Shoujian Wang

Thrombospondin-1-Deficient Mice Exhibit Increased Vascular Density During Retinal Vascular Development and Are Less Sensitive to Hyperoxia-Mediated Vessel Obliteration

Thrombospondin‐1 (TSP1) is a natural inhibitor of angiogenesis. Its expression is most prominent during the late stages of vascular development and in the adult vasculature. Our previous studies have shown that TSP1 expression promotes a quiescent, differentiated phenotype of vascular endothelial cells. However, the physiological role TSP1 plays during vascular development and neovascularization requires further delineation. Here, we investigated the role of TSP1 during development of retinal vasculature and retinal neovascularization during oxygen‐induced ischemic retinopathy. The retinal vascular density was increased in TSP1‐deficient (TSP1‐/‐) mice compared with wild‐type mice. This finding was mainly attributed to increased number of retinal endothelial cells in TSP1‐/‐ mice. During oxygen‐induced ischemic retinopathy, the developing retinal vasculature of TSP1‐/‐ mice was less sensitive to vessel obliteration induced by hyperoxia but exhibited a similar level of neovascularization induced by normoxia compared with wild‐type mice. This finding is consistent with the similar pattern of VEGF expression detected in wild‐type and TSP1‐/‐ mice. Furthermore, the increased expression of TSP1 during development of retinal vasculature was not affected by oxygen‐induced ischemic retinopathy. In addition, the regression of ocular embryonic (hyaloid) vessels, as well as the newly formed retinal vessels during oxygen‐induced ischemic retinopathy, was delayed in TSP1‐/‐ mice. Therefore, TSP1 is a modulator of vascular homeostasis and its expression is essential for appropriate remodeling and maturation of retinal vasculature. Developmental Dynamics 228:630–642, 2003.

PEDF-deficient mice exhibit an enhanced rate of retinal vascular expansion and are more sensitive to hyperoxia-mediated vessel obliteration

Pigment epithelium derived factor (PEDF) is an endogenous inhibitor of angiogenesis. However, its physiological role during vascular development and neovascularization remains elusive. Here we investigated the role of PEDF in normal postnatal vascularization of retina and retinal neovascularization during oxygen-induced ischemic retinopathy (OIR) using PEDF-deficient (PEDF-/-) mice. The beta-galactosidase staining of eye sections from PEDF-/- mice confirmed the expression pattern of endogenous PEDF previously reported in mouse retina. However, strongest staining was observed in the retinal outer plexiform layer. Retinal trypsin digests indicated that the ratio of endothelial cells (EC) to pericytes (PC) was significantly higher in PEDF-/- mice compared to wild type (PEDF+/+) mice at postnatal day 21 (P21). This was mainly attributed to increased numbers of EC in the absence of PEDF. There was no significant difference in the number of PC. We observed an increased rate of proliferation in retinal vasculature of PEDF-/- mice, which was somewhat compensated for by an increase in the rate of apoptosis. Staining of the retinal wholemounts and eye frozen sections indicated postnatal retinal vascularization expansion occurred at a faster rate in the absence of PEDF, and was more prominent at early time points (prior to P21). The retinal vascularization in PEDF+/+ mice reaches that of PEDF-/- mice such that no significant difference in vascular densities was observed by P42. Lack of PEDF had a minimal effect on the regression of hyaloid vasculature and VEGF levels. PEDF-/- mice also exhibited enhanced sensitivity to hyperoxia-mediated vessel obliteration during OIR compared to PEDF+/+ mice despite higher levels of VEGF. However, there was no significant difference in the degree of retinal neovascularization. Our studies indicate that PEDF is an important modulator of early postnatal retinal vascularization and in its absence retinal vascularization proceeds at a faster rate and is more susceptible to hyperoxia-mediated vessel obliteration.

