Pericyte-derived sphingosine 1-phosphate induces the expression of adhesion proteins and modulates the retinal endothelial cell barrier.

OBJECTIVE: The mechanisms that regulate the physical interaction of pericytes and endothelial cells and the effects of these interactions on interendothelial cell junctions are not well understood. We determined the extent to which vascular pericytes could regulate pericyte-endothelial adhesion and the consequences that this disruption might have on the function of the endothelial barrier. METHODS AND RESULTS: Human retinal microvascular endothelial cells were cocultured with pericytes, and the effect on the monolayer resistance of endothelial cells and expression of the cell junction molecules N-cadherin and VE-cadherin were measured. The molecules responsible for the effect of pericytes or pericyte-conditioned media on the endothelial resistance and cell junction molecules were further analyzed. Our results indicate that pericytes increase the barrier properties of endothelial cell monolayers. This barrier function is maintained through the secretion of pericyte-derived sphingosine 1-phosphate. Sphingosine 1-phosphate aids in maintenance of microvascular stability by upregulating the expression of N-cadherin and VE-cadherin, and downregulating the expression of angiopoietin 2. CONCLUSIONS: Under normal circumstances, the retinal vascular pericytes maintain pericyte-endothelial contacts and vascular barrier function through the secretion of sphingosine 1-phosphate. Alteration of pericyte-derived sphingosine 1-phosphate production may be an important mechanism in the development of diseases characterized by vascular dysfunction and increased permeability.

A peptide inhibitor of the urokinase/urokinase receptor system inhibits alteration of the blood-retinal barrier in diabetes.

One of the major complications of diabetes is the alteration of the blood-retinal barrier, leading to retinal edema and consequent vision loss. The aim of this study was to evaluate the role of the urokinase plasminogen activator (uPA)/uPA receptor (uPAR) system in the regulation of retinal vascular permeability. Biochemical, molecular, and histological techniques were used to examine the role of uPA and uPAR in the regulation of retinal vascular permeability in diabetic rats and cultured retinal endothelial cells. The increased retinal vascular permeability in diabetic rats was associated with a decrease in vascular endothelial (VE) -cadherin expression in retinal vessels. Treatment with the uPA/uPAR-inhibiting peptide (A6) was shown to reduce diabetes-induced permeability and the loss of VE-cadherin. The increased permeability of cultured cells in response to advanced glycation end products (AGEs) was significantly inhibited with A6. Treatment of endothelial cells with specific matrix metalloproteinases or AGEs resulted in loss of VE-cadherin from the cell surface, which could be inhibited by A6. uPA/uPAR physically interacts with AGEs/receptor for advanced glycation end products on the cell surface and regulates its activity. uPA and its receptor uPAR play important roles in the alteration of the blood-retinal barrier through proteolytic degradation of VE-cadherin. The ability of A6 to block retinal vascular permeability in diabetes suggests a potential therapeutic approach for the treatment of diabetic macular edema.

A potential role for angiopoietin 2 in the regulation of the blood-retinal barrier in diabetic retinopathy.

PURPOSE: Although VEGF has been identified as an important mediator of the blood-retinal barrier alteration in diabetic retinopathy, the hypothesis for this study was that that other molecules, including the angiopoietins (Ang-1 and -2), may play a role. The expression of angiopoietins was analyzed in an animal model of diabetic retinopathy, and the role of Ang-2 in the regulation of diabetes-induced alterations of vascular permeability was characterized. METHODS: Diabetes was induced in rats, and human retinal endothelial cells (HRECs) were grown in media with 5.5 or 30.5 mM glucose. Levels of Ang-1 and -2 mRNA and protein were analyzed. Fluorescence-based assays were used to assess the effect of Ang-2 on vascular permeability in vivo and in vitro. The effect of Ang-2 on VE-cadherin function was assessed by measuring the extent of tyrosine phosphorylation. RESULTS: Ang-2 mRNA and protein increased in the retinal tissues after 8 weeks of diabetes and in high-glucose-treated cells. Intravitreal injection of Ang-2 in rats produced a significant increase in retinal vascular permeability. Ang-2 increased HREC monolayer permeability that was associated with a decrease in VE-cadherin and a change in monolayer morphology. High glucose and Ang-2 produced a significant increase in VE-cadherin phosphorylation. CONCLUSIONS; Ang-2 is upregulated in the retina in an animal model of diabetes, and hyperglycemia induces the expression of Ang-2 in isolated retinal endothelial cells. Increased Ang-2 alters VE-cadherin function, leading to increased vascular permeability. Thus, Ang-2 may play an important role in increased vasopermeability in diabetic retinopathy.

