5,2′‐dibromo‐2,4′,5′‐trihydroxydiphenylmethanone attenuates LPS‐induced inflammation and ROS production in EA.hy926 cells via HMBOX1 induction

Inflammation and reactive oxygen species (ROS) are important factors in the pathogenesis of atherosclerosis (AS). 5,2′‐dibromo‐2,4′,5′‐trihydroxydiphenylmethanone (TDD), possess anti‐atherogenic properties; however, its underlying mechanism of action remains unclear. Therefore, we sought to understand the therapeutic molecular mechanism of TDD in inflammatory response and oxidative stress in EA.hy926 cells. Microarray analysis revealed that the expression of homeobox containing 1 (HMBOX1) was dramatically upregulated in TDD‐treated EA.hy926 cells. According to the gene ontology (GO) analysis of microarray data, TDD significantly influenced the response to lipopolysaccharide (LPS); it suppressed the LPS‐induced adhesion of monocytes to EA.hy926 cells. Simultaneously, TDD dose‐dependently inhibited the production or expression of IL‐6, IL‐1β, MCP‐1, TNF‐α, VCAM‐1, ICAM‐1 and E‐selectin as well as ROS in LPS‐stimulated EA.hy926 cells. HMBOX1 knockdown using RNA interference attenuated the anti‐inflammatory and anti‐oxidative effects of TDD. Furthermore, TDD inhibited LPS‐induced NF‐κB and MAPK activation in EA.hy926 cells, but this effect was abolished by HMBOX1 knockdown. Overall, these results demonstrate that TDD activates HMBOX1, which is an inducible protective mechanism that inhibits LPS‐induced inflammation and ROS production in EA.hy926 cells by the subsequent inhibition of redox‐sensitive NF‐κB and MAPK activation. Our study suggested that TDD may be a potential novel agent for treating endothelial cells dysfunction in AS.     

Transient receptor potential canonical channels are required for in vitro endothelial tube formation

In endothelial cells Ca(2+) entry is an essential component of the Ca(2+) signal that takes place during processes such as cell proliferation or angiogenesis. Ca(2+) influx occurs via the store-operated Ca(2+) entry pathway, involving stromal interaction molecule-1 (STIM1) and Orai1, but also through channels gated by second messengers like the transient receptor potential canonical (TRPC) channels. The human umbilical vein-derived endothelial cell line EA.hy926 expressed STIM1 and Orai1 as well as several TRPC channels. By invalidating each of these molecules, we showed that TRPC3, TRPC4, and TRPC5 are essential for the formation of tubular structures observed after EA.hy926 cells were plated on Matrigel. On the contrary, the silencing of STIM1 or Orai1 did not prevent tubulogenesis. Soon after being plated on Matrigel, the cells displayed spontaneous Ca(2+) oscillations that were strongly reduced by treatment with siRNA against TRPC3, TRPC4, or TRPC5, but not siRNA against STIM1 or Orai1. Furthermore, we showed that cell proliferation was reduced upon siRNA treatment against TRPC3, TRPC5, and Orai1 channels, whereas the knockdown of STIM1 had no effect. On primary human umbilical vein endothelial cells, TRPC1, TRPC4, and STIM1 are involved in tube formation, whereas Orai1 has no effect. These data showed that TRPC channels are essential for in vitro tubulogenesis, both on endothelial cell line and on primary endothelial cells.     

G1对EA.hy926内皮细胞内质网应激的抑制作用

目的观察GPR30受体激动剂G1对高糖诱导的EA.hy926内皮细胞内质网应激(endoplasmic reticulum stress,ERS)的影响。方法选用EA.hy926内皮细胞为研究对象,分为3组:正常对照组(Con,17.51mmol/L葡萄糖)、高糖组(HG,33.3mmol/L葡萄糖)、高糖+G1组(HG+G1,HG+1umol/L G1),利用流式细胞术检测3组细胞凋亡率,Western blot法检测ERS相关分子Bip、IRE1、PERK及凋亡分子Bax、Bcl-2的表达变化,RT-PCR法检测Bip和CHOP的mRNA表达变化。结果 HG组与Con组比较,细胞凋亡率明显升高(P0.01),Bip、IRE1、PERK及凋亡分子Bax表达上调(P0.01,P0.05或P0.001),Bcl-2的表达下调(P0.01),Bip mRNA、CHOP mRNA表达上调(P0.001及P0.01);HG+G1组与HG组比较,细胞凋亡率明显降低(P0.05),Bip、IRE1、PERK及凋亡分子Bax表达下调(P0.05或P0.01),Bcl-2的表达上调(P0.05),Bip mRNA、CHOP mRNA表达下调(P0.001及P0.01)。结论 GPR30受体激动剂G1可抑制EA.hy926内皮细胞内质网应激。     

