Nader Sheibani

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.

High glucose promotes retinal endothelial cell migration through activation of Src, PI3K/Akt1/eNOS, and ERKs

Hyperglycemia impacts retinal vascular function and promotes the development and progression of diabetic retinopathy, which ultimately results in growth of new blood vessels and loss of vision. How high glucose affects retinal endothelial cell (EC) properties requires further investigation. Here we determined the impact of high glucose on mouse retinal EC function in vitro. High glucose significantly enhanced the migration of retinal EC without impacting their proliferation, apoptosis, adhesion, and capillary morphogenesis. The enhanced migration of retinal EC under high glucose was reversed in the presence of the antioxidant N-acetylcysteine, suggesting increased oxidative stress under high-glucose conditions. Retinal EC under high-glucose conditions also expressed increased levels of fibronectin, osteopontin, and alpha(v)beta(3)-integrin, and reduced levels of thrombospondin-1. These changes were concomitant with sustained activation of the downstream prosurvival and promigratory signaling pathways, including Src kinase, phosphatidylinositol 3-kinase/Akt1/endothelial nitric oxide synthase, and ERKs. The sustained activation of these signaling pathways was essential for enhanced migration of retinal EC under high-glucose conditions. Together, our results indicate the exposure of retinal EC to high glucose promotes a promigratory phenotype. Thus alterations in the proangiogenic properties of retinal EC during diabetes may contribute to the development and pathogenesis of diabetic retinopathy.

CYP1B1 expression promotes the proangiogenic phenotype of endothelium through decreased intracellular oxidative stress and thrombospondin-2 expression

Reactive species derived from cell oxygenation processes play an important role in vascular homeostasis and the pathogenesis of many diseases including retinopathy of prematurity. We show that CYP1B1-deficient (CYP1B1(-/-)) mice fail to elicit a neovascular response during oxygen-induced ischemic retinopathy. In addition, the retinal endothelial cells (ECs) prepared from CYP1B1(-/-) mice are less adherent, less migratory, and fail to undergo capillary morphogenesis. These aberrant cellular responses were completely reversed when oxygen levels were lowered or an antioxidant added. CYP1B1(-/-) ECs exhibited increased oxidative stress and expressed increased amounts of the antiangiogenic factor thrombospondin-2 (TSP2). Increased lipid peroxidation and TSP2 were both observed in retinas from CYP1B1(-/-) mice and were reversed by administration of an antioxidant. Reexpression of CYP1B1 in CYP1B1(-/-) ECs resulted in down-regulation of TSP2 expression and restoration of capillary morphogenesis. A TSP2 knockdown in CYP1B1(-/-) ECs also restored capillary morphogenesis. Thus, CYP1B1 metabolizes cell products that modulate intracellular oxidative stress, which enhances production of TSP2, an inhibitor of EC migration and capillary morphogenesis. Evidence is presented that similar changes occur in retinal endothelium in vivo to limit neovascularization.

Lipopolysaccharide-induced blood-brain barrier disruption: roles of cyclooxygenase, oxidative stress, neuroinflammation, and elements of the neurovascular unit

BackgroundDisruption of the blood-brain barrier (BBB) occurs in many diseases and is often mediated by inflammatory and neuroimmune mechanisms. Inflammation is well established as a cause of BBB disruption, but many mechanistic questions remain.MethodsWe used lipopolysaccharide (LPS) to induce inflammation and BBB disruption in mice. BBB disruption was measured using 14C-sucrose and radioactively labeled albumin. Brain cytokine responses were measured using multiplex technology and dependence on cyclooxygenase (COX) and oxidative stress determined by treatments with indomethacin and N-acetylcysteine. Astrocyte and microglia/macrophage responses were measured using brain immunohistochemistry. In vitro studies used Transwell cultures of primary brain endothelial cells co- or tri-cultured with astrocytes and pericytes to measure effects of LPS on transendothelial electrical resistance (TEER), cellular distribution of tight junction proteins, and permeability to 14C-sucrose and radioactive albumin.ResultsIn comparison to LPS-induced weight loss, the BBB was relatively resistant to LPS-induced disruption. Disruption occurred only with the highest dose of LPS and was most evident in the frontal cortex, thalamus, pons-medulla, and cerebellum with no disruption in the hypothalamus. The in vitro and in vivo patterns of LPS-induced disruption as measured with 14C-sucrose, radioactive albumin, and TEER suggested involvement of both paracellular and transcytotic pathways. Disruption as measured with albumin and 14C-sucrose, but not TEER, was blocked by indomethacin. N-acetylcysteine did not affect disruption. In vivo, the measures of neuroinflammation induced by LPS were mainly not reversed by indomethacin. In vitro, the effects on LPS and indomethacin were not altered when brain endothelial cells (BECs) were cultured with astrocytes or pericytes.ConclusionsThe BBB is relatively resistant to LPS-induced disruption with some brain regions more vulnerable than others. LPS-induced disruption appears is to be dependent on COX but not on oxidative stress. Based on in vivo and in vitro measures of neuroinflammation, it appears that astrocytes, microglia/macrophages, and pericytes play little role in the LPS-mediated disruption of the BBB.

