Caveolin-1调节内皮祖细胞介导的血管新生在糖尿病和伤口愈合中的作用与机制
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摘要
研究背景
糖尿病血管病变包括大血管病变及微血管病变,是糖尿病的慢性并发症之一,导致心脑血管病变、视网膜病变、糖尿病肾病、糖尿病足等并发症,是影响糖尿病患者生活质量及预后的重要因素。糖尿病血管病变一个重要特点是血管新生受损,组织损伤局部血液供应不足,导致组织难以得到有效修复,如局部组织持续缺血、坏死、感染,可能导致坏疽的严重后果,目前仍有大量糖尿病患者为此需要施行截肢手术。所以,探索糖尿病血管新生受损的机制并寻找改善糖尿病血管新生的方法是治疗糖尿病足、糖尿病伤口不愈等糖尿病血管并发症的重要手段。
内皮祖细胞(endothelial progenitor cells, EPCs)被证实在出生后血管新生中发挥重要作用。EPCs参与血管新生的方式主要有两种：一是EPCs直接整合到血管新生网络,通过分化成内皮细胞,参与新生血管内皮层的形成,二是在血管新生局部通过旁分泌方式分泌一些促血管生成的细胞因子和生长因子,如VEGF、HGF、IGF-1等,促进血管新生。EPCs数量已被作为心血管疾病诊断及预后评价的重要指标,EPCs介导的细胞和基因治疗心血管疾病中有着广阔的应用前景。然而研究表明糖尿病时内皮祖细胞数量减少、功能受损,不能有效地发挥促血管新生的作用,限制了其在糖尿病血管病变中的应用。到目前为止,糖尿
病EPCs功能受损的机制仍未完全明了。因此探索糖尿病状态下内皮祖细胞功能损伤的机制,寻求改善EPCs功能的方法,这对于改善血管新生、治疗糖尿病血管病变具有重要意义。
研究表明,一氧化氮合成酶(eNOS)及eNOS催化生成的NO在心血管系统中有重要作用,可促进EPCs介导的血管新生。然而,糖尿病状态下,内皮NO生成减少,导致血管功能失调,这可能与小凹蛋白1(caveolin-1, Cav-1)表达增加,抑制eNOS的激活有关。Cav-1通过蛋白与蛋白相互作用,抑制内皮eNOS的活性,使细胞处于较低的NO水平,当受到外界刺激时,eNOS与Cav-1脱离并被激活,催化产生NO,从而发挥调节血管张力、抑制血小板聚集、减少氧化应激损伤、抗炎、抗动脉粥样硬化和促进血管新生等作用。EPCs同内皮细胞一样表达Cav-1和eNOS,糖尿病是否增加EPCs (?)的Cav-1表达并抑制eNOS/NO信号通路,导致EPCs(?)功能受损尚不明了。
基于以上背景,我们提出如下假说：糖尿病EPCs的Cav-1表达增加,抑制eNOS/NO信号通路,使EPCs血管新生功能受损,而降低糖尿病EPCs的Cav-1表达可改善血管新生。为验证我们的假设,我们将首先检测2型糖尿病db/db小鼠EPCs的Cav-1表达变化,并找其变化的原因；检测糖尿病eNOS/NO通路及EPCs功能的改变;通过慢病毒转染降低糖尿病EPCs Cav-1表达,增加leNOS(?)的磷酸化及NO的生成,以期改善EPCs的血管新生功能。
研究目的
1.研究Cav-1在2型糖尿病小鼠EPCs (?)的表达变化,并探索变化的原因。
2.探讨Cav-1对2型糖尿病小鼠EPCs功能的调节作用及机制。
研究方法
1.动物模型
12周龄的2型雄性糖尿病小鼠db/db (BKS.Cg-m+/+Leprdb/J)及其非糖尿病对照小鼠db/+(Leprdb m/++)用于实验。实验前检测db/db小鼠血糖,300mg/dl以上者作为糖尿病模型。
2. EPCs的分离、培养与鉴定
分离小鼠股骨、胫骨、髋骨,提取骨髓单个核细胞,接种在已包被玻璃粘连蛋白(vitronectin)的六孔板中,在37℃,5%CO2环境中培养。72小时后第一次完全换液,去除未贴壁的细胞,第五天时半量换液,继续培养到第七天,即可用于实验。为鉴定培养细胞为EPCs,细胞培养7天后,用Hoechst33258、Dil-acLDL和isolectin染色并在荧光显微镜下观察。为进一步鉴定EPCs,对培养7天的细胞采用流式细胞术分析其表面干细胞表面标记物CD34和Sca-1、内皮细胞表面标记物CD144和VEGFR2(Flk-1)及单核细胞表面标记物CDllb的表达。
3. EPCs功能的检测
采用小管形成试验(tube formation assay)和细胞迁移实验(migration assay)来检测EPCs的小管形成能力及迁移能力。
4.分子生物学检测
(1) Real-time PCR
提取EPCs的总RNA,进行逆转录反应,获得cDNA,最后用试剂盒进行定量PCR反应检测EPCs的Cav-1表达,GAPDH作为内参。
(2) Western blot
用特异性抗体检测EPCs中Cav-1、p-eNOS1177、eNOS和β-actin的蛋白表达,在ImageQuant4000凝胶成像系统上扫描,用Quantity-One图像软件分析条带的光密度值。
5.慢病毒转染EPCs
用40MOI的Lenti-Cav-1siRNA转染EPCs,等量的Lenti-scrambled作为对照,转染24小时后换液,继续培养48小时后进行后续实验。
6.NO的测定
用Griess法检测培养基中硝酸盐的含量,间接反应NO的生成量。
7.统计学分析
计量资料以平均值±标准误(mean±SEM)表示。两样本间比较采用Studentt-检验,多样本比较采用单因素方差分析(one way ANOVA),并用Student-Newman-Keuls方法进行组间两两比较分析检验。所有结果采用GraphpadPrism5.0软件包进行分析并作图。P<0.05认为有显著性统计学差异。
研究结果
1.瘦素上调正常EPCs的Cav-1表达
db/db小鼠血清瘦素(leptin)水平为正常对照组的4.4倍。Real-time PCR和Western blot(?)结果表明,用生理浓度(20ng/ml)的leptin刺激db/+小鼠EPCs即可使Cav-1表达表达增加。随着leptin (?)浓度的增加,Cav-1的表达也持续增长,在100ng/ml浓度(接近db/db血清leptin浓度)时,Cav-1的mRNA和蛋白水平比0浓度分别增加94.65%和144.63%。
2. db/db小鼠EPCs的Cav-1表达增加且不依赖于高瘦素
Western blot结果显示,db/db小鼠EPCs比db/+组EPCs表达更高的Cav-1,约增加55.30%(P<0.05)。然而用100ng/ml leptin刺激db/db小鼠EPCs后,Cav-1的表达并没有明显变化,可能由于db/db小鼠EPCs瘦素受体缺失所致。
3.高糖上调EPCs的Cav-1表达
体外给予db/+和db/db小鼠的EPCs(?)高糖刺激,并给予甘露醇做等渗对照。结果显示,高糖刺激72小时后db/+小鼠EPCs的Cav-1农达增加了94.77%,db/db小鼠增加了39.36%(P<0.05),渗透压并不影响Cav-1的表达。
4.下调db/db小鼠EPCs的Cav-1表达改善细胞的血管新生功能
用携载Cav-1siRNA的慢病毒转染db/db小鼠EPCs后,Cav-1蛋白表达下降了53.63%。小管形成实验和干细胞迁移实验的结果表明,下调Cav-1表达能使db/db EPCs的小竹形成能力和细胞迁移能力明显提高。
5.下调db/db小鼠EPCs的Cav-1表达促进eNOS磷酸化和NO生成
EPCs给予50ng/ml VEGF-A刺激30分钟后,收集培养基检测NO的生成,并收集细胞检测eNOS磷酸化水平Western blot结果显示,db/db EPCs中磷酸化的eNOS比例比db/+EPCs明显降低,而慢病毒下调Cav-1标达后,eNOS磷酸化增加了88.72%(P<0.05),同时NO的生成也增加了1.78倍。
结论
1.在2型糖尿病情况下,高糖可以上调EPCs的Cav-1表达,这种效应独立于高瘦素的作用：
2.下调2型糖尿病db/db小鼠EPCs的Cav-1表达能提高改善EPCs的小管形成和迁移能力;
3.下调Cav-1改善糖尿病EPCs的功能的机制可能与促进eNOS磷酸化和增加NO生成有关。
研究背景
上世纪50年代,由于电子显微镜技术的发展,细胞的许多超微结构为人所知,在细胞膜上发现有许多50-100nm的烧瓶样凹陷,被称为小凹(caveolae)。尽管小凹的发现已有半个多世纪,但人们对小凹本质及功能的认识主要集中在近20年,这得益于小凹上最重要的蛋白成分—小凹蛋白(caveolin)的发现。这一发现使人们了解了小凹的许多生理功能,如胆固醇转运、信号转导、细胞内吞、肿瘤抑制等。小凹蛋白有三种亚型,分别是小凹蛋白1(caveolin-1, Cav-1)、小凹蛋白2(caveolin-2, Cav-2)、小凹蛋白3(caveolin-3, Cav-3)。