源自天蚕素A和爪蟾素2的杂合肽P18体外抑制内皮细胞血管生成

目的探讨杂合肽P18体外对内皮细胞EA.hy926血管生成的抑制作用.方法采用MTT法检测P18对EA.hy926细胞增殖的影响;应用Matrigel实验检测P18对内皮细胞形成管状结构的影响;利用流式细胞术分析P18对内皮细胞的损伤作用.结果 MTT结果显示P18可明显抑制EA.hy926细胞的增殖,且抑制率存在剂量依赖性;Matrigel实验表明P18具有抑制EA.hy926细胞体外分化成管状结构的作用;流式结果显示15 μM P18作用内皮细胞6 h后,所诱导的细胞坏死比例达到81.4%.结论体外实验结果表明,杂合肽P18具有体外抑制EA.hy926细胞血管生成的作用.     

Differential roles of PKCalpha and PKCepsilon in controlling the gene expression of Nox4 in human endothelial cells

NADPH oxidases are major sources of superoxide in the vascular wall. This study investigates the role of protein kinase C (PKC) in regulating gene expression of NADPH oxidases. Treatment of human umbilical vein endothelial cells (HUVEC) and HUVEC-derived EA.hy 926 endothelial cells with phorbol 12-myristate 13-acetate (PMA) or phorbol 12,13-dibutyrate led to a PKC-dependent biphasic expression of the gp91phox homolog Nox4. A downregulation of Nox4 was observed at 6 h and an upregulation at 48 h after phorbol ester treatment. The early Nox4 downregulation was associated with a reduced superoxide production, whereas the late Nox4 upregulation was accompanied by a clear enhancement of superoxide. PMA activated the PKC isoforms alpha and epsilon in HUVEC and EA.hy 926 cells. Knockdown of PKCepsilon by siRNA prevented the early downregulation of Nox4, whereas knockdown of PKCalpha selectively abolished the late Nox4 upregulation. Vascular endothelial growth factor (VEGF), which activates PKCalpha but not PKCepsilon in HUVEC, increased Nox4 expression without the initial downregulation. VEGF-induced Nox4 upregulation was associated with an enhanced proliferation and angiogenesis of HUVEC. Both effects could be reduced by inhibition of NADPH oxidase. Thus, a selective inhibition/knockdown of PKCalpha may represent a novel therapeutic strategy for vascular disease.     

Paracrine or virus-mediated induction of decorin expression by endothelial cells contributes to tube formation and prevention of apoptosis in collagen lattices

Resting endothelial cells express the small proteoglycan biglycan, whereas sprouting endothelial cells also synthesize decorin, a related proteoglycan. Here we show that decorin is expressed in endothelial cells in human granulomatous tissue. For in vitro investigations, the human endothelium-derived cell line, EA.hy 926, was cultured for 6 or more days in the presence of 1% fetal calf serum on top of or within floating collagen lattices which were also populated by a small number of rat fibroblasts. Endothelial cells aligned in cord-like structures and developed cavities that were surrounded by human decorin. About 14% and 20% of endothelial cells became apoptotic after 6 and 12 days of co-culture, respectively. In the absence of fibroblasts, however, the extent of apoptosis was about 60% after 12 days, and cord-like structures were not formed nor could decorin production be induced. This was also the case when lattices populated by EA.hy 926 cells were maintained under one of the following conditions: 1) 10% fetal calf serum; 2) fibroblast-conditioned media; 3) exogenous decorin; or 4) treatment with individual growth factors known to be involved in angiogenesis. The mechanism(s) by which fibroblasts induce an angiogenic phenotype in EA.hy 926 cells is (are) not known, but a causal relationship between decorin expression and endothelial cell phenotype was suggested by transducing human decorin cDNA into EA.hy 926 cells using a replication-deficient adenovirus. When the transduced cells were cultured in collagen lattices, there was no requirement of fibroblasts for the formation of capillary-like structures and apoptosis was reduced. Thus, decorin expression seems to be of special importance for the survival of EA.hy 926 cells as well as for cord and tube formation in this angiogenesis model.     

