Breakdown of the blood-retinal barrier in a model of retinal neovascularization

Breakdown in the blood-retinal barrier occurs in retinal neovascularization in a number of diseases. To study the anatomic basis of this breakdown, we examined retinal neovascularization induced by injection of 250,000 homologous fibroblasts into the vitreous cavity of pigmented rabbits. Neovascularization is evident by electron microscopy in this model 3 days after fibroblast injection. Fluorescein angiography followed by intravenous horseradish peroxidase (HRP) injection was performed prior to enucleation on 2, 3, 5, 7, and 14 days after fibroblast injection. Fluorescein leakage from retinal vessels occurs early (at day 1) and persists as the neovascularization progresses. The leakage in the early stages is concentrated near puckers from the medullary wings. In the later stages, fluorescein leakage is most prominent in the developing tips of the new vessels. Horseradish peroxidase was not observed to leak from the lumen of new vessels. "Gaps" or separations in the endothelial cell junctions were not observed in developing vessels. The breakdown of the blood-retinal barrier in this model of retinal neovascularization is therefore selective, (ie, fluorescein leaks but not HRP) and it is not due to gaps or fenestrations between endothelial cells in developing vessels.     

牛视网膜微血管周细胞和内皮细胞的选择性培养

目的 建立视网膜微血管周细胞（ＰＣ）和内皮细胞（ＥＣ）选择性培养的简便方法。方法 采用含２０％胎牛血清（ＦＢＳ）的ＤＭＥＭ和在该培养基加入５％贫血小板人血浆（ＰＰＰ）及牛视网膜浸出液（２０ｕｌ／ｍｌ）,结合原代培养细胞克隆的分离、除杂。结果 获得的ＰＣ和ＥＣ能连续传代,纯净度〉９５％。ＰＣ形态不规则,无接触性抑制,对３Ｇ５和α－肌动蛋白单克隆抗体染色阳性,Ⅷ因子抗体染色阴性;ＥＣ呈鹅卵石状生长,有接触性抑制,对Ⅷ因子抗体染色阳性,３Ｇ５和α－肌动蛋白抗体染色阴性。结论 用２０％ＦＢＳ的ＤＭＥＭ或在该培养液中加入５％ＰＰＰ及视网膜浸出液,结合原代细胞克隆的分离、除杂,可分别获得较纯净的ＰＣ和ＥＣ培养物。     

基于内皮细胞/周细胞构建大鼠视网膜血管新生体外三维模型

目的：构建基于视网膜微血管内皮细胞(ECs)和周细胞(RMPs)的大鼠视网膜血管新生体外三维模型。

方法：分离、纯化、培养大鼠视网膜微血管内皮细胞及视网膜微血管周细胞,选用第3～7代经鉴定后的细胞用于研究,采用细胞示踪剂对细胞进行染色,混合后按表面培养法接种于Matrigel,动态观察,并检测血管新生期间血管内皮生长因子A(VEGF-A)的表达。

结果：培养12h时,RMPs被ECs招募,聚集成大小不等细胞团块; 24h时,ECs/RMPs共同形成复杂的三维血管样条索网; 48h时,网状结构出现明显崩解,仅保持少量不完整的简单网状结构; 72h时,血管样条索网完全崩解。在三维模型发生过程中,VEGF-A的表达量升高; 退化时,VEGF-A的表达量下降。