Attenuation of Retinal Vascular Development and Neovascularization in PECAM-1 Deficient Mice

Platelet-endothelial cell adhesion molecule-1 (PECAM-1/CD31) is expressed on the surface of endothelial cells (EC) at high levels with important roles in angiogenesis and inflammation. However, the physiological role PECAM-1 plays during vascular development and angiogenesis remains largely unknown. Here we determined the role of PECAM-1 in the postnatal development of retinal vasculature and retinal neovascularization during oxygen-induced ischemic retinopathy (OIR) using PECAM-1-deficient (PECAM-1-/-) mice. A significant decrease in retinal vascular density was observed in PECAM-1-/- mice compared with PECAM-1+/+ mice. This was attributed to a decreased number of EC in the retinas of PECAM-1-/- mice. An increase in the rate of apoptosis was observed in retinal vessels of PECAM-1-/- mice, which was compensated, in part, by an increase in the rate of proliferation. However, the development and regression of hyaloid vasculature were not affected in the absence of PECAM-1. We did not observe a significant defect in astrocytes, the number of endothelial tip cell filopodias, and the rate of developing retinal vasculature progression in PECAM-1-/- mice. However, we observed aberrant organization of arterioles and venules, decreased secondary branching, and dilated vessels in retinal vasculature of PECAM-1-/- mice. In addition, retinal neovascularization was attenuated in PECAM-1-/- mice during OIR despite an expression of VEGF similar to that of PECAM-1+/+ mice. Mechanistically, these changes were associated with an increase in EphB4 and ephrin B2, and a decrease in eNOS, expression in retinal vasculature of PECAM-1-/- mice. These results suggest that PECAM-1 expression and its potential interactions with EphB4/ephrin B2 and eNOS are important for survival, migration, and functional organization of EC during retinal vascular development and angiogenesis.

Regulation of angiogenesis by histone chaperone HIRA-mediated incorporation of lysine 56-acetylated histone H3.3 at chromatin domains of endothelial genes.

Angiogenesis is critically dependent on endothelial cell-specific transcriptional mechanisms. However, the molecular processes that regulate chromatin domains and thereby dictate transcription of key endothelial genes are poorly understood. Here, we report that, in endothelial cells, angiogenic signal-mediated transcriptional induction of Vegfr1 (vascular endothelial growth factor receptor 1) is dependent on the histone chaperone, HIRA (histone cell cycle regulation-defective homolog A). Our molecular analyses revealed that, in response to angiogenic signals, HIRA is induced in endothelial cells and mediates incorporation of lysine 56 acetylated histone H3.3 (H3acK56) at the chromatin domain of Vegfr1. HIRA-mediated incorporation of H3acK56 is a general mechanism associated with transcriptional induction of several angiogenic genes in endothelial cells. Depletion of HIRA inhibits H3acK56 incorporation and transcriptional induction of Vegfr1 and other angiogenic genes. Our functional analyses revealed that depletion of HIRA abrogates endothelial network formation on Matrigel and inhibits angiogenesis in an in vivo Matrigel plug assay. Furthermore, analysis in a laser-induced choroidal neovascularization model showed that depletion of HIRA significantly inhibits neovascularization. Our results for the first time decipher a histone chaperone (HIRA)-dependent molecular mechanism in endothelial gene regulation and indicate that histone chaperones could be new targets for angiogenesis therapy.

Attenuation of retinal vascular development and neovascularization in transgenic mice over‐expressing thrombospondin‐1 in the lens

Thrombospondin‐1 (TSP1) is an endogenous inhibitor of angiogenesis and induces endothelial cell (EC) apoptosis. To study the role TSP1 plays during vascular development and neovascularization, we assessed the effects of ectopic TSP1 expression in the lens on retinal vascularization in transgenic mice. The TSP1 over‐expressing mice showed abnormalities in the development of retinal vasculature. There was a dramatic decrease in the density of superficial and deep vascular plexuses of the retina in transgenic mice. The retinal vessels in TSP1 transgenic mice also appeared nonuniform and abnormal in maturation. We detected an increase in the number of EC undergoing apoptosis, which was compensated, in part, by an increase in cell proliferation in retinal vasculature of TSP1 transgenic mice. The TSP1 transgenic mice also exhibited increased levels of vessel obliteration and a limited preretinal neovascularization during oxygen‐induced ischemic retinopathy (OIR). Our results indicate increased expression of TSP1 attenuates normal retinal vascularization and preretinal neovascularization during OIR. Therefore, modulation of TSP1 expression may provide an effective mechanism for regulation of ocular angiogenesis. Developmental Dynamics 235:1908–1920, 2006.

