An ensemble of bias-adjusted CMIP6 climate simulations based on a high-resolution North American reanalysis.

ESPO-G6-R2 v1.0 is a set of statistically downscaled and bias-adjusted climate simulations based on the Coupled Model Intercomparison Project 6 (CMIP6) models. The dataset is composed of daily timeseries of three variables: daily maximum temperature, daily minimum temperature and daily precipitation. Data are available from 1950 to 2100 over North America. The simulation ensemble is comprised of 14 models driven by two emissions scenarios (SSP2-4.5 and SSP3-7.0). In this paper, we describe the workflow used for the bias-adjustment, which relies on the detrended quantile mapping method and the Regional Deterministic Reforecast System (RDRS) v2.1 reference dataset. Using the framework defined in the VALUE project, we show the improvements made by the bias-adjustment on marginal, temporal and multivariate aspects of the data. We also verify that the bias-adjusted climate data have similar climate change signal to the original climate model simulations. Finally, we provide guidance to users on how to use this dataset.

Insights into the Evolution of Ohnologous Sequences and Their Epigenetic Marks Post-WGD in Malus Domestica.

A Whole Genome Duplication (WGD) event occurred several Ma in a Rosaceae ancestor, giving rise to the Maloideae subfamily which includes today many pome fruits such as pear (Pyrus communis) and apple (Malus domestica). This complete and well-conserved genome duplication makes the apple an organism of choice to study the early evolutionary events occurring to ohnologous chromosome fragments. In this study, we investigated gene sequence evolution and expression, transposable elements (TE) density, and DNA methylation level. Overall, we identified 16,779 ohnologous gene pairs in the apple genome, confirming the relatively recent WGD. We identified several imbalances in QTL localization among duplicated chromosomal fragments and characterized various biases in genome fractionation, gene transcription, TE densities, and DNA methylation. Our results suggest a particular chromosome dominance in this autopolyploid species, a phenomenon that displays similarities with subgenome dominance that has only been described so far in allopolyploids.

Human Lung Tissue Implanted on the Chick Chorioallantoic Membrane as a Novel In Vivo Model of Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease with no curative pharmacological treatment. Current preclinical models fail to accurately reproduce human pathophysiology and are therefore poor predictors of clinical outcomes. Here, we investigated whether the chick embryo chorioallantoic membrane (CAM) assay supports the implantation of xenografts derived from IPF lung tissue and primary IPF lung fibroblasts and can be used to evaluate the efficacy of antifibrotic drugs. We demonstrate that IPF xenografts maintain their integrity and are perfused with chick embryo blood. Size measurements indicate that the xenografts amplify on the CAM, and Ki67 and pro-collagen type I immunohistochemical staining highlight the presence of proliferative and functional cells in the xenografts. Moreover, the IPF phenotype and immune microenvironment of lung tissues are retained when cultivated on the CAM and the fibroblast xenografts mimic invasive IPF fibroblastic foci. Daily treatments of the xenografts with nintedanib and PBI-4050 significantly reduce their size, fibrosis-associated gene expression, and collagen deposition. Similar effects are found with GLPG1205 and fenofibric acid, two drugs that target the immune microenvironment. Our CAM-IPF model represents the first in vivo model of IPF that uses human lung tissue. This rapid and cost-effective assay could become a valuable tool for predicting the efficacy of antifibrotic drug candidates for IPF.

Variability in frost occurrence under climate change and consequent risk of damage to trees of western Quebec, Canada.