Advanced glycation end-products disrupt human endothelial cells redox homeostasis: new insights into reactive oxygen species production

Advanced glycation end-products (AGEs) trigger multiple metabolic disorders in the vessel wall that may in turn lead to endothelial dysfunction. The molecular mechanisms by which AGEs generate these effects are not completely understood. Oxidative stress plays a key role in the development of deleterious effects that occur in endothelium during diabetes. Our main objectives were to further understand how AGEs contribute to reactive oxygen species (ROS) overproduction in endothelial cells and to evaluate the protective effect of an antioxidant plant extract. The human endothelial cell line EA.hy926 was treated with native or modified bovine serum albumin (respectively BSA and BSA-AGEs). To monitor free radicals formation, we used H₂DCF-DA, dihydroethidium (DHE), DAF-FM-DA and MitoSOX Red dyes. To investigate potential sources of ROS, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and mitochondrial inhibitors were used. The regulation of different types of ROS by the polyphenol-rich extract from the medicinal plant Doratoxylon apetalum was also studied for a therapeutic perspective. BSA-AGEs exhibited not only less antioxidant properties than BSA, but also pro-oxidant effects. The degree of albumin glycoxidation directly influenced oxidative stress through a possible communication between NADPH oxidase and mitochondria. D. apetalum significantly decreased intracellular hydrogen peroxide and superoxide anions mainly detected by H₂DCF-DA and DHE respectively. Our results suggest that BSA-AGEs promote a marked oxidative stress mediated at least by NADPH oxidase and mitochondria. D. apetalum plant extract appeared to be an effective antioxidant compound to protect endothelial cells.     

Variability in E-selectin expression, mRNA levels and sE-selectin release between endothelial cell lines and primary endothelial cells

Endothelial cell lines express markers and are assumed to exhibit other endothelial cell responses. We investigated E-selectin expression from human umbilical vein endothelial cells, the spontaneously transformed ECV304 line and the hybrid line EA.hy926 by flow cytometry and immunofluorescence, mRNA and soluble E-selectin release. In cells exposed to tumour necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1beta), median (range) percentage of E-selectin-positive HUVECs increased from 1.6(0.9-6. 2)% to 91.4(83.0-96.1)%, (P=0.001) using flow cytometry. In contrast, E-selectin expression by ECV304 and EA.hy926 cell lines was 100-fold lower. E-selectin mRNA was detectable after 2 h, maximal at 6 h in HUVECs and undetectable in EA.hy926 and ECV304 cell lines after exposure to TNF-alpha/IL-1beta. sE-selectin accumulation increased (P=0.004) in HUVECs only. Neutrophil adherence to ECV304 and EA.hy926 cells was poor compared to HUVECs (P=0.004). The cell lines ECV304 and EA.hy926 do not exhibit normal endothelium expression of E-selectin, and may not be appropriate for studies of adhesion.     

Plasma membrane microdomains regulate TACE-dependent TNFR1 shedding in human endothelial cells

Upon stimulation by histamine, human vascular endothelial cells (EC) shed a soluble form of tumour necrosis factor receptor 1 (sTNFR1) that binds up free TNF, dampening the inflammatory response. Shedding occurs through proteolytic cleavage of plasma membrane-expressed TNFR1 catalysed by TNF-α converting enzyme (TACE). Surface expressed TNFR1 on EC is largely sequestered into specific plasma membrane microdomains, the lipid rafts/caveolae. The purpose of this study was to determine the role of these domains in TACE-mediated TNFR1 shedding in response to histamine. Human umbilical vein endothelial cells derived EA.hy926 cells respond to histamine via H1 receptors to shed TNFR1. Both depletion of cholesterol by methyl-β-cyclodextrin and small interfering RNA knockdown of the scaffolding protein caveolin-1 (cav-1), treatments that disrupt caveolae, reduce histamine-induced shedding of membrane-bound TNFR1. Moreover, immunoblotting of discontinuous sucrose gradient fractions show that TACE, such as TNFR1, is present within low-density membrane fractions, concentrated within caveolae, in unstimulated EA.hy926 endothelial cells and co-immunoprecipitates with cav-1. Silencing of cav-1 reduces the levels of both TACE and TNFR1 protein and displaces TACE, from low-density membrane fractions where TNFR1 remains. In summary, we show that endothelial lipid rafts/caveolae co-localize TACE to surface expressed TNFR1, promoting efficient shedding of sTNFR1 in response to histamine.     

