張敏, 孫玉, 杜大鵬, 王小波, 于建新, 王漢琴△
水通道蛋白1在切應力調(diào)節(jié)血管內(nèi)皮細胞遷移和血管生成中的作用及機制*
張敏1,2, 孫玉1, 杜大鵬1,2, 王小波1, 于建新1, 王漢琴1,2△
(1湖北醫(yī)藥學院附屬隨州醫(yī)院轉(zhuǎn)化醫(yī)學研究中心,湖北 隨州 441300;2湖北醫(yī)藥學院基礎醫(yī)學院解剖學教研室,湖北 十堰 442000)
探討流體切應力作用下水通道蛋白1(AQP1)的表達對血管內(nèi)皮細胞遷移和血管生成的影響及可能的機制。取雄性C57BL/6小鼠主動脈弓和胸主動脈血管壁組織,用qPCR和Western blot檢測體內(nèi)不同切應力作用部位血管壁AQP1表達的差異。原代培養(yǎng)人臍靜脈內(nèi)皮細胞(HUVECs),體外采用平行平板流動腔系統(tǒng)給HUVECs分別加載層流(LF,15 dyn/cm2單向?qū)忧袘Γ┖蛿_流[DF,(0.5±4) dyn/cm2振蕩切應力],用特異性小干擾RNA(siRNA)轉(zhuǎn)染技術沉默基因,采用Transwell實驗和Matrigel小管形成實驗檢測HUVECs遷移和血管生成能力;用Western blot檢測內(nèi)皮型一氧化氮合酶(eNOS)Ser1177和Ser633磷酸化水平。在體胸主動脈中AQP1的mRNA和蛋白表達顯著高于主動脈弓(<0.05)。HUVECs靜態(tài)下敲減可以顯著抑制細胞遷移和血管生成(<0.01)。與DF組相比,LF顯著上調(diào)HUVECs中AQP1的mRNA和蛋白表達(<0.05),促進HUVECs遷移(<0.01)和血管生成能力(<0.05),同時顯著增加eNOS Ser1177(<0.01)和Ser633(<0.05)磷酸化水平。轉(zhuǎn)染siAQP1后,LF誘導的AQP1表達增強被抑制(<0.01),HUVECs的遷移和血管生成能力也隨之降低(<0.01),同時eNOS Ser1177和Ser633的磷酸化水平降低(<0.05或<0.01)。eNOS抑制劑G-硝基-L-精氨酸甲酯(L-NAME)預處理HUVECs后,LF誘導的細胞遷移和血管生成能力被抑制(<0.01)。AQP1在流體切應力調(diào)節(jié)血管內(nèi)皮細胞遷移和血管生成中發(fā)揮作用,其機制可能和eNOS信號有關。
水通道蛋白1;切應力;血管生成;血管內(nèi)皮細胞;內(nèi)皮型一氧化氮合酶
水通道蛋白1(aquaporin 1, AQP1)是細胞膜上的疏水跨膜蛋白,也是目前唯一發(fā)現(xiàn)在內(nèi)皮有表達的水通道蛋白[1]。研究表明,AQP1在惡性腫瘤的微血管內(nèi)皮高表達,能促進內(nèi)皮細胞遷移影響腫瘤血管形成[2];還有研究證實AQP1與血管內(nèi)皮生長因子各自獨立地調(diào)節(jié)視網(wǎng)膜微血管內(nèi)皮細胞的血管新生[3]。除微血管內(nèi)皮外,AQP1表達水平顯著影響體外培養(yǎng)的小鼠主動脈內(nèi)皮細胞遷移和血管生成能力[2]。顯然,AQP1對血管內(nèi)皮細胞的功能調(diào)節(jié)值得深入探討。
在人體生理狀態(tài)下,血流在動脈直段流體多為層流(laminar flow, LF),對血管內(nèi)皮有保護作用,能抵抗動脈粥樣硬化發(fā)生;而在動脈分支、分叉和彎曲等處血流為擾流(disturbed flow, DF),易損傷內(nèi)皮,誘發(fā)動脈粥樣硬化發(fā)生[4]。Fontijn等[5]在動物實驗中觀察到AQP1在沒有斑塊發(fā)生的血管內(nèi)膜高表達,而在有斑塊的內(nèi)膜表達缺失。體外實驗顯示,12 dyn/cm2的LF切應力可以上調(diào)人臍靜脈內(nèi)皮細胞(human umbilical vein endothelial cells, HUVECs)中AQP1的表達,并在細胞遷移中起作用[6]。近期還發(fā)現(xiàn)靜水壓力通過AQP1促進HUVECs小管形成[7]。以上研究都說明了機械應力對血管內(nèi)皮細胞AQP1表達的調(diào)節(jié)作用,但是,AQP1對流體切應力的響應及下游的信號機制尚不完全清楚。