Climate change affects timings, frequency, and intensity of frost events in northern ecosystems. However, our understanding of the impacts that frost will have on growth and survival of plants is still limited. When projecting the occurrence of frost, the internal variability and the different underlying physical formulations are two major sources of uncertainty of climate models. We use 50 climate simulations produced by a single-initial large climate ensemble and five climate simulations produced by different pairs of global and regional climate models based on the concentration pathway (RCP 8.5) over a latitudinal transect covering the temperate and boreal ecosystems of western Quebec, Canada, during 1955-2099 to provide a first-order estimate of the relative importance of these two sources of uncertainty on the occurrence of frost, i.e. when air temperature is < 0 °C, and their potential damage to trees. The variation in the date of the last spring frost was larger by 21 days (from 46 to 25 days) for the 50 climate simulations compared to the 5 different pairs of climate models. When considering these two sources of uncertainty in an eco-physiological model simulating the timings of budbreak for trees of northern environment, results show that 20% of climate simulations expect that trees will be exposed to frost even in 2090. Thus, frost damage to trees remains likely under global warming.

An Overview of Duplicated Gene Detection Methods: Why the Duplication Mechanism Has to Be Accounted for in Their Choice.

Gene duplication is an important evolutionary mechanism allowing to provide new genetic material and thus opportunities to acquire new gene functions for an organism, with major implications such as speciation events. Various processes are known to allow a gene to be duplicated and different models explain how duplicated genes can be maintained in genomes. Due to their particular importance, the identification of duplicated genes is essential when studying genome evolution but it can still be a challenge due to the various fates duplicated genes can encounter. In this review, we first describe the evolutionary processes allowing the formation of duplicated genes but also describe the various bioinformatic approaches that can be used to identify them in genome sequences. Indeed, these bioinformatic approaches differ according to the underlying duplication mechanism. Hence, understanding the specificity of the duplicated genes of interest is a great asset for tool selection and should be taken into account when exploring a biological question.

Fatty acid mimetic PBI-4547 restores metabolic homeostasis via GPR84 in mice with non-alcoholic fatty liver disease.

Non-alcoholic Fatty Liver Disease (NAFLD) is the most common form of liver disease and is associated with metabolic dysregulation. Although G protein-coupled receptor 84 (GPR84) has been associated with inflammation, its role in metabolic regulation remains elusive. The aim of our study was to evaluate the potential of PBI-4547 for the treatment of NAFLD and to validate the role of its main target receptor, GPR84. We report that PBI-4547 is a fatty acid mimetic, acting concomitantly as a GPR84 antagonist and GPR40/GPR120 agonist. In a mouse model of diet-induced obesity, PBI-4547 treatment improved metabolic dysregulation, reduced hepatic steatosis, ballooning and NAFLD score. PBI-4547 stimulated fatty acid oxidation and induced gene expression of mitochondrial uncoupling proteins in the liver. Liver metabolomics revealed that PBI-4547 improved metabolic dysregulation induced by a high-fat diet regimen. In Gpr84-/- mice, PBI-4547 treatment failed to improve various key NAFLD-associated parameters, as was observed in wildtype littermates. Taken together, these results highlight a detrimental role for the GPR84 receptor in the context of meta-inflammation and suggest that GPR84 antagonism via PBI-4547 may reflect a novel treatment approach for NAFLD and its related complications.

PBI-4050 via GPR40 activation improves adenine-induced kidney injury in mice.

PBI-4050 (3-pentylbenzenacetic acid sodium salt), a novel first-in-class orally active compound that has completed clinical Phases Ib and II in subjects with chronic kidney disease (CKD) and metabolic syndrome respectively, exerts antifibrotic effects in several organs via a novel mechanism of action, partly through activation of the G protein receptor 40 (GPR40) receptor. Here we evaluate the effects of PBI-4050 in both WT and Gpr40-/- mice on adenine-induced tubulointerstitial injury, anemia and activation of the unfolded protein response (UPR) pathway. Adenine-induced CKD was achieved in 8-week-old C57BL/6 mice fed a diet supplemented with 0.25% adenine. After 1 week, PBI-4050 or vehicle was administered daily by oral-gavage for 3 weeks. Gpr40-/- mice were also subjected to adenine-feeding, with or without PBI-4050 treatment. PBI-4050 improved renal function and urine concentrating ability. Anemia was present in adenine-fed mice, while PBI-4050 blunted these effects and led to significantly higher plasma erythropoietin (EPO) levels. Adenine-induced renal fibrosis, endoplasmic reticulum (ER) stress and apoptosis were significantly decreased by PBI-4050. In parallel, Gpr40-/- mice were more susceptible to adenine-induced fibrosis, renal function impairment, anemia and ER stress compared with WT mice. Importantly, PBI-4050 treatment in Gpr40-/- mice failed to reduce renal injury in this model. Taken together, PBI-4050 prevented adenine-induced renal injury while these beneficial effects were lost upon Gpr40 deletion. These data reinforce PBI-4050's use as a renoprotective therapy and identify GPR40 as a crucial mediator of its beneficial effects.

