Pericyte Dropout Research Articles

Characterizing placental pericytes: Hypoxia and proangiogenic signalling

IntroductionPericytes wrap microvessels and interact with endothelial cells to regulate vascular growth. Though pericyte dropout has been reported in pathological human placentae and mouse models of placental pathology, there has been limited investigation of the role and function of placental pericytes in vascular health and pathology. This study aimed to investigate the angiogenic potential of human placental pericytes relative to other villous cell populations. MethodsPrimary human placental pericytes, human umbilical vein endothelial cells (HUVEC), and BeWo cells ( ± 20 μM forskolin) were cultured in 1 % O2 or ambient air, followed by analysis of secreted angiogenic factors (ELISA). Additionally, the placental pericytes and HUVECs were co-cultured in a 3D sprouting assay to assess the capacity of pericytes to contribute to vascular sprouts. Results1 % O2 affected secretion of angiogenic factors in placental pericytes, HUVECs, and syncytialized BeWo cells. Specifically, in placental pericytes, angiopoietin-1 (ANG1) and soluble fms-like tyrosine kinase-1 (sFLT1) were decreased, while vascular endothelial growth factor (VEGF) was increased. In HUVECS, matrix metalloproteinase-2 (MMP2), VEGF, angiopoietin-2 (ANG2), platelet-derived growth factor beta (PDGFB), placental growth factor (PlGF), and sFLT1 were increased. In syncytialized BeWo cells, VEGF, MMP2, PDGFB, PlGF, and sFLT1 secretion were increased. Placental pericytes and HUVECS colocalized to vessel sprouts in the 3-D sprouting assay. DiscussionHypoxic conditions altered placental pericyte, endothelial, and syncytialized BeWo secretion of angiogenic factors. We speculate that pericyte dropout and, by extension, the loss of pericyte-derived angiogenic factors in hypoxic conditions may contribute to compromised fetal vascular development observed in placental pathologies.

Modeling early pathophysiological phenotypes of diabetic retinopathy in a human inner blood-retinal barrier-on-a-chip

Diabetic retinopathy (DR) is a microvascular disorder characterized by inner blood-retinal barrier (iBRB) breakdown and irreversible vision loss. While the symptoms of DR are known, disease mechanisms including basement membrane thickening, pericyte dropout and capillary damage remain poorly understood and interventions to repair diseased iBRB microvascular networks have not been developed. In addition, current approaches using animal models and in vitro systems lack translatability and predictivity to finding new target pathways. Here, we develop a diabetic iBRB-on-a-chip that produces pathophysiological phenotypes and disease pathways in vitro that are representative of clinical diagnoses. We show that diabetic stimulation of the iBRB-on-a-chip mirrors DR features, including pericyte loss, vascular regression, ghost vessels, and production of pro-inflammatory factors. We also report transcriptomic data from diabetic iBRB microvascular networks that may reveal drug targets, and examine pericyte-endothelial cell stabilizing strategies. In summary, our model recapitulates key features of disease, and may inform future therapies for DR.

Blockade of Annexin A2 Prevents Early Microvasculopathy in Murine Models of Diabetic Retinopathy.

We examined the role of annexin A2 (A2) in the development of diabetic retinal vasculopathy by testing the effect of Anxa2 gene deletion as well as administration of anti-A2 antibodies on pericyte dropout and retinal neovascularization in diabetic Akita mice, and in mice subjected to oxygen-induced retinopathy. We analyzed diabetic Ins2AKITA mice with or without global deletion of Anxa2, as well as Ins2AKITA mice that received intravitreal anti-A2 IgG or control antibody at 2, 4, and 6 months, for retinal pericyte dropout at 7 months of age. In addition, we assessed the effect of intravitreal anti-A2 on oxygen-induced retinopathy (OIR) in neonatal mice by quantifying retinal neovascular and vaso-obliterative area, and by enumeration of neovascular tufts. Both deletion of the Anxa2 gene and immunologic blockade of A2 prevented pericyte depletion in retinas of diabetic Ins2AKITA mice. Blockade of A2 also reduced vaso-obliteration and neovascularization in the OIR model of vascular proliferation. This effect was amplified when a combination of antivascular endothelial growth factor (VEGF) and anti-A2 antibodies was used. Therapeutic approaches that target A2, alone or in combination with anti-VEGF therapy, are effective in mice, and may also curtail the progression of retinal vascular disease in humans with diabetes.