Cav-1与内皮细胞功能的关系已有较广泛的研究,已知Cav-1可影响内皮的通透性、胆固醇的内吞、内皮依赖的血管新生、细胞增殖等等,但Cav-1与内皮祖细胞(endothelial progenitor cells, EPCs)关系的研究较少,Cav-1对于EPCs及EPCs参与的病理生理作用有待进一步研究。随着EPCs研究的深入,EPCs在缺血性疾病及血管新生领域的作用日益受到重视,已成为诊断和预测某些疾病的重要指标,EPCs介导的细胞和基因治疗有着广阔的应用前景。
研究表明,在小鼠皮肤造成伤口模型后,血中的EPCs数量开始增加,到第三天时达到峰值,表明EPCs从骨髓中动员出来参与伤口的修复。我们以往的研究表明,糖尿病小鼠的EPCs功能受损,不能有效地促进血管新生,是糖尿病小鼠伤口愈合延迟的重要原因,因此EPCs功能对于维持正常伤口J愈合至关重要。本研究以伤口愈合的小鼠作为模型,研究Cav-1尤其是EPCs上Cav-1的表达对伤口愈合的影响,并试图发现其内在的联系机制。为了达到研究目的,我们使用了两种基因技术的小鼠：Cav-1(?)基因敲除小鼠(Cav-1KO mice)和内皮特异爪组Cav-1小鼠(Cav-1RC mice),前者体内的Cav-1完全缺失,后者是在前者的基础上的改良,在Cav-1敲除的背景下将内皮又重组Cav-1,因此这种小鼠在内皮以外的其他细胞类型都不表达Cav-1。由于EPCs是内皮的始祖细胞,因此Cav-1RC小鼠的EPCs也应该表达Cav-1,我们在研究中也将证实。Sbaa等人研究表明Cav-1KO小鼠的血清SDF-1α水平异常。SDF-1α是一种趋化因子,参与干细胞和祖细胞在骨髓的驻留及从骨髓的动员和归巢,SDF-1α已用于多种缺血性疾病的治疗。SDF-1α可趋化EPCs到缺血部位参与血管新生,这也是在病理情况比如糖尿病时SDF-1α促进伤口愈合机制之一。鉴于SDF-1α对EPCs介导血管新生的重要性,我们在本实验中探讨Cav-1α的表达对SDF-1α水平的影响,并且用腺相关病毒转染的方法增加伤口局部SDF-1α的表达,观察不同基因背景小鼠对SDF-1α治疗的反应,从而阐明Cav-I在EPCs介导的血管新生和伤口愈合中的作用。
研究目的
1.研究Cav-I缺失对内皮祖细胞功能的影响。
2.研究Cav-I表达对小鼠皮肤伤口愈合的作用。
3.探讨内皮祖细胞的Cav-I表达在SDF-1α促进的血管新生及伤口愈合中的作用。
研究方法
1.动物模型
本实验所用动物为8-12周龄的雄性小鼠,其中Cav-I基因敲除(Cav-I KO)小鼠和内皮特异性表达Cav-I(Cav-I RC)小鼠由Dr.Michael Bauer实验室提供,B6129SF2/J小鼠为对照,购自The Jackson Laboratory,实验动物饲养于美国匹兹堡大学医学院血管外科动物中心,自由饮食饮水,每日12小时光照。所有动物操作均符合美国匹兹堡大学实验动物保护与利用委员会的规定。
2.EPCs的分离、培养与鉴定
分离小鼠股骨、胫骨、髋骨,提取骨髓单个核细胞,接种在已包被玻璃粘连蛋白(vitronectin)的六孔板中,在37℃,5%CO2中环境中培养。72小时后第一次完全换液,去除未贴壁的细胞,第五天时半量换液,继续培养到第七天,可用于实验。
3. EPCs功能的检测
采用小管形成试验(tube formation assay)和细胞划痕实验(in vitro wound healing assay)来检测EPCs的小管形成能力及迁移能力。
4.伤口模型
将小鼠麻醉,用剃毛器将小鼠背部正中至尾巴根部的毛发剔除,用退毛膏将残余细微毛发去除干净。消毒后,盖上无菌洞巾,将6mm直径的打孔器定位至小鼠两大腿上缘与背中线的交点处,向下按压并旋转打孔器,切除一圆形皮肤,包括表皮和真皮层。每隔一日采集伤口照片并测量伤口大小。
5.腺相关病毒转染伤口组织
伤口模型建立后,沿伤口切缘滴加100μl含有1012病毒颗粒的SDF-1α/AAV或者GFP/AAV液,孵育30分钟后,拭去多余的病毒液。伤口愈合过程中仅在第0天孵育病毒一次。
6.统计学分析
计量资料以平均值±标准误(mean±SEM)表示。两样本间比较采Student t-检验,多样本比较采用单因素方差分析(one way ANOVA),并用Student-Newman-Keuls方法进行组间两两比较分析检验,对于多组间不同时期伤口愈合情况的比较用双囚素方差分析(two way ANOVA)。所有结果采用Graphpad Prism5.0软件包进行分析并作图。P<0.05认为有显著性统计学差异。
研究结果
1.内皮重组Cav-1使Cav-1敲除小鼠EPCs的小管形成能力恢复
敲除Cav-1后,EPCs形成的小管数量明显减少,约为WT组的51.88%,然而重组Cav-1后EPCs形成的小管数量明显增加,达到了WT组的95.97%,说明Cav-1的表达对EPCs的小管形成功能十分重要。我们又用划痕实验来评价细胞的迁移功能,WT, Cav-1KO和Cav-1RC三种小鼠的EPCs在12小时、24小时两个时间点迁移的数量并没有明显差异,说明Cav-1(?)的表达与否并不明显影响EPCs的迁移能力。
2.内皮重组Cav-1后不能加快Cav-1敲除小鼠的伤口愈合
从第6天开始,Cav-1KO小鼠的伤口愈合开始明显落后于WT组,一直持续到第16天,此时WT组的皮肤伤口基本愈合,但Cav-1KO小鼠的皮肤只愈合到89.02%,全程愈合率与WT组比较有统计学差异(P<0.05)。尽管重组Cav-1使EPCs(?)的血管新生功能得到恢复,但是伤口愈合并没有得到改善,在第16天时仅愈合了90.25%,与Cav-1KO组并无明显差别,全程愈合率也明显低于WT组(P<0.05)。
3.内皮重组Cav-1未明显增加Cav-1KO小鼠伤口的血管新生数目
免疫组化结果显示,Cav-1KO小鼠皮肤的毛细血管密度明显低于WT组,Cav-1RC的新生血管数量与KO组相当,没有明显改善。以上结果提示,Cav-1KO小鼠皮肤伤口愈合延迟可能与血管新生受损有关,尽管重组Cav-1能够使EPCs的功能得到恢复,但并不足以明显改善皮肤伤口局部的血管新生。
4.局部SDF-1αAAV转染改善Cav-1RC/J、鼠皮肤的伤口愈合
我们将Cav-1KO和Cav-1RC小鼠实施6mm的全层皮肤切除后,分别给予SDF-1α/AAV在伤口局部,GFP/AAV作为对照,20分钟的转染后将剩余病毒液体拭去,贴好敷料,检测伤口愈合情况。结果显示,给予AAV-SDF-1α)后,Cav-1KO组的伤口愈合速度与对照组比较并无明显改变,仍然慢于WT组,而Cav-1RC小鼠伤口在AAV-SDF-1α治疗后愈合明显改善,在第16天达到了WT愈合水平。以上结果提示,内皮Cav-1的表达在SDF-1α促进伤口愈合的过程中有重要作用。
5. SDF-1α AAV治疗使Cav-1RC皮肤血管新生数目增加,但Cav-1KO组无明显改善
WT和Cav-1KO经过SDF-1α AAV转染后的新生血管数目与其对照组并没有明显改变,而Cav-1RC组比其对照组有更多的新生毛细血管数量,与伤口愈合的结果一致。以上结果提示,SDF-1α AAV改善Cav-1RC伤口愈合可能与招募更多功能正常的EPCs促进血管新生有关。
结论
1.Cav-1的表达对EPCs功能十分重要,Cav-1缺失导致EPCs的小管形成能力明显下降；
2.Cav-1缺失使伤口愈合延迟,仅仅内皮和内皮祖细胞上Cav-1恢复表达不能改善受损的伤口愈合；
3.SDF-l1α增加伤口局部EPCs的数量,从而促进血管新生和伤口愈合,这个过程需要EPCs上Cav-1的适量表达。
Background
Diabetic vascular diseases, among chronic complications in diabetes mellitus, are related with atherosclerosis, diabetic retinopathy, nephropathy and diabetic foot. Ineffective vascular repair is likely to be an important contributor to the diabetic vascular disease, and this effect is linked with impaired neovascularization in diabetes. Efforts to investigate the mechanisms of impaired neovascularization in diabetes and find new ways to improve it are significant to therapies of diabetic vascular complications.
