核受体LXR对损伤血管内皮修复的影响及机制研究
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摘要
1.研究背景
血管内皮损伤是介入术后的血栓形成和血管再狭窄的关键，也是高血压、动脉粥样硬化和糖尿病血管病变等多种血管损伤性疾病共同的病理生理基础。血管损伤后的内皮再生修复可以有效防止血栓形成和抑制血管平滑肌细胞的异常增生，因此，尽早促进损伤血管的再内皮化、恢复血管内皮功能，是防止介入术后血栓形成和血管再狭窄，预防和治疗血管损伤性疾病的有效策略。
肝X受体(liver X receptor, LXR)是配体激活的核受体超家族成员，被其内源性配体/合成激动剂激活后，可以通过调节一系列靶基因的转录，参与调节胆固醇代谢、糖代谢和炎症反应。近来的研究发现，LXR在心脑血管疾病防治中可能发挥着一定的作用：(1)LXR激动剂可以明显抑制动脉粥样硬化的发生发展过程；(2)LXR激动剂可以显著抑制血管损伤后的新生内膜形成；(3)LXR激动剂可以促进缺血脑组织的新生血管形成。损伤血管内皮的再生是上述心脑血管疾病防治的共同环节，因此我们猜测，LXR激活后是否可以通过促进血管内皮的再生，从而发挥对上述病理生理过程的调节作用？
在预实验中，我们采用小鼠颈动脉损伤模型观察LXR对损伤血管的内皮再生的影响，结果显示，LXR激动剂T0901317干预后，小鼠的损伤颈动脉的再内皮化面积明显增加。那么LXR激活后又是如何促进损伤血管内皮的再生修复呢？血管内皮的再生依赖于：(1)邻近内皮细胞(endothelial cells, ECs)的增殖和迁移；(2)骨髓、脾脏等来源的内皮祖细胞(endothelial progenitor cells, EPCs)，EPCs能从骨髓、脾脏等动员到外周血，迁移、归巢于血管损伤区域，增殖和分化为ECs，还可通过旁分泌机制(分泌促血管生成因子)促进邻近ECs增殖和迁移，从而促进内皮修复。
介于以上的研究背景，我们推测，激活LXR后可能通过调节ECs与EPCs的生物学功能(如：增殖、迁移能力等)，促进损伤血管内皮的再生与修复。
2.方法
2.1. LXR激动剂对小鼠颈动脉损伤血管内皮修复的影响
构建小鼠颈动脉损伤模型，采用LXR激动剂T0901317[30mg/(Kg·d)]于术前2天开始喂养小鼠，通过伊文氏蓝染色观察损伤颈动脉再内皮化的情况，以阐明激活内源性LXR对损伤血管内皮修复的影响。
2.2. LXR对内皮细胞的增殖、迁移能力的影响及机制研究
从健康新生儿脐带分离和培养原代人脐静脉内皮细胞(human umbilical veinendothelial cells, HUVECs)，显微镜下观察HUVECs的形态学特征，免疫荧光检测vWF进行内皮细胞鉴定；第3～5代HUVECs用于实验。
LXR激动剂T0901317(0、0.5、2、5μmol/L)处理HUVECs后，MTS方法检测HUVECs的增殖能力，Transwell迁移实验检测HUVECs的迁移能力, Western blot检测p-Akt、总Akt、p-eNOS、总eNOS的表达。
先用PI3K抑制剂LY294002(10μmol/L)或eNOS抑制剂L-NAME (100μmol/L)预处理HUVECs30min后，再用LXR激动剂T0901317干预，MTS方法检测HUVECs的增殖能力，Transwell迁移实验检测HUVECs的迁移能力, Western blot检测p-Akt、总Akt、p-eNOS、总eNOS的表达。
2.3. LXR对内皮祖细胞的生物学功能的影响及机制探讨
选用大鼠骨髓源性内皮祖细胞(BM-EPCs)和小鼠脾源性内皮祖细胞(Spleen-EPCs)这两种不同种属和来源的EPCs作为研究模型。密度梯度离心法分离大鼠骨髓单个核细胞(MNCs)和小鼠脾脏MNCs，诱导分化为EPCs，显微镜下观察细胞的形态学特征，通过DiI-ac-LDL、FITC-UEA-I荧光双染和流式细胞分析检测细胞分子表面标记进行EPCs的鉴定。培养第5～7天的大鼠BM-EPCs和小鼠Spleen-EPCs用于实验。
采用RT-PCR、western blot和免疫荧光检测LXRα、LXRβ在大鼠BM-EPCs和小鼠Spleen-EPCs中的表达和定位。
LXR激动剂T0901317(0、0.5、2、5μmol/L)或GW3965(0、0.5、2、5μmol/L)干预EPCs后，MTS方法检测EPCs的增殖能力，Transwell迁移实验检测EPCs的迁移能力，Western blot检测p-Akt、总Akt、p-eNOS、总eNOS的表达。
先用PI3K抑制剂LY294002(10μmol/L)或eNOS抑制剂L-NAME (100μmol/L)预处理EPCs30min后，再用LXR激动剂T0901317或GW3965干预，MTS方法检测BM-EPCs的增殖能力，Transwell迁移实验检测EPCs的迁移能力，Western blot检测p-Akt、总Akt、p-eNOS、总eNOS的表达。
LXR激动剂T0901317(2μmol/L)处理EPCs24h后，RT-PCR检测EPCs分泌的常见促血管生长因子(VEGF、SDF-1、HGF、IGF-1和G-CSF)mRNA的表达。采用LXR激动剂T0901317(0、0.5、2、5μmol/L)或GW3965(0、0.5、2、5μmol/L)处理EPCs，不同时间点收取细胞和细胞上清，Real-Time PCR、western blot、ELISA检测VEGF的mRNA、蛋白的表达和分泌。
2.4.统计学处理
所有计量资料以平均值±标准差(x±s)表示，使用SPSS18.0统计学软件进行分析，组间比较选用单因素方差分析(One-Way ANOVA)。P＜0.05为有统计学差异。
3.结果
3.1. LXR激动剂对小鼠颈动脉损伤血管内皮修复的影响
LXR激动剂T0901317干预后可明显增加颈动脉损伤后第4天、7天和14天的再内皮化面积(P＜0.05，n=8)，提示激活内源性LXR可以明显地促进损伤血管内皮的再生和修复。
3.2. LXR对内皮细胞的增殖、迁移能力的影响及机制研究
3.2.1. HUVECs的分离、培养和鉴定
细胞呈单层生长，互不重叠，形态呈多角形、椭圆形或梭形，呈铺路石样镶嵌排列；激光共聚焦显微镜下显示细胞质vWF阳性(红色荧光)即为HUVECs，鉴定阳性率＞95%(n=6)。
3.2.2. LXR激动剂增强HUVECs增殖、迁移能力
不同浓度的T0901317(0.5、2、5μmol/L)处理后，HUVECs的增殖和迁移能力明显增强，并与T0901317呈一定的剂量依赖关系(P＜0.05，P＜0.01，n=4-6)；表明LXR激动剂可以促进HUVECs的增殖和迁移。
3.2.3. LXR激动剂调节HUVECs增殖、迁移能力的机制研究
3.2.3.1. LXR激动剂对HUVECs中PI3K/Akt/eNOS信号通路的影响
T0901317处理后，呈时间和剂量依赖的上调HUVECs中p-Akt-Ser473和p-eNOS-Ser1177的表达(P＜0.05，P＜0.01，n=3-5)，PI3K抑制剂LY294002(10μmol/L)可以明显阻断上述作用，提示T0901317通过PI3K激活Akt/eNOS信号通路。然而在T0901317干预的早期(＜1h)，未见明显的p-Akt-Ser473和p-eNOS-Ser1177水平上调，提示LXR激活后可能通过间接作用导致了HUVECs中PI3K/Akt/eNOS信号途径的活化。