PBI-4050 Reduces Stellate Cell Activation and Liver Fibrosis through Modulation of Intracellular ATP Levels and the Liver Kinase B1/AMP-Activated Protein Kinase/Mammalian Target of Rapamycin Pathway.

Hepatic fibrosis is a major cause of morbidity and mortality for which there is currently no effective therapy. We previously showed that 2-(3-pentylphenyl)acetic acid (PBI-4050) is a dual G protein-coupled receptor GPR40 agonist/GPR84 antagonist that exerts antifibrotic, anti-inflammatory, and antiproliferative action. We evaluated PBI-4050 for the treatment of liver fibrosis in vivo and elucidated its mechanism of action on human hepatic stellate cells (HSCs). The antifibrotic effect of PBI-4050 was evaluated in carbon tetrachloride (CCl4)- and bile duct ligation-induced liver fibrosis rodent models. Treatment with PBI-4050 suppressed CCl4-induced serum aspartate aminotransferase levels, inflammatory marker nitric oxide synthase, epithelial to mesenchymal transition transcription factor Snail, and multiple profibrotic factors. PBI-4050 also decreased GPR84 mRNA expression in CCl4-induced injury, while restoring peroxisome proliferator-activated receptor γ (PPARγ) to the control level. Collagen deposition and α-smooth muscle actin (α-SMA) protein levels were also attenuated by PBI-4050 treatment in the bile duct ligation rat model. Transforming growth factor-ß-activated primary HSCs were used to examine the effect of PBI-4050 and its mechanism of action in vitro. PBI-4050 inhibited HSC proliferation by arresting cells in the G0/G1 cycle phase. Subsequent analysis demonstrated that PBI-4050 signals through a reduction of intracellular ATP levels, activation of liver kinase B1 (LKB1) and AMP-activated protein kinase (AMPK), and blockade of mammalian target of rapamycin (mTOR), resulting in reduced protein and mRNA levels of α-SMA and connective tissue growth factor and restored PPARγ mRNA expression. Our findings suggest that PBI-4050 may exert antifibrotic activity in the liver through a novel mechanism of action involving modulation of intracellular ATP levels and the LKB1/AMPK/mTOR pathway in stellate cells, and PBI-4050 may be a promising agent for treating liver fibrosis.

Fatty acid receptor modulator PBI-4050 inhibits kidney fibrosis and improves glycemic control.

