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.

The failure of blood vessels to revascularize ischemic neural tissue represents a significant challenge for vascular biology. Examples include proliferative retinopathies (PRs) such as retinopathy of prematurity and proliferative diabetic retinopathy, which are the leading causes of blindness in children and working-age adults. PRs are characterized by initial microvascular degeneration, followed by a compensatory albeit pathologic hypervascularization mounted by the hypoxic retina attempting to reinstate metabolic equilibrium. Paradoxically, this secondary revascularization fails to grow into the most ischemic regions of the retina. Instead, the new vessels are misdirected toward the vitreous, suggesting that vasorepulsive forces operate in the avascular hypoxic retina. In the present study, we demonstrate that the neuronal guidance cue semaphorin 3A (Sema3A) IntroductionProliferative retinopathies (PRs) are traditionally perceived as disorders limited to the microvasculature because of the characteristic profuse and deregulated growth of retinal vessels. 1 The mechanisms by which neovessels grow toward the vitreous and fail to revascularize ischemic zones are thought to result from high concentrations of proangiogenic factors such as VEGF in the vitreous of PR patients. However, if such an explanation were sufficient, retinal glial cells (astrocytes and Müller cells) 2 and neurons 3 that produce vast amounts of growth factors under hypoxic conditions should retain vessels on the retinal surface and ensure revascularization of the retina proper. It is, therefore, compelling to hypothesize the presence of a vasorepulsive force originating from the significantly hypoxic avascular retina that repels neovessels away from the vaso-obliterated retina and grows toward the vitreous.Neurovascular cross-talk shapes vascular development but has received limited attention in the pathology setting. In PRs, evidence points to an early decline in the function of ischemic regions of the neural retina, as shown by multifocal electroretinogram (mfERG). 4,5 Throughout the vaso-obliterative phase of retinopathy, the local retinal environment is hostile to both vasculature and neurons. 6 After blood vessel degeneration, neurons are metabolically starved and undergo several adaptive cellular changes to counter the ischemic state of the tissue. 3,6 If adequate vascular supply is not reinstated in time to salvage deprived neurons, it is conceivable that these severely hypoxic cells may mount a repulsive front in an attempt to shunt metabolic resources away from the perishing ischemic tissue toward less affected regions of the retina. In the process, excessive production of VEGF 7 induces exaggerated neovascularization at the periphery of the ischemic and repulsive zones into the pre-retinal region (normally devoid of vasculature), because reestablishing a vascular network to neurons that are unsalvageable would be wasteful.Given their established role in influencing endothelial cell (EC) behavior, classic neuronal guidance cues may ...

The effects of prostaglandin E 2 are thought to be mediated via G protein-coupled plasma membrane receptors, termed EP. However recent data implied that prostanoids may also act intracellularly. We investigated if the ubiquitous EP 3 and the EP 4 receptors are localized in nuclear membranes. Radioligand binding studies on isolated nuclear membrane fractions of neonatal porcine brain and adult rat liver revealed the presence of EP 3 and EP 4 . A perinuclear localization of EP 3␣ and EP 4 receptors was visualized by indirect immunocytofluorescence and confocal microscopy in porcine cerebral microvascular endothelial cells and in transfected HEK 293 cells that stably overexpress these receptors. Immunoelectron microscopy clearly revealed EP 3␣ and EP 4 receptors localization in the nuclear envelope of endothelial cells; this is the first demonstration of the nuclear localization of these receptors. Data also reveal that nuclear EP receptors are functional as they affect transcription of genes such as inducible nitric-oxide synthase and intranuclear calcium transients; this appears to involve pertussis toxin-sensitive G proteins. These results define a possible molecular mechanism of action of nuclear EP 3 receptors.Prostaglandin E 2 (PGE 2 ) 1 is one of the most abundant prostanoids in the brain (1) and plays an important role in many cerebral functions, particularly in the newborn (2). PGE 2 also influences mitogenesis (3), promotes growth and metastasis of tumors (4), and stimulates gene transcription (5). To date, the biological actions of PGE 2 have been attributed to result from its interaction with plasma membrane G protein-coupled receptors termed EP, which include EP 1 , EP 2 , EP 3 , and EP 4 subtypes (6). Recent studies have shown that the nuclear membrane contains high levels of cyclooxygenase-1 and -2 and of PGE 2 (7). Possible intracellular sites of action for prostanoids are also suggested by other data. For example, a transporter that mediates the influx of prostanoid has been identified (8). Cytosolic phospholipase A 2 undergoes a calcium-dependent translocation to the nuclear envelope (9), and cyclooxygenase-2 has been shown to translocate to the nucleus in response to certain growth factors (10). It is thus possible that prostanoids may exert some of their effects via intracellular EP receptors, to have a direct nuclear action as recently proposed by Goetzl et al. (11), and Morita et al. (12).It has generally been assumed that the signal transduction cascades are initiated at the plasma membrane and not the nuclear membranes. However, recent studies have disclosed that the nuclear envelope plays a major role in signal transduction cascades (13,14). In fact, a novel nuclear lipid metabolism that is a part of unique nuclear signaling cascade termed NEST (nuclear envelope signal transduction) has been hypothesized (15). Both heterotrimeric and low molecular weight G proteins (15, 16), phospholipase C (13), phospholipase D (15), and adenylate cyclase (17) have shown to be localized at the nucleus. Th...

Prostaglandin E 2 receptors (EP) were detected by radioligand binding in nuclear fractions isolated from porcine brain and myometrium. Intracellular localization by immunocytofluorescence revealed perinuclear localization of EPs in porcine cerebral microvascular endothelial cells. Nuclear association of EP 1 was also found in fibroblast Swiss 3T3 cells stably overexpressing EP 1 and in human embryonic kidney 293 (Epstein–Barr virus-encoded nuclear antigen) cells expressing EP 1 fused to green fluorescent protein. High-resolution immunostaining of EP 1 revealed their presence in the nuclear envelope of isolated (cultured) endothelial cells and in situ in brain (cortex) endothelial cells and neurons. Stimulation of these nuclear receptors modulate nuclear calcium and gene transcription.