Pattern recognition receptor gene expression in ischemia-induced flap revascularization.

BACKGROUND: The innate immune system is the major contributor to acute inflammation induced by microbial infection or tissue damage. Germline-encoded pattern recognition receptors (PRRs) are responsible for sensing the presence of micro-organisms and endogenous molecules released from damaged cells. We performed microarray analyses on ischemic wound tissue to investigate the temporal relationship between PRR gene expression, wound perfusion, and flap revascularization. METHODS: A cranial-based, peninsular-shaped myocutaneous flap was surgically created on the dorsum of C57BL6 mice (n = 25 total; n = 20 with flap). Laser speckle contrast imaging was utilized to study the pattern of flap ischemia and return of functional revascularization. Flap microvascular density was determined by image analysis of CD-31-immunostained sections. Total RNA was isolated from homogenized flap tissue and was converted to cDNA (RT), which was hybridized to a microarray of pathway-focused genes. Microarray results were validated with quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). RESULTS: Laser speckle contrast imaging predicted the spatial and temporal pattern of ischemia and functional revascularization. Histologic analysis demonstrated early leukocyte infiltration and later engraftment, resulting in flap revascularization by new blood vessel growth from the recipient bed and dilatation of preexisting proximal flap vasculature. qRT-PCR demonstrated significant early gene expression of select PRRs, cytokines, chemokines, and growth factors, peaking by 48 hours, and returning toward baseline but remaining elevated at 10 days. CONCLUSION: Surgical and ischemic tissue injury resulted in the early gene expression of select PRRs, which may bind with endogenous molecules released from ischemic or necrotic cells, leading to transcription of genes involved in wound inflammation and angiogenesis.

Plasminogen activator inhibitor-1 (PAI-1) facilitates retinal angiogenesis in a model of oxygen-induced retinopathy.

PURPOSE: Angiogenesis, or the formation of new retinal blood vessels, is a key feature of many proliferative retinal diseases including diabetic retinopathy, retinal vein occlusion, and retinopathy of prematurity. The aim of the present study was to investigate the role of the serine proteinase inhibitor plasminogen activator inhibitor -1 (PAI-1) in facilitating retinal angiogenesis. METHODS: The temporal expression of PAI-1 was examined by real-time PCR, Western blot analysis, and immunohistochemistry in retinal tissues from mice with oxygen-induced retinopathy. The requirement for PAI-1 in facilitating the retinal angiogenic response in this model was examined by quantitating the angiogenic response with wild-type and PAI-1 null mice. The mechanism by which PAI-1 mediates angiogenesis was further investigated with isolated human retinal vascular endothelial cells. RESULTS: PAI-1 expression was upregulated in the retinas of mice with oxygen-induced retinopathy, which coincided with a significant increase in the expression of vitronectin in the retina of the experimental mice. There was significant reduction in the angiogenic response of PAI-1(-/-) mice compared with wild-type mice. PAI-1 promotes endothelial cell migration in vitro and facilitates the migration of cells on a vitronectin substrate by regulating alpha v integrin cell surface expression. CONCLUSIONS: These observations suggest a role for PAI-1 during retinal angiogenesis and point to a potential new therapeutic target in the prevention or treatment of retinal neovascularization seen in many ocular diseases.

Suppression of retinal neovascularization with an antagonist to vascular endothelial cadherin.

OBJECTIVES: To examine the role of vascular endothelial cadherin (VE-cadherin) in cellular processes underlying angiogenesis and the effects of VE-cadherin inhibition on retinal angiogenesis. METHODS: Retinal neovascularization was induced in newborn mice by exposure to 75% oxygen (postnatal days 7-12) followed by room air and quantitated from histological sections. Mice received daily intraperitoneal injections of either a VE-cadherin antagonist or a control peptide from postnatal days 12 to 17. In vitro cell migration, proliferation, and tubule formation were examined in the presence of the VE-cadherin antagonist. The effect of antagonist treatment on the integrity of established cell junctions was examined by fluorescein isothiocyanate-dextran monolayer permeability and VE-cadherin immunocytochemistry. RESULTS: Treatment with the VE-cadherin antagonist significantly reduced retinal angiogenesis. Inhibition of VE-cadherin function suppressed tubule formation in endothelial cells. The antagonist treatment also decreased cell migration and proliferation. The antagonist treatment did not affect the integrity of existing cell junctions. Immunostaining for VE-cadherin and rates of monolayer permeability were comparable to those in untreated controls. CONCLUSION: Our study points to a pivotal role played by VE-cadherin in the angiogenic process. CLINICAL RELEVANCE: Inhibition of VE-cadherin might be an effective strategy for pharmacological inhibition in proliferative retinopathies.