HIF1α-dependent hypoxia response in myeloid cells requires IRE1α.

Cellular adaptation to low oxygen tension triggers primitive pathways that ensure proper cell function. Conditions of hypoxia and low glucose are characteristic of injured tissues and hence successive waves of inflammatory cells must be suited to function under low oxygen tension and metabolic stress. While Hypoxia-Inducible Factor (HIF)-1α has been shown to be essential for the inflammatory response of myeloid cells by regulating the metabolic switch to glycolysis, less is known about how HIF1α is triggered in inflammation. Here, we demonstrate that cells of the innate immune system require activity of the inositol-requiring enzyme 1α (IRE1α/XBP1) axis in order to initiate HIF1α-dependent production of cytokines such as IL1ß, IL6 and VEGF-A. Knockout of either HIF1α or IRE1α in myeloid cells ameliorates vascular phenotypes in a model of retinal pathological angiogenesis driven by sterile inflammation. Thus, pathways associated with ER stress, in partnership with HIF1α, may co-regulate immune adaptation to low oxygen.

Myeloid-resident neuropilin-1 promotes choroidal neovascularization while mitigating inflammation.

Age-related macular degeneration (AMD) in its various forms is a leading cause of blindness in industrialized countries. Here, we provide evidence that ligands for neuropilin-1 (NRP1), such as Semaphorin 3A and VEGF-A, are elevated in the vitreous of patients with AMD at times of active choroidal neovascularization (CNV). We further demonstrate that NRP1-expressing myeloid cells promote and maintain CNV. Expression of NRP1 on cells of myeloid lineage is critical for mitigating production of inflammatory factors such as IL6 and IL1ß. Therapeutically trapping ligands of NRP1 with an NRP1-derived trap reduces CNV. Collectively, our findings identify a role for NRP1-expressing myeloid cells in promoting pathological angiogenesis during CNV and introduce a therapeutic approach to counter neovascular AMD.

miR-106b suppresses pathological retinal angiogenesis.

MicroRNAs are small non-coding RNAs that post-transcriptionally regulate gene expression. We recently demonstrated that levels of miR-106b were significantly decreased in the vitreous and plasma of patients with neovascular age-related macular degeneration (AMD). Here we show that expression of the miR-106b-25 cluster is negatively regulated by the unfolded protein response pathway of protein kinase RNA-like ER kinase (PERK) in a mouse model of neovascular AMD. A reduction in levels of miR-106b triggers vascular growth both in vivo and in vitro by inducing production of pro-angiogenic factors. We demonstrate that therapeutic delivery of miR-106b to the retina with lentiviral vectors protects against aberrant retinal angiogenesis in two distinct mouse models of pathological retinal neovascularization. Results from this study suggest that miRNAs such as miR-106b have the potential to be used as multitarget therapeutics for conditions characterized by pathological retinal angiogenesis.

Neuropilin-1 expression in adipose tissue macrophages protects against obesity and metabolic syndrome.

Obesity gives rise to metabolic complications by mechanisms that are poorly understood. Although chronic inflammatory signaling in adipose tissue is typically associated with metabolic deficiencies linked to excessive weight gain, we identified a subset of neuropilin-1 (NRP1)-expressing myeloid cells that accumulate in adipose tissue and protect against obesity and metabolic syndrome. Ablation of NRP1 in macrophages compromised lipid uptake in these cells, which reduced substrates for fatty acid ß-oxidation and shifted energy metabolism of these macrophages toward a more inflammatory glycolytic metabolism. Conditional deletion of NRP1 in LysM Cre-expressing cells leads to inadequate adipose vascularization, accelerated weight gain, and reduced insulin sensitivity even independent of weight gain. Transfer of NRP1+ hematopoietic cells improved glucose homeostasis, resulting in the reversal of a prediabetic phenotype. Our findings suggest a pivotal role for adipose tissue-resident NRP1+-expressing macrophages in driving healthy weight gain and maintaining glucose tolerance.

Senescence-associated secretory phenotype contributes to pathological angiogenesis in retinopathy.

Pathological angiogenesis is the hallmark of diseases such as cancer and retinopathies. Although tissue hypoxia and inflammation are recognized as central drivers of vessel growth, relatively little is known about the process that bridges the two. In a mouse model of ischemic retinopathy, we found that hypoxic regions of the retina showed only modest rates of apoptosis despite severely compromised metabolic supply. Using transcriptomic analysis and inducible loss-of-function genetics, we demonstrated that ischemic retinal cells instead engage the endoplasmic reticulum stress inositol-requiring enzyme 1α (IRE1α) pathway that, through its endoribonuclease activity, induces a state of senescence in which cells adopt a senescence-associated secretory phenotype (SASP). We also detected SASP-associated cytokines (plasminogen activator inhibitor 1, interleukin-6, interleukin-8, and vascular endothelial growth factor) in the vitreous humor of patients suffering from proliferative diabetic retinopathy. Therapeutic inhibition of the SASP through intravitreal delivery of metformin or interference with effectors of senescence (semaphorin 3A or IRE1α) in mice reduced destructive retinal neovascularization in vivo. We conclude that the SASP contributes to pathological vessel growth, with ischemic retinal cells becoming prematurely senescent and secreting inflammatory cytokines that drive paracrine senescence, exacerbate destructive angiogenesis, and hinder reparative vascular regeneration. Reversal of this process may be therapeutically beneficial.