3.2.3.1. LXR激动剂通过PI3K/Akt/eNOS途径增强HUVECs的增殖、迁移能力
采用PI3K抑制剂LY294002(10μmol/L)或eNOS抑制剂L-NAME(100μmol/L)预处理HUVECs30min后，T0901317(5μmol/L)促进HUVECs增殖和迁移的效应明显减弱(P＜0.05，P＜0.01，n=4-6)，而LY294002和L-NAME并没有改变HUVECs的细胞活力和基础迁移水平(P=N.S.，n=4-6)；说明LXR激动剂可以通过PI3K/Akt/eNOS信号途径增强HUVECs的增殖、迁移能力。
3.3. LXR对内皮祖细胞的生物学功能的影响及机制探讨
3.3.1.大鼠BM-EPCs的分离、培养和鉴定
成功分离大鼠BM-EPCs，呈梭形、椭圆形或三角形，早期可见典型的克隆单位“集落”形成；晚期呈“线型”、“管型”或者“网状”血管样生长趋势，密度较大时呈铺路石样排列。DiI-ac-LDL(红色)和FITC-UEA-I(绿色)双染阳性即为正在分化的EPCs，所分离培养的细胞双染阳性率大于90%。流式细胞技术检测到CD133、CD34、VEGFR-2和CD31在所培养细胞(5~7天)中的阳性率分别为75.4%、82.3%、64.9%、86.2%和84.6%，符合EPCs的特征。
3.3.2.小鼠Spleen-EPCs的分离、培养和鉴定
成功分离小鼠Spleen-EPCs，呈梭形、椭圆形或三角形，早期可见典型的克隆单位“集落”形成；晚期呈“线型”、“管型”或者“网状”血管样生长趋势，密度较大时呈铺路石样排列。DiI-ac-LDL(红色)和FITC-UEA-I(绿色)双染阳性率大于90%，流式细胞技术检测到Sca-1、CD34、c-kit、VEGFR-2及CD31在所培养细胞(5~7天)中的阳性率分别为89.2%、75.4%、92.9%和91.9%，符合EPCs的特征。
3.3.3.检测LXR在EPCs的表达
培养的不同时间点(4天、7天、10天、14天)的大鼠BM-EPCs和小鼠Spleen-EPCs中，我们均检测到LXRα、LXRβ mRNA和蛋白的表达，免疫荧光提示LXRα、LXRβ在EPCs的细胞核和细胞浆均有表达，但主要表达于细胞核中；采用LXR激动剂T0901317或GW3965处理EPCs后，LXR的靶基因ABCA1的转录水平均明显上调，提示LXR在EPCs是具有转录活性和生物学功能的。
3.3.3. LXR激动剂增强EPCs的增殖、迁移能力
两种不同的LXR激动剂T0901317(0.5、2、5μmol/L)或GW3965(0.5、2、5μmol/L)处理后，大鼠BM-EPCs和小鼠Spleen-EPCs的增殖和迁移能力均明显增强，并与LXR激动剂呈一定的剂量依赖关系(P＜0.05，P＜0.01，n=4-6)；表明LXR激动剂可以促进EPCs的增殖和迁移。
3.3.4. LXR激动剂调节EPCs增殖、迁移能力的机制研究
3.3.4.1. LXR激动剂对EPCs中PI3K/Akt/eNOS信号通路的影响
LXR激动剂T0901317或GW3965处理后，呈时间和剂量依赖的上调EPCs(大鼠BM-EPCs和小鼠Spleen-EPCs)中p-Akt-Ser473和p-eNOS-Ser1177的表达(P＜0.05，P＜0.01，n=3-5)，PI3K抑制剂LY294002(10μmol/L)可以明显阻断上述作用，提示激活LXR通过PI3K导致Akt/eNOS信号通路的活化。然而在LXR激动剂干预的早期(＜1h)，未见明显的p-Akt-Ser473和p-eNOS-Ser1177水平上调，提示LXR激活后可能通过间接作用导致了EPCs中PI3K/Akt/eNOS信号途径的活化。
3.3.4.2. LXR激动剂通过PI3K/Akt/eNOS途径增强EPCs的增殖、迁移能力
采用PI3K抑制剂LY294002(10μmol/L)或eNOS抑制剂L-NAME(100μmol/L)预处理EPCs(大鼠BM-EPCs和小鼠Spleen-EPCs)30min后，T0901317(2μmol/L)和GW3965(5μmol/L)促进EPCs增殖和迁移的效应明显减弱(P＜0.05，P＜0.01，n=4-6)，而LY294002和L-NAME并没有改变EPCs的细胞活力和基础迁移水平(P=N.S.，n=4-6)；说明LXR激动剂可以通过PI3K/Akt/eNOS信号途径增强EPCs的增殖、迁移能力。
3.3.5. LXR激动剂对EPCs分泌促血管生长因子的影响
3.3.5.1. LXR激动剂对EPCs中促血管生长因子(VEGF、SDF-1、HGF、IGF-1和G-CSF)mRNA表达的影响
LXR激动剂T0901317(2μmol/L)作用EPCs24h后，VEGF mRNA的表达水平显著上调(P＜0.05，n=4)，而SDF-1、HGF、IGF-1和G-CSF mRNA表达水平未见明显变化，提示LXR激动剂可以上调EPCs中VEGF mRNA的表达。
3.3.5.2. LXR激动剂对EPCs中VEGF表达和分泌的影响
LXR激动剂T0901317或GW3965作用于EPCs后，呈时间和剂量依赖的上调VEGF mRNA的表达(P＜0.05，P＜0.01，n=4-6)，呈剂量依赖的上调VEGF蛋白的表达(P＜0.05， n=6)和促进VEGF的分泌(P＜0.05，P＜0.01，n=5)，说明LXR激活后可以上调EPCs中VEGF的表达和促进VEGF的分泌。
4.结论
4.1. LXR激动剂通过PI3K/Akt/eNOS信号途径增强内皮细胞的增殖和迁移能力；
4.2. LXRα与LXRβ表达于内皮祖细胞，LXR激动剂可以通过PI3K/Akt/eNOS信号途径增强内皮祖细胞增殖和迁移能力，LXR激动剂上调内皮祖细胞中促血管生长因子VEGF的表达和促进VEGF的分泌；
4.3.激活内源性LXR通过调节内皮细胞和内皮祖细胞的生物学功能，从而促进损伤血管内皮的再生与修复。
1. Background
Vascular endothelial injury, which is the common pathophysiological basis ofhypertension, atherosclerosis and vascular disease in diabetes, is also the major cause ofpost-angioplasty restenosis and thrombosis. Endothelial regeneration and repair couldprevent thrombosis and suppress vascular smooth muscle cell proliferation after arterialinjury. Hence, accelerating re-endothelialization of injured arteries and restoring endothelialfuction are useful strategies for inhibiting post-angioplasty restenosis and thrombosis andpreventing vascular injured diseases.
