骨髓来源细胞在脉络膜新生血管发生发展中的作用及其治疗潜能
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摘要
研究背景脉络膜新生血管(choroidal neovascularization, CNV)是近40种眼病的共有体征,视网膜下病理性新生血管的生长不但损伤正常脉络膜视网膜结构,而且常发生出血渗漏,往往造成严重视力损害。CNV的发生机制复杂,涉及多种细胞、因子和信号通路,确切机制尚未完全阐明。
成年个体新生血管的形成包含两种机制:血管生成(angiogenesis)指原有的血管细胞发生增生、移行,形成新的血管;血管发生(vasculogenesis)指由干细胞分化而来的血管细胞构成新血管。干细胞在一般情况下处于“休眠”状态,在外因刺激或病理状态下会进入血液循环,移行至周边组织,表现出不同程度的更新和分化能力,参与组织修复和新生物形成。近期的研究表明,CNV的生成兼具这两种方式,既有原位组织细胞的增殖,又有骨髓来源细胞(bone marrow-derived cells, BMCs)的参与,但BMCs究竟以何种方式参与CNV生成、BMCs对CNV发展有何影响有待进一步阐明。此外,BMCs是一个异质性细胞群体,包含多种干/祖细胞,究竟是哪种干/祖细胞参与了CNV的生成、它们在CNV发生发展中发挥着怎样的作用、是否可作为CNV治疗的靶点等诸多问题有待研究。
目的和内容探讨BMCs在CNV发生发展中的作用,在危险因素影响CNV发展中的介导作用,及作为干细胞疗法细胞载体的潜能。⑴建立C57-绿色荧光蛋白(green fluorescent protein, GFP)嵌合体小鼠,观察在嵌合体CNV发生发展过程中,BMCs在CNV聚集、分化和表达蛋白情况;⑵探讨尼古丁对参与CNV生成的BMCs的趋化、分化和蛋白表达能力的影响;⑶观察骨髓来源基质干细胞(mesenchymal stem cells, MSCs)向CNV的趋化特异性和时间窗,观察MSCs参与CNV生成情况,探讨MSCs是否具备作为药物载体的潜能;⑷构建表达人源性色素上皮衍生因子(pigment epithelium derived factor, PEDF)的腺病毒,观察腺病毒转导的MSCs对CNV的抑制作用,探讨利用MSCs靶向治疗CNV的新策略。
方法⑴通过GFP转基因小鼠与野生型C57小鼠的骨髓移植建立嵌合体小鼠,利用532nm激光建立小鼠CNV模型,脉络膜铺片观察GFP-BMCs向CNV趋化、参与血管结构组成的情况,免疫荧光染色观察CNV中BMCs分化的细胞类型(包括血管内皮细胞、血管平滑肌细胞和巨噬细胞)及表达血管内皮生长因子(vascular endothelial growth factor, VEGF)和碱性成纤维细胞生长因子(basic fibroblast cell growth factor, bFGF)的情况;⑵激光光凝建立C57-GFP嵌合体小鼠CNV模型后,即日在其饮水中添加尼古丁(100μg/ml),对照组嵌合体小鼠建立CNV模型后普通饲养,4周后眼球切片HE染色观察CNV厚度与直径,脉络膜铺片观察CNV表面积、GFP-BMCs的趋化和参与构成血管情况,免疫荧光染色观察CNV中BMCs分化情况及各因子表达变化;⑶体外培养GFP转基因小鼠骨髓来源MSCs,野生型小鼠激光建模后0.5-1小时尾静脉注射4.0×106 GFP-BMCs或GFP-MSCs,激光后1、3、7天,心、肝、脾、肺、心脏切片荧光观察比较两种细胞在器官内的滞留情况;野生型小鼠激光后0.5-1小时、3天及6天,分别注射4.0×106 GFP-MSCs,激光后7天观察比较单次注射、双次注射和三次注射后GFP-MSCs在体内移行和向CNV趋化的情况;酶联免疫吸附测定(enzyme linked immunosorbent assay, ELISA)和免疫荧光染色检测激光后不同时间眼内干细胞趋化因子基质细胞衍生因子-1(stromal cell derived factor-1, SDF-1)的表达;脉络膜铺片检测参与构成新血管的GFP-MSCs,免疫荧光染色分析CNV中GFP-MSCs分化的细胞类型;⑷构建表达人源性PEDF、含报告基因GFP、E1 E3缺陷的5型腺病毒(adenovirus, Ad),转导体外培养的野生型小鼠骨髓来源MSCs,倒置荧光显微镜观察及流式细胞仪测定感染效率,ELISA检测体外人源性PEDF表达情况;激光后0.5-1小时小鼠尾静脉分别注射4.0×106 Ad-PEDF/MSCs、对照病毒Ad-GFP/MSCs、GFP-MSCs及0.4ml磷酸盐缓冲液(Phosphate buffered saline, PBS),分别于激光后1、3、5、7天利用免疫荧光染色和ELISA检测眼内人源性PEDF表达,激光后7天眼球切片组织学分析和脉络膜铺片分析比较各组CNV厚度、直径及表面积。体外建立视网膜色素上皮(retinal pigment epithelium, RPE)细胞与MSCs的共培养体系,分析MSCs分泌的PEDF对RPE细胞增生和移行的影响。