The rs2442598 Polymorphism in ANGPT-2 Gene Is Associated With Risk for Diabetic Retinopathy in Patients With Type 1 Diabetes Mellitus From a Brazilian Population

Introduction: Angiopoietin-2 (ANGPT-2) is a member of a growth factor family that binds to the endothelial receptor tyrosine kinase 2 and regulates vascular development and function. Under hyperglycemic conditions, ANGPT-2 is upregulated and leads to vascular destabilization, pericyte dropout, and aggravation of inflammation. In this context, studies have reported the involvement of ANGPT-2 in the pathogenesis of diabetic retinopathy (DR). Thus, polymorphisms in ANGPT-2 gene may be of interest in this field. Objective: To investigate the association between the rs2442598 polymorphism in the ANGPT-2 gene and DR. Methods: This case-control study comprised 107 patients with type 1 diabetes mellitus (T1DM) and DR (cases) and 129 patients with T1DM without DR (controls) and with ≥10 years of DM. The ANGPT-2 rs2442598 (G/A) polymorphism was genotyped by real-time PCR using TaqMan MGB probes. Results: Genotype distributions of this polymorphism were consistent with Hardy-Weinberg Equilibrium. Frequency of the rs2442598 A allele was higher in cases compared to controls (P= 0.011). Moreover, the A/A genotype was higher in cases than controls (P= 0.017) and was associated with risk for DR after adjustment for duration of DM, HbA1c, triglycerides, estimated glomerular filtration rate, and presence of hypertension (OR= 5.19, IC95% 1.21 - 22.27). This association was maintained under the recessive (OR= 4.78, IC95% 1.14 - 19.99) and additive (OR= 6.861, IC95% 1.45 - 32.38) inheritance models. Conclusion: Our data demonstrate, for the first time, the association of the ANGPT-2 rs2442598 A allele with risk for DR in T1DM patients from Southern Brazil.

Pro-angiogenic Activity Discriminates Human Adipose-Derived Stromal Cells From Retinal Pericytes: Considerations for Cell-Based Therapy of Diabetic Retinopathy.

Diabetic retinopathy (DR) is a frequent diabetes-associated complication. Pericyte dropout can cause increased vascular permeability and contribute to vascular occlusion. Adipose-derived stromal cells (ASC) have been suggested to replace pericytes and restore microvascular support as potential therapy of DR. In models of DR, ASC not only generated a cytoprotective and reparative environment by the secretion of trophic factors but also engrafted and integrated into the retina in a pericyte-like fashion. The aim of this study was to compare the pro-angiogenic features of human ASC and human retinal microvascular pericytes (HRMVPC) in vitro. The proliferation and the expression of ASC and HRMVPC markers were compared. Adhesion to high glucose-conditioned endothelial extracellular matrix, mimicking the diabetic microenvironment, was measured. The angiogenesis-promoting features of both cell types and their conditioned media on human retinal endothelial cells (EC) were assessed. To identify a molecular basis for the observed differences, gene expression profiling was performed using whole-genome microarrays, and data were validated using PCR arrays and flow cytometry. Based on multiplex cytokine results, functional studies on selected growth factors were performed to assess their role in angiogenic support. Despite a distinct heterogeneity in ASC and HRMVPC cultures with an overlap of expressed markers, ASC differed functionally from HRMVPC. Most importantly, the pro-angiogenic activity was solely featured by ASC, whereas HRMVPC actively suppressed vascular network formation. HRMVPC, in contrast to ASC, showed impaired adhesion and proliferation on the high glucose-conditioned endothelial extracellular matrix. These data were supported by gene expression profiles with differentially expressed genes. The vessel-stabilizing factors were more highly expressed in HRMVPC, and the angiogenesis-promoting factors were more highly expressed in ASC. The vascular endothelial growth factor receptor-2 inhibition efficiently abolished the ASC angiogenic supportive capacities, whereas the addition of angiopoietin-1 and angiopoietin-2 did not alter these effects. Our results clearly show that ASC are pro-angiogenic, whereas HRMVPC are marked by anti-angiogenic/EC-stabilizing features. These data support ASC as pericyte replacement in DR but also suggest a careful risk-to-benefit analysis to take full advantage of the ASC therapeutic features.

