Retinal Vascular Regeneration

We discuss the potential use of stem cells for therapeutic angiogenesis in the treatment of retinal diseases. We demonstrate that the clinical utility of these EPC may be not limited in the treatment of ischemic retinal diseases but may also have application for the treatment of retinal degenerative disorders and for a form of cell-based gene therapy. One of the greatest potential benefits of bone marrow derived EPC therapy is the possible use of autologous grafts. Nonetheless, potential toxicities and unregulated cell growth will need to be carefully evaluated before this approach is brought to the clinics.     

Retinal vascular regeneration

We discuss the potential use of stem cells for therapeutic angiogenesis in the treatment of retinal diseases. We demonstrate that the clinical utility of these EPC may be not limited in the treatment of ischemic retinal diseases but may also have application for the treatment of retinal degenerative disorders and for a form of cell-based gene therapy. One of the greatest potential benefits of bone marrow derived EPC therapy is the possible use of autologous grafts. Nonetheless, potential toxicities and unregulated cell growth will need to be carefully evaluated before this approach is brought to the clinics.     

HIF-2alpha-haploinsufficient mice have blunted retinal neovascularization due to impaired expression of a proangiogenic gene battery

PURPOSE: To characterize the effect of HIF-2alpha haploinsufficiency on retinal neovascularization and angiogenic signaling in neonatal mice. METHODS: Retinal samples were obtained from HIF-2alpha-haploinsufficient (Epas1+/-) and wild-type (Epas1+/+) neonatal mice subjected to an oxygen-induced retinopathy (OIR) protocol. Histologic and molecular studies were performed immediately, 12 hours, or 5 days after initiation of the hypoxia phase of the OIR protocol. Molecular profiling was performed in mouse brain endothelial cells maintained in normoxia or hypoxia. Transfection studies assessed the response of isolated promoter regions from proangiogenic genes to HIF-1alpha or -2alpha overexpression. RESULTS: Epas1+/- mice exhibited no significant differences in retinal vasculature during normal development but had reduced retinal neovascularization in an OIR protocol. Multiple proangiogenic factors were induced during the hypoxia phase in Epas1+/+ OIR retinal samples, whereas Epas1+/- OIR retinal samples had absent or blunted induction of these same factors. Several, but not all, proangiogenic factors were induced in mouse brain endothelial cells after hypoxia. In transfection assays, most proangiogenic promoter regions were preferentially activated by HIF-2alpha relative to HIF-1alpha. CONCLUSIONS: HIF-2alpha deficiency results in reduced neovascularization and blunted inducibility of multiple proangiogenic factors in the retinas of mice with OIR. The authors propose that HIF-2alpha is a master regulator of proangiogenic factors in retinal vascular endothelial cells, the predominant cell type of the retina in which HIF-2alpha is expressed. Future studies will address whether the molecular and functional roles for HIF-2alpha identified from these studies can be generalized to other pathophysiological states involving neovascularization.     

细胞骨架相关蛋白2(CKAP2)在db/db小鼠视网膜组织中的表达及其与HIF-1α/VEGF信号通路的关系

目的 观察细胞骨架相关蛋白2(CKAP2)在db/db小鼠视网膜组织中的表达及分布特点,分析其与缺氧诱导因子1α(HIF-1α)/血管内皮生长因子(VEGF)信号通路的关系.方法 选取雄性db/db糖尿病小鼠(db/db组)及同窝正常野生型wt/wt小鼠(wt/wt组)各15只.采用HE染色观察两组小鼠视网膜结构变化,...     

Impacts of hypoxia-inducible factor-1 knockout in the retinal pigment epithelium on choroidal neovascularization

Purpose. Hypoxia-inducible factor (HIF)-1 is a key oxygen sensor and is believed to play an important role in neovascularization (NV). The purpose of this study is to determine the role of retinal pigment epithelium (RPE)-derived HIF-1α on ocular NV. Methods. Conditional HIF-1α knockout (KO) mice were generated by crossing transgenic mice expressing Cre in the RPE with HIF-1α floxed mice, confirmed by immunohistochemistry, Western blot analysis, and fundus fluorescein angiography. The mice were used for the oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularization (CNV) models. Results. HIF-1α levels were significantly decreased in the RPE layer of ocular sections and in primary RPE cells from the HIF-1α KO mice. Under normal conditions, the HIF-1α KO mice exhibited no apparent abnormalities in retinal histology or visual function as shown by light microscopy and electroretinogram recording, respectively. The HIF-1α KO mice with OIR showed no significant difference from the wild-type (WT) mice in retinal levels of HIF-1α and VEGF as well as in the number of preretinal neovascular cells. In the laser-induced CNV model, however, the disruption of HIF-1α in the RPE attenuated the over expression of VEGF and the intercellular adhesion molecule 1 (ICAM-1), and reduced vascular leakage and CNV area. Conclusions. RPE-derived HIF-1α plays a key role in CNV, but not in ischemia-induced retinal NV.     