Extensive kidney fibrosis occurs in several types of chronic kidney diseases. PBI-4050, a potentially novel first-in-class orally active low-molecular weight compound, has antifibrotic and antiinflammatory properties. We examined whether PBI-4050 affected the progression of diabetic nephropathy (DN) in a mouse model of accelerated type 2 diabetes and in a model of selective tubulointerstitial fibrosis. eNOS-/- db/db mice were treated with PBI-4050 from 8-20 weeks of age (early treatment) or from 16-24 weeks of age (late treatment). PBI-4050 treatment ameliorated the fasting hyperglycemia and abnormal glucose tolerance tests seen in vehicle-treated mice. In addition, PBI-4050 preserved (early treatment) or restored (late treatment) blood insulin levels and increased autophagy in islets. PBI-4050 treatment led to significant improvements in lifespan in the diabetic mice. Both early and late PBI-4050 treatment protected against progression of DN, as indicated by reduced histological glomerular injury and albuminuria, slow decline of glomerular filtration rate, and loss of podocytes. PBI-4050 inhibited kidney macrophage infiltration, oxidative stress, and TGF-ß-mediated fibrotic signaling pathways, and it also protected against the development of tubulointerstitial fibrosis. To confirm a direct antiinflammatory/antifibrotic effect in the kidney, further studies with a nondiabetic model of EGFR-mediated proximal tubule activation confirmed that PBI-4050 dramatically decreased the development of the associated tubulointerstitial injury and macrophage infiltration. These studies suggest that PBI-4050 attenuates development of DN in type 2 diabetes through improvement of glycemic control and inhibition of renal TGF-ß-mediated fibrotic pathways, in association with decreases in macrophage infiltration and oxidative stress.

A Newly Discovered Antifibrotic Pathway Regulated by Two Fatty Acid Receptors: GPR40 and GPR84.

Numerous clinical conditions can lead to organ fibrosis and functional failure. There is a great need for therapies that could effectively target pathophysiological pathways involved in fibrosis. GPR40 and GPR84 are G protein-coupled receptors with free fatty acid ligands and are associated with metabolic and inflammatory disorders. Although GPR40 and GPR84 are involved in diverse physiological processes, no evidence has demonstrated the relevance of GPR40 and GPR84 in fibrosis pathways. Using PBI-4050 (3-pentylbenzeneacetic acid sodium salt), a synthetic analog of a medium-chain fatty acid that displays agonist and antagonist ligand affinity toward GPR40 and GPR84, respectively, we uncovered an antifibrotic pathway involving these receptors. In experiments using Gpr40- and Gpr84-knockout mice in models of kidney fibrosis (unilateral ureteral obstruction, long-term post-acute ischemic injury, and adenine-induced chronic kidney disease), we found that GPR40 is protective and GPR84 is deleterious in these diseases. Moreover, through binding to GPR40 and GPR84, PBI-4050 significantly attenuated fibrosis in many injury contexts, as evidenced by the antifibrotic activity observed in kidney, liver, heart, lung, pancreas, and skin fibrosis models. Therefore, GPR40 and GPR84 may represent promising molecular targets in fibrosis pathways. We conclude that PBI-4050 is a first-in-class compound that may be effective for managing inflammatory and fibrosis-related diseases.

Restoration of renal function by a novel prostaglandin EP4 receptor-derived peptide in models of acute renal failure.

Acute renal failure (ARF) is a serious medical complication characterized by an abrupt and sustained decline in renal function. Despite significant advances in supportive care, there is currently no effective treatment to restore renal function. PGE(2) is a lipid hormone mediator abundantly produced in the kidney, where it acts locally to regulate renal function; several studies suggest that modulating EP(4) receptor activity could improve renal function following kidney injury. An optimized peptidomimetic ligand of EP(4) receptor, THG213.29, was tested for its efficacy to improve renal function (glomerular filtration rate, renal plasma flow, and urine output) and histological changes in a model of ARF induced by either cisplatin or renal artery occlusion in Sprague-Dawley rats. THG213.29 modulated PGE(2)-binding dissociation kinetics, indicative of an allosteric binding mode. Consistently, THG213.29 antagonized EP(4)-mediated relaxation of piglet saphenous vein rings, partially inhibited EP(4)-mediated cAMP production, but did not affect Gα(i) activation or ß-arrestin recruitment. In vivo, THG213.29 significantly improved renal function and histological changes in cisplatin- and renal artery occlusion-induced ARF models. THG213.29 increased mRNA expression of heme-oxygenase 1, Bcl2, and FGF-2 in renal cortex; correspondingly, in EP(4)-transfected HEK293 cells, THG213.29 augmented FGF-2 and abrogated EP(4)-dependent overexpression of inflammatory IL-6 and of apoptotic death domain-associated protein and BCL2-associated agonist of cell death. Our results demonstrate that THG213.29 represents a novel class of diuretic agent with noncompetitive allosteric modulator effects on EP(4) receptor, resulting in improved renal function and integrity following acute renal failure.