Liver X receptors (LXRs) are members of nuclear receptor superfamily. Onceactivated by endogenous ligands or synthetic agonists, LXR can regulate cholesterolmetabolism, glucose metabolism and inflammations by influencing the expression of anarray of LXR target genes. Studies performed recently have showed that LXR might playimportant roles in preventing cardiovascular diseases:(1) LXR agonists inhibited thedevelopment of atherosclerosis,(2) LXR agonists suppressed neointima formation inballoon-injured rat carotid arteries,(3) LXR activation promoted angiogenesis afterischemic stroke. Since endothelial regeneration and repair after vascular injury are thecommon steps in preventing above cardiovascular diseases, we hypothesize that activationof endogenous LXR might regulate all above pathophysiology processes by stimulatingendothelial regeneration.
In our preliminary experiments, we have found that treatment of LXR agonistT0901317increased re-endothelialized area in a mouse model of carotid injury, suggestingthat activation of endogenous LXR could promote endothelial regeneration and repair of injured arteries. However, the mechanisms are not known. Endothelial regenerationdepends on:(1) neighbouring endothelial cells (ECs) proliferation and migration;(2)endothelial progenitor cells (EPCs) derived from bone marrow, spleen and etc.Accumulating evidence has demonstrated that EPCs, which mobilized to circulation byischemia, physical training, and the administration of statins, estrogen, and a variety ofcytokines, can be recruited to sites of endothelial injury. There, they promotere-endothelialization directly by incorporating into the recovering endothelium at the sitesof injury and indirectly by producing and releasing angiogenic growth factors.
Based on these observations, we speculate that activation of LXR might promoteendothelial regeneration and repair after vascular injury via regulating ECs and EPCsbiological functions (such as proliferation, migration and etc.).
2. Methods
2.1. Effect of LXR activation on endothelial repair after vascular injury
To explain the effect of LXR activation on endothelial repair after injury, weestablished an mouse model of carotid injury as previously described. Mice were orallyadministered of LXR agonist T0901317(30mg/kg per day) or vehicle starting2daysbefore the injury. To measure the re-endothelialized area, animals were perfused in vivowith Evans blue dye4,7, and14days after the injury.
2.2. Effect and mechanism of LXR on ECs proliferation and migration
Human umbilical vein endothelial cells (HUVECs) were obtained and cultured by ourestablished methods. Immunostaining for von Willebrand factor (vWF) confirmed that thecells were endothelial. Cells used for experiments were from passages3through5.
HUVECs were treated with different concentrations of LXR agonist T0901317(0μmol/L,0.5μmol/L,2μmol/L,5μmol/L) for different time course. HUVECs proliferationwas evaluated using a colorimetric MTS assay kit and HUVECs migration assay wasperformed using a Transwell chamber (8μm pore size). In addition, western blot analysiswas used to determine the expression of phosphorylated Akt (Ser473), total Akt,phosphorylated eNOS (Ser1177) and total eNOS. LY294002(10μmol/L) and L-NAME (100μmol/L), when used, were added30minutes prior to the addition of LXR agonist.
2.3. Effect and mechanism of LXR on EPCs biological functions
Two distinct species and sources of EPCs, rat bone marrow-derived EPCs (BM-EPCs) and mouse spleen-derived EPCs (spleen-EPCs), were used for research. The EPCs wereisolated, cultured and characterized following our established protocol. After5~7days ofculture, the EPCs were used for experiments.
To determine the expression of LXR in rat BM-EPCs and mouse spleen-EPCs, weused reverse transcriptase-PCR (RT-PCR), western blot, and confocal immunofluorescence.
Rat BM-EPCs and mouse spleen-EPCs were treated with different dosages of LXRagonists T0901317(0μmol/L,0.5μmol/L,2μmol/L,5μmol/L) or GW3965(0μmol/L,0.5μmol/L,2μmol/L,5μmol/L) for different time course. EPCs proliferation was evaluatedusing a colorimetric MTS assay kit and EPCs migration assay was performed using aTranswell chamber (8μm pore size). In addition, western blot analysis was used todetermine the expression of phosphorylated Akt (Ser473), total Akt, phosphorylated eNOS(Ser1177) and total eNOS. LY294002(10μmol/L) and L-NAME (100μmol/L), when used,were added30minutes prior to the addition of LXR agonists.
EPCs were treated with LXR agonist T0901317(2μmol/L) for24hours, and then,semi-quantitative RT-PCR was used to determine the mRNA expressions of angiogenicgrowth factors (VEGF, SDF-1, HGF, IGF-1and G-CSF).
EPCs were treated with different concentrations of LXR agonists T0901317(0μmol/L,0.5μmol/L,2μmol/L,5μmol/L) or GW3965(0μmol/L,0.5μmol/L,2μmol/L,5μmol/L)for different time course. In addition, to detect the VEGF mRNA, protein expression andsecretion, we used Real Time-PCR, Western blot, and ELISA.
2.4. Statistical analysis
All values are expressed as the means±S.D. of at least three experiments.Comparisons between the groups were analyzed using one-way ANOVA followed by theappropriate post-hoc test. A value of P <0.05was considered to be statistically significant.
3. Results
3.1. Effect of LXR activation on endothelial repair after vascular injury
Treatment of LXR agonist T0901317increased re-endothelialized area as comparedwith control at all time points (4d,7d,14d) after arterial injury (P＜0.05，n=8), suggestingthat activation of endogenous LXR could promote endothelial regeneration and repair ofinjured vessels.
3.2. Effect and mechanism of LXR on ECs proliferation and migration
3.2.1. Characterization of HUVECs
HUVECs were grown as confluent monolayers with a “cobblestone” morphology. Thecells were homogenous, closely apposed, large, flat, and polygonal. Cells showing positivestaining for von Willebrand factor were HUVECs (＞95%, n=6).
3.2.2. LXR agonist enhances HUVECs proliferation and migration
Treatment with LXR agonist T0901317(0.5μmol/L,2μmol/L,5μmol/L) profoundlyincreased the HUVEC proliferation and migration in dose-dependent manners (P＜0.05，P＜0.01，n=4-6). These findings suggested that LXR agonist could augment HUVECsproliferation and migration.