缺氧诱导因子-1α、诱导型一氧化氮合成酶及环氧合酶-2在慢性高眼压大鼠视网膜中的表达

目的探讨缺氧诱导因子-1α（HIF-1α）、诱导型一氧化氮合成酶（iNOS）、环氧合酶-2（COX-2）的表达与高眼压视网膜损伤的关系,为青光眼视网膜缺氧提供证据。方法用巩膜静脉烧烙法在50只SPF级Wistar大鼠的双眼制作慢性高眼压模型,眼压高于造模前40%以上者为造模成功。按照视网膜取材时间的不同将动物模型眼随机分为高眼压3、7、14、21、28 d组,每组10只大鼠20只眼。另选取10只匹配大鼠作为假手术对照组,仅作球结膜切开。应用逆转录-聚合酶链反应（RT-PCR）法及Western blot法检测各组视网膜组织中HIF-1α、iNOS、COX-2 mRNA及其蛋白在大鼠视网膜中的表达。结果 HIF-1α、iNOS、COX-2 mRNA及蛋白在假手术组大鼠视网膜中呈微弱表达,造模眼HIF-1α、iNOS、COX-2 mRNA及其蛋白在视网膜中的表达水平明显升高,于7 d时达到高峰,随后有所下降,但仍明显高于正常组的表达水平。造模后3、7、14、21、28 d组大鼠视网膜中HIF-1α、iNOS、COX-2 mRNA及其蛋白的表达与假手术组相比,差异均有统计学意义（P〈0.05）。结论 HIF-1α、iNOS、COX-2的低氧信号途径是青光眼神经变性发生发展中重要的病理生理机制。     

早期糖尿病NOD小鼠视网膜HIF-1的表达

目的:探讨糖尿病NOD小鼠视网膜HIF-1α的表达。方法:NOD小鼠随机分为正常对照组和糖尿病组,分别于糖尿病2、4、6、8和12周处死正常对照组和糖尿病组小鼠,摘除眼球后分离视网膜。Westernblot方法检测视网膜HIF-1α的表达,免疫组化方法检测视网膜上HIF-1α的表达。结果:与正常对照组相比,糖尿病组小鼠血糖明显增高,体重明显下降(P<0.01);Westernblot显示糖尿病6周时视网膜HIF-1α低水平表达,12周时HIF-1α表达增高;免疫组化显示糖尿病组12周时视网膜内层HIF-1α表达增加,而未见视网膜外层HIF-1α表达。结论:糖尿病早期视网膜HIF-1α表达增高,说明在糖尿病早期视网膜发生缺血缺氧;HIF-1α可能参与了糖尿病早期视网膜缺血缺氧的继发损害过程。     

Biology and therapeutic potential of adult retinal stem cells

Retinal degeneration encompasses a constellation of common pathologies for which there is no regenerative treatment. Vision loss has a devastating impact on quality of life and activities of daily living. Pharmacologic treatments serve to stave off disease progression but do not represent a restorative approach. Cellular transplantation is considered to be a promising approach for future therapy for retinal degeneration. There are, however, significant barriers that must be overcome if cell transplantation is to become a clinical reality. In this review, we focus on the need for a cellular replacement therapy for retinal disease and the promise of stem cells as candidate cellular therapeutics. In particular, we discuss the origins of stem cells in the retina, the discovery and characterization of retinal stem cells isolated from adult humans, and their transplantation potential and clinical implications.     

Beyond intraocular pressure: neuroprotective strategies for future glaucoma therapy.

BACKGROUND: All currently approved glaucoma medications are directed toward lowering intraocular pressure. However, it is apparent that there are pressure-independent mechanisms associated with the development of glaucomatous optic neuropathy. There has been considerable effort to develop therapeutics that rescue the retinal ganglion cells from undergoing secondary degeneration after the original insult has occurred. This therapeutic strategy has been termed neuroprotection. METHODS: The literature was reviewed to examine the current knowledge of the degenerative cascade involved in glaucomatous damage, with emphasis on potential therapeutic targets for neuroprotective strategies. RESULTS: There are a number of promising areas of research for new glaucoma therapies including glutamate antagonists, calcium channel blockers, antioxidants, nitric oxide synthase inhibitors, neurotrophins, and anti-apoptotic agents. CONCLUSIONS: Glaucoma is a complex disease with a number of risk factors and mechanisms leading to ganglion cell death. Future glaucoma therapy will likely include neuroprotectants that could be used as an adjunct therapy with other medications designed to lessen the initial insult (i.e., intraocular pressure-lowering compounds). As the word neuroprotection becomes more popular, care must be taken in evaluating the research literature for clinically effective therapies.     