Endoglin regulates the activation and quiescence of endothelium by participating in canonical and non-canonical TGF-β signaling pathways

Summary Endoglin (Eng) is an auxiliary receptor for transforming growth factor-&bgr; (TGF&bgr;), with important roles in vascular function. TGF&bgr; regulates angiogenesis through balancing the pro-proliferative and pro-differentiation signaling pathways of endothelial cells (EC). However, the contribution of endoglin to these TGF&bgr; activities, and more specifically modulation of EC phenotype, remains elusive. Mutations in endoglin cause hereditary hemorrhagic telangiectasia-1 in humans. The Eng+/− mice are viable and exhibit some of the vascular defects seen in humans with endoglin haploinsufficiency. In the present study we show that haploinsufficiency of endoglin results in attenuation of retinal neovascularization during oxygen-induced ischemic retinopathy. Although the importance of endoglin expression in angiogenesis and vascular development has been demonstrated, the underlying mechanisms remain obscure. To gain detailed insight into the cell autonomous regulatory mechanisms that affect angiogenic properties of EC, we prepared retinal EC from Eng+/+ and Eng+/− Immorto mice. The Eng+/− EC were more adherent, less migratory, and failed to undergo capillary morphogenesis. Aortic sprouting angiogenesis was similarly attenuated in aortas from Eng+/− mice. In addition, Eng+/− EC expressed increased levels of VEGF but reduced expression of endothelial NO synthase and NO production. Mechanistically, these changes were consistent with sustained activation of mitogen-activated protein kinase (MAPK) pathways, and aberrant Smad-dependent signaling pathways in Eng+/− EC. Taken together, our results underscore the importance of endoglin in both canonical and non-canonical TGF&bgr; signaling pathways modulating both the activation and quiescence of the endothelium during angiogenesis.

Lack of Thrombospondin 1 and Exacerbation of Choroidal Neovascularization

OBJECTIVES To assess the impact of thrombospondin 1(TSP1) deficiency on choroidal neovascularization (CNV)and to determine whether administration of a TSP1 antiangiogenic mimetic peptide attenuates CNV. METHODS The impact of TSP1 deficiency on laser induced CNV was assessed using wild-type (TSP1 +/+) and TSP1-deficient (TSP1 −/−) mice. Three laser burns were placed in each eye of TSP1 +/+ and TSP1 −/− mice to induce CNV. Intravitreal injection of the TSP1 mimetic peptide was performed on days 1 and 7 postlaser in the mice.For quantitative measurements of neovascularization, intercellular adhesion molecule 2 staining was performed at 14 days postlaser of the choroidal-sclera flat mounts. The recruitment of macrophages to the sites of damage was investigated by immunohistochemistry. The CNV area was measured by intercellular adhesion molecule 2 staining and use of ImageJ software. RESULTS The TSP1 −/− mice exhibited significantly larger areas of neovascularization on choroidal flat mounts compared with TSP1 +/ mice. This was consistent with enhanced recruitment of macrophages in TSP1 −/− mice compared with TSP1 +/+ mice 3 days postlaser. The development of CNV was significantly attenuated in mice receiving the TSP1 antiangiogenic mimetic peptide compared with those receiving vehicle alone. CONCLUSIONS Deficiency of TSP1 contributes to enhanced choroidal neovascularization. This is consistent with the anti-inflammatory and antiangiogenic activity of TSP1. The TSP1 antiangiogenic peptide was effective in attenuation of CNV. CLINICAL RELEVANCE Intravitreal injection of TSP1 antiangiogenic mimetic peptides may provide alternative treatment for CNV.