3.2.3. Mechanism of LXR activation on ECs proliferation and migration
3.2.3.1. LXR agonist influences PI3K/Akt/eNOS signaling in HUVECs
LXR agonist T0901317induced significant increases in the Ser473Akt phosphorylationand Ser1177eNOS phosphorylation in time-and dose-dependent manners in HUVECs (P＜0.05，P＜0.01，n=3-5), which were abolished by the PI3K inhibitor LY294002. Theseobservations suggested that LXR activation could influence PI3K/Akt/eNOS signaling inHUVECs. However, T0901317did not change the levels of phosphorylated Akt andphosphorylated eNOS within1hour, suggesting that LXR activation might activate thePI3K/Akt/eNOS pathway via an indirect mechanism.
3.2.3.2. Activation of LXR promote HUVECs proliferation and migration via thePI3K/Akt/eNOS signaling pathway
It was showed that both the PI3K inhibitor LY294002(10μmol/L) and the eNOSinhibitor L-NAME (100μmol/L) significantly attenuated T0901317(5μmol/L)-stimulatedHUVECs proliferation and migration (P＜0.05， P＜0.01， n=4-6), whereas neitherLY294002nor L-NAME treatment influenced basal HUVECs proliferation and migration inthe absence of LXR agonist (P=N.S.， n=4-6). These data suggested that thePI3K/Akt/eNOS signaling pathway was involved in LXR activation-induced HUVECsproliferation and migration.
3.3. Effect and mechanism of LXR on EPCs biological functions
3.3.1. Characterization of rat bone marrow-derived EPCs (BM-EPCs)
The adherent rat bone marrow-derived mononuclear cells (BM-MNCs) wereoval-shaped, spindle-like, and polygonal. Cell clusters and colonies appeared in early phase (4d~14d). In late phase (>14d), cord-like structures and network formation were observedwhen cell density was low, where as the cells exhibited typical “cobblestone” morphologyat high cell density conditions. The majority of the adherent BM-MNCs (>90%) werepositive for both ac-LDL (red) uptake and UEA-I lectin binding (green), which indicatedendothelial cell characteristics. These adherent BM-MNCs were further characterized bydemonstrating the expression of the stem cell marker CD133(89.2%)，CD34(75.4%), theendothelial cell lineage antigen CD31(91.9%) and VEGFR-2(92.9%) by FCM.
3.3.2. Characterization of mouse spleen-derived EPCs (spleen-EPCs)
The adherent mouse spleen-derived mononuclear cells (spleen-MNCs) wereoval-shaped, spindle-like, and polygonal. Cell clusters and colonies could be found in earlyphase (4d~14d). In late phase (>14d), cord-like structures and network formation wereobserved when cell density was low, where as the cells exhibited typical “cobblestone”morphology at high cell density conditions. The majority of the adherent spleen-MNCs(>90%) were positive for both ac-LDL (red) uptake and UEA-I lectin binding (green),consistent with endothelial lineage cells. These adherent spleen-MNCs were furthercharacterized by demonstrating the expression of the stem cell marker Sca-1(75.4%)，CD34(82.3%) and c-kit (64.9%), the endothelial cell lineage antigen CD31(84.6%) andVEGFR-2(86.2%) by FCM.
3.3.3. LXRα and LXRβ are expressed in EPCs
LXRα and LXRβ mRNA and protein were present in the rat BM-EPCs and mousespleen-EPCs as determined by RT-PCR and western blot. Additionally, LXRα and LXRβimmunofluorescence was observed primarily in the nucleus of the EPCs, whereas onlyweak staining was observed throughout the cytoplasm. Treatment of the EPCs with theLXR agonists T0901317or GW3965led to a significant up-regulation of the LXR targetgene ABCA1, which indicated that the LXRs expressed in EPCs are biologically active.
3.3.3. LXR agonists enhance EPCs proliferation and migration
After treatment of synthetic LXR agonists T0901317(0.5μmol/L,2μmol/L,5μmol/L)or GW3965(0.5μmol/L,2μmol/L,5μmol/L), the proliferation and migration of EPCs (ratBM-EPCs and mouse spleen-EPCs) increased profoundly in dose-dependent manners (P＜0.05，P＜0.01，n=4-6). These findings suggested that LXR activation could enhance EPCsproliferation and migration.
3.3.4. Mechanism of LXR activation on EPCs proliferation and migration
3.3.4.1. LXR activation influences PI3K/Akt/eNOS signaling in EPCs
Both synthetic LXR agonists T0901317and GW3965induced significant increases inthe Ser473Akt phosphorylation and Ser1177eNOS phosphorylation in time-anddose-dependent manners in EPCs (rat BM-EPCs and mouse spleen-EPCs)(P＜0.05，P＜0.01，n=3-5), which were diminished by the PI3K inhibitor LY294002. These observationssuggested LXR activation influences PI3K/Akt/eNOS signaling in EPCs. Nevertheless,treatment with T0901317did not change the levels of phosphorylated Akt andphosphorylated eNOS within1hour, showing that LXR activation might activate thePI3K/Akt/eNOS pathway through an indirect mechanism.
3.3.4.2. LXR agonists promote EPCs proliferation and migration via thePI3K/Akt/eNOS signaling pathway
We found that both the PI3K inhibitor LY294002(10μmol/L) and the eNOS inhibitorL-NAME (100μmol/L) significantly attenuated T0901317(2μmol/L)-or GW3965(5μmol/L)-enhanced EPCs (rat BM-EPCs and mouse spleen-EPCs) proliferation andmigration (P＜0.05，P＜0.01，n=4-6), whereas neither LY294002nor L-NAME treatmentinfluenced basal EPC proliferation and migration in the absence of LXR agonists (P=N.S.，n=4-6). These data demonstrated suggested that the PI3K/Akt/eNOS signaling pathway wasinvolved in LXR activation-promoted EPCs proliferation and migration.
3.3.5. Effect of LXR on the production of angiogenic growth factors by EPCs
3.3.5.1. Effect of LXR activation on the mRNA expressions of angiogenic growthfactors
LXR agonist T0901317(2μmol/L) treatment didn’t change the mRNA levels ofSDF-1, HGF, IGF-1and G-CSF, but increased the VEGF mRNA expression (P＜0.05，n=4),suggesting that LXR activation might up-regulate VEGF mRNA expression in EPCs.
3.3.5.2. LXR agonists up-regulate VEGF expression and induces VEGF secretion inEPCs
Treatment of EPCs with either T0901317or GW3965led to an increased level ofVEGF mRNA expression in time-and dose-dependent manners (P＜0.05, P＜0.01, n=4-6).In addition, T0901317or GW3965treatment similarly increased the VEGF protein level (P＜0.05, n=6) and stimulated VEGF secretion (P＜0.01, n=5). These observations demonstrated that LXR agonists could up-regulate VEGF expression and induce VEGFsecretion in EPCs.
4. Conclusion
4.1. LXR activation increases ECs proliferation and migration throughPI3K/Akt/eNOS signaling pathway.
4.2. LXRα and LXRβ are expressed in EPCs. LXR agonists enhance EPCsproliferation and migration via PI3K/Akt/eNOS pathway. In addition, LXR agonistsup-regulate VEGF expression and induce VEGF secretion by EPCs.
4.3. Activation of LXR could promote endothelial regeneration and repair aftervascular injury through regulating the functions of ECs and EPCs.