Neovascularization is composed of angiogenesis and vasculogenesis, which involves distinct mechanisms. Vasculogenesis occurs in embryogenesis, and has been found to contribute to postnatal neovascularization due to the discovery of endothelial progenitor cells (EPCs). Asahara et al. reported for the first time the existence of EPCs in1997. Subsequent studies supported that EPCs played an crucial role in postnatal vasculogenesis. In the last decade, the important role played by EPCs in cardiovascular health is becoming increasing appreciated, especially for their crucial role in maintenance of endothelial integrity and function, as well as postnatal neovascularization.
There is accumulating evidence showing reduced number and impaired EPC function in the presence of diabetes mellitus, and dissatisfactory effects of diabetic EPC therapy. which limits the use of EPCs in practice. However, the mechanisms of EPC dysfunction in diabetes are incompletely understood. Nitric oxide (NO) was reported to play an important role in EPC mediated vasculogenesis. However, eNOS-derived NO decreases in the setting of diabetes, which contributes to impaired EPC function. Evidence has showed that eNOS resides in caveolae and Cav-1could inhibit the activity of eNOS via direct interaction with eNOS. Though the expression of Cav-1was upregulated in several tissues in diabetes, the change of Cav-1in diabetic EPCs is not clear. In current study, we hypothesize that Cav-1may be also upregulated in diabetic EPCs and involves in dysregulated eNOS/NO pathway. We determined the expression of Cav-1in EPCs from type2diabetic db/db mice, and tried to reveal the relationship between Cav-1and metabolic abnormalities, like hyperglycemia and hyperleptinemia. Specifically, we generated lentivirus carrying Cav-1siRNA to elucidate the role of Cav-1in EPC function and the underlying mechanisms in the setting of diabetes.
Objectives
1. To measure the changes of expression of Cav-1in type2diabetic EPCs, and to investgate the underlying mechanisms.
2. To investigate the role and mechanisms of Cav-1in regulating EPC functions in type2diabetes.
Methods
1. Animal model
Male db/db (BKS.Cg-m+/+Leprdbh/J) mice and their control mice(db/+), aged12weeks, were purchased from Model Animal Research Center of Nanjing University. In the current study, db/db mice with a blood glucose level>300mg/dl were considered diabetic and were used for EPC isolation.
2. EPC isolation, culture and characterization
Bone-marrow mononuclear cells were isolated from the femurs, tibias and pelvis of mice and then cultured with endothelial cell growth medium2in37℃5%CO2. After72hours, nonadherent cells were removed by changing medium and adherent cells were cultured continuously till day7for experimental use. The EPCs after7-day culture were characterized by Hoechst33258, DiI-acLDL and FITC-conjugated isolectin staining. To further characterize the phenotype of EPCs, the expressions of CD34, Sca-1, CD144, Flk-1and CD11b were analyzed by flow cytometry.
3. EPC function assays
The tube formation and migration assays were performed to determine the angiogenic function of EPCs. For tube formation assay,5×104EPCs were plated in a well of48-well plate pre-coated with growth factor-reduced Matrigel. After24-hour incubation at37℃5%CO2with EGM-2plus5%FBS, images were taken by an inverted microscope and tube lengths were measured. EPC migration was measured using Boyden chambers. Briefly,5×104EPCs were plated in the upper chamber of Transwell and allowed to migrate in a37℃5%CO2incubator. The upper cells were removed and the cells in the lower side were fixed and counted with a microscope.
4. Lentivirus constructs
We constructed a siRNA for mouse caveolin-1and a control (scrambled) that does not affect any protein expression using oligo pairs. Lentiviruses encoding scrambled and caveolin-1siRNA were produced by transfection of HEK293T which is performed commercially by GENECHEM.
5. Western Blot Analysis
20-50μg protein was subjected to SDS-polyacrylamide gel electrophoresis. The PVDF membranes were probed with primary antibodies against Cav-1, p-eNOS1177, eNOS and Actin. The blots were scanned with an ImageQuant4000system and band intensity was quantified with Quantity One System.
6. Real-time PCR
For each sample,1μg RNA was reverse transcribed using a reagent kit. Real-time PCR was performed using SYBR Green PCR Master Mix on a iCycler iQ Real-time PCR System. Variation in transcription levels was calculated using2△CT.
7. Measurement of NO release
After being serum-starved for24hours, EPCs were treated with VEGF (50ng/ml) for30minutes. Nitrite concentration in the culture medium was measured using the Griess reaction, and sodium nitrite served as a standard. The optical density of samples was measured in a reader system.
8. Statistical Analysis
Data were presented as means±SEM. Differences between two groups were compared by Student's t-test, and one-way ANOVA with Newman-Keuls multiple comparison tests was used for multiple groups. In all the tests, a value of P<0.05was taken as statistically significant.
Results
1. Leptin regulated Cav-1mRNA and protein expression
It was reported that leptin could upregulate the expression of Cav-1in HCAEC and HUVEC. To test whether leptin could regulate expression of Cav-1in EPCs, db/+EPCs were incubated with increasing concentration of leptin (0.20,50,100ng/ml) for24h, then cells were harvested for mRNA and protein analysis. Our data showed the mRNA and protein of Cav-1was upregulated by leptin at the concentration of20ng/ml. When the concentration of leptin was increased, Cav-1expression rose accordingly.
2. Cav-1protein expression was upregulated in db/db EPCs independent of high leptin level
Compared with db/+EPCs, db/db EPCs showed higher expression of Cav-1. We treated db/db EPCs with100ng/ml leptin to measure the Cav-1expression. The result showed a high level of leptin could not upregulate the expression of Cav-1in db/db EPCs.
3. High glucose upregulated Cav-1protein expression in db/db and db/+EPCs
As compared with that in control medium (5mmol/l glucose), the expression of Cav-I in db/+EPCs assessed by Western blot was significantly increased, by94.77%in high-glucose medium. High glucose also upregulated Cav-1expression in db/db EPCs by39.36%, though db/db EPCs had a higher expression of Cav-1originally. By contrast, the osmotic control with mannitol did not change the expression of Cav-1.
4. Cav-1knockdown improved functions of db/db EPCs
Our result showed db/db EPCs formed significantly fewer networks than db/+EPCs. In contrast, when Cav-1expression was knocked down by lentivirus-mediated siRNA, Matrigel tube formation was increased by69.32%in db/db EPCs. Cav-1knockdown also significantly increased migration of db/db EPCs.
5. Cav-1knockdown increased phosphorylation of eNOS and NO production
EPCs were incubated in EGM-2supplemented with VEGF (50ng/ml) for30minutes, then the medium was collected for NO detection and cells were harvested for protein analysis. The eNOS phosphorylation at Ser1177shown by immunoblotting was significantly decreased in db/db EPCs compared with that in db/+EPCs. This reduction in eNOS phosphorylation was associated with a decrease in EPC-derived NO production. Nevertheless, Cav-1knockdown significantly increased eNOS phosphorylation by88.72%in db/db EPCs coupled with significantly augmented NO production.
