphorylation of eNOS. Oxidized LDL, but not native LDL, or HDL depleted caveolae of cholesterol by serving as an acceptor for cholesterol. Cyclodextrin also depleted caveolae of cholesterol and caused eNOS and caveolin to translocate from caveolae. Furthermore, removal of oxLDL allowed eNOS and caveolin to return to caveolae. We conclude that oxLDL-induced depletion of caveola cholesterol causes eNOS to leave caveolae and inhibits acetylcholine-induced activation of the enzyme. This process may be an important mechanism in the early pathogenesis of atherosclerosis.     

Functional and cellular interactions between nitric oxide and prostacyclin

Nitric oxide (NO) and prostacyclin (PGI(2)) can be released by vascular agents to synergize their effects on vascular relaxation. In the present study we assess whether NO could affect PGI(2) production. We evaluated the effect of NO on PGI(2)-mediated arachidonic acid (AA)-induced relaxation in the perfused heart. We used cultured endothelial cells to characterize the mechanism involved in the NO effect on PGI(2) synthesis. AA-induced PGI(2) synthesis was enhanced when NO synthesis was inhibited. NO inhibited AA-induced relaxation and PGI(2) release in the coronary circulation. S-Nitroso-acetyl-DL-penicillamine (SNAP) decreased PGI(2) production in cultured endothelial cells. The SNAP effect was blunted by the inhibitor of soluble guanylate cyclase (LY-83,583) and the blocker of cGMP-dependent protein kinases (H-9). Specific cyclooxygenase-1 (COX-1) immunoprecipitation was associated to co-precipitation of four proteins. COX-1 showed neither serine nor threonine phosphorylation. One of the proteins that co-precipitated with COX-1 presented increased serine phosphorylation in the presence of SNAP. This effect was inhibited by the H-9. We suggest that NO, through cGMP-dependent protein kinases, produces the phosphorylation of a 104-kDa protein that is associated with inhibition in the activity of the COX-1, decreasing PGI(2) synthesis and thereby decreasing coronary PGI(2)-mediated vasodilatation.     

Hypergravity induces expression of cyclooxygenase-2 in the heart vessels

Cyclooxygenase-2 (COX-2), a rate-limiting enzyme for prostaglandin biosynthesis, is induced by various stimuli including mechanical stress and plays important roles in pathophysiological conditions. For example, gravitational stress has been shown to induce expression of COX-2 in bone tissues, which is essential for bone homeostasis. To investigate whether COX-2 is induced by gravitational loading in other tissues than bone, we exposed mice to hypergravity at 2G and 3G for 4 h. We demonstrate here that COX-2 is induced in the mouse heart vessels by hypergravity. Moreover, hypoxia-inducible factor (HIF)-1alpha and its downstream genes such as inducible nitric oxide synthase, vascular endothelial growth factor, and heme oxygenase-1 were induced in the heart simultaneously, while none of these genes were induced in the COX-2(-/-) mouse heart. Therefore, COX-2 induced in the heart helps protect the heart function against hypoxia under hypergravity condition through HIF-1alpha induction.     

eNOS, COX-2, and prostacyclin production are impaired in endothelial cells from diabetics

The vascular endothelium is a well-recognized target of damage for factors leading to increased cardiovascular risk. Among the agents playing an important role in cardiovascular homeostasis, nitric oxide and prostacyclin represent key markers of endothelial integrity. In the present work, we report for the first time the reduced expression of both endothelial nitric oxide synthase and cyclooxygenase-2 (COX-2) proteins, as well as decreased prostacyclin production, in unstimulated human endothelial cells from insulin-dependent diabetic mothers when compared to cells from non-diabetic, control subjects. According to a major role of COX-2 as a source of prostacyclin production even in unstimulated endothelial cells, prostacyclin production was concentration-dependently inhibited by the selective COX-2 inhibitor SC236. Overall, our results suggest a possible link between reduced endothelial COX-2 and NO-synthase expression and the increased risk of cardiovascular diseases affecting diabetic patients, and point to the use of endothelial cells from diabetic patients as a tool for investigating early dysfunction in pathological endothelium.     