血管内皮损伤是介入术后的血栓形成和血管再狭窄的关键，也是高血压、动脉粥样硬化和糖尿病血管病变等多种血管损伤性疾病共同的病理生理基础。血管损伤后的内皮再生修复可以有效防止血栓形成和抑制血管平滑肌细胞的异常增生，因此，尽早促进损伤血管的再内皮化、恢复血管内皮功能，是防止介入术后血栓形成和血管再狭窄，预防和治疗血管损伤性疾病的有效策略。
肝X受体(liver X receptor, LXR)是配体激活的核受体超家族成员，被其内源性配体/合成激动剂激活后，可以通过调节一系列靶基因的转录，参与调节胆固醇代谢、糖代谢和炎症反应。近来的研究发现，LXR在心脑血管疾病防治中可能发挥着一定的作用：(1)LXR激动剂可以明显抑制动脉粥样硬化的发生发展过程；(2)LXR激动剂可以显著抑制血管损伤后的新生内膜形成；(3)LXR激动剂可以促进缺血脑组织的新生血管形成。损伤血管内皮的再生是上述心脑血管疾病防治的共同环节，因此我们猜测，LXR激活后是否可以通过促进血管内皮的再生，从而发挥对上述病理生理过程的调节作用？
在预实验中，我们采用小鼠颈动脉损伤模型观察LXR对损伤血管的内皮再生的影响，结果显示，LXR激动剂T0901317干预后，小鼠的损伤颈动脉的再内皮化面积明显增加。那么LXR激活后又是如何促进损伤血管内皮的再生修复呢？血管内皮的再生依赖于：(1)邻近内皮细胞(endothelial cells, ECs)的增殖和迁移；(2)骨髓、脾脏等来源的内皮祖细胞(endothelial progenitor cells, EPCs)，EPCs能从骨髓、脾脏等动员到外周血，迁移、归巢于血管损伤区域，增殖和分化为ECs，还可通过旁分泌机制(分泌促血管生成因子)促进邻近ECs增殖和迁移，从而促进内皮修复。
介于以上的研究背景，我们推测，激活LXR后可能通过调节ECs与EPCs的生物学功能(如：增殖、迁移能力等)，促进损伤血管内皮的再生与修复。
2.方法
2.1. LXR激动剂对小鼠颈动脉损伤血管内皮修复的影响
构建小鼠颈动脉损伤模型，采用LXR激动剂T0901317[30mg/(Kg·d)]于术前2天开始喂养小鼠，通过伊文氏蓝染色观察损伤颈动脉再内皮化的情况，以阐明激活内源性LXR对损伤血管内皮修复的影响。
2.2. LXR对内皮细胞的增殖、迁移能力的影响及机制研究
从健康新生儿脐带分离和培养原代人脐静脉内皮细胞(human umbilical veinendothelial cells, HUVECs)，显微镜下观察HUVECs的形态学特征，免疫荧光检测vWF进行内皮细胞鉴定；第3～5代HUVECs用于实验。
LXR激动剂T0901317(0、0.5、2、5μmol/L)处理HUVECs后，MTS方法检测HUVECs的增殖能力，Transwell迁移实验检测HUVECs的迁移能力, Western blot检测p-Akt、总Akt、p-eNOS、总eNOS的表达。
先用PI3K抑制剂LY294002(10μmol/L)或eNOS抑制剂L-NAME (100μmol/L)预处理HUVECs30min后，再用LXR激动剂T0901317干预，MTS方法检测HUVECs的增殖能力，Transwell迁移实验检测HUVECs的迁移能力, Western blot检测p-Akt、总Akt、p-eNOS、总eNOS的表达。
2.3. LXR对内皮祖细胞的生物学功能的影响及机制探讨
选用大鼠骨髓源性内皮祖细胞(BM-EPCs)和小鼠脾源性内皮祖细胞(Spleen-EPCs)这两种不同种属和来源的EPCs作为研究模型。密度梯度离心法分离大鼠骨髓单个核细胞(MNCs)和小鼠脾脏MNCs，诱导分化为EPCs，显微镜下观察细胞的形态学特征，通过DiI-ac-LDL、FITC-UEA-I荧光双染和流式细胞分析检测细胞分子表面标记进行EPCs的鉴定。培养第5～7天的大鼠BM-EPCs和小鼠Spleen-EPCs用于实验。
采用RT-PCR、western blot和免疫荧光检测LXRα、LXRβ在大鼠BM-EPCs和小鼠Spleen-EPCs中的表达和定位。
LXR激动剂T0901317(0、0.5、2、5μmol/L)或GW3965(0、0.5、2、5μmol/L)干预EPCs后，MTS方法检测EPCs的增殖能力，Transwell迁移实验检测EPCs的迁移能力，Western blot检测p-Akt、总Akt、p-eNOS、总eNOS的表达。
先用PI3K抑制剂LY294002(10μmol/L)或eNOS抑制剂L-NAME (100μmol/L)预处理EPCs30min后，再用LXR激动剂T0901317或GW3965干预，MTS方法检测BM-EPCs的增殖能力，Transwell迁移实验检测EPCs的迁移能力，Western blot检测p-Akt、总Akt、p-eNOS、总eNOS的表达。
LXR激动剂T0901317(2μmol/L)处理EPCs24h后，RT-PCR检测EPCs分泌的常见促血管生长因子(VEGF、SDF-1、HGF、IGF-1和G-CSF)mRNA的表达。采用LXR激动剂T0901317(0、0.5、2、5μmol/L)或GW3965(0、0.5、2、5μmol/L)处理EPCs，不同时间点收取细胞和细胞上清，Real-Time PCR、western blot、ELISA检测VEGF的mRNA、蛋白的表达和分泌。
2.4.统计学处理
所有计量资料以平均值±标准差(x±s)表示，使用SPSS18.0统计学软件进行分析，组间比较选用单因素方差分析(One-Way ANOVA)。P＜0.05为有统计学差异。
3.结果
3.1. LXR激动剂对小鼠颈动脉损伤血管内皮修复的影响
LXR激动剂T0901317干预后可明显增加颈动脉损伤后第4天、7天和14天的再内皮化面积(P＜0.05，n=8)，提示激活内源性LXR可以明显地促进损伤血管内皮的再生和修复。
3.2. LXR对内皮细胞的增殖、迁移能力的影响及机制研究
3.2.1. HUVECs的分离、培养和鉴定
细胞呈单层生长，互不重叠，形态呈多角形、椭圆形或梭形，呈铺路石样镶嵌排列；激光共聚焦显微镜下显示细胞质vWF阳性(红色荧光)即为HUVECs，鉴定阳性率＞95%(n=6)。
3.2.2. LXR激动剂增强HUVECs增殖、迁移能力
不同浓度的T0901317(0.5、2、5μmol/L)处理后，HUVECs的增殖和迁移能力明显增强，并与T0901317呈一定的剂量依赖关系(P＜0.05，P＜0.01，n=4-6)；表明LXR激动剂可以促进HUVECs的增殖和迁移。
3.2.3. LXR激动剂调节HUVECs增殖、迁移能力的机制研究
3.2.3.1. LXR激动剂对HUVECs中PI3K/Akt/eNOS信号通路的影响
T0901317处理后，呈时间和剂量依赖的上调HUVECs中p-Akt-Ser473和p-eNOS-Ser1177的表达(P＜0.05，P＜0.01，n=3-5)，PI3K抑制剂LY294002(10μmol/L)可以明显阻断上述作用，提示T0901317通过PI3K激活Akt/eNOS信号通路。然而在T0901317干预的早期(＜1h)，未见明显的p-Akt-Ser473和p-eNOS-Ser1177水平上调，提示LXR激活后可能通过间接作用导致了HUVECs中PI3K/Akt/eNOS信号途径的活化。