Can adipose stromal cells replace retinal pericytes in diabetic retinopathy? In vitro comparison of pro-angiogenic capacities of human ASC and retinal pericytes

Background & Aim Pericyte dropout is a significant contribution to the microvascular complications leading to diabetic retinopathy (DR). The similarities with pericytes render adipose stromal cells (ASC) candidates to prevent vessel destabilization and control neoangiogenesis. In models of DR, ASC not only generate a cytoprotective and reparative environment by secretion of trophic factors acting on endothelial, neuronal and glial cells, but also engraft and integrate into the retina in a pericyte-like fashion. Concerns, however, are that AC may not withstand the hyperglycemic stress and may worsen DR by secretion of proangiogenic factors. Thus, we compared the angiogenic features of human ASC and retinal pericytes. Methods, Results & Conclusion We compared growth curves and marker expression of both cells. As angiogeneic features, we first assessed adhesion to endothelial extracellular matrix, modified by high-glucose. Next, we tested pro-angiogeneic properties of both cell types and their conditioned media on human retinal endothelial cells by means of vascular network formation. Further, we used whole genome microarrays to compare gene expression profiles. Based on multiplex cytokine results, inhibitors of certain cytokines used to assess their role in the angiogenic support. Although cells were not to be distinguished based on their phenotype, the pro-angiogenic activity was solely featured by ASC, whereas pericytes suppressed vascular network formation. Pericytes, in contrast to ASC, adhered and proliferated at reduced rates on high-glucose modified endothelial extracellular matrix. Differentially expressed genes clustered to cell adhesion and cell migration. Pericytes expressed predominantly genes related to vessel stabilisation, whereas ASC showed higher expression of genes related to angiogenesis. This was linked to cytokine secretion. In fact, VEGFR-2 was required for ASC angiogenic supportive capacities, whereas Ang-1 appeared not to be required for pericytes inhibitory action. Our results clearly document that the pro-angiogeneic activity discriminates ASC from retinal pericytes. Pericytes are marked by endothelial stabilizing properties whereas ASC rather show angiogenic supportive characteristics. These striking pro-angiogenic discrepancies prompts to investigate the ASC angiogeneic and potential pericyte replacement properties in more detail. Pericyte dropout is a significant contribution to the microvascular complications leading to diabetic retinopathy (DR). The similarities with pericytes render adipose stromal cells (ASC) candidates to prevent vessel destabilization and control neoangiogenesis. In models of DR, ASC not only generate a cytoprotective and reparative environment by secretion of trophic factors acting on endothelial, neuronal and glial cells, but also engraft and integrate into the retina in a pericyte-like fashion. Concerns, however, are that AC may not withstand the hyperglycemic stress and may worsen DR by secretion of proangiogenic factors. Thus, we compared the angiogenic features of human ASC and retinal pericytes. We compared growth curves and marker expression of both cells. As angiogeneic features, we first assessed adhesion to endothelial extracellular matrix, modified by high-glucose. Next, we tested pro-angiogeneic properties of both cell types and their conditioned media on human retinal endothelial cells by means of vascular network formation. Further, we used whole genome microarrays to compare gene expression profiles. Based on multiplex cytokine results, inhibitors of certain cytokines used to assess their role in the angiogenic support.

Pericyte Bridges in Homeostasis and Hyperglycemia.