结论：成功基于视网膜微血管全成分细胞ECs和RMPs构建了大鼠视网膜血管新生体外三维模型。     

周细胞对缺氧诱导下视网膜微血管内皮细胞增生的影响

目的探讨视网膜微血管内皮细胞（RMECs）与共培养的周细胞（PCs）及PCs条件培养液（PCM）对常氧和缺氧条件下RMECs增生的影响。方法传代培养大鼠RMECs和PCs,采用兔抗鼠Ⅷ因子多克隆抗体和小鼠抗大鼠CD31单克隆抗体免疫组织化学法鉴定RMECs,PCs采用小鼠抗大鼠desmin多克隆抗体和兔抗鼠抗血小板衍生生长因子受体-β（PDGFR-β）进行免疫荧光双标鉴定。Millicell小室建立PCs和RMECs共培养系统,PCs培养近融合期后制备PCM,向共培养系统或PCM中加入终浓度为200μmol/L的CoCl2诱导形成细胞化学缺氧模型,观察常氧和缺氧条件下PCs和PCM对RMECs增生的影响。使用MTT法、3H-TdR掺入法检测RMECs在不同培养条件下的增生数量,并用流式细胞仪检测RMECs在不同细胞周期的细胞率。结果近融合期的RMECs呈类纺锤状、单层生长,可见明显的接触抑制,Ⅷ因子和CD31均阳性表达。PCs形状不规则,细胞重叠生长无接触抑制,PDGFR-β和desmin抗体双标阳性。缺氧3～9d缺氧RMECs单独培养组较常氧RMECs单独培养组各时间点的RMECs均明显增生,缺氧6d时增生达高峰（P〈0.01）,细胞生长抑制率（GIR）为-24.9%。缺氧共培养组较缺氧RMECs单独培养组RMECs增生下降（P〈0.05）。缺氧3、6、9d时常氧共培养组较常氧RMECs单独培养组RMECs数目均明显下降（P〈0.05,P〈0.01,P〈0.01）。缺氧6d时S期RMECs数量明显增加,占各期细胞总数的（43.9±0.8）%;常氧或缺氧共培养组,PCs均可抑制RMECs的增生,S期细胞分别占各期细胞总数的（3.6±0.1）%、（15.1±0.9）%（P〈0.05,P〈0.01）。常氧PCM组RMECs的吸光度（A）值和3H-TdR掺入量4～24h均低于对照组（P〈0.05）。结论缺氧和常氧条件下RMECs和PCs共培养时PCs均可抑制RMECs的增生,常氧条件下PCM可抑制RMECs的增生。     

Interaction of retinal pericytes, capillary endothelial cells and pigment epithelial cells in vasoproliferative retinopathies

Degeneration of intramural pericytes is the first histopathological lesion seen in diabetic retinopathy. Since pericytes from the optic nerve and brain are spared, such degeneration appears to be specific to the retinal circulation. Recent studies have shown that when retinal pericytes and microvascular endothelial cells are co-cultured in vitro , direct contact between the two types of cells leads to the inhibition of proliferation of endothelial cells. Previously it was shown that when pericytes are cultured in the absence of microvascular endothelial cells, the cultured medium conditioned by the pericytes is actively mitogenic for retinal capillary endothelial cells. Furthermore, conditioned medium from human retinal pigment epithelial cells is also mitogenic for retinal capillary endothelial cells and pericytes. On the basis of these results, a new working hypothesis is proposed for the mechanism of action of photocoagulation used in vasoproliferative retinopathies.     

Interaction of retinal pericytes, capillary endothelial cells and pigment epithelial cells in vasoproliferative retinopathies

Degeneration of intramural pericytes is the first histopathological lesion seen in diabetic retinopathy. Since pericytes from the optic nerve and brain are spared, such degeneration appears to be specific to the retinal circulation. Recent studies have shown that when retinal pericytes and microvascular endothelial cells are co-cultured in vitro, direct contact between the two types of cells leads to the inhibition of proliferation of endothelial cells. Previously it was shown that when pericytes are cultured in the absence of microvascular endothelial cells, the cultured medium conditioned by the pericytes is actively mitogenic for retinal capillary endothelial cells. Furthermore, conditioned medium from human retinal pigment epithelial cells is also mitogenic for retinal capillary endothelial cells and pericytes. On the basis of these results, a new working hypothesis is proposed for the mechanism of action of photocoagulation used in vasoproliferative retinopathies.     