Cortactin activation by FVIIa/tissue factor and PAR2 promotes endothelial cell migration.

Cellular migration is a complex process that requires the polymerization of actin filaments to drive cellular extension. Smooth muscle and cancer cell migration has been shown to be affected by coagulation factors, notably the factor VII (FVIIa) and tissue factor (TF) complex. The present studies delineated mediators involved with the process of FVIIa/TF-induced cell migration and utilized a simple, precise, and reproducible, migration assay. Both FVIIa and protease-activated receptor-2 (PAR2)-activating peptide, SLIGRL, increased the migration rate of porcine cerebral microvascular endothelial cells (pCMVECs) overexpressing human TF. Ras homolog gene family member A (RhoA) and cortactin were upregulated during the process; expression of HIF, actin polymerization nuclear diaphanous-related formin-1 and -2 (Dia1, and Dia2) were unaffected. Gene silencing by shRNA to PAR2, RhoA, and cortactin attenuated this gene upregulation and migration induced by FVIIa/TF. Utilizing immunocellular localization, we demonstrate that during FVIIa/TF and PAR2 activation, cortactin molecules translocate from the cytoplasm to the cell periphery and assist in lamellipodia formation of pCMVECs. Overall, we demonstrate a novel regulation and role for cortactin in FVIIa/TF-mediated endothelial cell migration that occurs through a PAR2 and RhoA dependent mechanism.

Functional selectivity of natural and synthetic prostaglandin EP4 receptor ligands.

Classically, the prostaglandin E(2) (PGE(2)) receptor EP(4) has been classified as coupling to the Galpha(s) subunit, leading to intracellular cAMP increases. However, EP(4) signaling has been revealed to be more complex and also involves coupling to pertussis toxin-sensitive Galpha(i) proteins and beta-arrestin-mediated effects. There are now many examples of selective activation of independent pathways by G protein-coupled receptor (GPCR) ligands, a concept referred to as functional selectivity. Because most EP(4) ligands had thus far only been functionally characterized by their ability to stimulate cAMP production, we systematically determined the potencies and efficacies of a panel of EP(4) ligands for activation of Galpha(s), Galpha(i), and beta-arrestin relative to the endogenous ligand PGE(2). For this purpose, we adapted three bioluminescence resonance energy transfer (BRET) assays to evaluate the respective pathways in living cells. Our results suggest considerable functional selectivity among the tested, structurally related agonists. PGE(2) was the most selective in activating Galpha(s), whereas PGF(2alpha) and PGE(1) alcohol were the most biased for activating Galpha(i1) and beta-arrestin, respectively. We observed reversal in order of potencies between beta-arrestin 2 and Galpha(i1) functional assays comparing PGE(1) alcohol and either PGF(2alpha), PGD(2), or 7-[(1R,2R)-2-[(E,3R)-3-hydroxy-4-(phenoxy)but-1-enyl]-5-oxocyclopentyl]heptanoic acid (M&B28767). Most ligands were full agonists for the three pathways tested. Our results have implications for the use of PGE(2) analogs in experimental and possibly clinical settings, because their activity spectra on EP(4) differ from that of the native agonist. The BRET-based methodology used for this first systematic assessment of a set of EP(4) agonists should be applicable for the study of other GPCRs.

The succinate receptor GPR91 in neurons has a major role in retinal angiogenesis.