Diabetic retinopathy is a potentially blinding eye disease that threatens the vision of one-ninth of patients with diabetes. Progression of the disease has long been attributed to an initial dropout of pericytes that enwrap the retinal microvasculature. Revealed through retinal vascular digests, a subsequent increase in basement membrane bridges was also observed. Using cell-specific markers, we demonstrate that pericytes rather than endothelial cells colocalize with these bridges. We show that the density of bridges transiently increases with elevation of Ang-2, PDGF-BB, and blood glucose; is rapidly reversed on a timescale of days; and is often associated with a pericyte cell body located off vessel. Cell-specific knockout of KLF4 in pericytes fully replicates this phenotype. In vivo imaging of limbal vessels demonstrates pericyte migration off vessel, with rapid pericyte filopodial-like process formation between adjacent vessels. Accounting for off-vessel and on-vessel pericytes, we observed no pericyte loss relative to nondiabetic control retina. These findings reveal the possibility that pericyte perturbations in location and process formation may play a role in the development of pathological vascular remodeling in diabetic retinopathy.

TRPC proteins contribute to development of diabetic retinopathy and regulate glyoxalase 1 activity and methylglyoxal accumulation

ObjectiveDiabetic retinopathy (DR) is induced by an accumulation of reactive metabolites such as ROS, RNS, and RCS species, which were reported to modulate the activity of cation channels of the TRPC family. In this study, we use Trpc1/4/5/6−/− compound knockout mice to analyze the contribution of these TRPC proteins to diabetic retinopathy. MethodsWe used Nanostring- and qPCR-based analysis to determine mRNA levels of TRPC channels in control and diabetic retinae and retinal cell types. Chronic hyperglycemia was induced by Streptozotocin (STZ) treatment. To assess the development of diabetic retinopathy, vasoregression, pericyte loss, and thickness of individual retinal layers were analyzed. Plasma and cellular methylglyoxal (MG) levels, as well as Glyoxalase 1 (GLO1) enzyme activity and protein expression, were measured in WT and Trpc1/4/5/6−/− cells or tissues. MG-evoked toxicity in cells of both genotypes was compared by MTT assay. ResultsWe find that Trpc1/4/5/6−/− mice are protected from hyperglycemia-evoked vasoregression determined by the formation of acellular capillaries and pericyte drop-out. In addition, Trpc1/4/5/6−/− mice are resistant to the STZ-induced reduction in retinal layer thickness. The RCS metabolite methylglyoxal, which represents a key mediator for the development of diabetic retinopathy, was significantly reduced in plasma and red blood cells (RBCs) of STZ-treated Trpc1/4/5/6−/− mice compared to controls. GLO1 is the major MG detoxifying enzyme, and its activity and protein expression were significantly elevated in Trpc1/4/5/6-deficient cells, which led to significantly increased resistance to MG toxicity. GLO1 activity was also increased in retinal extracts from Trpc1/4/5/6−/− mice. The TRPCs investigated here are expressed at different levels in endothelial and glial cells of the retina. ConclusionThe protective phenotype in diabetic retinopathy observed in Trpc1/4/5/6−/− mice is suggestive of a predominant action of TRPCs in Müller cells and microglia because of their central position in the retention of a proper homoeostasis of the neurovascular unit.

TGFβ induces BIGH3 expression and human retinal pericyte apoptosis: a novel pathway of diabetic retinopathy.

PurposeOne of the earliest hallmarks of diabetic retinopathy is the loss of retinal pericytes. However, the mechanisms that promote pericyte dropout are unknown. In the present study, we propose a novel pathway in which pericyte apoptosis is mediated by macrophages, TGFβ and pro-apoptotic BIGH3 (TGFβ-induced Gene Human Clone 3) protein.Patients and methodsTo elucidate this pathway, we assayed human retinal pericyte (HRP) apoptosis by TUNEL assay, BIGH3 mRNA expression by qPCR, and BIGH3 protein expression by western blot analysis. HRP were treated with BIGH3 protein, TGFβ1 and TGFβ2 and inhibition assays were carried out by blocking with antibodies against BIGH3. The distribution of BIGH3 and CD68+ macrophages were compared in a post-mortem donor eye with 7-year history of Type II diabetes and histopathogically confirmed non-proliferative diabetic retinopathy (NPDR).ResultsTGFβ induced a significant increase in BIGH3 mRNA and protein expression, and HRP apoptosis. BIGH3 treatment showed HRP undergo apoptosis in a dose-dependent manner. At 5 μg/ml, BIGH3 induced 3.5-times more apoptosis in HRP than in retinal endothelial cells. TGFβ induced apoptosis was inhibited by blocking with antibodies against BIGH3. In an example of NPDR, BIGH3 accumulated within the walls of the inner retina arterioles. Macrophage infiltrates were frequently associated with these vessels and the inner nuclear layer.ConclusionTogether with our previously published results on macrophage-induced retinal endothelial cell apoptosis, the present study supports a novel inflammatory pathway mediated by macrophages and the BIGH3 protein leading to HRP apoptosis. As shown in human post-mortem globes, these observations are clinically relevant, suggesting a new mechanism underlying pericyte dropout during NPDR.