Modulation of Thrombospondin 1 and Pigment Epithelium-Derived Factor Levels in Vitreous Fluid of Patients With Diabetes

OBJECTIVE To determine the levels of 2 endogenous inhibitors of angiogenesis, thrombospondin 1 (TSP-1) and pigment epithelium-derived factor (PEDF), in the vitreous fluid from patients with and without diabetes. METHODS The levels of TSP-1 and PEDF in vitreous samples from diabetic and age-matched nondiabetic patients were determined by Western blot analysis. RESULTS We observed significant amounts of TSP-1 and PEDF in the vitreous samples of control eyes. The TSP-1 levels varied in samples from patients with diabetes. In contrast, PEDF levels showed little or no change in vitreous samples from patients with or without diabetes. However, the PEDF protein exhibited variation in its molecular weight among the samples. We consistently observed lower levels of TSP-1 in diabetic patients who expressed the higher-molecular-weight PEDF isoform. CONCLUSIONS In diabetes, changes in the TSP-1 level may play a role in shifting the angiogenic balance and contributing to the pathogenesis of diabetic retinopathy. Although the PEDF level did not change, the diabetic samples with the higher-molecular-weight PEDF isoform consistently showed lower levels of TSP-1. CLINICAL RELEVANCE The presence of the higher-molecular-weight PEDF isoform may be associated with greater risk of severe diabetic retinopathy.

Bim is Responsible for the Inherent Sensitivity of the Developing Retinal Vasculature to Hyperoxia

Apoptosis plays an important role in development and remodeling of vasculature during organogenesis. Coordinated branching and remodeling of the retinal vascular tree is essential for normal retinal function. Bcl-2 family members, such as bim not only influence apoptosis, but also cell adhesive and migratory properties essential during vascular development. Here we examined the impact of bim deficiency on postnatal retinal vascularization, as well as retinal neovascularization during oxygen-induced ischemic retinopathy (OIR) and laser-induced choroidal neovascularization. Loss of bim expression was associated with increased retinal vascular density in mature animals. This was mainly attributed to increased numbers of pericytes and endothelial cells. However, the initial spread of the superficial layer of retinal vasculature and, the appearance and density of the tip cells were similar in bim+/+ and bim-/- mice. In addition, hyaloid vessel regression was attenuated in the absence of bim. Furthermore, in the absence of bim retinal vessel obliteration and neovascularization did not occur during OIR. Instead, normal inner retinal vascularization proceeded independent of changes in oxygen levels. In contrast, choroidal neovascularization occurred equally well in bim+/+ and bim-/- mice. Together our data suggest bim expression may be responsible for the inherent sensitivity of the developing retinal vasculature to changes in oxygen levels, and promotes vessel obliteration in response to hyperoxia.

Development of choroidal neovascularization in rats with advanced intense cyclic light-induced retinal degeneration.

OBJECTIVES To study the progressive changes of intense cyclic light-induced retinal degeneration and to determine whether it results in choroidal neovascularization (CNV). METHODS Albino rats were exposed to 12 hours of 3000-lux cyclic light for 1, 3, or 6 months. Fundus examination, fundus photography, fluorescein and indocyanine green angiography, and optical coherence tomography were performed prior to euthanization. Light-exposed animals were euthanized after 1, 3, or 6 months for histopathological evaluation. Retinas were examined for the presence of 4-hydroxy-2-nonenal- and nitrotyrosine-modified proteins by immunofluorescence staining. RESULTS Long-term intense cyclic light exposure resulted in retinal degeneration with loss of the outer segments of photoreceptors and approximately two-thirds of the outer nuclear layer as well as development of subretinal pigment epithelium neovascularization after 1 month. Almost the entire outer nuclear layer was absent with the presence of CNV, which penetrated the Bruch membrane and extended into the outer retina after 3 months. Absence of the outer nuclear layer, multiple foci of CNV, retinal pigment epithelial fibrous metaplasia, and connective tissue bands containing blood vessels extending into the retina were observed after 6 months. All intense light-exposed animals showed an increased presence of 4-hydroxy-2-nonenal and nitrotyrosine staining. Optical coherence tomographic and angiographic studies confirmed retinal thinning and leakiness of the newly formed blood vessels. CONCLUSIONS Our results suggest that albino rats develop progressive stages of retinal degeneration and CNV after long-term intense cyclic light exposure, allowing the detailed study of the pathogenesis and treatment of age-related macular degeneration. CLINICAL RELEVANCE The ability to study the progressive pathogenesis of age-related macular degeneration and CNV will provide detailed knowledge about the disease and aid in the development of target-specific therapy.