结果⑴激光后大量GFP-BMCs聚集于激光斑处并整合入CNV中的血管结构内,由其组成的血管面积约占CNV表面积的16.22%。GFP-BMCs主要分布于CNV区域(包括CNV深层的脉络膜),少量散布于CNV表层的视网膜、角巩膜缘、睫状体、视盘周围及远离CNV的视网膜、脉络膜和巩膜。CD31或αSMA阳性的GFP+细胞仅出现于CNV区域,F4/80+/GFP+细胞不仅出现在CNV区域,还出现在CNV表层的视网膜、角巩膜缘和睫状体内。CNV内CD31/GFP、αSMA/GFP和F4/80/GFP双阳性细胞在全部GFP+细胞中的构成比随CNV进展而变化,CD31/GFP或F4/80/GFP双阳性细胞构成比的最高值出现于激光后第2周,而αSMA+/GFP+细胞构成比在激光后4周仍处上升趋势。CNV区域的一些GFP-BMCs可表达促血管形成因子VEGF和bFGF。⑵尼古丁处理组小鼠CNV长度和表面积增大,CNV内GFP-BMCs的面积和密度增加,GFP-BMCs来源的血管细胞面积增加,F4/80+/GFP+细胞构成比下降,CNV内VEGF和bFGF的表达及CNV下脉络膜内VCAM-1的表达上调。⑶在整个观察期间,肺、肝和脾内可见GFP+BMCs滞留,但未观测到GFP+MSCs。单次、双次或三次MSCs注射后趋化至CNV的MSCs的数量没有明显差异,双次和三次注射组小鼠的脾内发现大量GFP-MSCs。SDF-1免疫荧光染色显示,激光后初期,SDF-1在激光斑处RPE层表达,随着CNV生成,CNV内出现阳性染色;ELISA结果显示,SDF-1表达水平在最初24小时内迅速增加,在第2天时达顶峰,然后迅速下降。MSCs移植后外周血和骨髓的流式分析显示,MSCs仅于第1天内在骨髓中作短暂停留。脉络膜铺片显示,激光后第1天,GFP-MSCs散布于激光斑周围;第3天时,GFP-MSCs组成的“细胞环”围绕激光斑,显示出向CNV靠近的定向移行趋势;第7天,GFP-MSCs进入CNV内并加入血管组成中。眼球切片显示绝大多数GFP-MSCs位于激光斑处脉络膜与光感受器间的CNV内。在这些MSCs中发现了血管内皮细胞、血管平滑肌细胞、巨噬细胞、上皮细胞和成纤维细胞的标记物(CD31,αSMA, F4/80, keratin,和vimentin)的阳性表达。⑷Ad转导小鼠MSCs后24小时,倒置荧光显微镜检测到报告基因GFP的表达,流式分析显示(73.6±5.3)%的细胞转导成功,AdPEDF转导的MSCs在体外表达PEDF可持续至少8天。AdPEDF组小鼠的眼球切片中,人PEDF的阳性染色出现于MSCs内及其周围的细胞外基质中;ELISA结果显示,在整个观察期间,眼内人PEDF呈稳定表达,表达量是抑制CNV阈值的4倍多;其CNV的厚度、长度和表面积明显减小。与AdPEDF/MSCs共培养的RPE细胞较之其他共培养条件下的RPE细胞增生和移行更快。
结论⑴BMCs分化为多种细胞类型参与CNV细胞构成,分泌促血管形成因子促进CNV发展,在CNV生成过程中发挥着重要作用。CNV微环境趋化BMCs,并支持和调控BMCs的分化方向。BMCs与CNV微环境可能存在相互作用。⑵尼古丁促进BMCs向CNV趋化和参与CNV生成,并影响CNV内BMCs的分化。尼古丁对BMCs的这种作用可能部分通过调节局部因子(如VEGF、VCAM-1)表达实现。⑶MSCs仅出现于CNV而不在其他器官停留(骨髓中一过性停留),提示其在CNV模型中具有比BMCs更为卓越的特异趋化能力;一次激光光凝引起的MSCs趋化具有有限的时间窗,其原因可能是激光后CNV区域趋化因子的表达规律。激光后MSCs逐步趋化至CNV内,并分化为CNV生成所需的多种细胞类型。MSCs具备作为细胞载体的潜能。⑷经基因修饰的MSCs到达CNV部位并在局部表达抗新生血管形成因子,从而抑制了CNV的生长,该效应可能部分通过在CNV发展中发挥重要作用的RPE细胞介导。MSCs有望作为抗新生血管药物的释放系统用于CNV相关疾病的治疗。
Background choroidal neovascularization (CNV), which refers to the formation of new blood vessels that arise from the choriocapillaries through Bruch's membrane into the subretinal space causing damage of topic choriod and retina, exudation of fluid and hemorrhage, is now known to occur as a final common pathway in nearly 40 ophthalmic diseases leading to irreversible visual loss. The pathogenesis of CNV, which includes multiple cell-types, cytokines and signal transduction pathways, is quite complicated and still poorly understood.