Conclusions
1. High glucose upregulated Cav-1protein expression independent of high leptin level.
2. Cav-1knockdown in db/db EPCs improved the angiogenic function.
3. Cav-1knockdown increased phosphorylation of eNOS and NO production in db/db EPCs
Background
Caveolae are50-100nm flask-shaped invaginations of the plasma membrane which are implicated in cholesterol transport, signal transduction. endocytosis. Caveolin, an important protein component of caveolae membrane coats, is known to have three isoforms which are caveolin-1(Cav-1), Cav-2and Cav-3. Cav-1has been widely and intensively studied, especially after the generation of Cav-1knockout mice. The putative importance of Cav-1in cardiovascular system have been validated in several physiological and pathological models, in which Cav-1influences vascular permeability, angiogenesis and progress of atherosclerosis. Notably, endothelial expression of Cav-1is closely involved in these conditions, and both lower and higher levels of Cav-1expression may impair vascular functions.
Mature endothelial cells possess limited regenerative capacity, therefore, there is growing interest into circulating endothelial progenitor cells (EPCs). Not only have EPCs been used to diagnose and predict cardiovascular diseases, EPCs have also served as a useful tool for cell and gene therapy in ischemic diseases. Though Cav-1has been well investigated in endothelial cells, there is few reports concerning its role in EPCs. Sbaa et al. reported that Cav-1knockout mice presented impaired EPC mobilization from bone marrow, which resulted in defective neovascularization in a hindlimb ischemia model. This study suggested that Cav-1might play an important role in EPC functions. In current study, we would investigate the impact of Cav-1expression on EPC functions. Our previous study showed EPCs isolated form type2diabetic db/db mice presented impaired angiogenic functions, which caused deficient new vessel formation in skin repair, and subsequently delayed wound healing. This suggests EPC is an essential opponent participating in wound healing. Herein, we mainly examined the effects of Cav-1expression in EPCs on neovascularization and cutaneous wound healing. To address this question, Cav-1KO mice and Cav-1KO mice reconstituted with a transgene expressing Cav-1specifically in endothelial cells (Cav-1RC mice) were used. We assessed neovascularization and wound healing in WT, Cav-1KO and RC mice, and examined whether reconstitution of Cav-1regulates wound healing via EPC mediated vasculogenesis.
It is well known that stromal cell-derived factor-1alpha (SDF-1α) is a predominant chemokine which is upregulated in response to ischemic stimuli and acts as a homing signal for EPCs. SDF-1α gene transfer has become a novel chemokine therapy for ischemic disease and tissue repair via recruiting EPCs to ischemic sites. It was proved that SDF-1α treatment accelerated wound closure in diabetes, while inhibition of SDF-1α further impaired diabetic wound healing, and the main mechanism was closely related with EPC mediated vasculogenesis. It was found in Cav-1KO mice plasma level of SDF-1α was2-fold higher than WT mice, which suggested Cav-1might regulate SDF-1α. Based on the evidence that SDF-la therapy effectively improves the injury repair in several models, our study also determined whether SDF-la therapy achieved by SDF-1α/AAV treatment enhances wound healing in Cav-1KO and RC mice, and whether the effects of SDF-la is Cav-1dependent.
Objectives
1. To determine the role of Cav-1in EPC function;
2. To investigate the role of Cav-1in cutaneous would healing
3. To investigate the role and mechanisms of EPCs' Cav-1in SDF-1α enhanced neovascularization and would healing;
Methods
1. Animals
All animal studies were performed with approval of the University of Pittsburgh Institutional Animal Care and Use Committee. The generation of Cav-1KO mice and Cav-1RC mice was previously described. All KO and RC mice were supplied by Dr. Michael Bauer. The WT mice (B6129SF2/J) were purchased from The Jackson Laboratory. Male mice with8-12weeks old were used in this study.
2. Full-thickness excisional wounds
Mice were anesthetized using isoflurane and then depilated with an electric shaver and depilatory creamin dorsum. Mouse skin was cleaned by Betadine and70%alcohol. Then full-thickness skins, containing epidermis and dermis, were removed by a6-mm punch. Wounds were covered with a bioclusive transparent oxygen permeable wound dressing. Wound closure rate was measured every other day till day16on which WT mice were almost cured completely.
4. EPC function assays
The tube formation assay and in vitro wound healing assay were performed to determine the angiogenic function of EPCs. For tube formation assay,5×104EPCs were plated in a well of48-well plate pre-coated with growth factor-reduced Matrigel. After24-hour incubation, images were taken by an inverted microscope and tube lengths were measured. For in vitro wound healing assay, EPCs ware seeded at1.5×105cells/well into a24-well plate. After24-hour incubation, EPCs were almost confluent, and scratched using a10μl pipette tip. After being washed3times using PBS, EPCs were kept on culturing. Images were taken at the time of wounding0h,12h and24h. Migration was estimated by counting cell numbers within wounded region.
5. Enzyme-linked immunosorbent assay (ELISA) SDF-1α protein levels in serum and bone marrow were determined by using the mouse SDF-1α Quantikine ELISA Kit based on the manufacturer's instructions. Peripheral blood was obtained by cardiac aspiration and then centrifuged to collect serum. BM was obtained from femurs, tibias and pelvis. The bones were flushed in500μl PBS. After centrifugation, the supernatant was collected for SDF-la ELISA and total protein quantification.
6. SDF-la AAV topical treatment
AAV vectors carrying SDF-1α gene has been used to treat wounds in mice. In this study, human SDF-la/AAV and control vector, green fluorescent protein (GFP)/AAV, which were gifts from Dr. Yanfang Chen (Wright State University, Ohio), were constructed by inserting the human SDF-la or GFP gene into AAV2/9vector. After wounds were created,100μl AAV at1012particle/ml in PBS was injected into the wound beds. Mice were maintained under anesthesia for30minutes, and then a bioclusive dressing was placed over the wound. Wounds were measured every other day till day16.
7. Statistical Analysis
Data were presented as means±SEM. Differences between two groups were compared by Student's t-test, and one-way ANOVA with Newman-Keuls multiple comparison tests was used for multiple groups. The rate of wound healing among groups was analyzed by2-way ANOVA, followed by Bonferroni's correction to control type I error. In all the tests, a value of P<0.05was taken as statistically significant.
Results
1. Reconstitution of Cav-1restores tube formation of Cav-1KO EPCs
Cav-1KO EPCs formed significantly fewer networks than WT EPCs, and relative tube length decreased by nearly50%. And this impairment was completely restored in Cav-1RC group by reconstitution of Cav-1in EPCs. In vitro wound healing assay showed these three groups presented similar migrating pattern which was analyzed at the time point of12hours and24hours after creating wound.
2. Reconstitution of Cav-1in endothelium is insufficient to restore impaired wound healing in global Cav-1knockout mice
These three strains of mice underwent a single6-mm dorsal punch biopsy to create a full-thickness excisional wound, and then the rate of wound closure was measured every other day till day16. Cav-1KO mice exhibited slower healing rate than WT mice, which was significant from day6to day16. Nevertheless, reexprcssion of Cav-1in endothelium failed to rescue the impaired wound healing in global Cav-1knockout background, and exhibited similar recovery pattern with Cav-1KO mice. At day16, wounds in WT mice were almost entirely closed, whereas unhealed in Cav-1KO and Cav-1RC mice (89.02%and90.25%respectively).
3. Wounded skins in Cav-1KO and RC mice showed decreased new vessel formation
We wondered whether deficient neovascularization led to impaired wound healing in Cav-1KO and RC mice, so we measured the vessel number in skins16days after wound creation. As showed by immunofluorescence staining, the percentage of vessel density in Cav-1KO and RC mice wounded skin were32.64%and38.03%respectively compared with that of WT skin. The impaired neovascularization in wounded skin might lead to the delayed wound healing in Cav-1KO and RC mice.
4. Topical treatment of SDF-1α AAV accelerated wound healing in Cav-1RC mice coupled with increased neovascularization in skin
Following wounding,1011viral particles of SDF-1α/AAV in100μl PBS was injected onto wound base for30minutes in WT, Cav-1KO, and Cav-1RC mice, while same amount of GFP/AAV was administered to control groups. During16-day observation, Cav-1RC mice showed enhanced wound healing rate and were restored to the WT level, however. Cav-1KO mice didn't respond to the treatment. We next measured the neovascularization of wounded skins treated with SDF-1α/AAV or GFP/AAV. Results showed Cav-1RC mice treated with SDF-1α/AAV had more new vessel formation than those treated with GFP/AAV, while Cav-1KO mice showed no response to SDF-la treatment.
Conclusions
1. Expression of Cav-1is essential to the angiogenic function of FPCs.
2. Cav-1is required for cutaneous wound healing, and reconstitution only in endothelium is insufficient to restore the impaired wound healing in Cav-1KO mice.