3.2.3.1. LXR激动剂通过PI3K/Akt/eNOS途径增强HUVECs的增殖、迁移能力
采用PI3K抑制剂LY294002(10μmol/L)或eNOS抑制剂L-NAME(100μmol/L)预处理HUVECs30min后，T0901317(5μmol/L)促进HUVECs增殖和迁移的效应明显减弱(P＜0.05，P＜0.01，n=4-6)，而LY294002和L-NAME并没有改变HUVECs的细胞活力和基础迁移水平(P=N.S.，n=4-6)；说明LXR激动剂可以通过PI3K/Akt/eNOS信号途径增强HUVECs的增殖、迁移能力。
3.3. LXR对内皮祖细胞的生物学功能的影响及机制探讨
3.3.1.大鼠BM-EPCs的分离、培养和鉴定
成功分离大鼠BM-EPCs，呈梭形、椭圆形或三角形，早期可见典型的克隆单位“集落”形成；晚期呈“线型”、“管型”或者“网状”血管样生长趋势，密度较大时呈铺路石样排列。DiI-ac-LDL(红色)和FITC-UEA-I(绿色)双染阳性即为正在分化的EPCs，所分离培养的细胞双染阳性率大于90%。流式细胞技术检测到CD133、CD34、VEGFR-2和CD31在所培养细胞(5~7天)中的阳性率分别为75.4%、82.3%、64.9%、86.2%和84.6%，符合EPCs的特征。
3.3.2.小鼠Spleen-EPCs的分离、培养和鉴定
成功分离小鼠Spleen-EPCs，呈梭形、椭圆形或三角形，早期可见典型的克隆单位“集落”形成；晚期呈“线型”、“管型”或者“网状”血管样生长趋势，密度较大时呈铺路石样排列。DiI-ac-LDL(红色)和FITC-UEA-I(绿色)双染阳性率大于90%，流式细胞技术检测到Sca-1、CD34、c-kit、VEGFR-2及CD31在所培养细胞(5~7天)中的阳性率分别为89.2%、75.4%、92.9%和91.9%，符合EPCs的特征。
3.3.3.检测LXR在EPCs的表达
培养的不同时间点(4天、7天、10天、14天)的大鼠BM-EPCs和小鼠Spleen-EPCs中，我们均检测到LXRα、LXRβ mRNA和蛋白的表达，免疫荧光提示LXRα、LXRβ在EPCs的细胞核和细胞浆均有表达，但主要表达于细胞核中；采用LXR激动剂T0901317或GW3965处理EPCs后，LXR的靶基因ABCA1的转录水平均明显上调，提示LXR在EPCs是具有转录活性和生物学功能的。
3.3.3. LXR激动剂增强EPCs的增殖、迁移能力
两种不同的LXR激动剂T0901317(0.5、2、5μmol/L)或GW3965(0.5、2、5μmol/L)处理后，大鼠BM-EPCs和小鼠Spleen-EPCs的增殖和迁移能力均明显增强，并与LXR激动剂呈一定的剂量依赖关系(P＜0.05，P＜0.01，n=4-6)；表明LXR激动剂可以促进EPCs的增殖和迁移。
3.3.4. LXR激动剂调节EPCs增殖、迁移能力的机制研究
3.3.4.1. LXR激动剂对EPCs中PI3K/Akt/eNOS信号通路的影响
LXR激动剂T0901317或GW3965处理后，呈时间和剂量依赖的上调EPCs(大鼠BM-EPCs和小鼠Spleen-EPCs)中p-Akt-Ser473和p-eNOS-Ser1177的表达(P＜0.05，P＜0.01，n=3-5)，PI3K抑制剂LY294002(10μmol/L)可以明显阻断上述作用，提示激活LXR通过PI3K导致Akt/eNOS信号通路的活化。然而在LXR激动剂干预的早期(＜1h)，未见明显的p-Akt-Ser473和p-eNOS-Ser1177水平上调，提示LXR激活后可能通过间接作用导致了EPCs中PI3K/Akt/eNOS信号途径的活化。
3.3.4.2. LXR激动剂通过PI3K/Akt/eNOS途径增强EPCs的增殖、迁移能力
采用PI3K抑制剂LY294002(10μmol/L)或eNOS抑制剂L-NAME(100μmol/L)预处理EPCs(大鼠BM-EPCs和小鼠Spleen-EPCs)30min后，T0901317(2μmol/L)和GW3965(5μmol/L)促进EPCs增殖和迁移的效应明显减弱(P＜0.05，P＜0.01，n=4-6)，而LY294002和L-NAME并没有改变EPCs的细胞活力和基础迁移水平(P=N.S.，n=4-6)；说明LXR激动剂可以通过PI3K/Akt/eNOS信号途径增强EPCs的增殖、迁移能力。
3.3.5. LXR激动剂对EPCs分泌促血管生长因子的影响
3.3.5.1. LXR激动剂对EPCs中促血管生长因子(VEGF、SDF-1、HGF、IGF-1和G-CSF)mRNA表达的影响
LXR激动剂T0901317(2μmol/L)作用EPCs24h后，VEGF mRNA的表达水平显著上调(P＜0.05，n=4)，而SDF-1、HGF、IGF-1和G-CSF mRNA表达水平未见明显变化，提示LXR激动剂可以上调EPCs中VEGF mRNA的表达。
3.3.5.2. LXR激动剂对EPCs中VEGF表达和分泌的影响
LXR激动剂T0901317或GW3965作用于EPCs后，呈时间和剂量依赖的上调VEGF mRNA的表达(P＜0.05，P＜0.01，n=4-6)，呈剂量依赖的上调VEGF蛋白的表达(P＜0.05， n=6)和促进VEGF的分泌(P＜0.05，P＜0.01，n=5)，说明LXR激活后可以上调EPCs中VEGF的表达和促进VEGF的分泌。
4.结论
4.1. LXR激动剂通过PI3K/Akt/eNOS信号途径增强内皮细胞的增殖和迁移能力；
4.2. LXRα与LXRβ表达于内皮祖细胞，LXR激动剂可以通过PI3K/Akt/eNOS信号途径增强内皮祖细胞增殖和迁移能力，LXR激动剂上调内皮祖细胞中促血管生长因子VEGF的表达和促进VEGF的分泌；
4.3.激活内源性LXR通过调节内皮细胞和内皮祖细胞的生物学功能，从而促进损伤血管内皮的再生与修复。
1. Background
Vascular endothelial injury, which is the common pathophysiological basis ofhypertension, atherosclerosis and vascular disease in diabetes, is also the major cause ofpost-angioplasty restenosis and thrombosis. Endothelial regeneration and repair couldprevent thrombosis and suppress vascular smooth muscle cell proliferation after arterialinjury. Hence, accelerating re-endothelialization of injured arteries and restoring endothelialfuction are useful strategies for inhibiting post-angioplasty restenosis and thrombosis andpreventing vascular injured diseases.