Postnatal Vessels are formed through two distinct processes. Angiogenesis involve the remodeling of established capillary networks and arterioles, while vasculogenesis involves the differentiation of stem/progenitor cells into mature vascular cells. Though being‘quiescence’generally, these stem/progenitor cells can be mobilized into circulation by physiological or pathologic stimulus to contribute to tissue repair and neoplasia formation at perienchyma. Recent studies have indicated that CNV occur through not only angiogenesis, but also vasculogenesis, that is, both bone marrow-derived cells (BMCs) and cells in situ participate in CNV development. However, little is known about the dynamic conduct of BMCs in the CNV microenvironment. BMCs, a heterogeneous cell population, is comprise of multiple stem/progenitor cell-types. Which types of them contribute to CNV and what role they play in the process? Whether they could be the new targets for CNV treatment? Lots of issues need to be studied.
Objectives This study aims⑴to investigate the contribution of BMCs to CNV, and the dynamic conduct of BMCs in CNV microenvironment;⑵to investigate the effects of nicotine on BMCs’contribution to CNV and the underlying mechanism;⑶to investigate the specific recruitment of MSCs to CNV and their contribution to CNV formation, to confirm the potential of MSCs being cell vectors;⑷to explore a noval therapeutic strategy of applicating MSCs as delivery vehicles for CNV treatment.
Methods⑴Green fluorescent protein (GFP) chimeric mice were developed by transplanting unpurified bone marrow cells from GFP +/+ transgenic mice to wild-type adult C57 mice. The qualified chimeric mice underwent laser rupture of Bruch membrane to induce CNV. Choroidal flatmount was performed to detect BMCs recruitment to and participation in CNV lesions, and immunofluorescent staining was performed to detect the phenotype of GFP+ cells (including vascular endothelial cells (VEC), vascular smooth muscle cells (VSMC) and macrophages) and expression of vascular endothelia growth factor (VEGF) and basic fibroblast cell growth factor (bFGF) in CNV;⑵GFP chimeric mice were developed. CNV was induced by lasering, and nicotine was administered orally in drinking water of mice in nicotine group on that very day. The chimeric mice in control group were normal raised. Four weeks later, histopathologic study and choroidal flatmount were performed to measure the CNV severity and BMCs recruitment. The differentiation of BMCs in CNV and local expressions of VEGF, bFGF and vascular cell adhesion molecule-1 (VCAM-1) were detected by immunofluorescent staining;⑶MSCs derived from GFP transgenic C57 mice were enriched and cultured. 4.0×106 GFP-MSCs or unpurified bone marrow cells were injected into wild type C57 mice through tail veins 0.5-1 hour after laser photocoagulation induction of CNV. To compare the migration of these two cell-types, GFP+ cells were detected in peripheral blood, bone marrow, heart, liver, spleen and lung. To study the time window of MSCs recruitment, mice were randomly divided into three groups. Mice in one group received single injection of 4.0×106 GFP-MSCs 0.5-1 hours after laser photocoagulation. The second group received twice injection at 0.5-1 hours and 3 days after lasering, and mice in another group received three times injections at 0.5-1 hours, 3 days and 6 days after lasering, respectively. Migration of GFP-MSCs in vivo was observed and compared. Expression of SDF-1, an important chemotactic factor for stem cells, in eyes after lasering was detected by using ELISA and immunofluorescent staining. Seven days after laser, choroidal flatmount was performed to investigate the participate of MSCs in CNV, and the differentiation of MSCs in CNV were analyzed by cell-markers immunofluorescent staining;⑷Adenoviral vectors (Ad) expressing human pigment epithelial-derived factor (PEDF) were generated with a reporter gene (GFP). Control vectors that do not express PEDF were constructed and produced in parallel (AdNull). MSCs were transduced. Transduction efficiency was detected by inverted microscopy and flow cytometry. Concentrations of human PEDF released by the transduced MSCs were measured by ELISA. AdPEDF/ MSCs, AdNull/MSCs, GFP-MSCs (4.0×106 cells/mouse) or 0.4ml PBS were injected into C57 mice tail veins 0.5-1 hour after laser photocoagulation. Human PEDF expression in mice eyes was examined by ELISA and immunofluorescent staining. One week after laser, CNV lesion severity was measured by quantitative analysis of both histopathology and choroidal flatmount. To test the impact of PEDF secreted by transduced MSCs on the RPE proliferation and migration, coculture system of AdPEDF/MSCs and RPE cells was established.