Inhibition of proliferation of retinal microvascular endothelial cells by pericytes through down-regulating KDR/Flk-1 in a co-culture system

To investigate the role of pericytes in growth of retinal microvascular endothelial cells(RMECs) in a co-culture system in order to understand some mechanism of angiogenesis in hypoxia induced retinal neovascular disorders. · METHODS: RMECs were isolated by a modified protocol using CD31 coated Dynabeads, and identified by immunocytochemical staining with anti-Factor VIII and CD31 antibodies. Rat retinal pericytes were isolated and characterized by immunofluorescent staining with PDGFR-β; and desmin antibodies. Pericytes and RMECs were cultured in a contact co-culture system both under normoxia and hypoxia by Millicell chamber. RMECs proliferation was evaluated by MTT and cell cycle assay with flow cytometry. RT-PCR was used to detect the alteration of KDR/Flk-1 mRNA level in RMECs under normoxia or hypoxia in the co-culture system. · RESULTS: Highly pured rat RMECs and pericytes were harvested with the modified isolating method. The two cell types were identified by positive Factor VIII, CD31 and PDGFR-β, desmin cytochemical staining respectively. RMECs proliferated significantly under hypoxia from 3 to 9 day with a maximal rate on day 6 (24.9%, P < 0.01) by MTT. In the co-culture system, the proliferation of RMECs was inhibited by pericytes. After 6 days exposure to hypoxia, the fraction of S-phase RMECs number was greatly increased by 43.9%(P < 0.01). In the co-culture system, RMECs proliferation was inhibited by pericytes through decreasing the fraction of S-phase cell number both under normoxia (3.6%, P <0.05) and under hypoxia (15.1%,P <0.01). KDR/Flk-1 mRNA level in single cultured RMECs was shown to increase approximately 1.3-fold when exposed to hypoxia. Compared with single cultured RMECs, co-culture with pericytes could decrease KDR/Flk-1 mRNA by 45.1% (P <0.05) and 27.7% (P <0.05) under normoxia and hypoxia condition respectively. · CONCLUSION: The present study demonstrated that pericytes could inhibit proliferation of RMECs under both normoxia and hypoxia. The inhibition effects of pericytes maybe, at least in part, due to downregulation of KDR/Flk-1 of RMECs. These findings confirm that pericytes could be a potential inhibitor in the pathogenesis of retinal neovascularization(RNV).     

共培养系统中视网膜微血管周细胞经KDR/Flk-1途径对内皮细胞增生的影响

目的:采用细胞共培养模型研究缺氧诱导视网膜新生血管生成过程中周细胞对微血管内皮细胞增生的作用及其相关血管内皮细胞生长因子受体2(KDR/Flk-1)调节机制。方法:免疫磁珠法原代分离培养大鼠视网膜微血管内皮细胞和周细胞,分别采用抗VIII因子、CD31抗体以及抗血小板源性生长因子受体β、结蛋白抗体免疫细胞化学法鉴定内皮细胞和周细胞。通过Millicell小室建立周细胞和内皮细胞共培养模型,使用MTT法和流式细胞仪检测内皮细胞增殖数量及周期,观察缺氧和常氧下周细胞对血管内皮细胞增生的影响,并采用RT-PCR法检测内皮细胞KDR/Flk-1的mRNA水平变化,探讨相关调节机制。结果:经免疫细胞化学法鉴定,免疫磁珠法可获得高纯度的视网膜微血管内皮细胞和周细胞。MTT法显示,缺氧3～9d内皮细胞增生明显,6d增生达高峰(24.9%,P<0.01);共培养时内皮细胞的增生可被周细胞抑制。流式细胞仪检测发现,缺氧6dS期内皮细胞数量明显增加(43.9%,P<0.01);常氧(3.6%,P<0.05)或缺氧(15.1%,P<0.01)共培养时,周细胞均可抑制内皮细胞增生。缺氧下内皮细胞KDR/Flk-1的mRNA水平是常氧下的1.3倍;常氧(45.1%,P<0.05)和缺氧(27.7%,P<0.05)共培养下,周细胞均可下调内皮细胞KDR/Flk-1的mRNA表达。结论:缺氧和常氧共培养时周细胞均可抑制微血管内皮细胞增生,此抑制作用部分是通过下调内皮细胞KDR/Flk-1mRNA表达实现。     

