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Astrocytes (ACs) are the most abundant cells in the central nervous system. Retinal ACs play an important role in maintaining the integrity of retinal neurovascular function, and their dysfunction contributes to the pathogenesis of various eye diseases including diabetic retinopathy. Cytochrome P450 1B1 (CYP1B1) expression in the neurovascular structures of the central nervous system including ACs has been reported. We previously showed that CYP1B1 expression is a key regulator of redox homeostasis in retinal vascular cells. Its deficiency in mice resulted in increased oxidative stress and attenuation of angiogenesis in vivo and proangiogenic activity of retinal vascular cells in vitro. Here, using retinal ACs prepared from wild-type (Cyp1b1+/+) and Cyp1b1-deficient (Cyp1b1-/-) mice, we determined the impact of Cyp1b1 expression on retinal AC function. We showed that Cyp1b1-/- retinal ACs were more proliferative and migratory. These cells also produced increased amounts of fibronectin and its receptors, αvβ3- and α5β1- integrin. These results were consistent with the increased adhesive properties of Cyp1b1-/- ACs and their lack of ability to form a network in Matrigel. This was reversed by reexpression of Cyp1b1 in Cyp1b1-/- ACs. Although no significant changes were observed in Akt/SRC/MAPK signaling pathways, production of inflammatory mediators bone morphogenetic protein-7 (BMP-7) and monocyte chemoattractant protein-1 (MCP-1) was decreased in Cyp1b1-/- ACs. Cyp1b1-/- ACs also showed increased levels of connexin 43 phosphorylation and cluster of differentiation 38 expression when challenged with H2O2. These results are consistent with increased proliferation and diminished oxidative stress in Cyp1b1-/- cells. Thus, Cyp1b1 expression in ACs plays an important role in retinal neurovascular homeostasis. [ABSTRACT FROM AUTHOR]

Age-related macular degeneration (AMD) is the leading cause of vision loss among the elderly population, and is associated with severe macular degeneration and choroidal neovascularization (CNV). Although the pathogenesis of AMD is associated with choroidal dysfunction and CNV, the detailed underlying mechanisms remain unresolved. Altered production of pigment epithelium-derived factor (PEDF), a neuroprotective and antiangiogenic factor, contributes to CNV. Furthermore, exogenous PEDF mitigates angiogenesis in preclinical CNV models. How PEDF expression affects choroidal endothelial cell (ChEC) function is unknown. Here we isolated ChECs from PEDF+/+ and PEDF-deficient (PEDF−/−) mice and determined the impact of PEDF expression on the proangiogenic and pro-inflammatory properties of ChECs. We showed that PEDF expression significantly affects the proliferation, migration, adhesion, and oxidative and inflammatory state of ChECs. The PEDF−/− ChECs were, however, more sensitive to H2O2 challenge and exhibited increased rate of apoptosis and oxidative stress. We also observed a significant increase in production of cytokines with a primary role in inflammation and angiogenesis including vascular endothelial growth factor (VEGF) and osteopontin, and a reprograming of chemokines and cytokines expression profiles in PEDF−/− ChECs. Collectively, our results indicate that PEDF expression has a significant impact on oxidative and inflammatory properties of ChECs, whose alteration could contribute to pathogenesis of chronic inflammatory diseases including exudative AMD. [ABSTRACT FROM AUTHOR]

Pigment epithelium-derived factor (PEDF) is an endogenous inhibitor of angiogenesis. Although various ocular cell types including retinal endothelial cells (EC) produce PEDF, we know very little about cell autonomous effects of PEDF in these cell types. Here we determined how PEDF expression affects retinal EC proangiogenic properties. Retinal EC were prepared from wild-type (PEDF+/+) and PEDF-deficient (PEDF-/-) mice. The identity of EC was confirmed by staining for specific markers including vascular endothelial cadherin, CD31, and B4-lectin. Retinal EC also expressed VEGF receptor 1 and endoglin, as well as ICAM-1, ICAM-2, and VCAM-1. PEDF-/- retinal EC were more proliferative, less apoptotic when challenged with H2O2, less migratory, and less adherent compared with PEDF+/+ EC. These changes could be associated, at least in part, with increased levels of tenascin-C, fibronectin, thrombospondin-1 and collagen IV, and lower amounts of osteopontin. PEDF-/- EC also exhibited alterations in expression of a number of integrins including α2, αv, β1, β8, and αvβ3, and cell-cell adhesion molecules including CD31, zonula occluden-1, and occludin. These observations correlated with attenuation of capillary morphogenesis and increased levels of oxidative stress in PEDF-/- EC. PEDF-/- EC also produced lower levels of VEGF compared with PEDF+/+ cells. Thus, PEDF deficiency has a significant impact on retinal EC adhesion and migration, perhaps through altered production of extracellular matrix and junctional proteins in response to increased oxidative stress affecting their proangiogenic activity. [ABSTRACT FROM AUTHOR]