Results⑴Large number of GFP-BMCs appeared in the CNV lesions and integrated into CNV, constituting 16.22% of the total vascular area. Most GFP-BMCs appeared in CNV lesions (including choroid beneath CNV lesion), and a few GFP-BMCs were in neurosensory retina over CNV, corneoscleral limbus, ciliary body, optic disc and sclera, retina and choroid distant from CNV. GFP+ cells, which were immunoreactive forαSMA or CD31, appeared in CNV lesions only. However, F4/80+/GFP+ cells can be also detected in neurosensory retina over CNV, corneoscleral limbus and ciliary body. The constituent ratio of those three cell-types in total GFP-BMCs in CNV altered as CNV developed. The maximal ratios of CD31-labeled cells and F4/80-labeled cells presented at 2 week, while the ratio ofαSMA-labeled cells upgraded continuously. Immunofluorescent staining showed some BMCs in CNV were VEGF or bFGF positive.⑵Nicotine administration resulted in larger diameter and surface area of CNV. Nicotine-exposed mice demonstrated increased area and density of GFP+BMCs, increased GFP+ vascular cells area, and decreased ratio of BMCs expressing F4/80 in CNV. Furthermore, the expression of VEGF and bFGF within CNV and VCAM-1 in choroid beneath CNV was up-regulated in nicotine-exposed mice.⑶GFP-labeled BMCs , rather than MSCs, were found in lungs, livers and spleens at all time point. The quantity of MSCs recruited to CNV after singer, twice, or thrice MSCs-injections was compared, and there was no statistic difference between each two groups, while lots of GFP-MSCs were found in spleens of mice in the latter two groups. SDF-1 expression in eyes of mice models was examined. Initially, SDF-1 was expressed in RPE layer. As CNV formed, positive staining presented in CNV. The level of SDF-1 expression increased rapidly in the first 24h, reached peak at the second day, and then decreased sharply. Flow cytometry analysis showed MSCs underwent a transient retention in bone marrow. Choroidal flatmount indicated that 1 day after laser, GFP-MSCs were found dispersed around the laser sites. Subsequently, a directional movement of GFP-MSCs coming closer toward the CNV lesions presented. On day 7, GFP-MSCs were found in CNV and appeared to participate in vascular structure formation. Most GFP-MSCs were located in CNV between choroid and photoreceptors. In differentiation analysis, positive expression was found for cell-markers of VEC, VSMC, macrophage, fibroblast and epithelial cell in MSCs examined.⑷Reporter GFP expression in MSCs was found 24 hours after transduction by using inverted fluorescent microscope. Flow cytometry analysis further showed (73.6±5.3)% of the total cell population was GFP positive. Human PEDF production by AdPEDF/MSCs persisted for at least 8 days in vitro. After transplantation of AdPEDF/MSCs, human PEDF positive staining was found in MSCs and the nearby extracellular matrix in CNV. ELISA analysis showed that PEDF expression persisted for at least one week, the average production of human PEDF in the eyes was steady and the level was 4 folds higher than the quantity required to elicit anti-angiogenic effects for CNV. Histopathology and flatmount analysis indicated that there was significant reduction in CNV thickness, diameter and surface area in the AdPEDF group. The proliferation and migration of RPE cells which cocultured with AdPEDF/MSCs were enhanced.
Conclusion⑴BMCs play an important role in CNV by differentiating into multiple cell-types to compose CNV and secreting angiogenic factors. CNV microenvironment recruits BMCs, supports and regulates the differentiation of BMCs. There may be interaction between BMCs and the CNV microenvironment.⑵Nicotine promotes recruitment and incorporation of BMCs into CNV, and affects differentiation of BMCs in CNV. These effects may be partly due to regulation of related factors (e.g. VEGF or VCAM-1) expression by nicotin.⑶Comparing with unpurified BMCs, MSCs could be specifically recruited into CNV lesions, without stagnation in other organs, indicating their predominant potential for specific recruitment to CNV. Single lasering induction results in a finite time window for MSCs recruitment, it may due to the expression pattern of chemotatic factor, such as SDF-1, in CNV. After laser photocoagulation, MSCs were specifically recruited into CNV lesions gradually, and differentiated into multiple cell types required for CNV formation.⑷Genetic engineered MSCs localized and produced anti-angiogenic factor in CNV lesion. Thus, the growth of CNV was inhibited in vivo. The effect may be mediated, partly at least, by RPE cells, which function as an important regulator for CNV development. MSCs could serve as delivery vehicles of anti-angiogenic agents for the treatment of a range of CNV-associated diseases.