Increased Expression and Activity of 12-Lipoxygenase in Oxygen-Induced Ischemic Retinopathy and Proliferative Diabetic Retinopathy: Implications in Retinal Neovascularization

OBJECTIVE Arachidonic acid is metabolized by 12-lipoxygenase (12-LOX) to 12-hydroxyeicosatetraenoic acid (12-HETE) and has an important role in the regulation of angiogenesis and endothelial cell proliferation and migration. The goal of this study was to investigate whether 12-LOX plays a role in retinal neovascularization (NV). RESEARCH DESIGN AND METHODS Experiments were performed using retinas from a murine model of oxygen-induced ischemic retinopathy (OIR) that was treated with and without the LOX pathway inhibitor, baicalein, or lacking 12-LOX. We also analyzed vitreous samples from patients with and without proliferative diabetic retinopathy (PDR). Western blotting and RT-PCR were used to assess the expression of 12-LOX, vascular endothelial growth factor (VEGF), and pigment epithelium–derived factor (PEDF). Liquid chromatography–mass spectrometry was used to assess the amounts of HETEs in the murine retina and human vitreous samples. The effects of 12-HETE on VEGF and PEDF expression were evaluated in Müller cells (rMCs), primary mouse retinal pigment epithelial cells, and astrocytes. RESULTS Retinal NV during OIR was associated with increased 12-LOX expression and 12-, 15-, and 5-HETE production. The amounts of HETEs also were significantly higher in the vitreous of diabetic patients with PDR. Retinal NV was markedly abrogated in mice treated with baicalein or mice lacking 12-LOX. This was associated with decreased VEGF expression and restoration of PEDF levels. PEDF expression was reduced in 12-HETE–treated rMCs, astrocytes, and the retinal pigment epithelium. Only rMCs and astrocytes showed increased VEGF expression by 12-HETE. CONCLUSIONS 12-LOX and its product HETE are important regulators of retinal NV through modulation of VEGF and PEDF expression and could provide a new therapeutic target to prevent and treat ischemic retinopathy.

Pericytes Regulate Vascular Basement Membrane Remodeling and Govern Neutrophil Extravasation during Inflammation

During inflammation polymorphonuclear neutrophils (PMNs) traverse venular walls, composed of the endothelium, pericyte sheath and vascular basement membrane. Compared to PMN transendothelial migration, little is known about how PMNs penetrate the latter barriers. Using mouse models and intravital microscopy, we show that migrating PMNs expand and use the low expression regions (LERs) of matrix proteins in the vascular basement membrane (BM) for their transmigration. Importantly, we demonstrate that this remodeling of LERs is accompanied by the opening of gaps between pericytes, a response that depends on PMN engagement with pericytes. Exploring how PMNs modulate pericyte behavior, we discovered that direct PMN-pericyte contacts induce relaxation rather than contraction of pericyte cytoskeletons, an unexpected response that is mediated by inhibition of the RhoA/ROCK signaling pathway in pericytes. Taking our in vitro results back into mouse models, we present evidence that pericyte relaxation contributes to the opening of the gaps between pericytes and to the enlargement of the LERs in the vascular BM, facilitating PMN extravasation. Our study demonstrates that pericytes can regulate PMN extravasation by controlling the size of pericyte gaps and thickness of LERs in venular walls. This raises the possibility that pericytes may be targeted in therapies aimed at regulating inflammation.

Attenuation of proliferation and migration of retinal pericytes in the absence of thrombospondin-1

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)1 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 TSP1 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-beta, 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 TSP1 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 alpha(v)beta(3)-integrin on their surface. Thus, TSP1 plays a significant role in retinal PC proliferation and migration impacting retinal vascular development and homeostasis.

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.

A combined method to quantify the retinal metabolic rate of oxygen using photoacoustic ophthalmoscopy and optical coherence tomography

Quantitatively determining physiological parameters at a microscopic level in the retina furthers the understanding of the molecular pathways of blinding diseases, such as diabetic retinopathy and glaucoma. An essential parameter, which has yet to be quantified noninvasively, is the retinal oxygen metabolic rate (rMRO2). Quantifying rMRO2 is challenging because two parameters, the blood flow rate and hemoglobin oxygen saturation (sO2), must be measured together. We combined photoacoustic ophthalmoscopy (PAOM) with spectral domain-optical coherence tomography (SD-OCT) to tackle this challenge, in which PAOM measured the sO2 and SD-OCT mapped the blood flow rate. We tested the integrated system on normal wild-type rats, in which the measured rMRO2 was 297.86 ± 70.23 nl/minute. This quantitative method may shed new light on both fundamental research and clinical care in ophthalmology in the future.