Stem cell therapies in the treatment of diabetic retinopathy and keratopathy.

Nonproliferative diabetic retinopathy (DR) is characterized by multiple degenerative changes that could be potentially corrected by stem cell therapies. Most studies so far have attempted to alleviate typical abnormalities of early retinopathy, including vascular hyperpermeability, capillary closure and pericyte dropout. Success was reported with adult stem cells (vascular progenitors or adipose stem cells), as well as induced pluripotent stem cells from cord blood. The cells were able to associate with damaged vessels in both pericyte and endothelial lining positions in models of DR and ischemia-reperfusion. In some diabetic models, functional amelioration of vasculature and electroretinograms was noted. Another approach for endogenous progenitor cell therapy is to normalize dysfunctional diabetic bone marrow and residing endothelial progenitors using NO donors, PPAR-δ and -γ agonists, or inhibition of TGF-β. A potentially important strategy would be to reduce neuropathy by stem cell inoculations, either naïve (e.g., paracrine-acting adipose stem cells) or secreting specific neuroprotectants, such as ciliary neurotrophic factor or brain-derived neurotrophic factor that showed benefit in amyotrophic lateral sclerosis and Parkinson's disease. Recent advances in stem cell therapies for diabetic retinal microangiopathy may form the basis of first clinical trials in the near future. Additionally, stem cell therapies may prove beneficial for diabetic corneal disease (diabetic keratopathy) with pronounced epithelial stem cell dysfunction.

Molecular mechanisms of extracellular vesicle-induced vessel destabilization in diabetic retinopathy.

Diabetic retinopathy (DR) is characterized by early dropout of capillary pericytes, leading to loss of control on endothelial proliferation and, subsequently, angiogenesis. We have demonstrated that extracellular vesicles (EV) derived from mesenchymal stem cells (MSC) maintained in diabetic-like conditions may play a role in vessel destabilization, thus contributing to angiogenesis through paracrine signalling. In particular, a role for MMP-2 was described. This study was aimed at further investigating the molecular mechanisms of EV-induced vessel destabilization. We evaluated miR-126 expression, the subsequent HIF-1α and VEGF modulation, Ang-2 and PDGF signalling pathways in human retinal pericytes (HRP) after exposure to MSC-derived EV obtained in diabetic-like conditions (high glucose and/or hypoxia). HRP express miR-126, and this expression is down-regulated in intermittent high glucose. MSC-derived EV obtained in hyperglycaemic/hypoxic conditions down-regulate miR-126 expression in pericytes, leading to increased expression of angiogenic molecules, such as VEGF and HIF-1α. No modulation of Ang-2 and PDGF signalling pathways in pericytes was observed following EV exposure. HRP express miR-126, and this expression is down-regulated in diabetic-like conditions. Exposure of HRP to EV obtained in diabetic-like conditions is able to decrease miR-126 expression, consistently with previous observations of its involvement in DR and providing further insights into the role of EV in vessel destabilization. In contrast, PDGF and Ang-2 signalling pathways do not seem to be involved in these mechanisms.

Experimental Branch Retinal Vein Occlusion Induces Upstream Pericyte Loss and Vascular Destabilization.