Subretinal transplantation of bone marrow mesenchymal stem cells delays retinal degeneration in the RCS rat model of retinal degeneration

Because there is no effective treatment for this retinal degeneration, potential application of cell-based therapy has attracted considerable attention. Several investigations support that bone marrow mesenchymal stem cells (MSCs) can be used for a broad spectrum of indications. Bone marrow MSCs exert their therapeutic effect in part by secreting trophic factors to promote cell survival. The current study investigates whether bone marrow MSCs secrete factor(s) to promote photoreceptor cell survival and whether subretinal transplantation of bone marrow MSCs promotes photoreceptor survival in a retinal degeneration model using Royal College of Surgeons (RCS) rats. In vitro, using mouse retinal cell culture, it was demonstrated that the conditioned medium of the MSCs delays photoreceptor cell apoptosis, suggesting that the secreted factor(s) from the MSCs promote photoreceptor cell survival. In vivo, the MSCs were injected into the subretinal space of the RCS rats and histological analysis, real-time RT-PCR and electrophysiological analysis demonstrated that the subretinal transplantation of MSCs delays retinal degeneration and preserves retinal function in the RCS rats. These results suggest that MSC is a useful cell source for cell-replacement therapy for some forms of retinal degeneration.     

大鼠视网膜缺血再灌注后HIF-1α的表达与视网膜细胞凋亡的研究

目的:探讨大鼠视网膜细胞缺血再灌注后低氧诱导因子(HIF-1α)的表达、视网膜细胞凋亡的发生以及二者之间的关系.方法:采用前房灌注生理盐水升高大鼠眼压到110mmHg(1kPa=7.5mmHg)持续1 h的方法制作实验性视网膜缺血再灌注损伤模型,分别于再灌注不同时间点取材,行免疫组织化学法染色观察HIF-1α在视网膜细胞的阳性表达;采用DNA原位末端标记(terminal dUTP nick end labelling,TUNEL)法定位凋亡的视网膜细胞来检测视网膜凋亡细胞.结果:缺血再灌注2h组视网膜神经节细胞层及内核层细胞出现HIF-1α弱阳性表达,12h组阳性表达至高峰,24h组HIF-1α表达渐减少.视网膜组织细胞凋亡见于缺血再灌注12,24及48h组,凋亡细胞主要位于内核层,且24h组凋亡阳性表达最强.结论:缺血再灌注后大鼠视网膜HIF-1α的表达增加.参与视网膜缺血再灌注损伤;视网膜细胞的损伤部分以凋亡的形式发生,HIF-1α的表达可能与视网膜细胞凋亡有密切的关系.     

缺氧时视网膜血管内皮细胞缺氧诱导因子-1α及其mRNA的时序性表达

目的探讨在缺氧状态下视网膜血管内皮细胞缺氧诱导因子-1α(HIF-1α)及其mRNA表达的时空规律.方法对分离的牛视网膜血管内皮细胞分别进行常规和CoCl2模拟缺氧培养,采用免疫组化和逆转录多聚酶链反应(RT-PCR)检测不同缺氧时间HIF-1α及其mRNA的表达.结果正常对照组HIF-1α有极低表达,在缺氧1h时表达量显著上升,4h达到高峰,16h后下降.与对照组比较,各缺氧组HIF-1α的表达均有显著性差异(P＜0.01).但各缺氧组HIF-1αmRNA的表达无明显差异.结论视网膜血管内皮细胞在缺氧早期HIF-1 α表达有短暂、迅速的增加,但其mRNA的表达无明显变化,提示缺氧可以促进视网膜血管内皮细胞HIF-1α的表达,但不是在基因的转录水平.     

Ritonavir inhibits HIF-1α-mediated VEGF expression in retinal pigment epithelial cells in vitro

Purpose

Retinal hypoxia-mediated activation of the hypoxia-inducible factor (HIF pathway) leading to angiogenesis is a major signaling mechanism underlying a number of sight-threatening diseases. Inhibiting this signaling mechanism with an already approved therapeutic molecule may have promising anti-angiogenic role with fewer side effects. Hence, the primary objective of this study was to examine the expression of HIF-1α and VEGF in human retinal pigment epithelial cells treated with ritonavir under hypoxic and normoxic conditions.