Liver X receptors (LXRs) are members of nuclear receptor superfamily. Onceactivated by endogenous ligands or synthetic agonists, LXR can regulate cholesterolmetabolism, glucose metabolism and inflammations by influencing the expression of anarray of LXR target genes. Studies performed recently have showed that LXR might playimportant roles in preventing cardiovascular diseases:(1) LXR agonists inhibited thedevelopment of atherosclerosis,(2) LXR agonists suppressed neointima formation inballoon-injured rat carotid arteries,(3) LXR activation promoted angiogenesis afterischemic stroke. Since endothelial regeneration and repair after vascular injury are thecommon steps in preventing above cardiovascular diseases, we hypothesize that activationof endogenous LXR might regulate all above pathophysiology processes by stimulatingendothelial regeneration.
In our preliminary experiments, we have found that treatment of LXR agonistT0901317increased re-endothelialized area in a mouse model of carotid injury, suggestingthat activation of endogenous LXR could promote endothelial regeneration and repair of injured arteries. However, the mechanisms are not known. Endothelial regenerationdepends on:(1) neighbouring endothelial cells (ECs) proliferation and migration;(2)endothelial progenitor cells (EPCs) derived from bone marrow, spleen and etc.Accumulating evidence has demonstrated that EPCs, which mobilized to circulation byischemia, physical training, and the administration of statins, estrogen, and a variety ofcytokines, can be recruited to sites of endothelial injury. There, they promotere-endothelialization directly by incorporating into the recovering endothelium at the sitesof injury and indirectly by producing and releasing angiogenic growth factors.
Based on these observations, we speculate that activation of LXR might promoteendothelial regeneration and repair after vascular injury via regulating ECs and EPCsbiological functions (such as proliferation, migration and etc.).
2. Methods
2.1. Effect of LXR activation on endothelial repair after vascular injury
To explain the effect of LXR activation on endothelial repair after injury, weestablished an mouse model of carotid injury as previously described. Mice were orallyadministered of LXR agonist T0901317(30mg/kg per day) or vehicle starting2daysbefore the injury. To measure the re-endothelialized area, animals were perfused in vivowith Evans blue dye4,7, and14days after the injury.
2.2. Effect and mechanism of LXR on ECs proliferation and migration
Human umbilical vein endothelial cells (HUVECs) were obtained and cultured by ourestablished methods. Immunostaining for von Willebrand factor (vWF) confirmed that thecells were endothelial. Cells used for experiments were from passages3through5.
HUVECs were treated with different concentrations of LXR agonist T0901317(0μmol/L,0.5μmol/L,2μmol/L,5μmol/L) for different time course. HUVECs proliferationwas evaluated using a colorimetric MTS assay kit and HUVECs migration assay wasperformed using a Transwell chamber (8μm pore size). In addition, western blot analysiswas used to determine the expression of phosphorylated Akt (Ser473), total Akt,phosphorylated eNOS (Ser1177) and total eNOS. LY294002(10μmol/L) and L-NAME (100μmol/L), when used, were added30minutes prior to the addition of LXR agonist.
2.3. Effect and mechanism of LXR on EPCs biological functions
Two distinct species and sources of EPCs, rat bone marrow-derived EPCs (BM-EPCs) and mouse spleen-derived EPCs (spleen-EPCs), were used for research. The EPCs wereisolated, cultured and characterized following our established protocol. After5~7days ofculture, the EPCs were used for experiments.
To determine the expression of LXR in rat BM-EPCs and mouse spleen-EPCs, weused reverse transcriptase-PCR (RT-PCR), western blot, and confocal immunofluorescence.
Rat BM-EPCs and mouse spleen-EPCs were treated with different dosages of LXRagonists T0901317(0μmol/L,0.5μmol/L,2μmol/L,5μmol/L) or GW3965(0μmol/L,0.5μmol/L,2μmol/L,5μmol/L) for different time course. EPCs proliferation was evaluatedusing a colorimetric MTS assay kit and EPCs migration assay was performed using aTranswell chamber (8μm pore size). In addition, western blot analysis was used todetermine the expression of phosphorylated Akt (Ser473), total Akt, phosphorylated eNOS(Ser1177) and total eNOS. LY294002(10μmol/L) and L-NAME (100μmol/L), when used,were added30minutes prior to the addition of LXR agonists.
EPCs were treated with LXR agonist T0901317(2μmol/L) for24hours, and then,semi-quantitative RT-PCR was used to determine the mRNA expressions of angiogenicgrowth factors (VEGF, SDF-1, HGF, IGF-1and G-CSF).
EPCs were treated with different concentrations of LXR agonists T0901317(0μmol/L,0.5μmol/L,2μmol/L,5μmol/L) or GW3965(0μmol/L,0.5μmol/L,2μmol/L,5μmol/L)for different time course. In addition, to detect the VEGF mRNA, protein expression andsecretion, we used Real Time-PCR, Western blot, and ELISA.
2.4. Statistical analysis
All values are expressed as the means±S.D. of at least three experiments.Comparisons between the groups were analyzed using one-way ANOVA followed by theappropriate post-hoc test. A value of P <0.05was considered to be statistically significant.
3. Results
3.1. Effect of LXR activation on endothelial repair after vascular injury
Treatment of LXR agonist T0901317increased re-endothelialized area as comparedwith control at all time points (4d,7d,14d) after arterial injury (P＜0.05，n=8), suggestingthat activation of endogenous LXR could promote endothelial regeneration and repair ofinjured vessels.
3.2. Effect and mechanism of LXR on ECs proliferation and migration
3.2.1. Characterization of HUVECs
HUVECs were grown as confluent monolayers with a “cobblestone” morphology. Thecells were homogenous, closely apposed, large, flat, and polygonal. Cells showing positivestaining for von Willebrand factor were HUVECs (＞95%, n=6).
3.2.2. LXR agonist enhances HUVECs proliferation and migration
Treatment with LXR agonist T0901317(0.5μmol/L,2μmol/L,5μmol/L) profoundlyincreased the HUVEC proliferation and migration in dose-dependent manners (P＜0.05，P＜0.01，n=4-6). These findings suggested that LXR agonist could augment HUVECsproliferation and migration.