Blockade of endothelin-1 release contributes to the anti-angiogenic effect by pro-opiomelanocortin overexpression in endothelial cells

Pro-opiomelanocortin (POMC) is the precursor of several neuropeptides, such as corticotropin (ACTH), alpha-melanocyte-stimulating hormone (MSH), and the endogenous opioid, beta-endorphin (EP). ACTH-dependent Cushing's syndrome is characterized by ACTH overproduction and is associated with an increased risk of cardiovascular disease. Endothelial dysfunction has been recognized as an early marker of cardiovascular disease. However, the mechanism underlying endothelial dysfunction by ACTH overexpression in Cushing's patients remains elusive. Endothelial cells, the primary cells producing endothelin (ET)-1, are both the source and target of POMC-derived peptides. In the present study, we generated adenovirus vectors (Ad) encoding POMC (Ad-POMC) and green fluorescent protein (GFP; Ad-GFP) to investigate whether POMC gene transfer altered the ET-1 homeostasis and angiogenic functions in human EA.hy926 endothelial cells. Via adenovirus gene delivery, the POMC-transduced EA.hy926 cells released significantly elevated ACTH and beta-EP levels (P < 0.001). In addition, POMC gene delivery significantly decreased the ET-1 release (P < 0.001) without affecting the ET-1 messenger RNA (mRNA) level. Despite no effect on the secretion of matrix metalloproteinases (MMPs) and cell proliferation, POMC gene delivery significantly inhibited the migration (P < 0.01) and tube-forming capability (P < 0.01) of endothelial cells. Moreover, the POMC-induced inhibition of tube formation could be partially reversed by adding exogenous ET-1 (P < 0.05). In summary, the attenuated ET-1 release and angiogenic processes by POMC overexpression may contribute to endothelial dysfunction, thereby providing a link between Cushing's syndrome and cardiovascular diseases.     

Adaptive Induction of Growth Differentiation Factor 15 Attenuates Endothelial Cell Apoptosis in Response to High Glucose Stimulus

Growth differentiation factor 15 (GDF15), a direct target gene of p53, is a multifunctional member of the TGF-β/BMP superfamily. GDF15 can be induced and is implicated as a key secretory cytokine in response to multiple cellular stimuli. Accumulating evidence indicates that GDF15 is associated with the development and prognosis of diabetes mellitus, while whether GDF15 can be induced by high glucose is unknown. In the present study, we revealed that high glucose could induce GDF15 expression and secretion in cultured human umbilical vein endothelial cells in a ROS- and p53-dependent manner. Inhibition of high glucose-induced GDF15 expression by siRNA demonstrated that adaptively induced GDF15 played a protective role against high glucose-induced human umbilical vein endothelial cell apoptosis via maintaining the active state of PI3K/Akt/eNOS pathway and attenuating NF-κB/JNK pathway activation. The protective effects of GDF15 were probably achieved by inhibiting ROS overproduction in high glucose-treated human umbilical vein endothelial cells in a negative feedback manner. Our results suggest that high glucose can promote GDF15 expression and secretion in human umbilical vein endothelial cells, which in turn attenuates high glucose-induced endothelial cell apoptosis.     

Increased glyoxalase I levels inhibit accumulation of oxidative stress and an advanced glycation end product in mouse mesangial cells cultured in high glucose