Methods

ARPE-19 and D407 cells were cultured in normoxic or hypoxic conditions, alone or in the presence of ritonavir. Quantitative real-time polymerase chain reaction, immunoblot analysis, sandwich ELISA, endothelial cell proliferation, and cytotoxicity were performed.

Results

A 12-h hypoxic exposure resulted in elevated mRNA expression levels of both HIF-1α and VEGF in ARPE-19 and D407 cells. Hence, this time point was selected for subsequent experiments. Presence of ritonavir in the culture medium strongly inhibited VEGF expression in a concentration-dependent manner under hypoxic conditions. Immunoblot analysis demonstrated a substantially reduced protein expression of HIF-1α in the presence of ritonavir. Further, hypoxic exposure-induced VEGF secretion was also inhibited by ritonavir, as demonstrated using ELISA. Finally, ritonavir significantly diminished the proliferation of choroid-retinal endothelial (RF/6A) cells demonstrating potential anti-angiogenic activity. Cytotoxicity studies showed that ritonavir is non-toxic to RPE cells.

Conclusions

This study demonstrates for the first time that ritonavir can inhibit HIF-1α and VEGF in ARPE-19 and D407 cells. Such inhibition may form a platform for application of ritonavir in the treatment of various ocular diseases.     

缺氧诱导因子-1α与血管内皮生长因子在视网膜新生血管中的表达及相关性

目的:检测视网膜新生血管中缺氧诱导因子-1α(HIF-1α)的表达,研究其与血管内皮生长因子(VEGF)表达的相关性。方法:以高浓度氧诱导C57BL/6J小鼠建立视网膜新生血管模型,取对照组和给氧组幼鼠眼球作荧光素血管灌注,病理切片及免疫组织化学检测,分别观察视网膜血管的改变,视网膜新生血管内皮细胞数目及HIF-1α,VEGF蛋白的表达。结果:给氧组视网膜可见大量新生血管形成,新生血管内皮细胞核数为(23.38±1.07)个,与对照组相比差异有极显著性(P<0.01)。HIF-1α蛋白表达在神经节细胞层和突破视网膜内界膜的新生血管。VEGF蛋白表达在内核层,神经节细胞层和突破视网膜内界膜的新生血管。两者表达呈显著正相关(r=0.931,P<0.01)。结论:在视网膜新生血管形成中存在HIF-1α,VEGF的高表达,且两者表达密切相关。     

Gentherapie bei Netzhautdystrophien

Genetic mutations are the cause of inherited retinal dystrophies. The underlying genetic basis of these diseases suggests that a gene therapy approach is logical either to replace or reduce the expression of defective genes. The first proof-of-concept clinical studies in patients with Leber's congenital amaurosis have suggested that retinal gene therapy is safe and potentially effective, at least for specific disease entities. In contrast to pharmacological treatment gene therapy has the advantage of being able to express a protein within specific cell populations and is a potentially definitive therapy. Besides replacing deficient genes in inherited diseases, additional strategies that might broaden the application of retinal gene therapy are also being developed. These include the permanent expression of neuroprotective substances or photosensitive molecules (so-called optogenetics). This overview discusses the current clinical strategies and potential problems of retinal gene therapy.     

Embryonic stem cells: potential source for ocular repair

Many ocular diseases, such as retinitis pigmentosa and age-related macular degeneration, reflect damage to specific cells that are not normally repaired or replaced. Preliminary results of animal studies suggest that these degenerative diseases may be treatable by transplantation of healthy fetal cells. However, obtaining a sufficient number of suitable donor cells remains a problem. The isolation of human embryonic stem (ES) cells has drawn much attention because of their potential clinical application for patients with these degenerative diseases. Because ES cells have the potential to generate all adult cell types, ocular diseases resulting from the failure of specific cell types would be potentially treatable through the transplantation of differentiated cells derived from ES cells. In addition, because ES cells can proliferate indefinitely in their undifferentiated state, they are expected to alleviate the problem of the shortage of donor cells for cell-replacement therapy. Recently, reproducible and efficient differentiation methods for the generation of lens cells, retinal neurons, and retinal pigment epithelial (RPE) cells from ES cells have been developed. This review focuses especially on these ocular cells differentiated from ES cells. We will also discuss the potential therapeutic uses of ES cells for the treatment of ocular diseases.