本研究利用平行平板流動腔系統(tǒng),對體外原代培養(yǎng)的HUVECs施加流體切應力刺激,LF為加載15 dyn/cm2單向?qū)忧袘Γ╨aminar shear stress),DF為加載(0.5±4) dyn/cm2振蕩切應力(oscillatory shear stress),檢測AQP1的表達與HUVECs遷移和血管生成能力的改變,并觀察內(nèi)皮型一氧化氮合酶(endothelial nitric oxide synthase, eNOS)信號的激活,從力學生物學角度進一步探討AQP1對血管內(nèi)皮功能的調(diào)節(jié)作用。
胎牛血清(fetal bovine serum, FBS)和M199培養(yǎng)液購自Gibco;酸性成纖維細胞生長因子(acidic fibroblast growth factor, aFGF)購自Sigma;Trizol和Lipofectamine? 3000購自Invitrogen;逆轉(zhuǎn)錄試劑盒K1621購自Thermo;iTaqTMUniversal SYBR Green Supermix購自Bio-Rad。G-硝基-L-精氨酸甲酯(G-nitro-L-arginine methyl ester, L-NAME)購自TargetMol;AQP1抗體購自GeneTex;總eNOS抗體和p-eNOS (Ser1177)抗體購自Novus;p-eNOS (Ser633)抗體購自BD;β-actin抗體和BCIP/NBT堿性磷酸酶顯色試劑盒購自上海碧云天生物技術有限公司。Matrigel購自BD;Transwell小室購自Corning。
2.1HUVECs的培養(yǎng)及鑒定采用我們既往報道方法[8]。取新鮮新生兒臍帶(湖北醫(yī)藥學院附屬隨州醫(yī)院產(chǎn)科提供,標本采集經(jīng)過患者家屬知情同意及醫(yī)院倫理委員會批準)。找到臍靜脈后用0.125%胰酶消化,將細胞懸液接種在已用0.1 g/L多聚賴氨酸包被的培養(yǎng)瓶中,加入內(nèi)皮細胞培養(yǎng)液(含20% FBS的M199培養(yǎng)液中加入100 mmol/L HEPES、10 nmol/L胸苷、2 mmol/L谷氨酰胺、4 g/L aFGF和5 000 U/L肝素),置于5% CO2、37 ℃培養(yǎng)箱中靜置8 h,待細胞貼壁后換液,大約7 d長至融合狀態(tài)。胰酶消化后傳代,血管性血友病因子(von Willebrand factor, vWF)免疫熒光鑒定,取第2~4代用于實驗。
2.2動物及取材6~8周齡SPF級雄性C57BL/6小鼠28只,體質(zhì)量18~22 g,由湖北醫(yī)藥學院實驗動物中心提供,許可證號為SCXK(鄂)2017-0012。用戊巴比妥鈉經(jīng)腹腔注射麻醉后,固定、充分暴露小鼠胸腔,剪開右心耳分離出主動脈,取胸主動脈直段和主動脈弓小彎側(cè)血管壁組織,用EP管置于液氮中保存。