成年个体新生血管的形成包含两种机制:血管生成(angiogenesis)指原有的血管细胞发生增生、移行,形成新的血管;血管发生(vasculogenesis)指由干细胞分化而来的血管细胞构成新血管。干细胞在一般情况下处于“休眠”状态,在外因刺激或病理状态下会进入血液循环,移行至周边组织,表现出不同程度的更新和分化能力,参与组织修复和新生物形成。近期的研究表明,CNV的生成兼具这两种方式,既有原位组织细胞的增殖,又有骨髓来源细胞(bone marrow-derived cells, BMCs)的参与,但BMCs究竟以何种方式参与CNV生成、BMCs对CNV发展有何影响有待进一步阐明。此外,BMCs是一个异质性细胞群体,包含多种干/祖细胞,究竟是哪种干/祖细胞参与了CNV的生成、它们在CNV发生发展中发挥着怎样的作用、是否可作为CNV治疗的靶点等诸多问题有待研究。
目的和内容探讨BMCs在CNV发生发展中的作用,在危险因素影响CNV发展中的介导作用,及作为干细胞疗法细胞载体的潜能。⑴建立C57-绿色荧光蛋白(green fluorescent protein, GFP)嵌合体小鼠,观察在嵌合体CNV发生发展过程中,BMCs在CNV聚集、分化和表达蛋白情况;⑵探讨尼古丁对参与CNV生成的BMCs的趋化、分化和蛋白表达能力的影响;⑶观察骨髓来源基质干细胞(mesenchymal stem cells, MSCs)向CNV的趋化特异性和时间窗,观察MSCs参与CNV生成情况,探讨MSCs是否具备作为药物载体的潜能;⑷构建表达人源性色素上皮衍生因子(pigment epithelium derived factor, PEDF)的腺病毒,观察腺病毒转导的MSCs对CNV的抑制作用,探讨利用MSCs靶向治疗CNV的新策略。
方法⑴通过GFP转基因小鼠与野生型C57小鼠的骨髓移植建立嵌合体小鼠,利用532nm激光建立小鼠CNV模型,脉络膜铺片观察GFP-BMCs向CNV趋化、参与血管结构组成的情况,免疫荧光染色观察CNV中BMCs分化的细胞类型(包括血管内皮细胞、血管平滑肌细胞和巨噬细胞)及表达血管内皮生长因子(vascular endothelial growth factor, VEGF)和碱性成纤维细胞生长因子(basic fibroblast cell growth factor, bFGF)的情况;⑵激光光凝建立C57-GFP嵌合体小鼠CNV模型后,即日在其饮水中添加尼古丁(100μg/ml),对照组嵌合体小鼠建立CNV模型后普通饲养,4周后眼球切片HE染色观察CNV厚度与直径,脉络膜铺片观察CNV表面积、GFP-BMCs的趋化和参与构成血管情况,免疫荧光染色观察CNV中BMCs分化情况及各因子表达变化;⑶体外培养GFP转基因小鼠骨髓来源MSCs,野生型小鼠激光建模后0.5-1小时尾静脉注射4.0×106 GFP-BMCs或GFP-MSCs,激光后1、3、7天,心、肝、脾、肺、心脏切片荧光观察比较两种细胞在器官内的滞留情况;野生型小鼠激光后0.5-1小时、3天及6天,分别注射4.0×106 GFP-MSCs,激光后7天观察比较单次注射、双次注射和三次注射后GFP-MSCs在体内移行和向CNV趋化的情况;酶联免疫吸附测定(enzyme linked immunosorbent assay, ELISA)和免疫荧光染色检测激光后不同时间眼内干细胞趋化因子基质细胞衍生因子-1(stromal cell derived factor-1, SDF-1)的表达;脉络膜铺片检测参与构成新血管的GFP-MSCs,免疫荧光染色分析CNV中GFP-MSCs分化的细胞类型;⑷构建表达人源性PEDF、含报告基因GFP、E1 E3缺陷的5型腺病毒(adenovirus, Ad),转导体外培养的野生型小鼠骨髓来源MSCs,倒置荧光显微镜观察及流式细胞仪测定感染效率,ELISA检测体外人源性PEDF表达情况;激光后0.5-1小时小鼠尾静脉分别注射4.0×106 Ad-PEDF/MSCs、对照病毒Ad-GFP/MSCs、GFP-MSCs及0.4ml磷酸盐缓冲液(Phosphate buffered saline, PBS),分别于激光后1、3、5、7天利用免疫荧光染色和ELISA检测眼内人源性PEDF表达,激光后7天眼球切片组织学分析和脉络膜铺片分析比较各组CNV厚度、直径及表面积。体外建立视网膜色素上皮(retinal pigment epithelium, RPE)细胞与MSCs的共培养体系,分析MSCs分泌的PEDF对RPE细胞增生和移行的影响。