Vascular endothelial growth factor signals endothelial cell production of nitric oxide and prostacyclin through flk-1/KDR activation of c-Src.

Vascular endothelial growth factor (VEGF) is a potent endothelial cell-specific mitogen that promotes angiogenesis, vascular hyperpermeability, and vasodilation by autocrine mechanisms involving nitric oxide (NO) and prostacyclin (PGI(2)) production. These experiments used immunoprecipitation and immunoassay procedures to characterize the signaling pathways by which VEGF induces NO and PGI(2) formation in cultured endothelial cells. The data showed that VEGF stimulates complex formation of the flk-1/kinase-insert domain-containing receptor (KDR) VEGF receptor with c-Src and that Src activation is required for VEGF induction of phospholipase C gamma1 activation and inositol 1,4,5-trisphosphate formation. Reporter cell assays showed that VEGF promotes a approximately 50-fold increase in NO formation, which peaks at 5-20 min. This effect is mediated by a signaling cascade initiated by flk-1/KDR activation of c-Src, leading to phospholipase C gamma1 activation, inositol 1,4,5-trisphosphate formation, release of [Ca(2+)](i) and nitric oxide synthase activation. Immunoassays of VEGF-induced 6-keto prostaglandin F(1alpha) formation as an indicator of PGI(2) production revealed a 3-4-fold increase that peaked at 45-60 min. The PGI(2) signaling pathway follows the NO pathway through release of [Ca(2+)](i), but diverges prior to NOS activation and also requires activation of mitogen-activated protein kinase. These results suggest that NO and PGI(2) function in parallel in mediating the effects of VEGF.     

Extracellular histones disarrange vasoactive mediators release through a COX‐NOS interaction in human endothelial cells

Extracellular histones are mediators of inflammation, tissue injury and organ dysfunction. Interactions between circulating histones and vascular endothelial cells are key events in histone‐mediated pathologies. Our aim was to investigate the implication of extracellular histones in the production of the major vasoactive compounds released by human endothelial cells (HUVECs), prostanoids and nitric oxide (NO). HUVEC exposed to increasing concentrations of histones (0.001 to 100 μg/ml) for 4 hrs induced prostacyclin (PGI2) production in a dose‐dependent manner and decreased thromboxane A2 (TXA2) release at 100 μg/ml. Extracellular histones raised cyclooxygenase‐2 (COX‐2) and prostacyclin synthase (PGIS) mRNA and protein expression, decreased COX‐1 mRNA levels and did not change thromboxane A2 synthase (TXAS) expression. Moreover, extracellular histones decreased both, eNOS expression and NO production in HUVEC. The impaired NO production was related to COX‐2 activity and superoxide production since was reversed after celecoxib (10 μmol/l) and tempol (100 μmol/l) treatments, respectively. In conclusion, our findings suggest that extracellular histones stimulate the release of endothelial‐dependent mediators through an up‐regulation in COX‐2‐PGIS‐PGI2 pathway which involves a COX‐2‐dependent superoxide production that decreases the activity of eNOS and the NO production. These effects may contribute to the endothelial cell dysfunction observed in histone‐mediated pathologies.     

Cyclic stretch induces cyclooxygenase-2 gene expression in vascular endothelial cells via activation of nuclear factor kappa-β

Vascular endothelial cells respond to biomechanical forces, such as cyclic stretch and shear stress, by altering gene expression. Since endothelial-derived prostanoids, such as prostacyclin and thromboxane A₂, are key mediators of endothelial function, we investigated the effects of cyclic stretch on the expression of genes in human umbilical vein endothelial cells controlling prostanoid synthesis: cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), prostacyclin synthase (PGIS) and thromboxane A₂ synthase (TXAS). COX-2 and TXAS mRNAs were upregulated by cyclic stretch for 24 h. In contrast, PGIS mRNA was decreased and stretch had no effect on COX-1 mRNA expression. We further show that stretch-induced upregulation of COX-2 is mediated by activation of the NF-κβ signaling pathway.     