Chronic high glucose levels lead to the formation of advanced glycation end-products (AGEs) as well as AGE precursors, such as methylglyoxal (MG) and glyoxal, via non-enzymatic glycation reactions in patients with diabetic mellitus. Glyoxalase 1 (GLO-1) detoxifies reactive dicarbonyls that form AGEs. To investigate the interaction between AGEs and GLO-1 in mesangial cells (MCs) under diabetic conditions, AGE levels and markers of oxidative stress were measured in GLO-1-overexpressing MCs (GLO-1-MCs) cultured in high glucose. Furthermore, we also examined levels of high glucose-induced apoptosis in GLO-1-MCs. In glomerular MCs, high glucose levels increased the formation of both MG and argpyrimidine (an MG-derived adduct) as well as GLO-1 expression. GLO-1-MCs had lower intracellular levels of MG accumulation, 8-hydroxy-deoxyguanosine (an oxidative DNA damage marker), 4-hydroxyl-2-nonenal (a lipid peroxidation product), and nitrosylated protein (a marker of oxidative-nitrosative stress) compared to control cells. Expression of mitochondrial oxidative phosphorylation complexes I, II, and III was also decreased in GLO-1-MCs. Furthermore, fewer GLO-1-MCs showed evidence of apoptosis as determined by terminal deoxynucleotidyl transferase-mediated dUTP nick labeling assay, and activation of both poly (ADP-ribose) polymerase 1 cleavage and caspase-3 was lower in GLO-1-MCs than in control cells cultured in high glucose. These results suggest that GLO-1 plays a role in high glucose-mediated signaling by reducing MG accumulation and oxidative stress in diabetes mellitus.     

Adrenomedullin promotes human endothelial cell proliferation via HIF-1α

Adrenomedullin (ADM) and hypoxia-inducible factor-1α (HIF-1α) are important pro-proliferation genes in response to hypoxic stress. Although it was reported that ADM is a target gene for HIF-1, recent studies also showed that ADM regulates HIF-1 expression and its activity; however, the mechanism of action remains unknown. Two stable human endothelial cell lines with HIF-1α knockdown by hy926-siHIF-1α or HMEC-siHIF-1α were established. mRNA and protein expression of ADM and HIF-1α in EA.hy926 and HMEC1 cells were examined under hypoxic stress. Upon ADM treatment, cell proliferation was investigated and the expression profiles of HIF-1α and its target genes (VEGF, PFKP, PGK1, and AK1) were examined. Furthermore, the proline hydroxylase (PHD) mRNA level and its activity were investigated. We observed that mRNA and protein expression of ADM in hypoxia are earlier events than HIF-1α in EA.hy926 and HMEC1 cells. ADM-promoted cell proliferation of endothelial cells, which was HIF-1α dependent. We also found that ADM up-regulated the mRNA and protein expressions of HIF-1α- and HIF-1-targeted genes, and ADM up-regulated the protein expressions of HIF-1α through down-regulation of PHD mRNA expression and PHD activity.     

Proteomic differences between microvascular endothelial cells and the EA.hy926 cell line forming three‐dimensional structures

Proteomic changes of two types of human endothelial cells (ECs) were determined and compared to morphological alterations occurring during the scaffold‐free in vitro formation of 3D structures resembling vascular intimas. The EA.hy926 cell line and human microvascular ECs (HMVECs) were cultured on a random positioning machine or static on ground (normal gravity) for 5 and 7 days, before their morphology was examined and their protein content was analysed by MS after free‐flow electrophoretic separation. A total of 1175 types of proteins were found in EA.hy926 cells and 846 in HMVEC forming 3D structures faster than the EA.hy926 cells. Five hundred and eighty‐four of these kinds of proteins were present in both types of cells. They included a number of metabolic enzymes, of structure‐related and stress proteins. Comparing proteins of EA.hy926 cells growing either adherently on ground or in 3D aggregates on the random positioning machine revealed that ribosomal proteins were enhanced, while tubes are formed and various components of 26S proteasomes remained prevalent in static normal gravity control cells only. The fast developing tube‐like 3D structures of HMVEC suggested a transient augmentation of ribosomal proteins during the 3D assembling of ECs.     

Production of prostacyclin and prostaglandin E2 in resting and IL-1beta-stimulated A549, HUVEC and hybrid EA.HY 926 cells.

Production of arachidonic acid (AA) metabolites - prostacyclin (PGI(2)) in large vessels and prostaglandin E(2) (PGE(2)) in microcirculation is intrinsically involved in maintenance of vascular wall homeostasis. EA.hy 926 is a hybrid cell line, is derived by fusion of HUVEC with A549 cells. The aim of this study was to examine the production of prostacyclin and PGE2 in resting and IL-1beta-stimulated EA.ha 926 cells, in comparison with its progenitor cells. Non-stimulated EA.hy 926 cells has been found to produce much lower amounts of prostacyclin than resting HUVEC. Resting hybrid cells produced more PGE(2) than prostacyclin, despite they expressed high levels of COX-1 and PGI(2) synthase. On the contrary to HUVEC and A549, EA.hy 926 cells did not respond to IL-1beta with COX-2 induction and increase of prostaglandin production, however they did it in response to lysophosphatidylcholine (LPC). The characteristics of EA.hy 926 cells in terms of the pattern of prostanoid formation could facilitate studies on endothelial metabolism and role of these important lipid mediators.     