3. SDF-1α improved neovascularization and wound healing, in which expression of Cav-1in EPCs played an essential role.
糖尿病血管病变包括大血管病变及微血管病变,是糖尿病的慢性并发症之一,导致心脑血管病变、视网膜病变、糖尿病肾病、糖尿病足等并发症,是影响糖尿病患者生活质量及预后的重要因素。糖尿病血管病变一个重要特点是血管新生受损,组织损伤局部血液供应不足,导致组织难以得到有效修复,如局部组织持续缺血、坏死、感染,可能导致坏疽的严重后果,目前仍有大量糖尿病患者为此需要施行截肢手术。所以,探索糖尿病血管新生受损的机制并寻找改善糖尿病血管新生的方法是治疗糖尿病足、糖尿病伤口不愈等糖尿病血管并发症的重要手段。
内皮祖细胞(endothelial progenitor cells, EPCs)被证实在出生后血管新生中发挥重要作用。EPCs参与血管新生的方式主要有两种：一是EPCs直接整合到血管新生网络,通过分化成内皮细胞,参与新生血管内皮层的形成,二是在血管新生局部通过旁分泌方式分泌一些促血管生成的细胞因子和生长因子,如VEGF、HGF、IGF-1等,促进血管新生。EPCs数量已被作为心血管疾病诊断及预后评价的重要指标,EPCs介导的细胞和基因治疗心血管疾病中有着广阔的应用前景。然而研究表明糖尿病时内皮祖细胞数量减少、功能受损,不能有效地发挥促血管新生的作用,限制了其在糖尿病血管病变中的应用。到目前为止,糖尿
病EPCs功能受损的机制仍未完全明了。因此探索糖尿病状态下内皮祖细胞功能损伤的机制,寻求改善EPCs功能的方法,这对于改善血管新生、治疗糖尿病血管病变具有重要意义。
研究表明,一氧化氮合成酶(eNOS)及eNOS催化生成的NO在心血管系统中有重要作用,可促进EPCs介导的血管新生。然而,糖尿病状态下,内皮NO生成减少,导致血管功能失调,这可能与小凹蛋白1(caveolin-1, Cav-1)表达增加,抑制eNOS的激活有关。Cav-1通过蛋白与蛋白相互作用,抑制内皮eNOS的活性,使细胞处于较低的NO水平,当受到外界刺激时,eNOS与Cav-1脱离并被激活,催化产生NO,从而发挥调节血管张力、抑制血小板聚集、减少氧化应激损伤、抗炎、抗动脉粥样硬化和促进血管新生等作用。EPCs同内皮细胞一样表达Cav-1和eNOS,糖尿病是否增加EPCs (?)的Cav-1表达并抑制eNOS/NO信号通路,导致EPCs(?)功能受损尚不明了。
基于以上背景,我们提出如下假说：糖尿病EPCs的Cav-1表达增加,抑制eNOS/NO信号通路,使EPCs血管新生功能受损,而降低糖尿病EPCs的Cav-1表达可改善血管新生。为验证我们的假设,我们将首先检测2型糖尿病db/db小鼠EPCs的Cav-1表达变化,并找其变化的原因；检测糖尿病eNOS/NO通路及EPCs功能的改变;通过慢病毒转染降低糖尿病EPCs Cav-1表达,增加leNOS(?)的磷酸化及NO的生成,以期改善EPCs的血管新生功能。
研究目的
1.研究Cav-1在2型糖尿病小鼠EPCs (?)的表达变化,并探索变化的原因。
2.探讨Cav-1对2型糖尿病小鼠EPCs功能的调节作用及机制。
研究方法
1.动物模型
12周龄的2型雄性糖尿病小鼠db/db (BKS.Cg-m+/+Leprdb/J)及其非糖尿病对照小鼠db/+(Leprdb m/++)用于实验。实验前检测db/db小鼠血糖,300mg/dl以上者作为糖尿病模型。
2. EPCs的分离、培养与鉴定
分离小鼠股骨、胫骨、髋骨,提取骨髓单个核细胞,接种在已包被玻璃粘连蛋白(vitronectin)的六孔板中,在37℃,5%CO2环境中培养。72小时后第一次完全换液,去除未贴壁的细胞,第五天时半量换液,继续培养到第七天,即可用于实验。为鉴定培养细胞为EPCs,细胞培养7天后,用Hoechst33258、Dil-acLDL和isolectin染色并在荧光显微镜下观察。为进一步鉴定EPCs,对培养7天的细胞采用流式细胞术分析其表面干细胞表面标记物CD34和Sca-1、内皮细胞表面标记物CD144和VEGFR2(Flk-1)及单核细胞表面标记物CDllb的表达。
3. EPCs功能的检测
采用小管形成试验(tube formation assay)和细胞迁移实验(migration assay)来检测EPCs的小管形成能力及迁移能力。
4.分子生物学检测
(1) Real-time PCR
提取EPCs的总RNA,进行逆转录反应,获得cDNA,最后用试剂盒进行定量PCR反应检测EPCs的Cav-1表达,GAPDH作为内参。
(2) Western blot
用特异性抗体检测EPCs中Cav-1、p-eNOS1177、eNOS和β-actin的蛋白表达,在ImageQuant4000凝胶成像系统上扫描,用Quantity-One图像软件分析条带的光密度值。
5.慢病毒转染EPCs
用40MOI的Lenti-Cav-1siRNA转染EPCs,等量的Lenti-scrambled作为对照,转染24小时后换液,继续培养48小时后进行后续实验。
6.NO的测定
用Griess法检测培养基中硝酸盐的含量,间接反应NO的生成量。
7.统计学分析
计量资料以平均值±标准误(mean±SEM)表示。两样本间比较采用Studentt-检验,多样本比较采用单因素方差分析(one way ANOVA),并用Student-Newman-Keuls方法进行组间两两比较分析检验。所有结果采用GraphpadPrism5.0软件包进行分析并作图。P<0.05认为有显著性统计学差异。
研究结果
1.瘦素上调正常EPCs的Cav-1表达
db/db小鼠血清瘦素(leptin)水平为正常对照组的4.4倍。Real-time PCR和Western blot(?)结果表明,用生理浓度(20ng/ml)的leptin刺激db/+小鼠EPCs即可使Cav-1表达表达增加。随着leptin (?)浓度的增加,Cav-1的表达也持续增长,在100ng/ml浓度(接近db/db血清leptin浓度)时,Cav-1的mRNA和蛋白水平比0浓度分别增加94.65%和144.63%。
2. db/db小鼠EPCs的Cav-1表达增加且不依赖于高瘦素
Western blot结果显示,db/db小鼠EPCs比db/+组EPCs表达更高的Cav-1,约增加55.30%(P<0.05)。然而用100ng/ml leptin刺激db/db小鼠EPCs后,Cav-1的表达并没有明显变化,可能由于db/db小鼠EPCs瘦素受体缺失所致。
3.高糖上调EPCs的Cav-1表达
体外给予db/+和db/db小鼠的EPCs(?)高糖刺激,并给予甘露醇做等渗对照。结果显示,高糖刺激72小时后db/+小鼠EPCs的Cav-1农达增加了94.77%,db/db小鼠增加了39.36%(P<0.05),渗透压并不影响Cav-1的表达。
4.下调db/db小鼠EPCs的Cav-1表达改善细胞的血管新生功能
用携载Cav-1siRNA的慢病毒转染db/db小鼠EPCs后,Cav-1蛋白表达下降了53.63%。小管形成实验和干细胞迁移实验的结果表明,下调Cav-1表达能使db/db EPCs的小竹形成能力和细胞迁移能力明显提高。
5.下调db/db小鼠EPCs的Cav-1表达促进eNOS磷酸化和NO生成
EPCs给予50ng/ml VEGF-A刺激30分钟后,收集培养基检测NO的生成,并收集细胞检测eNOS磷酸化水平Western blot结果显示,db/db EPCs中磷酸化的eNOS比例比db/+EPCs明显降低,而慢病毒下调Cav-1标达后,eNOS磷酸化增加了88.72%(P<0.05),同时NO的生成也增加了1.78倍。
结论
1.在2型糖尿病情况下,高糖可以上调EPCs的Cav-1表达,这种效应独立于高瘦素的作用：
2.下调2型糖尿病db/db小鼠EPCs的Cav-1表达能提高改善EPCs的小管形成和迁移能力;
3.下调Cav-1改善糖尿病EPCs的功能的机制可能与促进eNOS磷酸化和增加NO生成有关。
研究背景
上世纪50年代,由于电子显微镜技术的发展,细胞的许多超微结构为人所知,在细胞膜上发现有许多50-100nm的烧瓶样凹陷,被称为小凹(caveolae)。尽管小凹的发现已有半个多世纪,但人们对小凹本质及功能的认识主要集中在近20年,这得益于小凹上最重要的蛋白成分—小凹蛋白(caveolin)的发现。这一发现使人们了解了小凹的许多生理功能,如胆固醇转运、信号转导、细胞内吞、肿瘤抑制等。小凹蛋白有三种亚型,分别是小凹蛋白1(caveolin-1, Cav-1)、小凹蛋白2(caveolin-2, Cav-2)、小凹蛋白3(caveolin-3, Cav-3)。