Gut microbiota influences pathological angiogenesis in obesity-driven choroidal neovascularization.

Age-related macular degeneration in its neovascular form (NV AMD) is the leading cause of vision loss among adults above the age of 60. Epidemiological data suggest that in men, overall abdominal obesity is the second most important environmental risk factor after smoking for progression to late-stage NV AMD To date, the mechanisms that underscore this observation remain ill-defined. Given the impact of high-fat diets on gut microbiota, we investigated whether commensal microbes influence the evolution of AMD Using mouse models of NV AMD, microbiotal transplants, and other paradigms that modify the gut microbiome, we uncoupled weight gain from confounding factors and demonstrate that high-fat diets exacerbate choroidal neovascularization (CNV) by altering gut microbiota. Gut dysbiosis leads to heightened intestinal permeability and chronic low-grade inflammation characteristic of inflammaging with elevated production of IL-6, IL-1ß, TNF-α, and VEGF-A that ultimately aggravate pathological angiogenesis.

Truncated netrin-1 contributes to pathological vascular permeability in diabetic retinopathy.

Diabetic retinopathy (DR) is a major complication of diabetes and a leading cause of blindness in the working-age population. Impaired blood-retinal barrier function leads to macular edema that is closely associated with the deterioration of central vision. We previously demonstrated that the neuronal guidance cue netrin-1 activates a program of reparative angiogenesis in microglia within the ischemic retina. Here, we provide evidence in both vitreous humor of diabetic patients and in retina of a murine model of diabetes that netrin-1 is metabolized into a bioactive fragment corresponding to domains VI and V of the full-length molecule. In contrast to the protective effects of full-length netrin-1 on retinal microvasculature, the VI-V fragment promoted vascular permeability through the uncoordinated 5B (UNC5B) receptor. The collagenase matrix metalloprotease 9 (MMP-9), which is increased in patients with diabetic macular edema, was capable of cleaving netrin-1 into the VI-V fragment. Thus, MMP-9 may release netrin-1 fragments from the extracellular matrix and facilitate diffusion. Nonspecific inhibition of collagenases or selective inhibition of MMP-9 decreased pathological vascular permeability in a murine model of diabetic retinal edema. This study reveals that netrin-1 degradation products are capable of modulating vascular permeability, suggesting that these fragments are of potential therapeutic interest for the treatment of DR.

Deficiency in the metabolite receptor SUCNR1 (GPR91) leads to outer retinal lesions.

Age-related macular degeneration (AMD) is a prominent cause of blindness in the Western world. To date, its molecular pathogenesis as well as the sequence of events leading to retinal degeneration remain largely ill-defined. While the invasion of choroidal neovessels in the retina is the primary mechanism that precipitates loss of sight, an earlier dry form precedes it. Here we provide the first evidence for the protective role of the Retinal Pigment Epithelium (RPE)-resident metabolite receptor, succinate receptor 1 (SUCNR1; G-Protein coupled Receptor-91 (GPR91), in preventing dry AMD-like lesions of the outer retina. Genetic analysis of 925 patients with geographic atrophy and 1199 AMD-free peers revealed an increased risk of developing geographic atrophy associated with intronic variants in theSUCNR1 gene. In mice, outer retinal expression of SUCNR1 is observed in the RPE as well as microglial cells and decreases progressively with age. Accordingly, Sucnr1-/- mice show signs of premature sub-retinal dystrophy with accumulation of oxidized-LDL, abnormal thickening of Bruch's membrane and a buildup of subretinal microglia. The accumulation of microglia in Sucnr1-deficient mice is likely triggered by the inefficient clearance of oxidized lipids by the RPE as bone marrow transfer of wild-type microglia into Sucnr1-/- mice did not salvage the patho-phenotype and systemic lipolysis was equivalent between wild-type and control mice. Our findings suggest that deficiency in SUCNR1 is a possible contributing factor to the pathogenesis of dry AMD and thus broaden our understanding of this clinically unmet need.

Neuronal ER stress impedes myeloid-cell-induced vascular regeneration through IRE1α degradation of netrin-1.

In stroke and proliferative retinopathy, despite hypoxia driven angiogenesis, delayed revascularization of ischemic tissue aggravates the loss of neuronal function. What hinders vascular regrowth in the ischemic central nervous system remains largely unknown. Using the ischemic retina as a model of neurovascular interaction in the CNS, we provide evidence that the failure of reparative angiogenesis is temporally and spatially associated with endoplasmic reticulum (ER) stress. The canonical ER stress pathways of protein kinase RNA-like ER kinase (PERK) and inositol-requiring enzyme-1α (IRE1α) are activated within hypoxic/ischemic retinal ganglion neurons, initiating a cascade that results in angiostatic signals. Our findings demonstrate that the endoribonuclease IRE1α degrades the classical guidance cue netrin-1. This neuron-derived cue triggers a critical reparative-angiogenic switch in neural macrophage/microglial cells. Degradation of netrin-1, by persistent neuronal ER stress, thereby hinders vascular regeneration. These data identify a neuronal-immune mechanism that directly regulates reparative angiogenesis.