结果⑴激光后大量GFP-BMCs聚集于激光斑处并整合入CNV中的血管结构内,由其组成的血管面积约占CNV表面积的16.22%。GFP-BMCs主要分布于CNV区域(包括CNV深层的脉络膜),少量散布于CNV表层的视网膜、角巩膜缘、睫状体、视盘周围及远离CNV的视网膜、脉络膜和巩膜。CD31或αSMA阳性的GFP+细胞仅出现于CNV区域,F4/80+/GFP+细胞不仅出现在CNV区域,还出现在CNV表层的视网膜、角巩膜缘和睫状体内。CNV内CD31/GFP、αSMA/GFP和F4/80/GFP双阳性细胞在全部GFP+细胞中的构成比随CNV进展而变化,CD31/GFP或F4/80/GFP双阳性细胞构成比的最高值出现于激光后第2周,而αSMA+/GFP+细胞构成比在激光后4周仍处上升趋势。CNV区域的一些GFP-BMCs可表达促血管形成因子VEGF和bFGF。⑵尼古丁处理组小鼠CNV长度和表面积增大,CNV内GFP-BMCs的面积和密度增加,GFP-BMCs来源的血管细胞面积增加,F4/80+/GFP+细胞构成比下降,CNV内VEGF和bFGF的表达及CNV下脉络膜内VCAM-1的表达上调。⑶在整个观察期间,肺、肝和脾内可见GFP+BMCs滞留,但未观测到GFP+MSCs。单次、双次或三次MSCs注射后趋化至CNV的MSCs的数量没有明显差异,双次和三次注射组小鼠的脾内发现大量GFP-MSCs。SDF-1免疫荧光染色显示,激光后初期,SDF-1在激光斑处RPE层表达,随着CNV生成,CNV内出现阳性染色;ELISA结果显示,SDF-1表达水平在最初24小时内迅速增加,在第2天时达顶峰,然后迅速下降。MSCs移植后外周血和骨髓的流式分析显示,MSCs仅于第1天内在骨髓中作短暂停留。脉络膜铺片显示,激光后第1天,GFP-MSCs散布于激光斑周围;第3天时,GFP-MSCs组成的“细胞环”围绕激光斑,显示出向CNV靠近的定向移行趋势;第7天,GFP-MSCs进入CNV内并加入血管组成中。眼球切片显示绝大多数GFP-MSCs位于激光斑处脉络膜与光感受器间的CNV内。在这些MSCs中发现了血管内皮细胞、血管平滑肌细胞、巨噬细胞、上皮细胞和成纤维细胞的标记物(CD31,αSMA, F4/80, keratin,和vimentin)的阳性表达。⑷Ad转导小鼠MSCs后24小时,倒置荧光显微镜检测到报告基因GFP的表达,流式分析显示(73.6±5.3)%的细胞转导成功,AdPEDF转导的MSCs在体外表达PEDF可持续至少8天。AdPEDF组小鼠的眼球切片中,人PEDF的阳性染色出现于MSCs内及其周围的细胞外基质中;ELISA结果显示,在整个观察期间,眼内人PEDF呈稳定表达,表达量是抑制CNV阈值的4倍多;其CNV的厚度、长度和表面积明显减小。与AdPEDF/MSCs共培养的RPE细胞较之其他共培养条件下的RPE细胞增生和移行更快。
结论⑴BMCs分化为多种细胞类型参与CNV细胞构成,分泌促血管形成因子促进CNV发展,在CNV生成过程中发挥着重要作用。CNV微环境趋化BMCs,并支持和调控BMCs的分化方向。BMCs与CNV微环境可能存在相互作用。⑵尼古丁促进BMCs向CNV趋化和参与CNV生成,并影响CNV内BMCs的分化。尼古丁对BMCs的这种作用可能部分通过调节局部因子(如VEGF、VCAM-1)表达实现。⑶MSCs仅出现于CNV而不在其他器官停留(骨髓中一过性停留),提示其在CNV模型中具有比BMCs更为卓越的特异趋化能力;一次激光光凝引起的MSCs趋化具有有限的时间窗,其原因可能是激光后CNV区域趋化因子的表达规律。激光后MSCs逐步趋化至CNV内,并分化为CNV生成所需的多种细胞类型。MSCs具备作为细胞载体的潜能。⑷经基因修饰的MSCs到达CNV部位并在局部表达抗新生血管形成因子,从而抑制了CNV的生长,该效应可能部分通过在CNV发展中发挥重要作用的RPE细胞介导。MSCs有望作为抗新生血管药物的释放系统用于CNV相关疾病的治疗。
Background choroidal neovascularization (CNV), which refers to the formation of new blood vessels that arise from the choriocapillaries through Bruch's membrane into the subretinal space causing damage of topic choriod and retina, exudation of fluid and hemorrhage, is now known to occur as a final common pathway in nearly 40 ophthalmic diseases leading to irreversible visual loss. The pathogenesis of CNV, which includes multiple cell-types, cytokines and signal transduction pathways, is quite complicated and still poorly understood.