牛视网膜微血管内皮细胞和周细胞的体外培养

目的 探讨牛视网膜微血管内皮细胞（ bovine retinal endothelial cells, BREC ）和周细胞（bovine retinal pericytes, BRP）的体外选择性培养方法。 方法 结合视网膜微血管的消化分离，采用含10％人血清、100 μg/ml 肝素（Heparin）的Dulbecco改良Eagle培养基(Dulbecco′s modified Eagle′s medium, DMEM)和含20％胎牛血清的DMEM培养基分别选择性培养BREC和BRP。通过荧光显微镜观察乙酰化低密度脂蛋白(acetylated low density lipoprotein, Dil-Ac-LDL)吞噬情况并应用Von Willebrand因子抗体通过免疫组织化学方法鉴定BREC；以及α-平滑肌肌动蛋白抗体免疫组织化学鉴定BRP。 结果 通过选择性培养获得的BREC和BRP的纯度达到98％以上，并能连续传代。BREC早期似小鲤鱼状聚集在微血管碎片周围，呈片状鹅卵石样单层生长，存在接触性抑制；周细胞多散布在距血管碎片稍远处，形状不规则，呈无接触性抑制的生长。BREC可吞噬Dil-Ac-LDL，细胞浆内有荧光表达 ；消化传代后BREC中Von Willebrand因子表达阳性，而α-平滑肌肌动蛋白表达阴性；BRP的α-平滑肌肌动蛋白表达阳性，而Von Willebrand因子表达阴性。 结论 选择性培养基的应用和培养皿的处理，可分别获得较高纯度的BREC和BRP，简单且具有良好的重复性，无需额外步骤来去除BREC中混杂的BRP。 （中华眼底病杂志，2004，20：23-26）     

The effects of high ambient glucose on the radiosensitivity of retinal microvascular endothelial cells and pericytes

PURPOSE: The metabolic peturbations of diabetes cause functional and structural changes in the retinal microvasculature which are termed diabetic retinopathy. Exposure of the eye to ionising radiation results in retinal vascular damage with a clinical manifestation known as radiation retinopathy. Anecdotal studies have suggested that exposure to even low levels of ionising radiation may accelerate development of pathological changes in the retinal vessels of patients with diabetes. This in vitro study was designed to test the hypothesis that the combination of a high ambient glucose environment (mimicking hyperglycaemia and diabetes) along with exposure to ionising radiation would result in more accentuated damage to cultured retinal vascular cells. METHODS: Retinal microvascular endothelial cells and pericytes were propagated for 5 days in either 5 mM (euglycaemia) or 15 mM (hyperglycaemia) glucose. Cells were irradiated with 250, 500 or 1000 cGy of ionising radiation using a 6 MV beam photon accelerator which was used for radiotherapy. Similarly treated but unirradiated cells were used as controls. DNA damage was assessed using the single-cell gel electrophoresis (comet) assay. RESULTS: Unirradiated control cells pre-exposed to glucose at either 5 mM or 15 mM for 5 days showed no significant difference in mean percentage tail DNA representing damage. However, in both pericytes and endothelial cells exposed to ionising radiation, cells cultured in 15 mM glucose showed significantly higher levels of DNA damage compared with those cultured in 5 mM glucose, with maximal differences being seen at the higher radiation doses (500 and 1000 cGy). CONCLUSIONS: This study has demonstrated that retinal microvascular cells cultured in high glucose express more DNA damage when exposed ionising radiation. These findings have important implications for the management of patients with diabetes if they require radiotherapy for neoplastic disease.     

Polyol formation and NADPH-dependent reductases in dog retinal capillary pericytes and endothelial cells