Recruitment of endothelial caveolae into mechanotransduction pathways by flow conditioning in vitro

The luminal surface of rat lung microvascular endothelial cells in situ is sensitive to changing hemodynamic parameters. Acute mechanosignaling events initiated in response to flow changes in perfused lung microvessels are localized within specialized invaginated microdomains called caveolae. Here we report that chronic exposure to shear stress alters caveolin expression and distribution, increases caveolae density, and leads to enhanced mechanosensitivity to subsequent changes in hemodynamic forces within cultured endothelial cells. Flow-preconditioned cells expressed a fivefold increase in caveolin (and other caveolar-residing proteins) at the luminal surface compared with no-flow controls. The density of morphologically identifiable caveolae was enhanced sixfold at the luminal cell surface of flow-conditioned cells. Laminar shear stress applied to static endothelial cultures (flow step of 5 dyn/cm2), enhanced the tyrosine phosphorylation of luminal surface proteins by 1.7-fold, including caveolin-1 by 1.3-fold, increased Ser1179 phosphorylation of endothelial nitric oxide synthase (eNOS) by 2.6-fold, and induced a 1.4-fold activation of mitogen-activated protein kinases (ERK1/2) over no-flow controls. The same shear step applied to endothelial cells preconditioned under 10 dyn/cm2 of laminar shear stress for 6 h and induced a sevenfold increase of total phosphotyrosine signal at the luminal endothelial cell surface enhanced caveolin-1 tyrosine phosphorylation 5.8-fold and eNOS phosphorylation by 3.3-fold over static control values. In addition, phosphorylated caveolin-1 and eNOS proteins were preferentially localized to caveolar microdomains. In contrast, ERK1/2 activation was not detected in conditioned cells after acute shear challenge. These data suggest that cultured endothelial cells respond to a sustained flow environment by directing caveolae to the cell surface where they serve to mediate, at least in part, mechanotransduction responses.     

Effects of shear stress on eicosanoid gene expression and metabolite production in vascular endothelium as studied in a novel biomechanical perfusion model

This study investigated the effects of shear stress on gene expression of prostacyclin synthesis-related enzymes cyclooxygenases (COX-1 and COX-2), prostacyclin synthase (PGS), and thromboxane synthase (TXS) and their metabolites prostaglandin (PGI(2)) and thromboxane A(2) (TXA(2)) in endothelium of intact conduit vessels. Paired human umbilical veins were perfused at high/low shear stress (25/<4 dyn/cm(2)) at identical intraluminal pressure (20 mmHg) for 1.5, 3, or 6 hours in a computerized vascular model. High shear perfusion induced a significant, monophasic upregulation of PGS and TXS gene expressions after 6 hours. COX-1 and COX-2 mRNA showed a biphasic response with peaks at 1.5 and 6 hours, with a nadir level at 3 hours. Shear-induced gene expression was associated with a significantly greater accumulation of 6-keto prostaglandin F(1alpha) and TXA(2) in the perfusion medium. Thus, shear stress independently of perfusion pressure alters the expression of prostacyclin synthesis-related enzymes and the biosynthesis of their vasoactive metabolites.     

Functional alterations in endothelial NO, PGI₂ and EDHF pathways in aorta in ApoE/LDLR⁻/⁻ mice

Adequate endothelial production of nitric oxide (NO), endothelium-derived hyperpolarizing factor (EDHF), and prostacyclin (PGI?) is critical to the maintenance of vascular homeostasis. However, it is not clear whether alterations in each of these vasodilatory pathways contribute to the impaired endothelial function in murine atherosclerosis. In the present study, we analyze the alterations in NO-, EDHF- and PGI?-dependent endothelial function in the thoracic aorta in relation to the development of atherosclerotic plaques in apoE/LDLR?/? mice. We found that in the aorta of 2-month-old apoE/LDLR?/? mice there was no lipid deposition, subendothelial macrophage accumulation; and matrix metalloproteinase (MMP) activity was low, consistent with the absence of atherosclerotic plaques. Interestingly, at this stage the endothelium was already activated and hypertrophic as evidenced by electron microscopy, while acetylcholine-induced NO-dependent relaxation in the thoracic aorta was impaired, with concomitant upregulation of cyclooxygenase-2 (COX-2)/PGI? and EDHF (epoxyeicosatrienoic acids, EETs) pathways. In the aorta of 3-6-month-old apoE/LDLR?/? mice, lipid deposition, macrophage accumulation and MMP activity in the intima were gradually increased, while impairment of NO-dependent function and compensatory upregulation of COX-2/PGI? and EDHF pathways were more accentuated. These results suggest that impairment of NO-dependent relaxation precedes the development of atherosclerosis in the aorta and early upregulation of COX-2/PGI? and EDHF pathways may compensate for the loss of the biological activity of NO.     