Vascularization is essential for tissue development and in restoration of tissue integrity after an ischemic injury. In studies of vascularization, the focus has largely been placed on vascular endothelial growth factor (VEGF), yet other factors may also orchestrate this process. Here we show that succinate accumulates in the hypoxic retina of rodents and, via its cognate receptor G protein-coupled receptor-91 (GPR91), is a potent mediator of vessel growth in the settings of both normal retinal development and proliferative ischemic retinopathy. The effects of GPR91 are mediated by retinal ganglion neurons (RGCs), which, in response to increased succinate levels, regulate the production of numerous angiogenic factors including VEGF. Accordingly, succinate did not have proangiogenic effects in RGC-deficient rats. Our observations show a pathway of metabolite signaling where succinate, acting through GPR91, governs retinal angiogenesis and show the propensity of RGCs to act as sensors of ischemic stress. These findings provide a new therapeutic target for modulating revascularization.

Development of a novel noncompetitive antagonist of IL-1 receptor.

IL-1 is a major proinflammatory cytokine which interacts with the IL-1 receptor I (IL-1RI) complex, composed of IL-1RI and IL-1R accessory protein subunits. Currently available strategies to counter pathological IL-1 signaling rely on a recombinant IL-1 receptor antagonist, which directly competes with IL-1 for its binding site. Presently, there are no small antagonists of the IL-1RI complex. Given this void, we derived 15 peptides from loops of IL-1R accessory protein, which are putative interactive sites with the IL-1RI subunit. In this study, we substantiate the merits of one of these peptides, rytvela (we termed "101.10"), as an inhibitor of IL-1R and describe its properties consistent with those of an allosteric negative modulator. 101.10 (IC(50) approximately 1 nM) blocked human thymocyte proliferation in vitro, and demonstrated robust in vivo effects in models of hyperthermia and inflammatory bowel disease as well as topically in contact dermatitis, superior to corticosteroids and IL-1ra; 101.10 did not bind to IL-1RI deficient cells and was ineffective in vivo in IL-1RI knockout mice. Importantly, characterization of 101.10, revealed noncompetitive antagonist actions and functional selectivity by blocking certain IL-1R pathways while not affecting others. Findings describe the discovery of a potent and specific small (peptide) antagonist of IL-1RI, with properties in line with an allosteric negative modulator.

[Hypercapnia- and trans-arachidonic acid-induced retinal microvascular degeneration: implications in the genesis of retinopathy of prematurity]. / La rétinopathie du prématuré: Rôle de l'acide trans-arachidonique dans la dégénérescence des micro-vaisseaux de la rétine.

High oxygen tension is a major factor in the genesis of retinopathy of prematurity (ROP). However, clinical and experimental evidence also suggest a significant role for high levels of carbon dioxide (CO(2)). Hypercapnia is a facilitator of nitration in vitro, and nitrative stress is known to have an important role in microvascular degeneration leading to ischemia in conditions such as ROP. We hereby present evidence that prolonged exposure to CO(2) impairs developmental retinal neovascularisation through a mechanism involving increased endothelial nitric oxide synthase and induction of a nitrative stress; effects of hypercapnia are independent of its hyperaemic effects. Moreover, in a model of oxygen-induced retinopathy, we demonstrate that an in vivo nitrative stress associated with retinal vasoobliteration results in nitration of cis-arachidonic acids into trans-arachidonic acids (TAAs). TAAs act in turn as mediators of nitrative stress by causing microvascular degeneration by inducing expression of the anti-angiogenic factor thrombospondin-1. These recent findings establish a previously unexplored means by which hypercapnia hinders efficient neovascularisation and provide new insight into the molecular mechanisms of nitrative stress on microvascular injury involving TAA, therefore opening new therapeutic avenues in the management of nitrative stress disorders such as in ischemic retinopathies (of prematurity and of diabetes) and encephalopathies.

Recombinant human butyrylcholinesterase from milk of transgenic animals to protect against organophosphate poisoning.