3.2.3. Mechanism of LXR activation on ECs proliferation and migration
3.2.3.1. LXR agonist influences PI3K/Akt/eNOS signaling in HUVECs
LXR agonist T0901317induced significant increases in the Ser473Akt phosphorylationand Ser1177eNOS phosphorylation in time-and dose-dependent manners in HUVECs (P＜0.05，P＜0.01，n=3-5), which were abolished by the PI3K inhibitor LY294002. Theseobservations suggested that LXR activation could influence PI3K/Akt/eNOS signaling inHUVECs. However, T0901317did not change the levels of phosphorylated Akt andphosphorylated eNOS within1hour, suggesting that LXR activation might activate thePI3K/Akt/eNOS pathway via an indirect mechanism.
3.2.3.2. Activation of LXR promote HUVECs proliferation and migration via thePI3K/Akt/eNOS signaling pathway
It was showed that both the PI3K inhibitor LY294002(10μmol/L) and the eNOSinhibitor L-NAME (100μmol/L) significantly attenuated T0901317(5μmol/L)-stimulatedHUVECs proliferation and migration (P＜0.05， P＜0.01， n=4-6), whereas neitherLY294002nor L-NAME treatment influenced basal HUVECs proliferation and migration inthe absence of LXR agonist (P=N.S.， n=4-6). These data suggested that thePI3K/Akt/eNOS signaling pathway was involved in LXR activation-induced HUVECsproliferation and migration.
3.3. Effect and mechanism of LXR on EPCs biological functions
3.3.1. Characterization of rat bone marrow-derived EPCs (BM-EPCs)
The adherent rat bone marrow-derived mononuclear cells (BM-MNCs) wereoval-shaped, spindle-like, and polygonal. Cell clusters and colonies appeared in early phase (4d~14d). In late phase (>14d), cord-like structures and network formation were observedwhen cell density was low, where as the cells exhibited typical “cobblestone” morphologyat high cell density conditions. The majority of the adherent BM-MNCs (>90%) werepositive for both ac-LDL (red) uptake and UEA-I lectin binding (green), which indicatedendothelial cell characteristics. These adherent BM-MNCs were further characterized bydemonstrating the expression of the stem cell marker CD133(89.2%)，CD34(75.4%), theendothelial cell lineage antigen CD31(91.9%) and VEGFR-2(92.9%) by FCM.
3.3.2. Characterization of mouse spleen-derived EPCs (spleen-EPCs)
The adherent mouse spleen-derived mononuclear cells (spleen-MNCs) wereoval-shaped, spindle-like, and polygonal. Cell clusters and colonies could be found in earlyphase (4d~14d). In late phase (>14d), cord-like structures and network formation wereobserved when cell density was low, where as the cells exhibited typical “cobblestone”morphology at high cell density conditions. The majority of the adherent spleen-MNCs(>90%) were positive for both ac-LDL (red) uptake and UEA-I lectin binding (green),consistent with endothelial lineage cells. These adherent spleen-MNCs were furthercharacterized by demonstrating the expression of the stem cell marker Sca-1(75.4%)，CD34(82.3%) and c-kit (64.9%), the endothelial cell lineage antigen CD31(84.6%) andVEGFR-2(86.2%) by FCM.
3.3.3. LXRα and LXRβ are expressed in EPCs
LXRα and LXRβ mRNA and protein were present in the rat BM-EPCs and mousespleen-EPCs as determined by RT-PCR and western blot. Additionally, LXRα and LXRβimmunofluorescence was observed primarily in the nucleus of the EPCs, whereas onlyweak staining was observed throughout the cytoplasm. Treatment of the EPCs with theLXR agonists T0901317or GW3965led to a significant up-regulation of the LXR targetgene ABCA1, which indicated that the LXRs expressed in EPCs are biologically active.
3.3.3. LXR agonists enhance EPCs proliferation and migration
After treatment of synthetic LXR agonists T0901317(0.5μmol/L,2μmol/L,5μmol/L)or GW3965(0.5μmol/L,2μmol/L,5μmol/L), the proliferation and migration of EPCs (ratBM-EPCs and mouse spleen-EPCs) increased profoundly in dose-dependent manners (P＜0.05，P＜0.01，n=4-6). These findings suggested that LXR activation could enhance EPCsproliferation and migration.
3.3.4. Mechanism of LXR activation on EPCs proliferation and migration
3.3.4.1. LXR activation influences PI3K/Akt/eNOS signaling in EPCs
Both synthetic LXR agonists T0901317and GW3965induced significant increases inthe Ser473Akt phosphorylation and Ser1177eNOS phosphorylation in time-anddose-dependent manners in EPCs (rat BM-EPCs and mouse spleen-EPCs)(P＜0.05，P＜0.01，n=3-5), which were diminished by the PI3K inhibitor LY294002. These observationssuggested LXR activation influences PI3K/Akt/eNOS signaling in EPCs. Nevertheless,treatment with T0901317did not change the levels of phosphorylated Akt andphosphorylated eNOS within1hour, showing that LXR activation might activate thePI3K/Akt/eNOS pathway through an indirect mechanism.
3.3.4.2. LXR agonists promote EPCs proliferation and migration via thePI3K/Akt/eNOS signaling pathway
We found that both the PI3K inhibitor LY294002(10μmol/L) and the eNOS inhibitorL-NAME (100μmol/L) significantly attenuated T0901317(2μmol/L)-or GW3965(5μmol/L)-enhanced EPCs (rat BM-EPCs and mouse spleen-EPCs) proliferation andmigration (P＜0.05，P＜0.01，n=4-6), whereas neither LY294002nor L-NAME treatmentinfluenced basal EPC proliferation and migration in the absence of LXR agonists (P=N.S.，n=4-6). These data demonstrated suggested that the PI3K/Akt/eNOS signaling pathway wasinvolved in LXR activation-promoted EPCs proliferation and migration.
3.3.5. Effect of LXR on the production of angiogenic growth factors by EPCs
3.3.5.1. Effect of LXR activation on the mRNA expressions of angiogenic growthfactors
LXR agonist T0901317(2μmol/L) treatment didn’t change the mRNA levels ofSDF-1, HGF, IGF-1and G-CSF, but increased the VEGF mRNA expression (P＜0.05，n=4),suggesting that LXR activation might up-regulate VEGF mRNA expression in EPCs.
3.3.5.2. LXR agonists up-regulate VEGF expression and induces VEGF secretion inEPCs
Treatment of EPCs with either T0901317or GW3965led to an increased level ofVEGF mRNA expression in time-and dose-dependent manners (P＜0.05, P＜0.01, n=4-6).In addition, T0901317or GW3965treatment similarly increased the VEGF protein level (P＜0.05, n=6) and stimulated VEGF secretion (P＜0.01, n=5). These observations demonstrated that LXR agonists could up-regulate VEGF expression and induce VEGFsecretion in EPCs.
4. Conclusion
4.1. LXR activation increases ECs proliferation and migration throughPI3K/Akt/eNOS signaling pathway.
4.2. LXRα and LXRβ are expressed in EPCs. LXR agonists enhance EPCsproliferation and migration via PI3K/Akt/eNOS pathway. In addition, LXR agonistsup-regulate VEGF expression and induce VEGF secretion by EPCs.
4.3. Activation of LXR could promote endothelial regeneration and repair aftervascular injury through regulating the functions of ECs and EPCs.
引文
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