Cav-1与内皮细胞功能的关系已有较广泛的研究,已知Cav-1可影响内皮的通透性、胆固醇的内吞、内皮依赖的血管新生、细胞增殖等等,但Cav-1与内皮祖细胞(endothelial progenitor cells, EPCs)关系的研究较少,Cav-1对于EPCs及EPCs参与的病理生理作用有待进一步研究。随着EPCs研究的深入,EPCs在缺血性疾病及血管新生领域的作用日益受到重视,已成为诊断和预测某些疾病的重要指标,EPCs介导的细胞和基因治疗有着广阔的应用前景。
研究表明,在小鼠皮肤造成伤口模型后,血中的EPCs数量开始增加,到第三天时达到峰值,表明EPCs从骨髓中动员出来参与伤口的修复。我们以往的研究表明,糖尿病小鼠的EPCs功能受损,不能有效地促进血管新生,是糖尿病小鼠伤口愈合延迟的重要原因,因此EPCs功能对于维持正常伤口J愈合至关重要。本研究以伤口愈合的小鼠作为模型,研究Cav-1尤其是EPCs上Cav-1的表达对伤口愈合的影响,并试图发现其内在的联系机制。为了达到研究目的,我们使用了两种基因技术的小鼠：Cav-1(?)基因敲除小鼠(Cav-1KO mice)和内皮特异爪组Cav-1小鼠(Cav-1RC mice),前者体内的Cav-1完全缺失,后者是在前者的基础上的改良,在Cav-1敲除的背景下将内皮又重组Cav-1,因此这种小鼠在内皮以外的其他细胞类型都不表达Cav-1。由于EPCs是内皮的始祖细胞,因此Cav-1RC小鼠的EPCs也应该表达Cav-1,我们在研究中也将证实。Sbaa等人研究表明Cav-1KO小鼠的血清SDF-1α水平异常。SDF-1α是一种趋化因子,参与干细胞和祖细胞在骨髓的驻留及从骨髓的动员和归巢,SDF-1α已用于多种缺血性疾病的治疗。SDF-1α可趋化EPCs到缺血部位参与血管新生,这也是在病理情况比如糖尿病时SDF-1α促进伤口愈合机制之一。鉴于SDF-1α对EPCs介导血管新生的重要性,我们在本实验中探讨Cav-1α的表达对SDF-1α水平的影响,并且用腺相关病毒转染的方法增加伤口局部SDF-1α的表达,观察不同基因背景小鼠对SDF-1α治疗的反应,从而阐明Cav-I在EPCs介导的血管新生和伤口愈合中的作用。
研究目的
1.研究Cav-I缺失对内皮祖细胞功能的影响。
2.研究Cav-I表达对小鼠皮肤伤口愈合的作用。
3.探讨内皮祖细胞的Cav-I表达在SDF-1α促进的血管新生及伤口愈合中的作用。
研究方法
1.动物模型
本实验所用动物为8-12周龄的雄性小鼠,其中Cav-I基因敲除(Cav-I KO)小鼠和内皮特异性表达Cav-I(Cav-I RC)小鼠由Dr.Michael Bauer实验室提供,B6129SF2/J小鼠为对照,购自The Jackson Laboratory,实验动物饲养于美国匹兹堡大学医学院血管外科动物中心,自由饮食饮水,每日12小时光照。所有动物操作均符合美国匹兹堡大学实验动物保护与利用委员会的规定。
2.EPCs的分离、培养与鉴定
分离小鼠股骨、胫骨、髋骨,提取骨髓单个核细胞,接种在已包被玻璃粘连蛋白(vitronectin)的六孔板中,在37℃,5%CO2中环境中培养。72小时后第一次完全换液,去除未贴壁的细胞,第五天时半量换液,继续培养到第七天,可用于实验。
3. EPCs功能的检测
采用小管形成试验(tube formation assay)和细胞划痕实验(in vitro wound healing assay)来检测EPCs的小管形成能力及迁移能力。
4.伤口模型
将小鼠麻醉,用剃毛器将小鼠背部正中至尾巴根部的毛发剔除,用退毛膏将残余细微毛发去除干净。消毒后,盖上无菌洞巾,将6mm直径的打孔器定位至小鼠两大腿上缘与背中线的交点处,向下按压并旋转打孔器,切除一圆形皮肤,包括表皮和真皮层。每隔一日采集伤口照片并测量伤口大小。
5.腺相关病毒转染伤口组织
伤口模型建立后,沿伤口切缘滴加100μl含有1012病毒颗粒的SDF-1α/AAV或者GFP/AAV液,孵育30分钟后,拭去多余的病毒液。伤口愈合过程中仅在第0天孵育病毒一次。
6.统计学分析
计量资料以平均值±标准误(mean±SEM)表示。两样本间比较采Student t-检验,多样本比较采用单因素方差分析(one way ANOVA),并用Student-Newman-Keuls方法进行组间两两比较分析检验,对于多组间不同时期伤口愈合情况的比较用双囚素方差分析(two way ANOVA)。所有结果采用Graphpad Prism5.0软件包进行分析并作图。P<0.05认为有显著性统计学差异。
研究结果
1.内皮重组Cav-1使Cav-1敲除小鼠EPCs的小管形成能力恢复
敲除Cav-1后,EPCs形成的小管数量明显减少,约为WT组的51.88%,然而重组Cav-1后EPCs形成的小管数量明显增加,达到了WT组的95.97%,说明Cav-1的表达对EPCs的小管形成功能十分重要。我们又用划痕实验来评价细胞的迁移功能,WT, Cav-1KO和Cav-1RC三种小鼠的EPCs在12小时、24小时两个时间点迁移的数量并没有明显差异,说明Cav-1(?)的表达与否并不明显影响EPCs的迁移能力。
2.内皮重组Cav-1后不能加快Cav-1敲除小鼠的伤口愈合
从第6天开始,Cav-1KO小鼠的伤口愈合开始明显落后于WT组,一直持续到第16天,此时WT组的皮肤伤口基本愈合,但Cav-1KO小鼠的皮肤只愈合到89.02%,全程愈合率与WT组比较有统计学差异(P<0.05)。尽管重组Cav-1使EPCs(?)的血管新生功能得到恢复,但是伤口愈合并没有得到改善,在第16天时仅愈合了90.25%,与Cav-1KO组并无明显差别,全程愈合率也明显低于WT组(P<0.05)。
3.内皮重组Cav-1未明显增加Cav-1KO小鼠伤口的血管新生数目
免疫组化结果显示,Cav-1KO小鼠皮肤的毛细血管密度明显低于WT组,Cav-1RC的新生血管数量与KO组相当,没有明显改善。以上结果提示,Cav-1KO小鼠皮肤伤口愈合延迟可能与血管新生受损有关,尽管重组Cav-1能够使EPCs的功能得到恢复,但并不足以明显改善皮肤伤口局部的血管新生。
4.局部SDF-1αAAV转染改善Cav-1RC/J、鼠皮肤的伤口愈合
我们将Cav-1KO和Cav-1RC小鼠实施6mm的全层皮肤切除后,分别给予SDF-1α/AAV在伤口局部,GFP/AAV作为对照,20分钟的转染后将剩余病毒液体拭去,贴好敷料,检测伤口愈合情况。结果显示,给予AAV-SDF-1α)后,Cav-1KO组的伤口愈合速度与对照组比较并无明显改变,仍然慢于WT组,而Cav-1RC小鼠伤口在AAV-SDF-1α治疗后愈合明显改善,在第16天达到了WT愈合水平。以上结果提示,内皮Cav-1的表达在SDF-1α促进伤口愈合的过程中有重要作用。
5. SDF-1α AAV治疗使Cav-1RC皮肤血管新生数目增加,但Cav-1KO组无明显改善
WT和Cav-1KO经过SDF-1α AAV转染后的新生血管数目与其对照组并没有明显改变,而Cav-1RC组比其对照组有更多的新生毛细血管数量,与伤口愈合的结果一致。以上结果提示,SDF-1α AAV改善Cav-1RC伤口愈合可能与招募更多功能正常的EPCs促进血管新生有关。
结论
1.Cav-1的表达对EPCs功能十分重要,Cav-1缺失导致EPCs的小管形成能力明显下降；
2.Cav-1缺失使伤口愈合延迟,仅仅内皮和内皮祖细胞上Cav-1恢复表达不能改善受损的伤口愈合；
3.SDF-l1α增加伤口局部EPCs的数量,从而促进血管新生和伤口愈合,这个过程需要EPCs上Cav-1的适量表达。
Background
Diabetic vascular diseases, among chronic complications in diabetes mellitus, are related with atherosclerosis, diabetic retinopathy, nephropathy and diabetic foot. Ineffective vascular repair is likely to be an important contributor to the diabetic vascular disease, and this effect is linked with impaired neovascularization in diabetes. Efforts to investigate the mechanisms of impaired neovascularization in diabetes and find new ways to improve it are significant to therapies of diabetic vascular complications.