Globular adiponectin counteracts VCAM-1-mediated monocyte adhesion via AdipoR1/NF-κB/COX-2 signaling in human aortic endothelial cells

Adiponectin (Ad) is an insulin-sensitizing adipocytokine with anti-inflammatory and vasoprotective properties. Cleavage of native full-length Ad (fAd) by elastases from activated monocytes generates globular Ad (gAd). Increased gAd levels are observed in the proximity of atherosclerotic lesions, but the physiological meaning of this proteolytic Ad fragment in the cardiovascular system is controversial. We compared molecular and biological properties of fAd and gAd in human aortic endothelial cells (HAEC). In control HAEC, both fAd and gAd acutely stimulated nitric oxide (NO) production by AMPK-dependent pathways. With respect to fAd, gAd more efficiently increased activation of NF-κB signaling pathways, resulting in cyclooxygenase-2 (COX-2) overexpression and COX-2-dependent prostacyclin 2 (PGI(2)) release. In contrast with fAd, gAd also increased p38 MAPK phosphorylation and VCAM-1 expression, ultimately enhancing adhesion of monocytes to endothelial cells. In HAEC lacking AdipoR1 (by siRNA), both activation of NF-κB as well as COX-2 overexpression by gAd were abrogated. Conversely, gAd-mediated p38MAPK activation and VCAM-1 expression were unaffected, and monocyte adhesion was greatly enhanced. In HAEC lacking COX-2 (by siRNA), reduced levels of PGI(2) further increased gAd-dependent monocyte adhesion. Our findings suggest that biological activities of fAd and gAd in endothelium do not completely overlap, with gAd possessing both AdipoR1-dependent ability to stimulate COX-2 expression and AdipoR1-independent effects related to expression of VCAM-1 and adhesion of monocytes to endothelium.     

An involvement of SR-B1 mediated PI3K-Akt-eNOS signaling in HDL-induced cyclooxygenase 2 expression and prostacyclin production in endothelial cells

It is well-known that sphingosine-1-phosphate (S1P), the phospholipid content of HDL, binding to S1P receptors can raise COX-2 expression and PGI(2) release through p38MAPK/CREB pathway. In the present study we assess the action of SR-B1 initiated PI3K-Akt-eNOS signaling in the regulation of COX-2 expression and PGI(2) production in response to HDL. We found that apoA1 could increase PGI(2) release and COX-2 expression in ECV 304 endothelial cells. Furthermore, SR-B1 was found to be involved in HDL induced up-regulation of COX-2 and PGI(2). Over-expressed SR-B1 did not significantly increase the expression of COX-2 and the PGI(2) levels, but knock-down of SR-B1 by siRNA could significantly attenuate COX-2 expression and PGI(2) release together with p38MAPK and CREB phosphorylation. Consistently, the declines of p-p38MAPK, p-CREB, COX-2 and PGI(2) were also observed after incubation with LY294002 (25μmol/L; PI3K special inhibitor) or L-NAME (50μmol/L; eNOS special inhibitor). In addition, we demonstrated the increases of PGI(2) release, COX-2 expression and p38MAPK phosphorylation, when nitric oxide level was raised through the incubation of L-arginine (10 or 20nmol/L) in endothelial cells. Taking together, our data support that SR-B1 mediated PI3K-Akt-eNOS signaling was involved in HDL-induced COX-2 expression and PGI(2) release in endothelial cells.