Defects in the outer blood-retinal barrier have significant impact on the pathogenesis of diabetic retinopathy and macular edema. However, the detailed mechanisms involved remain largely unknown. This is, in part, attributed to the lack of suitable animal and cell culture models, including those of mouse origin. We recently reported a method for the culture of retinal pigment epithelial (RPE) cells from wild-type and transgenic mice. The RPE cells are responsible for maintaining the integrity of the outer blood-retinal barrier whose dysfunction during diabetes has a significant impact on vision. Here we determined the impact of high glucose on the function of RPE cells. We showed that high glucose conditions resulted in enhanced migration and increased the level of oxidative stress in RPE cells, but minimally impacted their rate of proliferation and apoptosis. High glucose also minimally affected the cell-matrix and cell-cell interactions of RPE cells. However, the expression of integrins and extracellular matrix proteins including pigment epithelium-derived factor (PEDF) were altered under high glucose conditions. Incubation of RPE cells with the antioxidant N-acetylcysteine under high glucose conditions restored normal migration and PEDF expression. These cells also exhibited increased nuclear localization of the antioxidant transcription factor Nrf2 and ZO-1, reduced levels of β-catenin and phagocytic activity, and minimal effect on production of vascular endothelial growth factor, inflammatory cytokines, and Akt, MAPK, and Src signaling pathways. Thus high glucose conditions promote RPE cell migration through increased oxidative stress and expression of PEDF without a significant effect on the rate of proliferation and apoptosis. [ABSTRACT FROM AUTHOR]

Nuclear factor-κB (NF-κB) is a master regulator of genes that control a large number of cellular processes, including angiogenesis and inflammation. We recently demonstrated that cytochrome P-450 1B1 (Cyp1B1) deficiency in endothelial cells (EC) and pericytes (PC) results in increased oxidative stress, alterations in migration, attenuation of capillary morphogenesis, sustained activation of NF-κB, and increased expression of thrombospondin-2 (TSP2), an endogenous inhibitor of angiogenesis. On the basis of a growing body of evidence that phenethyl isothiocyanate (PEITC) and pyrrolidine dithiocarbamate (PDTC) function as antioxidants and suppressors of NF-κB activation, we investigated their potential ability to restore a normal phenotype in Cyp1B1-deficient (cyp1b1-/-) vascular cells. PEITC and PDTC inhibited NF-κB activity and expression in cyp1b1-/- EC and PC. We also observed restoration of migration and capillary morphogenesis of cyp1b1-/- EC and decreased cellular oxidative stress in cyp1b1-/- EC and PC without restoration to normal TSP2 levels. In addition, expression of a dominant-negative inhibitor κB, a suppressor of NF-κB activation, decreased NF-κB activity without affecting TSP2 expression in these cells. In contrast, knockdown of TSP2 expression resulted in attenuation of NF-κB activity in cyp1b1-/- vascular cells. Furthermore, expression of TSP2 in wild-type (cyp1b1+/+) cells resulted in increased NF-κB activity. Together, our results demonstrate an important role for TSP2 in modulation of NF-κB activity and attenuation of angiogenesis. Thus Cyp1B1 expression in vascular cells plays an important role in the regulation of vascular homeostasis through modulation of the cellular reductive state, TSP2 expression, and NF-κB activation. [ABSTRACT FROM AUTHOR]

Platelet endothehal cell adhesion molecule-1 (PECAM-l/CD3I) is a member of the immunoglobulin superfamily of cell adhesion molecules with important roles in angiogenesis and inflammation. However, the molecular and cellular mechanisms, and the role that specific PECAM-l isoforms play in these processes, remain elusive. We recently showed attenuation of retinal vascular development and neovascularization in PECAM-1-deficient (PECAM-1-/-) mice. To gain further insight into the role of PECAM- 1 in these processes, we isolated primary retinal endothelial cells (EC) from wild-type (PECAM-l+/+) and PECAM-l-/- mice. Lack of PECAM-1 had a significant impact on endothehial cell-cell and cell-matrix interactions, resulting in attenu- ation of cell migration and capillary morphogenesis. Mechanistically these changes were associated with a significant decrease in expres- sion of endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) bioavailability in PECAM-l-/- retinal EC. PECAM-l-/- retinal EC also exhibited a lower rate of apoptosis under basal and challenged conditions, consistent with their increased growth rate. Furthermore, reexpression of PECAM- 1 was sufficient to restore migration and capillary morphogenesis of null cells in an isoform-specific manner. Thus PECAM-l expression modulates proangiogenic properties of EC, and these activities are significantly influenced by alternative splicing of its cytoplasmic domain. [ABSTRACT FROM AUTHOR]