Neovascularization is composed of angiogenesis and vasculogenesis, which involves distinct mechanisms. Vasculogenesis occurs in embryogenesis, and has been found to contribute to postnatal neovascularization due to the discovery of endothelial progenitor cells (EPCs). Asahara et al. reported for the first time the existence of EPCs in1997. Subsequent studies supported that EPCs played an crucial role in postnatal vasculogenesis. In the last decade, the important role played by EPCs in cardiovascular health is becoming increasing appreciated, especially for their crucial role in maintenance of endothelial integrity and function, as well as postnatal neovascularization.
There is accumulating evidence showing reduced number and impaired EPC function in the presence of diabetes mellitus, and dissatisfactory effects of diabetic EPC therapy. which limits the use of EPCs in practice. However, the mechanisms of EPC dysfunction in diabetes are incompletely understood. Nitric oxide (NO) was reported to play an important role in EPC mediated vasculogenesis. However, eNOS-derived NO decreases in the setting of diabetes, which contributes to impaired EPC function. Evidence has showed that eNOS resides in caveolae and Cav-1could inhibit the activity of eNOS via direct interaction with eNOS. Though the expression of Cav-1was upregulated in several tissues in diabetes, the change of Cav-1in diabetic EPCs is not clear. In current study, we hypothesize that Cav-1may be also upregulated in diabetic EPCs and involves in dysregulated eNOS/NO pathway. We determined the expression of Cav-1in EPCs from type2diabetic db/db mice, and tried to reveal the relationship between Cav-1and metabolic abnormalities, like hyperglycemia and hyperleptinemia. Specifically, we generated lentivirus carrying Cav-1siRNA to elucidate the role of Cav-1in EPC function and the underlying mechanisms in the setting of diabetes.
Objectives
1. To measure the changes of expression of Cav-1in type2diabetic EPCs, and to investgate the underlying mechanisms.
2. To investigate the role and mechanisms of Cav-1in regulating EPC functions in type2diabetes.
Methods
1. Animal model
Male db/db (BKS.Cg-m+/+Leprdbh/J) mice and their control mice(db/+), aged12weeks, were purchased from Model Animal Research Center of Nanjing University. In the current study, db/db mice with a blood glucose level>300mg/dl were considered diabetic and were used for EPC isolation.
2. EPC isolation, culture and characterization
Bone-marrow mononuclear cells were isolated from the femurs, tibias and pelvis of mice and then cultured with endothelial cell growth medium2in37℃5%CO2. After72hours, nonadherent cells were removed by changing medium and adherent cells were cultured continuously till day7for experimental use. The EPCs after7-day culture were characterized by Hoechst33258, DiI-acLDL and FITC-conjugated isolectin staining. To further characterize the phenotype of EPCs, the expressions of CD34, Sca-1, CD144, Flk-1and CD11b were analyzed by flow cytometry.
3. EPC function assays
The tube formation and migration assays were performed to determine the angiogenic function of EPCs. For tube formation assay,5×104EPCs were plated in a well of48-well plate pre-coated with growth factor-reduced Matrigel. After24-hour incubation at37℃5%CO2with EGM-2plus5%FBS, images were taken by an inverted microscope and tube lengths were measured. EPC migration was measured using Boyden chambers. Briefly,5×104EPCs were plated in the upper chamber of Transwell and allowed to migrate in a37℃5%CO2incubator. The upper cells were removed and the cells in the lower side were fixed and counted with a microscope.
4. Lentivirus constructs
We constructed a siRNA for mouse caveolin-1and a control (scrambled) that does not affect any protein expression using oligo pairs. Lentiviruses encoding scrambled and caveolin-1siRNA were produced by transfection of HEK293T which is performed commercially by GENECHEM.
5. Western Blot Analysis
20-50μg protein was subjected to SDS-polyacrylamide gel electrophoresis. The PVDF membranes were probed with primary antibodies against Cav-1, p-eNOS1177, eNOS and Actin. The blots were scanned with an ImageQuant4000system and band intensity was quantified with Quantity One System.
6. Real-time PCR
For each sample,1μg RNA was reverse transcribed using a reagent kit. Real-time PCR was performed using SYBR Green PCR Master Mix on a iCycler iQ Real-time PCR System. Variation in transcription levels was calculated using2△CT.
7. Measurement of NO release
After being serum-starved for24hours, EPCs were treated with VEGF (50ng/ml) for30minutes. Nitrite concentration in the culture medium was measured using the Griess reaction, and sodium nitrite served as a standard. The optical density of samples was measured in a reader system.
8. Statistical Analysis
Data were presented as means±SEM. Differences between two groups were compared by Student's t-test, and one-way ANOVA with Newman-Keuls multiple comparison tests was used for multiple groups. In all the tests, a value of P<0.05was taken as statistically significant.
Results
1. Leptin regulated Cav-1mRNA and protein expression
It was reported that leptin could upregulate the expression of Cav-1in HCAEC and HUVEC. To test whether leptin could regulate expression of Cav-1in EPCs, db/+EPCs were incubated with increasing concentration of leptin (0.20,50,100ng/ml) for24h, then cells were harvested for mRNA and protein analysis. Our data showed the mRNA and protein of Cav-1was upregulated by leptin at the concentration of20ng/ml. When the concentration of leptin was increased, Cav-1expression rose accordingly.
2. Cav-1protein expression was upregulated in db/db EPCs independent of high leptin level
Compared with db/+EPCs, db/db EPCs showed higher expression of Cav-1. We treated db/db EPCs with100ng/ml leptin to measure the Cav-1expression. The result showed a high level of leptin could not upregulate the expression of Cav-1in db/db EPCs.
3. High glucose upregulated Cav-1protein expression in db/db and db/+EPCs
As compared with that in control medium (5mmol/l glucose), the expression of Cav-I in db/+EPCs assessed by Western blot was significantly increased, by94.77%in high-glucose medium. High glucose also upregulated Cav-1expression in db/db EPCs by39.36%, though db/db EPCs had a higher expression of Cav-1originally. By contrast, the osmotic control with mannitol did not change the expression of Cav-1.
4. Cav-1knockdown improved functions of db/db EPCs
Our result showed db/db EPCs formed significantly fewer networks than db/+EPCs. In contrast, when Cav-1expression was knocked down by lentivirus-mediated siRNA, Matrigel tube formation was increased by69.32%in db/db EPCs. Cav-1knockdown also significantly increased migration of db/db EPCs.
5. Cav-1knockdown increased phosphorylation of eNOS and NO production
EPCs were incubated in EGM-2supplemented with VEGF (50ng/ml) for30minutes, then the medium was collected for NO detection and cells were harvested for protein analysis. The eNOS phosphorylation at Ser1177shown by immunoblotting was significantly decreased in db/db EPCs compared with that in db/+EPCs. This reduction in eNOS phosphorylation was associated with a decrease in EPC-derived NO production. Nevertheless, Cav-1knockdown significantly increased eNOS phosphorylation by88.72%in db/db EPCs coupled with significantly augmented NO production.