PURPOSE: Dogs fed a diet containing 30% galactose experience retinal vascular changes similar to those in human diabetic retinopathy, with selective pericyte loss as an initial lesion. In the present study the relationship among reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reductases, polyol formation, and flux through the polyol pathway in cultured dog retinal capillary cells were investigated. METHODS: Pericytes and endothelial cells were cultured from retina of beagle dogs. NADPH-dependent reductases were characterized by chromatofocusing after gel filtration. Sugars in cultured cells were analyzed by gas chromatography, and flux through the polyol pathway was investigated by 19F nuclear magnetic resonance (NMR) with 3-fluoro-3-deoxy-D-glucose (3FG) as a substrate. The presence of aldose reductase and sorbitol dehydrogenase in these cells was examined by northern blot analysis. RESULTS: Two distinct peaks corresponding to aldose reductase and aldehyde reductase, the latter being dominant, were observed in pericytes by chromatofocusing. Culture in medium containing either 10 mM D-galactose or 30 mM D-glucose resulted in the accumulation of sugar alcohol in pericytes that was markedly reduced by aldose reductase inhibitors. 19F NMR spectra obtained from pericytes cultured for 5 days in medium containing 2 mM 3FG displayed the marked accumulation of 3-fluoro-deoxysorbitol but not 3-fluoro-deoxyfructose. No 3FG metabolism was observed in similarly cultured endothelial cells. With northern blot analysis, aldose reductase was detected in pericytes but not in endothelial cells. Sorbitol dehydrogenase was below the detectable limit in pericytes and endothelial cells. CONCLUSIONS: Aldose, aldehyde, and glyceraldehyde reductases are present in dog retinal capillary pericytes, with aldehyde reductase being the major reductase present. Polyol accumulation easily occurs in pericytes but not in endothelial cells.     

Simplified methods for consistent and selective culture of bovine retinal endothelial cells and pericytes

Different matrix components, in combination with various media and serum supplements, were evaluated for their ability to promote selectively the growth of bovine retinal endothelial cells in primary culture. The optimal setting for the selective growth of retinal endothelial cells was a fibronectin/hyaluronic acid matrix, Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% pooled human serum and 100 micrograms/ml heparin. These conditions consistently yielded virtually homogeneous cultures of endothelial cells, assessed using specific endothelial markers. Thus obtained, the retinal endothelial cells could be subcultured and maintained in phenotypically stable long-term serial cultivation. Homogeneous cultures of retinal pericytes were obtained when microvessel isolates were seeded to uncoated or gelatin-coated culture dishes and grown in DMEM supplemented with 20% fetal bovine serum. The retinal pericytes could also be subcultured and cultivated for numerous populations doublings. Additionally, observations from this study suggest that two populations of pericytes may be obtained in culture and distinguished on the basis of their relative size and antigenic properties.     

Mouse model of post-surgical breakdown of the blood-retinal barrier

PURPOSE: Post-surgical macular edema is an important clinical problem resulting from breakdown of the blood-retinal barrier (BRB) after surgery. This study was designed to develop a mouse model of post-surgical BRB breakdown. METHODS: Two 25-gauge needles, one for infusion and one for aspiration, were inserted through the limbus and into the lens of one eye of adult male C57BL/6 mice. The anterior portion of the lens was aspirated and the fellow eye was untreated. At several time points after surgery, the integrity of the BRB was assessed quantitatively, using [3H]mannitol as a tracer, or qualitatively, using immunohistochemical staining for albumin. RESULTS: Eyes with partial lens extraction had a significant increase in retinal vascular leakage one day after surgery, which persisted two and three days after surgery, but by five days, was not significantly different from controls. Immunohistochemical staining for albumin demonstrated that the breech in the barrier was sufficient to allow passage of a 60kDa protein into the retina, and was localized predominantly to retinal vessels. CONCLUSIONS: Partial lens extraction in mice results in BRB breakdown (primarily the inner BRB) that is highly reproducible in the severity of leakage and its time course. This provides a valuable tool for investigation of the molecular pathogenesis and new treatment approaches for post-surgical breakdown of the BRB.     

A study of the effects of angiotensins 1, 2, 3 and bradykinin on the replication of bovine retinal capillary endothelial cells and pericytes.

The renin-angiotensin system of the human eye may play a role in the regulation of retinal blood flow and the development of new vessels. We have investigated whether angiotensins 1, 2 and 3 or bradykinin, in concentrations ranging between 1 x 10(-12) and 1 x 10(-6), have any mitogenic activity on cultured bovine retinal endothelial cells (BREC) and pericytes (BRP). Cell replication was assessed by 3H-thymidine incorporation and, in the case of BREC and AT-2, also by cell counts. AT-2 was also tested on bovine aortic EC (BAEC). None of the substances elicited any response on BREC, BRP or BAEC. Whether angiotensin(s) induce angiogenesis in retinal vessels in vivo remains to be established but this does not appear to occur through direct stimulation of cell replication.     