Dangerous organophosphorus (OP) compounds have been used as insecticides in agriculture and in chemical warfare. Because exposure to OP could create a danger for humans in the future, butyrylcholinesterase (BChE) has been developed for prophylaxis to these chemicals. Because it is impractical to obtain sufficient quantities of plasma BChE to treat humans exposed to OP agents, the production of recombinant BChE (rBChE) in milk of transgenic animals was investigated. Transgenic mice and goats were generated with human BChE cDNA under control of the goat beta-casein promoter. Milk from transgenic animals contained 0.1-5 g/liter of active rBChE. The plasma half-life of PEGylated, goat-derived, purified rBChE in guinea pigs was 7-fold longer than non-PEGylated dimers. The rBChE from transgenic mice was inhibited by nerve agents at a 1:1 molar ratio. Transgenic goats produced active rBChE in milk sufficient for prophylaxis of humans at risk for exposure to OP agents.

Direct assessment of CXCR4 mutant conformations reveals complex link between receptor structure and G(alpha)(i) activation.

Ligand binding to G protein-coupled receptors (GPCRs) is thought to induce changes in receptor conformation that translate into activation of downstream effectors. The link between receptor conformation and activity is still insufficiently understood, as current models of GPCR activation fail to take an increasing amount of experimental data into account. To elucidate structure-function relationships in GPCR activation, we used bioluminescence resonance energy transfer to directly assess the conformation of mutants of the chemokine receptor CXCR4. We analyzed substitutions in the arginine cage DRY motif and in the conserved asparagine N(3.35)119, which are pivotal molecular switches for receptor conformation and activation. G(alpha)(i) activation of the mutants was either similar to wild-type CXCR4 (D133N, Y135A, and N119D) or resulted in loss of activity (R134A and N119K). Mutant N119S was constitutively active but further activated by agonist. Bioluminescence resonance energy transfer analysis suggested no simple correlation between conformational changes in response to ligand binding and activation of G(alpha)(i) by the mutants. Different conformations of active receptors were detected (for wild-type CXCR4, D133N, and N119S), suggesting that different receptor conformations are able to trigger G(alpha)(i) activity. Several conformations were also found for inactive mutants. These data provide biophysical evidence for different receptor conformations being active with respect to a single readout. They support models of GPCR structure-activity relationships that take this conformational flexibility of active receptors into account.

Potential role of microglia in retinal blood vessel formation.

PURPOSE: The role of microglia, present in the retina early in development before vascularization, remains ill defined. The authors investigated whether microglia are implicated in retinal blood vessel formation. METHODS: The microglia and vasculature of developing human fetal and rodent retinas were examined by labeling the endothelial cells with lectin and the microglia with CD18 antibody or green fluorescent protein driven by the promoter of the chemokine receptor CX(3)CR1. Rodent ischemic proliferative retinopathy induced by hyperoxia or hypercapnia, which model retinopathy of prematurity, and ex vivo retinal explants were used to assess microglial involvement in vascular pathology. Microglial participation in developmental retinal vessel formation was further studied in neonatal rats after pharmacologic macrophage depletion with the use of clodronate liposomes and subsequent intravitreal injection of microglia. RESULTS: Microglia intimately appose developing vessels of human and murine retinas. Ischemic retinopathy models exhibit decreased microglia concomitant with the characteristic reductions in vasculature observed in these retinopathies. Retinal explants exposed to conditions resulting in ischemic retinopathies (in vivo) reveal that antioxidants protect against microglial loss. Depletion of resident retinal microglia, but not systemic macrophages, reduced developmental vessel growth and density, which were restored by intravitreal microglial injection. CONCLUSIONS: These observations suggest that proper retinal blood vessel formation requires an adequate resident microglial population because diminished microglia are associated with decreased vascularity in models of ischemic retinopathy and retinal vascular development. In light of these findings, the traditional definition of microglia as merely immunocompetent cells should be reconsidered to encompass this new function related to blood vessel formation.