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Background— Upregulated by atheroprotective flow, the transcription factor Krüppel-like factor 2 (KLF2) is crucial for maintaining endothelial function. MicroRNAs (miRNAs) are noncoding small RNAs that regulate gene expression at the posttranscriptional level. We examined the role of miRNAs, particularly miR-92a, in the atheroprotective flow–regulated KLF2. Methods and Results— Dicer knockdown increased the level of KLF2 mRNA in human umbilical vein endothelial cells, suggesting that KLF2 is regulated by miRNA. In silico analysis predicted that miR-92a could bind to the 3′ untranslated region of KLF2 mRNA. Overexpression of miR-92a decreased the expression of KLF2 and the KLF2-regulated endothelial nitric oxide synthase and thrombomodulin at mRNA and protein levels. A complementary finding is that miR-92a inhibitor increased the mRNA and protein expression of KLF2, endothelial nitric oxide synthase, and thrombomodulin. Subsequent studies revealed that atheroprotective laminar flow downregulated the level of miR-92a precursor to induce KLF2, and the level of this flow-induced KLF2 was reduced by miR-92a precursor. Furthermore, miR-92a level was lower in human umbilical vein endothelial cells exposed to the atheroprotective pulsatile shear flow than under atheroprone oscillatory shear flow. Anti-Ago1/2 immunoprecipitation coupled with real-time polymerase chain reaction revealed that pulsatile shear flow decreased the functional targeting of miR-92a precursor/KLF2 mRNA in human umbilical vein endothelial cells. Consistent with these findings, mouse carotid arteries receiving miR-92a precursor exhibited impaired vasodilatory response to flow. Conclusions— Atheroprotective flow patterns decrease the level of miR-92a, which in turn increases KLF2 expression to maintain endothelial homeostasis.

Migration and proliferation of endothelial cells are involved in re-endothelialization and angiogenesis, two important cardiovascular processes that are increased in response to estrogens. RhoA, a small GTPase which controls multiple cellular processes, is involved in the control of cell migration and proliferation. Our aim was to study the role of RhoA on estradiol-induced migration and proliferation and its dependence on estrogen receptors activity. Human umbilical vein endothelial cells were stimulated with estradiol, in the presence or absence of ICI 182780 (estrogen receptors antagonist) and Y-27632 (Rho kinase inhibitor). Estradiol increased Rho GEF-1 gene expression and RhoA (gene and protein expression and activity) in an estrogen receptor-dependent manner. Cell migration, stress fiber formation and cell proliferation were increased in response to estradiol and were also dependent on the estrogen receptors and RhoA activation. Estradiol decreased p27 levels, and significantly raised the expression of cyclins and CDK. These effects were counteracted by the use of either ICI 182780 or Y-27632. In conclusion, estradiol enhances the RhoA/ROCK pathway and increases cell cycle-related protein expression by acting through estrogen receptors. This results in an enhanced migration and proliferation of endothelial cells.

Estradiol (E2) acts on the endothelium to promote vasodilatation through the release of several compounds, including prostanoids, which are products of arachidonic acid metabolism. Among these, prostacyclin (PGI2) and thromboxane A2 (TXA2) exert opposite effects on vascular tone. The role of different estrogen receptors (ERs) in the PGI2/TXA2 balance, however, has not been fully elucidated. Our study sought to uncover whether E2 enhances basal production of PGI2 or TXA2 in cultured human umbilical vein endothelial cells (HUVECs), to analyze the enzymatic mechanisms involved, and to evaluate the different roles of both types of ERs (ERα and ERβ). HUVECs were exposed to E2, selective ERα (1,3,5-tris(4-hydroxyphenyl)-4-propyl-1h-pyrazole, PPT) or ERβ (diarylpropionitrile, DPN) agonists and antagonists (unspecific: ICI 182 780; specific for ERα: methyl-piperidino-pyrazole, MPP). PGI2 and TXA2 production was measured by ELISA. Expression of phospholipases, cyclooxygenases (COX-1 and COX-2), PGI2 synthase (PGIS), and thromboxane synthase (TXAS) was analyzed by western blot and quantitative RT-PCR. E2 (1–100 nM) dose dependently increased PGI2 production (up to 50%), without affecting TXA2 production. COX-1 and PGIS protein and gene expressions were increased, whereas COX-2, phospholipases, and TXAS expression remained unaltered. All these effects were mediated through ERα, since they were produced not only in the presence of E2, but also in that of PPT, while they were abolished in the presence of MPP. In conclusion, E2, acting through ERα, up-regulates COX-1 and PGIS expression, thus directing prostanoid balance toward increased PGI2 production.

Oxidized low density lipoproteins (oxLDLs) act as an etiological factor in the development of atherosclerosis by modifying the biological properties of endothelial cells through mechanisms of vascular inflammation. To deepen the oxLDL changes at cellular level, a transcriptomic analysis of human umbilical artery endothelial cells (HUAECs) treated with oxLDL was performed to identify the modified signaling pathways. Total RNA was isolated from HUAECs treated with oxLDL (100 μg/ml). Gene expression analysis was carried out using Affymetrix oligonucleotide microarrays. Biological pathway analysis was performed using Ingenuity Pathway Analysis software. Microarray assay demonstrated that oxLDL strongly affected two metabolic and signaling transduction pathways: the biosynthesis of steroids, via modification of nine genes that act sequentially in this metabolic pathway, and the Jak-Stat signaling pathway acting through STAT1 and STAT2. By means of qPCR, immunoblot, RNA interference and inhibitors we demonstrate that the mechanism used by oxLDL to activate Jak-Stat signaling pathway in artery endothelial cells is mainly mediated by STAT1. These findings provide a new mechanistic framework to better understand the effects that oxLDLs exert in artery endothelial cell gene expression and provide a source of hypothesis to understand the involvement of oxLDL in diseases in which endothelial cells play a key role, such as atherosclerosis.

Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide (NO) synthase. ADMA accumulation, mainly due to a decreased dimethylarginine dimethylaminohydrolase (DDAH) activity, has been related to the development of cardiovascular diseases. We investigate whether estradiol prevents the changes induced by oxidized low density lipoprotein (oxLDL) on the DDAH/ADMA/NO pathway in human umbilical artery endothelial cells (HUAEC). HUAEC were exposed to estradiol, native LDL (nLDL), oxLDL and their combinations for 24 h. In some experiments, cells were also exposed to the unspecific estrogen receptor (ER) antagonist ICI 182780, the specific ERα antagonist MPP or specific agonists for ERα, ERβ and GPER. ADMA concentration was measured by HPLC and concentration of NO by amperometry. Protein expression and DDAH activity were measured by immunoblotting and an enzymatic method, respectively. oxLDL, but not nLDL, increased ADMA concentration with a concomitant decrease on DDAH activity. oxLDL reduced eNOS protein and NO production. Estradiol alone had no effects on DDAH/ADMA/NO pathway, but increased the attenuated endothelial NO production induced by oxLDL by reduction in ADMA and preventing loss of eNOS protein levels. ICI 182780 and MPP completely abolished these effects of estradiol on oxLDL-exposed cells. ERα agonist, but not ERβ and GPER agonists, mirrored estradiol effects on NO production. In conclusion, estradiol restores (1) DDAH activity, and therefore ADMA levels, and (2) NO production impaired by oxLDL in HUAEC acting through ERα.

Objectives Progestogens have been poorly studied concerning their roles in endothelial physiology. Prostanoids are vasoactive compounds, such as thromboxane A2, a potent vasoconstrictor, and prostacyclin, a vasodilator. We examined the effects of two progestogens used clinically, progesterone and medroxyprogesterone acetate, on thromboxane A2 production by cultured human umbilical vein endothelial cells (HUVEC) and investigated the role of progesterone receptors and the enzymes involved in production of thromboxane A2 and prostacyclin. Methods Cells were exposed to 1‐100 nmol/l of either progesterone or medroxyprogesterone acetate, and thromboxane A2 production was measured in culture medium by enzyme immunoassay. Gene expression of prostacyclin synthase and thromboxane synthase was analyzed by quantitative real-time polymerase chain reaction. Expression of prostacyclin synthase protein was analyzed by Western blot. Results Both progestogens decreased thromboxane A2 release after 24 h. Protein and gene expression of prostacyclin synthase were increased after exposure to both progestogens, without changes in thromboxane synthase expression. These effects induced by progestogens were mediated through progesterone receptors, since they were decreased in the presence of the progesterone receptor antagonist RU486. The cyclooxygenase-1 selective inhibitor reduced thromboxane release. Conclusion Progesterone and medroxyprogesterone acetate decreased HUVEC thromboxane release in a progesterone receptor-dependent manner, without changes in thromboxane synthase expression and enhanced prostacyclin synthase gene and protein expression.

Vascular effects of estradiol are being investigated because there are controversies among clinical and experimental studies. DNA microarrays were used to investigate global gene expression patterns in cultured human umbilical vein endothelial cells (HUVEC) exposed to 1 nmol/L estradiol for 24 hours. When compared to control, 187 genes were identified as differentially expressed with 1.9-fold change threshold. Supervised principal component analysis and hierarchical cluster analysis revealed the differences between control and estradiol-treated samples. Physiological concentrations of estradiol are sufficient to elicit significant changes in HUVEC gene expression. Notch signaling, actin cytoskeleton signaling, pentose phosphate pathway, axonal guidance signaling and integrin signaling were the top-five canonical pathways significantly regulated by estrogen. A total of 26 regulatory networks were identified as estrogen responsive. Microarray data were confirmed by quantitative RT-PCR in cardiovascular meaning genes; cyclooxigenase (COX)1, dimethylarginine dimethylaminohydrolase (DDAH)2, phospholipase A2 group IV (PLA2G4) B, and 7-dehydrocholesterol reductase were up-regulated by estradiol in a dose-dependent and estrogen receptor-dependent way, whereas COX2, DDAH1 and PLA2G4A remained unaltered. Moreover, estradiol-induced COX1 gene expression resulted in increased COX1 protein content and enhanced prostacyclin production. DDAH2 protein content was also increased, which in turn decreased asymmetric dimethylarginine concentration and increased NO release. All stimulated effects of estradiol on gene and protein expression were estrogen receptor-dependent, since were abolished in the presence of the estrogen receptor antagonist ICI 182780. This study identifies new vascular mechanisms of action by which estradiol may contribute to a wide range of biological processes.