糖基化终末产物对牛视网膜微血管内皮细胞和周细胞存活和形态的影响

目的 观察体外孵育的牛血清白蛋白(BSA)非酶促糖基化终末产物（AGEs）对牛视网膜微血管内皮细胞（BREC）和周细胞（BRP）存活和形态的影响。 方法 取终浓度为50mg/ml的牛血清白蛋白（BSA）、500 mmol/L D-葡萄糖，于37℃孵箱内避光孵育12周，制备外源性AGEs-BSA，经Sephacryl S-300层析纯化，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS PAGE）蛋白电泳鉴定AGEs-BSA和coomassie 蛋白质定量法测定蛋白浓度。分设不同浓度梯度的AGEs-BSA实验组和BSA对照组，以及空白对照组，分别观察体外孵育的AGEs-BSA对体外培养的BREC和BRP的毒性作用。相差倒置显微镜观察500μg/ml AGEs-BSA和BSA作用48 h对BREC和BRP形态的影响。 结果 随着AGEs-BSA剂量的增加，细胞被抑制呈上升趋势。500μg/ml AGEs-BSA抑制BREC为空白对照组的（72.8±15.9）%，抑制周细胞为空白对照组的（64.8±9.0）%。低浓度AGEs-BSA对BREC有一定促增生作用，但与空白对照组比较无统计学意义（P=0.231）。相差倒置显微镜观察结果显示AGEs-BSA处理组细胞增殖受抑制，失去正常细胞形态，而BSA对照组细胞同空白对照组，细胞形态正常。 结论 AGEs-BSA在高浓度时，无论是对BREC还是BRP，都产生生长抑制作用，从而导致BRP的丢失，损伤血管功能。进一步证实了非酶糖化是糖尿病微血管并发症的一个重要原因。 (中华眼底病杂志, 2006, 22: 11-15)     

选择性培养牛视网膜血管内皮细胞和周细胞

目的　建立选择性培养牛视网膜血管内皮细胞和周细胞的简要方法。方法　采用机械除杂法或等密度沉降分离法 ,培养皿用不同的基质成分包埋 ,培养液中加入有利于细胞生长的添加物。结果　原代培养的牛视网膜血管内皮细胞和周细胞纯净度 >95 % ,能连续传代。周细胞在传代后不规则形状及细胞内微丝更明显。视网膜血管内皮细胞融合后呈“铺路石”样改变。视网膜血管内皮细胞对Ⅷ因子相关抗体染色阳性。视网膜周细胞对单克隆抗体α 平滑肌肌动蛋白抗体染色阳性。结论　用明胶包被培养皿 ,在培养液中加入肝素、内皮细胞生长因子及高浓度的胎牛血清可获得较纯的内皮细胞。而周细胞最适合的培养液为DMEM加入 2 0 g·L-1胎牛血清。利用Percoll分离液也可分离培养出较纯的牛视网膜血管内皮细胞和周细胞     

TNF-α对培养视网膜微血管内皮细胞与周细胞PDGF-B/PDGFR-β表达的作用

目的:研究TNF-α对内皮细胞与周细胞PDGF-B/PDGFR-β表达的作用。方法:常规剂量100μg/L以及大剂量2450μg/LTNF-α作用于培养的视网膜微血管内皮细胞、周细胞,通过免疫组化、Western Blot、RT-PCR检测PDGF-B/PDGFR-β表达。结果:内皮细胞组大剂量TNF-α组,PDGF-B表达显著下调,8h表达开始出现下调,在12h,PDGF-B的表达量为原来的39%;在人视网膜微血管周细胞组,与对照组相比,常规TNF-α剂量组PDGFR-β表达并未出现显著改变;大剂量TNF-α组,与对照组相比,PDGFR-β表达在12h下调为45%。结论:TNF-α可引起内皮细胞与周细胞PDGF-B/PDGFR-β表达降低。