Postnatal Vessels are formed through two distinct processes. Angiogenesis involve the remodeling of established capillary networks and arterioles, while vasculogenesis involves the differentiation of stem/progenitor cells into mature vascular cells. Though being‘quiescence’generally, these stem/progenitor cells can be mobilized into circulation by physiological or pathologic stimulus to contribute to tissue repair and neoplasia formation at perienchyma. Recent studies have indicated that CNV occur through not only angiogenesis, but also vasculogenesis, that is, both bone marrow-derived cells (BMCs) and cells in situ participate in CNV development. However, little is known about the dynamic conduct of BMCs in the CNV microenvironment. BMCs, a heterogeneous cell population, is comprise of multiple stem/progenitor cell-types. Which types of them contribute to CNV and what role they play in the process? Whether they could be the new targets for CNV treatment? Lots of issues need to be studied.
Objectives This study aims⑴to investigate the contribution of BMCs to CNV, and the dynamic conduct of BMCs in CNV microenvironment;⑵to investigate the effects of nicotine on BMCs’contribution to CNV and the underlying mechanism;⑶to investigate the specific recruitment of MSCs to CNV and their contribution to CNV formation, to confirm the potential of MSCs being cell vectors;⑷to explore a noval therapeutic strategy of applicating MSCs as delivery vehicles for CNV treatment.
Methods⑴Green fluorescent protein (GFP) chimeric mice were developed by transplanting unpurified bone marrow cells from GFP +/+ transgenic mice to wild-type adult C57 mice. The qualified chimeric mice underwent laser rupture of Bruch membrane to induce CNV. Choroidal flatmount was performed to detect BMCs recruitment to and participation in CNV lesions, and immunofluorescent staining was performed to detect the phenotype of GFP+ cells (including vascular endothelial cells (VEC), vascular smooth muscle cells (VSMC) and macrophages) and expression of vascular endothelia growth factor (VEGF) and basic fibroblast cell growth factor (bFGF) in CNV;⑵GFP chimeric mice were developed. CNV was induced by lasering, and nicotine was administered orally in drinking water of mice in nicotine group on that very day. The chimeric mice in control group were normal raised. Four weeks later, histopathologic study and choroidal flatmount were performed to measure the CNV severity and BMCs recruitment. The differentiation of BMCs in CNV and local expressions of VEGF, bFGF and vascular cell adhesion molecule-1 (VCAM-1) were detected by immunofluorescent staining;⑶MSCs derived from GFP transgenic C57 mice were enriched and cultured. 4.0×106 GFP-MSCs or unpurified bone marrow cells were injected into wild type C57 mice through tail veins 0.5-1 hour after laser photocoagulation induction of CNV. To compare the migration of these two cell-types, GFP+ cells were detected in peripheral blood, bone marrow, heart, liver, spleen and lung. To study the time window of MSCs recruitment, mice were randomly divided into three groups. Mice in one group received single injection of 4.0×106 GFP-MSCs 0.5-1 hours after laser photocoagulation. The second group received twice injection at 0.5-1 hours and 3 days after lasering, and mice in another group received three times injections at 0.5-1 hours, 3 days and 6 days after lasering, respectively. Migration of GFP-MSCs in vivo was observed and compared. Expression of SDF-1, an important chemotactic factor for stem cells, in eyes after lasering was detected by using ELISA and immunofluorescent staining. Seven days after laser, choroidal flatmount was performed to investigate the participate of MSCs in CNV, and the differentiation of MSCs in CNV were analyzed by cell-markers immunofluorescent staining;⑷Adenoviral vectors (Ad) expressing human pigment epithelial-derived factor (PEDF) were generated with a reporter gene (GFP). Control vectors that do not express PEDF were constructed and produced in parallel (AdNull). MSCs were transduced. Transduction efficiency was detected by inverted microscopy and flow cytometry. Concentrations of human PEDF released by the transduced MSCs were measured by ELISA. AdPEDF/ MSCs, AdNull/MSCs, GFP-MSCs (4.0×106 cells/mouse) or 0.4ml PBS were injected into C57 mice tail veins 0.5-1 hour after laser photocoagulation. Human PEDF expression in mice eyes was examined by ELISA and immunofluorescent staining. One week after laser, CNV lesion severity was measured by quantitative analysis of both histopathology and choroidal flatmount. To test the impact of PEDF secreted by transduced MSCs on the RPE proliferation and migration, coculture system of AdPEDF/MSCs and RPE cells was established.