2.3流體切應力加載平行平板流動腔系統(tǒng)(購自上海泉眾機電科技有限公司)用于流體切應力加載。該系統(tǒng)包括恒流泵、儲液瓶、管道及流室,細胞沿中軸方向種植于7.62 cm×2.54 cm×0.12 cm(長×寬×高)經(jīng)過用0.1 g/L多聚賴氨酸包被的載玻片上。載玻片置入流室后,在載玻片上方形成高度0.02 cm、寬度2.30 cm的流道,采用=6/2計算切應力強度。為壁面切應力(Pa),為流量(cm3/s),為液體黏度(Pa·s),和分別為流動腔的寬和高(cm)。其中和恒定,平行平板流動腔系統(tǒng)所用灌流液均為含1%FBS的M199。通過調(diào)節(jié)恒流泵改變值,達到實驗所需15 dyn/cm2單向?qū)忧袘Γ↙F組);在灌流液進入流室前,增加一個擾流泵,通過調(diào)節(jié)擾流泵往復頻率,獲得(0.5±4) dyn/cm2振蕩切應力(DF組)。
2.4Transwell遷移實驗將預先處理好的HUVECs以每孔2×104的數(shù)量(總體積200 μL,含1%FBS)接種于Transwell小室的上室,下室每孔加入500 μL含20% FBS的M199完全培養(yǎng)液,常規(guī)培養(yǎng)12 h后取出小室,PBS清洗小室2遍,用棉簽輕輕擦去微孔膜上層未遷移的細胞。將遷移的細胞用4%多聚甲醛固定20 min,結(jié)晶紫染色10 min,三蒸水漂洗3次,晾干,倒置相差顯微鏡觀察并拍照計數(shù)。
2.5Matrigel小管形成實驗在96孔板中加入50 μL提前稀釋的Matrigel,放入37 ℃培養(yǎng)箱孵育30 min,盡可能避免氣泡產(chǎn)生。將預先處理好的HUVECs消化后用含1% FBS的M199培養(yǎng)液制成細胞懸液,以每孔2×104個細胞的密度接種于96孔板,置于37 ℃、5% CO2培養(yǎng)箱中孵育4 h,顯微鏡下觀察小管形成、拍照,使用ImageJ軟件測量成管數(shù)量、長度。
2.6siRNA干擾實驗依據(jù)GenBank中人基因全長cDNA序列,委托廣州銳博生物科技有限公司合成特異性靶向基因的siRNA片段。待HUVECs生長融合至80%時,用Lipofectamine? 3000介導siRNA轉(zhuǎn)染,實驗方法按說明書進行。轉(zhuǎn)染后36 h用qPCR檢測。陰性對照RNA(scrambled siRNA,siScr)序列正義鏈為5'-UUCUCCGAACGUGUCACGUTT-3',反義鏈為5'-ACGUGACACGUUCGGAGAATT-3'。篩選出最佳siAQP1序列正義鏈為5'-UGGCUGUACUCAUCUACGAdTdT-3',反義鏈為5'-UCGUAGAUGAGUACAGCCAdTdT-3'。
2.7實驗分組加載15 dyn/cm2單向?qū)忧袘Γ↙F組)為實驗組,加載(0.5±4) dyn/cm2振蕩切應力(DF組)為對照組。HUVECs分別轉(zhuǎn)染陰性對照亂序RNA(siScr)和有效干擾序列(siAQP1)后,移入流動腔系統(tǒng),siScr+LF和siAQP1+LF為實驗組,siScr+DF和siAQP1+DF為平行對照組。HUVECs用L-NAME(20 μmol/L)和DMSO孵育30 min后,移入流動腔系統(tǒng),DMSO+LF和L-NAME+LF為實驗組,DMSO+DF和L-NAME+DF為平行對照組。
2.8qPCR實驗使用Trizol試劑從細胞或組織中分離總RNA,使用逆轉(zhuǎn)錄試劑盒獲得cDNA,采用SYBR Green PCR試劑盒進行qPCR。GAPDH用于標準化AQP1的相對表達,采用2-ΔΔCt法計算相對表達量,每個樣品設3個復孔。人源AQP1的上游引物序列5'-GGACACCTCCTGGCTATTGA-3',下游引物序列5'-GAATGGCCCCACCCAGAAAA-3';人源GAPDH的上游引物序列5'-ATGGAAATCCCATCACCATCTT-3',下游引物序列5'-CGCCCCACTTGATTTTGG-3'。小鼠源AQP1的上游引物序列5'-GCGCCGAGACTTAGGTGG-3',下游引物序列5'-GCCAGTGTAGTCAATCGCCAG-3';小鼠源GAPDH的上游引物序列5'-GGGTTCCTATAAATACGGACTGC-3',下游引物序列5'-TCTACGGGACGAGGCTGG-3'。