Conclusions
1. High glucose upregulated Cav-1protein expression independent of high leptin level.
2. Cav-1knockdown in db/db EPCs improved the angiogenic function.
3. Cav-1knockdown increased phosphorylation of eNOS and NO production in db/db EPCs
Background
Caveolae are50-100nm flask-shaped invaginations of the plasma membrane which are implicated in cholesterol transport, signal transduction. endocytosis. Caveolin, an important protein component of caveolae membrane coats, is known to have three isoforms which are caveolin-1(Cav-1), Cav-2and Cav-3. Cav-1has been widely and intensively studied, especially after the generation of Cav-1knockout mice. The putative importance of Cav-1in cardiovascular system have been validated in several physiological and pathological models, in which Cav-1influences vascular permeability, angiogenesis and progress of atherosclerosis. Notably, endothelial expression of Cav-1is closely involved in these conditions, and both lower and higher levels of Cav-1expression may impair vascular functions.
Mature endothelial cells possess limited regenerative capacity, therefore, there is growing interest into circulating endothelial progenitor cells (EPCs). Not only have EPCs been used to diagnose and predict cardiovascular diseases, EPCs have also served as a useful tool for cell and gene therapy in ischemic diseases. Though Cav-1has been well investigated in endothelial cells, there is few reports concerning its role in EPCs. Sbaa et al. reported that Cav-1knockout mice presented impaired EPC mobilization from bone marrow, which resulted in defective neovascularization in a hindlimb ischemia model. This study suggested that Cav-1might play an important role in EPC functions. In current study, we would investigate the impact of Cav-1expression on EPC functions. Our previous study showed EPCs isolated form type2diabetic db/db mice presented impaired angiogenic functions, which caused deficient new vessel formation in skin repair, and subsequently delayed wound healing. This suggests EPC is an essential opponent participating in wound healing. Herein, we mainly examined the effects of Cav-1expression in EPCs on neovascularization and cutaneous wound healing. To address this question, Cav-1KO mice and Cav-1KO mice reconstituted with a transgene expressing Cav-1specifically in endothelial cells (Cav-1RC mice) were used. We assessed neovascularization and wound healing in WT, Cav-1KO and RC mice, and examined whether reconstitution of Cav-1regulates wound healing via EPC mediated vasculogenesis.
It is well known that stromal cell-derived factor-1alpha (SDF-1α) is a predominant chemokine which is upregulated in response to ischemic stimuli and acts as a homing signal for EPCs. SDF-1α gene transfer has become a novel chemokine therapy for ischemic disease and tissue repair via recruiting EPCs to ischemic sites. It was proved that SDF-1α treatment accelerated wound closure in diabetes, while inhibition of SDF-1α further impaired diabetic wound healing, and the main mechanism was closely related with EPC mediated vasculogenesis. It was found in Cav-1KO mice plasma level of SDF-1α was2-fold higher than WT mice, which suggested Cav-1might regulate SDF-1α. Based on the evidence that SDF-la therapy effectively improves the injury repair in several models, our study also determined whether SDF-la therapy achieved by SDF-1α/AAV treatment enhances wound healing in Cav-1KO and RC mice, and whether the effects of SDF-la is Cav-1dependent.
Objectives
1. To determine the role of Cav-1in EPC function;
2. To investigate the role of Cav-1in cutaneous would healing
3. To investigate the role and mechanisms of EPCs' Cav-1in SDF-1α enhanced neovascularization and would healing;
Methods
1. Animals
All animal studies were performed with approval of the University of Pittsburgh Institutional Animal Care and Use Committee. The generation of Cav-1KO mice and Cav-1RC mice was previously described. All KO and RC mice were supplied by Dr. Michael Bauer. The WT mice (B6129SF2/J) were purchased from The Jackson Laboratory. Male mice with8-12weeks old were used in this study.
2. Full-thickness excisional wounds
Mice were anesthetized using isoflurane and then depilated with an electric shaver and depilatory creamin dorsum. Mouse skin was cleaned by Betadine and70%alcohol. Then full-thickness skins, containing epidermis and dermis, were removed by a6-mm punch. Wounds were covered with a bioclusive transparent oxygen permeable wound dressing. Wound closure rate was measured every other day till day16on which WT mice were almost cured completely.
4. EPC function assays
The tube formation assay and in vitro wound healing assay were performed to determine the angiogenic function of EPCs. For tube formation assay,5×104EPCs were plated in a well of48-well plate pre-coated with growth factor-reduced Matrigel. After24-hour incubation, images were taken by an inverted microscope and tube lengths were measured. For in vitro wound healing assay, EPCs ware seeded at1.5×105cells/well into a24-well plate. After24-hour incubation, EPCs were almost confluent, and scratched using a10μl pipette tip. After being washed3times using PBS, EPCs were kept on culturing. Images were taken at the time of wounding0h,12h and24h. Migration was estimated by counting cell numbers within wounded region.
5. Enzyme-linked immunosorbent assay (ELISA) SDF-1α protein levels in serum and bone marrow were determined by using the mouse SDF-1α Quantikine ELISA Kit based on the manufacturer's instructions. Peripheral blood was obtained by cardiac aspiration and then centrifuged to collect serum. BM was obtained from femurs, tibias and pelvis. The bones were flushed in500μl PBS. After centrifugation, the supernatant was collected for SDF-la ELISA and total protein quantification.
6. SDF-la AAV topical treatment
AAV vectors carrying SDF-1α gene has been used to treat wounds in mice. In this study, human SDF-la/AAV and control vector, green fluorescent protein (GFP)/AAV, which were gifts from Dr. Yanfang Chen (Wright State University, Ohio), were constructed by inserting the human SDF-la or GFP gene into AAV2/9vector. After wounds were created,100μl AAV at1012particle/ml in PBS was injected into the wound beds. Mice were maintained under anesthesia for30minutes, and then a bioclusive dressing was placed over the wound. Wounds were measured every other day till day16.
7. Statistical Analysis
Data were presented as means±SEM. Differences between two groups were compared by Student's t-test, and one-way ANOVA with Newman-Keuls multiple comparison tests was used for multiple groups. The rate of wound healing among groups was analyzed by2-way ANOVA, followed by Bonferroni's correction to control type I error. In all the tests, a value of P<0.05was taken as statistically significant.
Results
1. Reconstitution of Cav-1restores tube formation of Cav-1KO EPCs
Cav-1KO EPCs formed significantly fewer networks than WT EPCs, and relative tube length decreased by nearly50%. And this impairment was completely restored in Cav-1RC group by reconstitution of Cav-1in EPCs. In vitro wound healing assay showed these three groups presented similar migrating pattern which was analyzed at the time point of12hours and24hours after creating wound.
2. Reconstitution of Cav-1in endothelium is insufficient to restore impaired wound healing in global Cav-1knockout mice
These three strains of mice underwent a single6-mm dorsal punch biopsy to create a full-thickness excisional wound, and then the rate of wound closure was measured every other day till day16. Cav-1KO mice exhibited slower healing rate than WT mice, which was significant from day6to day16. Nevertheless, reexprcssion of Cav-1in endothelium failed to rescue the impaired wound healing in global Cav-1knockout background, and exhibited similar recovery pattern with Cav-1KO mice. At day16, wounds in WT mice were almost entirely closed, whereas unhealed in Cav-1KO and Cav-1RC mice (89.02%and90.25%respectively).
3. Wounded skins in Cav-1KO and RC mice showed decreased new vessel formation
We wondered whether deficient neovascularization led to impaired wound healing in Cav-1KO and RC mice, so we measured the vessel number in skins16days after wound creation. As showed by immunofluorescence staining, the percentage of vessel density in Cav-1KO and RC mice wounded skin were32.64%and38.03%respectively compared with that of WT skin. The impaired neovascularization in wounded skin might lead to the delayed wound healing in Cav-1KO and RC mice.
4. Topical treatment of SDF-1α AAV accelerated wound healing in Cav-1RC mice coupled with increased neovascularization in skin
Following wounding,1011viral particles of SDF-1α/AAV in100μl PBS was injected onto wound base for30minutes in WT, Cav-1KO, and Cav-1RC mice, while same amount of GFP/AAV was administered to control groups. During16-day observation, Cav-1RC mice showed enhanced wound healing rate and were restored to the WT level, however. Cav-1KO mice didn't respond to the treatment. We next measured the neovascularization of wounded skins treated with SDF-1α/AAV or GFP/AAV. Results showed Cav-1RC mice treated with SDF-1α/AAV had more new vessel formation than those treated with GFP/AAV, while Cav-1KO mice showed no response to SDF-la treatment.
Conclusions
1. Expression of Cav-1is essential to the angiogenic function of FPCs.
2. Cav-1is required for cutaneous wound healing, and reconstitution only in endothelium is insufficient to restore the impaired wound healing in Cav-1KO mice.
3. SDF-1α improved neovascularization and wound healing, in which expression of Cav-1in EPCs played an essential role.
引文
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