Results⑴Large number of GFP-BMCs appeared in the CNV lesions and integrated into CNV, constituting 16.22% of the total vascular area. Most GFP-BMCs appeared in CNV lesions (including choroid beneath CNV lesion), and a few GFP-BMCs were in neurosensory retina over CNV, corneoscleral limbus, ciliary body, optic disc and sclera, retina and choroid distant from CNV. GFP+ cells, which were immunoreactive forαSMA or CD31, appeared in CNV lesions only. However, F4/80+/GFP+ cells can be also detected in neurosensory retina over CNV, corneoscleral limbus and ciliary body. The constituent ratio of those three cell-types in total GFP-BMCs in CNV altered as CNV developed. The maximal ratios of CD31-labeled cells and F4/80-labeled cells presented at 2 week, while the ratio ofαSMA-labeled cells upgraded continuously. Immunofluorescent staining showed some BMCs in CNV were VEGF or bFGF positive.⑵Nicotine administration resulted in larger diameter and surface area of CNV. Nicotine-exposed mice demonstrated increased area and density of GFP+BMCs, increased GFP+ vascular cells area, and decreased ratio of BMCs expressing F4/80 in CNV. Furthermore, the expression of VEGF and bFGF within CNV and VCAM-1 in choroid beneath CNV was up-regulated in nicotine-exposed mice.⑶GFP-labeled BMCs , rather than MSCs, were found in lungs, livers and spleens at all time point. The quantity of MSCs recruited to CNV after singer, twice, or thrice MSCs-injections was compared, and there was no statistic difference between each two groups, while lots of GFP-MSCs were found in spleens of mice in the latter two groups. SDF-1 expression in eyes of mice models was examined. Initially, SDF-1 was expressed in RPE layer. As CNV formed, positive staining presented in CNV. The level of SDF-1 expression increased rapidly in the first 24h, reached peak at the second day, and then decreased sharply. Flow cytometry analysis showed MSCs underwent a transient retention in bone marrow. Choroidal flatmount indicated that 1 day after laser, GFP-MSCs were found dispersed around the laser sites. Subsequently, a directional movement of GFP-MSCs coming closer toward the CNV lesions presented. On day 7, GFP-MSCs were found in CNV and appeared to participate in vascular structure formation. Most GFP-MSCs were located in CNV between choroid and photoreceptors. In differentiation analysis, positive expression was found for cell-markers of VEC, VSMC, macrophage, fibroblast and epithelial cell in MSCs examined.⑷Reporter GFP expression in MSCs was found 24 hours after transduction by using inverted fluorescent microscope. Flow cytometry analysis further showed (73.6±5.3)% of the total cell population was GFP positive. Human PEDF production by AdPEDF/MSCs persisted for at least 8 days in vitro. After transplantation of AdPEDF/MSCs, human PEDF positive staining was found in MSCs and the nearby extracellular matrix in CNV. ELISA analysis showed that PEDF expression persisted for at least one week, the average production of human PEDF in the eyes was steady and the level was 4 folds higher than the quantity required to elicit anti-angiogenic effects for CNV. Histopathology and flatmount analysis indicated that there was significant reduction in CNV thickness, diameter and surface area in the AdPEDF group. The proliferation and migration of RPE cells which cocultured with AdPEDF/MSCs were enhanced.
Conclusion⑴BMCs play an important role in CNV by differentiating into multiple cell-types to compose CNV and secreting angiogenic factors. CNV microenvironment recruits BMCs, supports and regulates the differentiation of BMCs. There may be interaction between BMCs and the CNV microenvironment.⑵Nicotine promotes recruitment and incorporation of BMCs into CNV, and affects differentiation of BMCs in CNV. These effects may be partly due to regulation of related factors (e.g. VEGF or VCAM-1) expression by nicotin.⑶Comparing with unpurified BMCs, MSCs could be specifically recruited into CNV lesions, without stagnation in other organs, indicating their predominant potential for specific recruitment to CNV. Single lasering induction results in a finite time window for MSCs recruitment, it may due to the expression pattern of chemotatic factor, such as SDF-1, in CNV. After laser photocoagulation, MSCs were specifically recruited into CNV lesions gradually, and differentiated into multiple cell types required for CNV formation.⑷Genetic engineered MSCs localized and produced anti-angiogenic factor in CNV lesion. Thus, the growth of CNV was inhibited in vivo. The effect may be mediated, partly at least, by RPE cells, which function as an important regulator for CNV development. MSCs could serve as delivery vehicles of anti-angiogenic agents for the treatment of a range of CNV-associated diseases.
引文
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