2.9Western blot檢測將預先處理好的細胞,用冷PBS清洗一次(組織用液氮碾磨后),加入蛋白裂解混合液(RIPA裂解液與蛋白酶抑制劑混合物的比例為49∶1),超聲波粉碎變性后備用;用10% SDS-PAGE分離,轉(zhuǎn)膜后用5%牛血清白蛋白室溫封閉至少2 h;Ⅰ抗[AQP1、eNOS、p-eNOS (Ser633)、p-eNOS (Ser1177)和β-actin,均按1∶1 000比例稀釋]4℃搖床孵育過夜;Ⅱ抗(堿性磷酸酶標記的山羊抗兔和山羊抗小鼠IgG,1∶2 000比例稀釋),室溫孵育2 h;TBST清洗膜后加入BCIP/NBT顯色液顯色。β-actin作為內(nèi)參照。
數(shù)據(jù)以均數(shù)±標準差(mean±SD)表示,應用GraphPad Prism 8.0軟件進行數(shù)據(jù)處理和分析。實驗數(shù)據(jù)均為至少3次獨立重復實驗結(jié)果,兩組間均數(shù)比較采用Student's檢驗,多組間比較采用單因素方差分析(one-way ANOVA)。以0.05表示差異具有統(tǒng)計學意義。
如圖1A所示,取C57BL/6小鼠胸主動脈直段(受層流作用部位)和主動脈弓小彎側(cè)(受擾流作用部位)血管壁組織。qPCR和Western blot結(jié)果顯示,胸主動脈直段AQP1的mRNA和蛋白表達顯著高于主動脈弓小彎側(cè)(<0.05),見圖1B。
體外靜態(tài)培養(yǎng)HUVECs(static, ST組),以及加載單向?qū)忧袘Υ碳ぃ↙F組)和振蕩切應力刺激(DF組)。首先,從形態(tài)上觀察(圖1C),ST組和DF組細胞呈現(xiàn)無規(guī)則排列,而LF組中細胞沿流體方向整齊排列并被拉長,細胞狀態(tài)良好;其次,qPCR和Western blot結(jié)果顯示,LF組AQP1的mRNA和蛋白表達顯著高于DF組和ST組(<0.01或<0.05),見圖1D。
Figure 1.Effect of shear stress on the AQP1 expression in aortic tissues of mice and in HUVECs cultured invitro. A: the diagram of aortic arches and straight segments of thoracic aortas obtained from C57BL/6 mice; B: AQP1 mRNA and protein levels in aortas were detected by qPCR and Western blot, respectively; C: representative morphological images of HUVECs exposed to static control (ST), laminar flow (LF) and disturbed flow (DF) for 24 h (the arrow points to the direction of the force on HUVECs; scale bar=10 μm); D: AQP1 mRNA and protein levels in HUVECs under ST, LF and DF conditions for 8 h. Mean±SD. n=3. △P<0.05 vs aortic arch group; *P<0.05, **P<0.01 vs ST group; #P<0.05 vs LF group.