AimsBranch retinal vein occlusion (BRVO) leads to extensive vascular remodeling and is important cause of visual impairment. Although the vascular morphological changes following experimental vein occlusion have been described in a variety of models using angiography, the underlying cellular events are ill defined.Methods and ResultsWe here show that laser-induced experimental BRVO in mice leads to a wave of TUNEL-positive endothelial cell (EC) apoptosis in the upstream vascular network associated with a transient edema and hemorrhages. Subsequently, we observe an induction of EC proliferation within the dilated vein and capillaries, detected by EdU incorporation, and the edema resolves. However, the pericytes of the upstream capillaries are severely reduced, which was associated with continuing EC apoptosis and proliferation. The vascular remodeling was associated with increased expression of TGFβ, TSP-1, but also FGF2 expression. Exposure of the experimental animals to hypoxia, when pericyte (PC) dropout had occurred, led to a dramatic increase in endothelial cell proliferation, confirming the vascular instability induced by the experimental BRVO.ConclusionExperimental BRVO leads to acute endothelial cells apoptosis and increased permeability. Subsequently the upstream vascular network remains destabilized, characterized by pericyte dropout, un-physiologically high endothelial cells turnover and sensitivity to hypoxia. These early changes might pave the way for capillary loss and subsequent chronic ischemia and edema that characterize the late stage disease.

Interruption of Wnt Signaling in Müller Cells Ameliorates Ischemia-Induced Retinal Neovascularization

Retinal Müller cells are major producers of inflammatory and angiogenic cytokines which contribute to diabetic retinopathy (DR). Over-activation of the Wnt/β-catenin pathway has been shown to play an important pathogenic role in DR. However, the roles of Müller cell-derived Wnt/β-catenin signaling in retinal neovascularization (NV) and DR remain undefined. In the present study, mice with conditional β-catenin knockout (KO) in Müller cells were generated and subjected to oxygen-induced retinopathy (OIR) and streptozotocin (STZ)-induced diabetes. Wnt signaling was evaluated by measuring levels of β-catenin and expression of its target genes using immunoblotting. Retinal vascular permeability was measured using Evans blue as a tracer. Retinal NV was visualized by angiography and quantified by counting pre-retinal nuclei. Retinal pericyte loss was evaluated using retinal trypsin digestion. Electroretinography was performed to examine visual function. No abnormalities were detected in the β-catenin KO mice under normal conditions. In OIR, retinal levels of β-catenin and VEGF were significantly lower in the β-catenin KO mice than in littermate controls. The KO mice also had decreased retinal NV and vascular leakage in the OIR model. In the STZ-induced diabetic model, disruption of β-catenin in Müller cells attenuated over-expression of inflammatory cytokines and ameliorated pericyte dropout in the retina. These findings suggest that Wnt signaling activation in Müller cells contributes to retinal NV, vascular leakage and inflammation and represents a potential therapeutic target for DR.

Peroxisome Proliferator–Activated Receptor α Protects Capillary Pericytes in the Retina

Pericyte degeneration is an early event in diabetic retinopathy and plays an important role in progression of diabetic retinopathy. Clinical studies have shown that fenofibrate, a peroxisome proliferator-activated receptor α (PPARα) agonist, has robust therapeutic effects on diabetic retinopathy in type 2 diabetic patients. We evaluated the protective effect of PPARα against pericyte loss in diabetic retinopathy. In streptozotocin-induced diabetic mice, fenofibrate treatment significantly ameliorated retinal acellular capillary formation and pericyte loss. In contrast, PPARα(-/-) mice with diabetes developed more severe retinal acellular capillary formation and pericyte dropout, compared with diabetic wild-type mice. Furthermore, PPARα knockout abolished the protective effect of fenofibrate against diabetes-induced retinal pericyte loss. In cultured primary human retinal capillary pericytes, activation and expression of PPARα both significantly reduced oxidative stress-induced apoptosis, decreased reactive oxygen species production, and down-regulated NAD(P)H oxidase 4 expression through blockade of NF-κB activation. Furthermore, activation and expression of PPARα both attenuated the oxidant-induced suppression of mitochondrial O2 consumption in human retinal capillary pericytes. Primary retinal pericytes from PPARα(-/-) mice displayed more apoptosis, compared with those from wild-type mice under the same oxidative stress. These findings identified a protective effect of PPARα on retinal pericytes, a novel function of endogenous PPARα in the retina.