We have recently shown that deletion of constitutively expressed CYP1B1 is associated with attenuation of retinal endothelial cell (EC) capillaiy morphogenesis (CM) in vitro and angiogenesis in vivo. This was largely caused by increased intracellular oxidative stress and increased production of thrombospondin-2, an endogenous inhibitor of angiogenesis. Here, we demonstrate that endothelium nitric oxide synthase (eNOS) expression is dramatically decreased in the ECs prepared from retina, lung, heart, and aorta of CYP1B1-deficient (CYP1B1-/-) mice compared with wild-type (CYP1B1+/+) mice. The eNOS expression was also decreased in retinal vasculature of CYP1B1-/- mice. Inhibition of eNOS activity in cultured CYP1B1+/+ retinal ECs blocked CM and was concomitant with increased oxidative stress, like in CYP1B1+/+ retinal ECs. In addition, expression of eNOS in CYP1B1-/- retinal ECs or their incubation with a nitric oxide (NO) donor enhanced NO levels, lowered oxidative stress, and improved cell migration and CM. Inhibition of CYP1B1 activity in the CYP1Bl-/- retinal ECs resulted in reduced NO levels and attenuation of CM. In contrast, expression of CYPIB1 increased NO levels and enhanced CM of CYP1B1-/- retinal ECs. Furthermore, attenuation of CYP1B1 expression with small interfering RNA proportionally lowered eNOS expression and NO levels in wild-type cells. Together, our results link CYP1B1 metabolism in retinal ECs with sustained eNOS activity and NO synthesis and/or bioavailability and low oxidative stress and thrombospondin-2 expression. Thus CYPIBI and eNOS cooperate in different ways to lower oxidative stress and thereby to promote CM in vitro and angiogenesis in vivo. [ABSTRACT FROM AUTHOR]

Perivascular supporting cells, including vascular smooth muscle cells (VSMCs) and pericytes (PCs), provide instructive signals to adjacent endothelial cells helping to maintain vascular homeostasis. These signals are provided through direct contact and by the release of soluble factors by these cells. Thrombospondin (TSP)l is a matricellular protein and an autocrine factor for VSMCs. TSP1 activity, along with that of PDGF, regulates VSMC proliferation and migration. However, the manner in which TSPI and PDGF impact retinal PC function requires further investigation. In the present study, we describe, for the first time, the isolation and culture of retinal PCs from wild-type (TSP1+/+) and TSP1-deficient (TSP1-/-) immortomice. We showed that these cells express early and mature markers of PCs, including NG2, PDGF receptor-p, and smooth muscle actin as well as desmin, calbindin, and mesenchymal stem cell markers. These cells were successfully passaged and maintained in culture for several months without significant loss of expression of these markers. TSP1+/+ PCs proliferated at a faster rate compared with TSP1-/- PCs. In addition, TSP1+/+ PCs, like VSMCs, responded to PDGF-BB with enhanced migration and proliferation. In contrast, TSP1-/- PCs failed to respond to the promigratory and proliferative activity of PDGF-BB. This may be attributed, at least in part, to the limited interaction of PDGF-BB with TSP I in null cells, which is essential for PDGF proliferative and migratory action. We observed no significant differences in the rates of apoptosis in these cells. TSP1-/- PCs were also less adherent, expressed increased levels of TSP2 and fibronectin, and had decreased amounts of N-cadherin and αv/β3-integrin on their surface. Thus, TSP1 plays a significant role in retinal PC proliferation and migration impacting retinal vascular development and homeostasis. [ABSTRACT FROM AUTHOR]

Apoptosis plays a critical role during development and in the maintenance of the vascular system. B-cell leukemia lymphoma 2 (bcl-2) protects endothelial cells (EC) from apoptosis in response to a variety of stimuli. Previous work from this laboratory demonstrated attenuation of postnatal retinal vascular development and retinal neovascularization during oxygen-induced ischemic retinopathy in bcl-2-deficient (bcl-2-/-) mice. To gain further insight into the function of bcl-2 in the endothelium, we isolated retinal EC from bcl-2-/- and bcl-2-/- mice. Retinal EC lacking bcl-2 demonstrated reduced cell migration, tenascin-C expression, and adhesion to vitronectin and fibronectin. The bc1-2-/- retinal EC also failed to undergo capillary morphogenesis in Matrigel. In addition, using an ex vivo angiogenesis assay, we observed reduced sprouting from aortic rings grown in culture from bcl-2-/- mice compared with bcl-2+/+ mice. Furthermore, reexpression of bcl-2-/- was sufficient to restore migration and capillary morphogenesis defects observed in bcl-2-/- retinal EC. Mechanistically, bcl-2-/- cells expressed significantly less endothelial nitric oxide synthase, an important downstream effecter of proangiogenic signaling. This may be attributed to increased oxidative stress in the absence of bcl-2-/-. In fact, incubation of retinal EC or aortic rings from bcl-2-/- mice with the antioxidant N-acetylcysteine rescued their capillary morphogenesis and sprouting defects. Thus, bcl-2-mediated cellular functions play important roles not only in survival but also in proangiogenic phenotype of EC with a significant impact on vascular development and angiogenesis. [ABSTRACT FROM AUTHOR]