HUVECs轉(zhuǎn)染特異性siRNA(siAQP1),Western blot結(jié)果顯示(圖2A),與siScr組相比較,siAQP1組AQP1蛋白表達顯著降低(<0.01)。Transwell遷移形成實驗表明(圖2B),siAQP1組HUVECs遷移細胞數(shù)量顯著低于siScr組(<0.01)。Matrigel小管形成實驗表明(圖2C),siAQP1組細胞成管數(shù)量顯著少于siScr組(<0.01)。
Figure 2.Effect of AQP1 knockdown on the migration and angiogenesis of HUVECs. HUVECs were treated with siScr or siAQP1 transfection. A: Western blot was used to confirm the down-regulation of AQP1 protein; B: the migration of HUVECs was detected by Transwell assay; C: the angiogenesis of HUVECs was determined by tube formation assay. The scale bar=50 μm. Mean±SD. n=4. **P<0.01 vs siScr group.
HUVECs加載DF和LF,Transwell遷移實驗顯示(圖3A),LF組遷移細胞數(shù)量顯著高于DF組(<0.01);Matrigel小管形成實驗顯示(圖3B),LF組細胞成管數(shù)量顯著高于DF組(<0.05)。
Figure 3.Effect of shear stress on the migration and angiogenesis of HUVECs. HUVECs were exposed to LF or DF for 8 h. A: Transwell assay was performed to detect the migration of HUVECs; B: tube formation assay was performed to assess the angiogenesis of HUVECs. The scale bar=50 μm. Mean±SD. n=3. *P<0.05, **P<0.01 vs DF group.
HUVECs轉(zhuǎn)染siAQP1和siScr后,加載LF和DF,Western blot結(jié)果顯示(圖4A),siScr+LF組細胞AQP1蛋白表達顯著高于siScr+DF組(<0.01);與siScr+LF組比較,siAQP1+LF組上調(diào)的AQP1蛋白表達顯著被抑制(<0.01)。Transwell遷移(圖4B)和Matrigel小管形成實驗(圖4C)顯示,siScr+LF組遷移細胞和成管數(shù)量均顯著高于siScr+DF組(<0.01);而與siScr+LF組比較,siAQP1+LF組遷移細胞和細胞成管數(shù)量均顯著被抑制(<0.01)。
Figure 4.Effectof AQP1 knockdown on the migration and angiogenesis of HUVECs induced by LF. HUVECs were treated with siScr or siAQP1 transfection and then exposed to DF or LF for 8 h. A: AQP1 protein level was detected by Western blot; B: the migration of HUVECs was determined by Transwell assay; C: the angiogenesis of HUVECs was assessed by tube formation assay. The scale bar=50 μm. Mean±SD. n=3. **P<0.01 vs siScr+DF group; ##P<0.01 vs siScr+LF group.
Western blot結(jié)果顯示(圖5A),與DF相比,LF能顯著上調(diào)eNOS Ser1177(<0.01)和Ser633(<0.05)磷酸化水平。HUVECs轉(zhuǎn)染siAQP1和siScr后,再加載LF和DF,Western blot結(jié)果顯示,siScr+LF組eNOS的Ser1177和Ser633磷酸化水平顯著高于siScr+DF組(<0.01);而siAQP1+LF組顯著抑制了siScr+LF組上調(diào)的eNOS Ser1177(<0.05)和Ser633(<0.01)磷酸化水平,見圖5B。
Figure 5.Effectof AQP1 knockdown on LF-induced phosphorylation of eNOS in HUVECs. A: HUVECs were exposed to LF or DF for 8 h, and the phosphorylation of eNOS at Ser1177 and Ser633 was determined by Western blot; B: HUVECs were treated with siScr or siAQP1 transfection and then exposed to DF or LF for 8 h, and the phosphorylation of eNOS at Ser1177 and Ser633 was assessed by Western blot. Mean±SD. n=3. △P<0.05, △△P<0.01 vs DF group; **P<0.01 vs siScr+DF group; #P<0.05, ##P<0.01 vs siScr+LF group.