Retinal Microangiopathy in a Mouse Model of Inducible Mural Cell Loss

Diabetes can lead to vision loss because of progressive degeneration of the neurovascular unit in the retina, a condition known as diabetic retinopathy. In its early stages, the pathology is characterized by microangiopathies, including microaneurysms, microhemorrhages, and nerve layer infarcts known as cotton-wool spots. Analyses of postmortem human retinal tissue and retinas from animal models indicate that degeneration of the pericytes, which constitute the outer layer of capillaries, is an early event in diabetic retinopathy; however, the relative contribution of specific cellular components to the pathobiology of diabetic retinopathy remains to be defined. We investigated the phenotypic consequences of pericyte death on retinal microvascular integrity by using nondiabetic mice conditionally expressing a diphtheria toxin receptor in mural cells. Five days after administering diphtheria toxin in these adult mice, changes were observed in the retinal vasculature that were similar to those observed in diabetes, including microaneurysms and increased vascular permeability, suggesting that pericyte cell loss is sufficient to trigger retinal microvascular degeneration. Therapies aimed at preventing or delaying pericyte dropout may avoid or attenuate the retinal microangiopathy associated with diabetes.

Molecular analysis of blood–retinal barrier loss in the Akimba mouse, a model of advanced diabetic retinopathy

The molecular mechanisms of vascular leakage in diabetic macular edema and proliferative retinopathy are poorly understood, mainly due to the lack of reliable in vivo models. The Akimba (Ins2AkitaVEGF+/−) mouse model combines retinal neovascularization with hyperglycemia, and in contrast to other models, displays the majority of signs of advanced clinical diabetic retinopathy (DR). To study the molecular mechanism that underlies the breakdown of the blood-retinal barrier (BRB) in diabetic macular edema and proliferative diabetic retinopathy, we investigated the retinal vasculature of Akimba and its parental mice Kimba (trVEGF029) and Akita (Ins2Akita). Quantitative PCR, immunohistochemistry and fluorescein angiography were used to characterize the retinal vasculature with special reference to the inner BRB. Correlations between the degree of fluorescein leakage and retinal gene expression were tested by calculating the Spearman's correlation coefficient. Fluorescein leakage demonstrating BRB loss was observed in Kimba and Akimba, but not in Akita or wild type mice. In Kimba and Akimba mice fluorescein leakage was associated with focal angiogenesis and correlated significantly with Plvap gene expression. PLVAP is an endothelial cell-specific protein that is absent in intact blood-retinal barrier, but its expression significantly increases in pathological conditions such as DR. Furthermore, in Akimba mice BRB disruption was linked to decreased expression of endothelial junction proteins, pericyte dropout and vessel loss. Despite fluorescein leakage, no alteration in BRB protein levels or pericyte coverage was detected in retinas of Kimba mice. In summary, our data not only demonstrate that hyperglycemia sensitizes retinal vasculature to the effects of VEGF, leading to more severe microvascular changes, but also confirm an important role of PLVAP in the regulation of BRB permeability.

Connective Tissue Growth Factor Is Involved in Structural Retinal Vascular Changes in Long-Term Experimental Diabetes

Early retinal vascular changes in the development of diabetic retinopathy (DR) include capillary basal lamina (BL) thickening, pericyte loss and the development of acellular capillaries. Expression of the CCN (connective tissue growth factor/cysteine-rich 61/nephroblastoma overexpressed) family member CCN2 or connective tissue growth factor (CTGF), a potent inducer of the expression of BL components, is upregulated early in diabetes. Diabetic mice lacking one functional CTGF allele (CTGF+/−) do not show this BL thickening. As early events in DR may be interrelated, we hypothesized that CTGF plays a role in the pathological changes of retinal capillaries other than BL thickening. We studied the effects of long-term (6-8 months) streptozotocin-induced diabetes on retinal capillary BL thickness, numbers of pericytes and the development of acellular capillaries in wild type and CTGF+/− mice. Our results show that an absence of BL thickening of retinal capillaries in long-term diabetic CTGF+/− mice is associated with reduced pericyte dropout and reduced formation of acellular capillaries. We conclude that CTGF is involved in structural retinal vascular changes in diabetic rodents. Inhibition of CTGF in the eye may therefore be protective against the development of DR.