Endothelin-1 is expressed and released by a human endothelial hybrid cell line (EA.hy 926).

A permanent vascular endothelial cell line, EA.hy 926, was shown to express endothelin-1 (ET-1) mRNA and to secrete big ET-1 and ET-1 into culture medium. The concentration of both big ET-1 and ET-1 was significantly increased in EA.hy 926 culture medium by phosphoramidon, a metalloproteinase inhibitor, suggesting that phosphoramidon sensitive protease(s) may be responsible for the degradation of ET-1 and big ET-1. EA.hy 926 cells responded to various regulators of ET-1 similarly as primary human vascular endothelial cells. The production of ET-1 was increased by thrombin and decreased by vasodilators such as atrial natriuretic peptide, brain natriuretic peptide and nitroprusside, and by 8-bromo cyclic GMP and papaverine. This continuous human endothelial hybrid cell line could facilitate studies of regulation of ET-1 production in human endothelial cells, which in primary cultures have limited replication potential.     

Inhibition of intracellular Angiotensin II formation blocks high glucose effect on mesangial matrix

High glucose causes increased matrix synthesis by glomerular mesangial cells and angiotensin II (Ang II) has been shown to mediate this effect of glucose. These studies investigate whether inhibition of Ang II formation can block high glucose-induced increase in mesangial matrix. Human mesangial cells were incubated with 25 mM glucose (HG) along with captopril, an ACE inhibitor, to block Ang II formation. In other experiments, cells were nucleofected with siRNA to knockdown angiotensinogen (Agt), the precursor of Ang II, and then exposed to high glucose. Captopril blocked high glucose-induced increase in Ang II levels in the cell media (extracellular) but failed to inhibit it in the cell lysate (intracellular). Moreover, captopril treatment did not block the stimulatory effect of high glucose on TGF-β1 and fibronectin. In contrast, knockdown of the Agt gene prevented high glucose-induced increase in both extracellular and intracellular Ang II levels, and was accompanied by normalization of TGF-β1 and fibronectin. These data suggest that intracellular Ang II may play an important role in the mediation of the high glucose effect on matrix and that ACE inhibitors may not be effective in blocking intracellular Ang II formation in mesangial cells.     

长链非编码RNA GAS5抑制内皮细胞功能

最新研究表明,长链非编码RNA GAS5（lncRNA GAS5）可调节血管内皮细胞的凋亡,但对内皮细胞其他功能的调控并不明确。本研究旨在了解lncRNA GAS5对内皮细胞的增殖、成血管、NO分泌及内皮标志分子CD31和vWF表达的影响及可能机制。将LncRNA GAS5干扰慢病毒（LV-GAS5-RNAi）转染人脐静脉内皮细胞株（EA.hy926）后,采用CCK8及Matrigel胶分别检测EA.hy926的增殖和成血管能力;硝酸还原酶法检测NO的分泌情况;real-time RT-PCR检测CD31、vWF及miR-21的表达;Western印迹检测PTEN在蛋白质水平的表达。结果显示：与对照组比较,LV-GAS5-RNAi组EA.hy926增殖能力无明显变化(0.34±0.01 vs. 0.34±0.04,P>0.05),而其成血管能力升高(133.70±12.64 vs. 100.00±4.65,P<0.05),NO的分泌量亦增加(28.54±2.75 μmol/L vs.15.11±1.19 μmol/L,P<0.01);内皮标志分子CD31（是对照组的1.46倍）及vWF（是对照组的2.94倍)的基因表达量均显著升高。同时,miR-21表达亦明显升高(是对照组的1.42倍),而miR-21下游靶基因PTEN蛋白质的表达量则显著降低（0.13±0.05 vs. 0.38±0.03,P<0.01）。以上结果提示,LncRNA GAS5抑制了内皮细胞的功能,miR-21、PTEN信号分子可能参与其中的调节。