為了進一步檢測eNOS對HUVECs遷移和血管生成能力的作用。細胞用eNOS抑制劑L-NAME(20 μmol/L)孵育30 min后,加載LF和DF,Transwell遷移和Matrigel小管形成實驗結(jié)果統(tǒng)計分析如圖6所示,DMSO+LF組遷移細胞和成管數(shù)量均顯著高于DMSO+DF組(<0.05,<0.01);而與DMSO+LF組相比較,L-NAME+LF組遷移細胞和細胞成管數(shù)量均顯著減少(<0.01)。
Figure 6.Effect of L-NAME on the migration and angiogenesis of HUVECs induced by LF. HUVECs were pretreated with L-NAME or DMSO for 30 min and then exposed to DF or LF for 8 h. A: the migration of HUVECs was determined by Transwell assay; B: the angiogenesis of HUVECs was assessed by tube formation assay. Mean±SD. n=3. *P<0.05, **P<0.01 vs DMSO+DF group; ##P<0.01 vs DMSO+LF group.
既往研究報道,各種形式的機械刺激作用血管內(nèi)皮細胞,比如流體切應力[6, 9]、靜水壓力[7, 10]、高血壓[11]等,都可以調(diào)節(jié)AQP1的表達,說明AQP1對機械應力敏感。在人體生理環(huán)境下血管流體切應力波動較大,動脈管壁切應力大小在10 dyn/cm2以上,流體為層流,能誘導動脈粥樣硬化保護性基因的表達;在動脈分支、分叉和彎曲處等動脈粥樣硬化好發(fā)部位,切應力低(<4 dyn/cm2)且多為擾流[4]。為了在體外探討流體切應力對血管內(nèi)皮細胞的影響,本研究選擇以加載15 dyn/cm2單向?qū)忧袘閷恿鞔碳?;加載(0.5±4) dyn/cm2(頻率1 Hz)的振蕩切應力為擾流刺激,模擬在體血流切應力環(huán)境。結(jié)果顯示,和靜止培養(yǎng)的HUVECs相比,層流切應力促進了AQP1表達,這一點與既往結(jié)果一致[6]。考慮到在體血管內(nèi)皮細胞始終受到血流刺激,一般不存在完全靜止狀態(tài),因此,以(0.5±4) dyn/cm2的振蕩切應力為對照,我們觀察到AQP1在層流組表達水平顯著高于擾流組,這一點在動物體內(nèi)也得到驗證(AQP1在受層流刺激的小鼠胸主動脈中的表達顯著高于受擾流刺激的主動脈弓)。以上結(jié)果提示,AQP1在具有內(nèi)皮保護作用的切應力刺激下高表達,而在促動脈粥樣硬化發(fā)生的擾流作用下表達被抑制,可能在動脈粥樣硬化發(fā)生過程中發(fā)揮作用。
和已有的研究結(jié)果一致[2, 12],敲減表達后,我們觀察到HUVECs遷移和血管生成能力均降低。和擾流刺激相比,層流在顯著上調(diào)AQP1表達的同時,HUVECs遷移和血管生成能力均顯著增強,的敲減,會抑制層流刺激下的HUVECs這些功能。說明AQP1的表達水平在內(nèi)皮細胞功能變化中起作用,流體切應力可以通過AQP1來調(diào)節(jié)血管內(nèi)皮細胞功能。
eNOS在調(diào)節(jié)內(nèi)皮細胞增殖、遷移和血管生成中發(fā)揮重要作用[13-14]。有研究報道AQP1參與了人主動脈內(nèi)皮細胞中eNOS的活化[15]。eNOS活性調(diào)節(jié)的一個重要方式就是磷酸化位點的調(diào)控,其中Ser1177、Ser615、Ser633和Tyr81位點的磷酸化水平增高可使該酶活性增強[8, 16-17]。我們首先檢測到層流切應力在上調(diào)HUVECs中AQP1蛋白表達同時還顯著增加磷酸化eNOS(Ser1177和Ser633位點)表達,敲減后,層流切應力上調(diào)的磷酸化eNOS(Ser1177和Ser633位點)均被抑制,這就說明層流切應力可能通過AQP1調(diào)節(jié)eNOS活性,而且對eNOSSer1177和Ser633位點都有調(diào)節(jié)作用。同時,我們用eNOS抑制劑后,發(fā)現(xiàn)層流切應力增加的HUVECs遷移和血管生成能力被抑制,說明AQP1可能是通過調(diào)節(jié)eNOS的活化而發(fā)揮作用。當然,有關AQP1對eNOS不同磷酸化位點的調(diào)控還值得深入研究。
綜上所述,本研究初步證實了生理層流切應力可通過AQP1調(diào)節(jié)HUVECs遷移和血管生成能力,其中可能與AQP1調(diào)節(jié)eNOS Ser1177和Ser633位點的磷酸化有關。
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Role of aquaporin 1 in shear stress-regulated endothelial cell migration and angiogenesis