Pericyte Loss in Diabetic Retinopathy: Mechanisms and Consequences

The onset of diabetic retinopathy is characterized by morphologic alterations of the microvessels, with thickening of the basement membrane, loss of inter-endothelial tight junctions and early and selective loss of pericytes, together with increased vascular permeability, capillary occlusions, microaneurysms and, later, loss of endothelial cells (EC). A key role in the evolution of the disease is played by pericytes, specialized contractile mesenchymal cells of mesodermal origin, that, in capillaries, exert a function similar to smooth muscle cells in larger vessels, regulating vascular tone and perfusion pressure. Thickening of the basement membrane, together with systemic and local hypertension, hyperglycaemia, advanced glycation end-product formation and hypoxia, may disrupt the tight link between pericytes and EC causing pericyte apoptosis, while endothelium, deprived of proliferation control, can give rise to new vessels. Pericyte dropout has great consequences on capillary remodelling and may cause the first abnormalities of the diabetic eye which can be observed clinically. Hyperglycaemia and local hypertension are known to be a direct cause of pericyte apoptosis and dropout, and intracellular biochemical pathways of the glucose metabolites have been explored. However, the exact mechanisms are not yet fully understood and need further clarification in order to develop new effective drugs for the prevention of retinopathy.

Pericytes in the eye

Pericytes in the retina differ from pericytes in many other organs by their high density and their cooperative role in the neurovascular unit. Their diverse ontogeny and the fact that not one pericyte marker identifies the entire population suggest also functional plurality in the retina, including invading cells of mesenchymal origin. Further, to establish factors determining pericyte recruitment, modifiers of pericyte adhesion and homeostasis, such as notch-3 and angptl-4, have been recently identified, expanding the understanding of pericyte function in the retina. Also, the role of pericytes as part of the neurovascular unit has been appreciated, given that the neuroglia determines pericyte survival and motility under disease conditions. Pericyte dropout is not unique in the diabetic retina, and non-diabetic animal models may prove useful in the search for mechanisms involved in disease-associated dysfunction of the neurovascular unit.

COMP‐Ang1 preserves vasculature and prevents vision loss in diabetic retinopathy

Diabetic retinopathy (DR) is the most common cause of blindness in the working‐age population. Vascular dysfunction is a hallmark of DR. Prolonged hyperglycemia results in loss of pericytes, the support cells that surround the capillary. An important pericyte derived trophic factor is angiopoietin‐1 (Ang‐1), which binds to the endothelial Tie2 receptor. Loss of pericytes and decreased Ang1 has been described as an early event in diabetic retinopathy. Here we show that vascular normalization with cartilage oligo matrix protein angiopoietin 1 (COMP‐Ang1) prevents endothelial dropout and the critical sequelae of ischemia and hyperpermeability in a mouse model of DR. COMP‐Ang1 increased VE‐cadherin expression and decreased VEGF‐A expression in the diabetic mouse retina. COMP‐Ang1 preserved vascular network area comparable to non‐diabetic control levels, despite persistent pericyte dropout. Additionally, COMP‐Ang1 decreased retinal inflammation, as noted by decreased leukocyte adhesion and infiltration. Furthermore, AAV2.COMP‐Ang1 therapy prevented retinal thinning and ganglion cell layer dropout. Most importantly, diabetic mice treated with COMP‐Ang1 retained near‐normal visual acuity and electroretinographic response. COMP‐Ang1 may be useful in promoting vascular normalization by reducing ischemia and hyperpermeability in diabetic retinopathy.