ZHANG Min1,2, SUN Yu1, Du Da-peng1,2, WANG Xiao-bo1, YU Jian-xin1, WANG Han-qin1,2△
(1,,,441300,;2,,,442000,)
To investigate the role of aquaporin 1 (AQP1) in shear stress-regulated migration and angiogenesis of endothelial cells, and to explore the underlying mechanisms.The aortic arches and straight segments of thoracic aortas were obtained from C57BL/6 mice. Human umbilical vein endothelial cells (HUVECs) were isolated from fresh human umbilical cord. The parallel plate flow chamber system was used to load unidirectional laminar flow (LF, 15 dyn/cm2laminar shear stress) and disturbed flow [DF, (0.5±4) dyn/cm2oscillatory shear stress] on HUVECs. qPCR and Western blot were used to detect the expression of AQP1 at mRNA and protein levels, respectively. Silencing ofwas performed by-specific siRNA. Assays of Transwell and tube formation in Matrigel were performed to detect cell migration and angiogenesis. The protein levels of endothelial nitric oxide synthase (eNOS) and phosphorylated eNOS at Ser633 and Ser1177 were measured by Western blot.The mRNA and protein expression of AQP1 was up-regulated in the straight thoracic aorta compared with the inner of the aortic arch (<0.05). Knockdown ofin HUVECs at static condition markedly reduced cell migration and angiogenesis (<0.01). Exposure of HUVECs to LF for 8 h obviously induced AQP1 expression and promoted HUVEC migration and angiogenesis (<0.05 or<0.01). LF also significantly increased eNOS phosphorylation both at Ser1177 (<0.05) and Ser633 (<0.01). As expected,silencing abolished the above effects of LF-stimulated HUVECs (<0.05 or<0.01).G-nitro-L-arginine methyl ester (L-NAME), an eNOS inhibitor, also prevented LF-induced HUVEC migration and angiogenesis (<0.01).AQP1 plays a crucial role in the regulation of migration and angiogenesis in shear stress-stimulated endothelial cells, and its mechanism may be related to the eNOS signaling pathway.
Aquaporin 1; Shear stress; Angiogenesis; Endothelial cells; Endothelial nitric oxide synthase
R363.2; R329.2+5
A
10.3969/j.issn.1000-4718.2022.03.003
1000-4718(2022)03-0403-09
2021-09-07
2022-01-04
[基金項目]國家自然科學基金資助項目(No. 31670961);湖北醫(yī)藥學院研究生科技創(chuàng)新項目(No. YC2019038)
Tel: 0722-3252556; E-mail: hanqin.wang@hbmu.edu.cn
(責任編輯:盧萍,羅森)