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.

Early-life peripheral infections reprogram retinal microglia and aggravate neovascular age-related macular degeneration in later life.

Pathological neovascularization in age-related macular degeneration (nvAMD) drives the principal cause of blindness in the elderly. While there is a robust genetic association between genes of innate immunity and AMD, genome-to-phenome relationships are low, suggesting a critical contribution of environmental triggers of disease. Possible insight comes from the observation that a past history of infection with pathogens such as Chlamydia pneumoniae, or other systemic inflammation, can predispose to nvAMD in later life. Using a mouse model of nvAMD with prior C. pneumoniae infection, endotoxin exposure, and genetic ablation of distinct immune cell populations, we demonstrated that peripheral infections elicited epigenetic reprogramming that led to a persistent memory state in retinal CX3CR1+ mononuclear phagocytes (MNPs). The immune imprinting persisted long after the initial inflammation had subsided and ultimately exacerbated choroidal neovascularization in a model of nvAMD. Single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) identified activating transcription factor 3 (ATF3) as a central mediator of retina-resident MNP reprogramming following peripheral inflammation. ATF3 polarized MNPs toward a reparative phenotype biased toward production of proangiogenic factors in response to subsequent injury. Therefore, a past history of bacterial endotoxin-induced inflammation can lead to immunological reprograming within CNS-resident MNPs and aggravate pathological angiogenesis in the aging retina.

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.

Neutrophil extracellular traps target senescent vasculature for tissue remodeling in retinopathy.

In developed countries, the leading causes of blindness such as diabetic retinopathy are characterized by disorganized vasculature that can become fibrotic. Although many such pathological vessels often naturally regress and spare sight-threatening complications, the underlying mechanisms remain unknown. Here, we used orthogonal approaches in human patients with proliferative diabetic retinopathy and a mouse model of ischemic retinopathies to identify an unconventional role for neutrophils in vascular remodeling during late-stage sterile inflammation. Senescent vasculature released a secretome that attracted neutrophils and triggered the production of neutrophil extracellular traps (NETs). NETs ultimately cleared diseased endothelial cells and remodeled unhealthy vessels. Genetic or pharmacological inhibition of NETosis prevented the regression of senescent vessels and prolonged disease. Thus, clearance of senescent retinal blood vessels leads to reparative vascular remodeling.

Fibrin fragment E potentiates TGF-ß-induced myofibroblast activation and recruitment.

Fibrin is an essential constituent of the coagulation cascade, and the formation of hemostatic fibrin clots is central to wound healing. Fibrin clots are over time degraded into fibrin degradation products as the injured tissue is replaced by granulation tissue. Our goal was to study the role of the fibrin degradation product fragment E (FnE) in fibroblast activation and migration. We present evidence that FnE is a chemoattractant for fibroblasts and that FnE can potentiate TGF-ß-induced myofibroblast formation. FnE forms a stable complex with αVß3 integrin, and the integrin ß3 subunit is required both for FnE-induced fibroblast migration and for potentiation of TGF-ß-induced myofibroblast formation. Finally, subcutaneous infusion of FnE in mice results in a fibrotic response in the hypodermis. These results support a model where FnE released from clots in wounded tissue promote wound healing and fibrosis by both recruitment and activation of fibroblasts. Fibrin fragment E could thus represent a therapeutic target for treatment of pathological fibrosis.

NOTCH1 signaling induces pathological vascular permeability in diabetic retinopathy.

Diabetic macular edema is a major complication of diabetes resulting in loss of central vision. Although heightened vessel leakiness has been linked to glial and neuronal-derived factors, relatively little is known on the mechanisms by which mature endothelial cells exit from a quiescent state and compromise barrier function. Here we report that endothelial NOTCH1 signaling in mature diabetic retinas contributes to increased vascular permeability. By providing both human and mouse data, we show that NOTCH1 ligands JAGGED1 and DELTA LIKE-4 are up-regulated secondary to hyperglycemia and activate both canonical and rapid noncanonical NOTCH1 pathways that ultimately disrupt endothelial adherens junctions in diabetic retinas by causing dissociation of vascular endothelial-cadherin from ß-catenin. We further demonstrate that neutralization of NOTCH1 ligands prevents diabetes-induced retinal edema. Collectively, these results identify a fundamental process in diabetes-mediated vascular permeability and provide translational rational for targeting the NOTCH pathway (primarily JAGGED1) in conditions characterized by compromised vascular barrier function.

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.

Endoplasmic reticulum stress enhances fibrosis through IRE1α-mediated degradation of miR-150 and XBP-1 splicing.

ER stress results in activation of the unfolded protein response and has been implicated in the development of fibrotic diseases. In this study, we show that inhibition of the ER stress-induced IRE1α signaling pathway, using the inhibitor 4µ8C, blocks TGFß-induced activation of myofibroblasts in vitro, reduces liver and skin fibrosis in vivo, and reverts the fibrotic phenotype of activated myofibroblasts isolated from patients with systemic sclerosis. By using IRE1α(-/-) fibroblasts and expression of IRE1α-mutant proteins lacking endoribonuclease activity, we confirmed that IRE1α plays an important role during myofibroblast activation. IRE1α was shown to cleave miR-150 and thereby to release the suppressive effect that miR-150 exerted on αSMA expression through c-Myb. Inhibition of IRE1α was also demonstrated to block ER expansion through an XBP-1-dependent pathway. Taken together, our results suggest that ER stress could be an important and conserved mechanism in the pathogenesis of fibrosis and that components of the ER stress pathway may be therapeutically relevant for treating patients with fibrotic diseases.

ER Stress and Angiogenesis.

Proper tissue vascularization is vital for cellular function as it delivers oxygen, nutrients, hormones, and immune cells and helps to clear cellular debris and metabolic waste products. Tissue angiogenesis occurs to satisfy energy requirements and cellular sensors of metabolic imbalance coordinate vessel growth. In this regard, the classical pathways of the unfolded protein response activated under conditions of ER stress have recently been described to generate angiomodulatory or angiostatic signals. This review elaborates on the link between angiogenesis and ER stress and discusses the implications for diseases characterized by altered vascular homeostasis, such as cancer, retinopathies, and atherosclerosis.

Identification and characterization of VEGF-A-responsive neutrophils expressing CD49d, VEGFR1, and CXCR4 in mice and humans.

Vascular endothelial growth factor A (VEGF-A) is upregulated during hypoxia and is the major regulator of angiogenesis. VEGF-A expression has also been found to recruit myeloid cells to ischemic tissues where they contribute to angiogenesis. This study investigates the mechanisms underlying neutrophil recruitment to VEGF-A as well as the characteristics of these neutrophils. A previously undefined circulating subset of neutrophils shown to be CD49d(+)VEGFR1(high)CXCR4(high) was identified in mice and humans. By using chimeric mice with impaired VEGF receptor 1 (VEGFR1) or VEGFR2 signaling (Flt-1tk(-/-), tsad(-/-)), we found that parallel activation of VEGFR1 on neutrophils and VEGFR2 on endothelial cells was required for VEGF-A-induced recruitment of circulating neutrophils to tissue. Intravital microscopy of mouse microcirculation revealed that neutrophil recruitment by VEGF-A versus by the chemokine macrophage inflammatory protein 2 (MIP-2 [CXCL2]) involved the same steps of the recruitment cascade but that an additional neutrophil integrin (eg, VLA-4 [CD49d/CD29]) played a crucial role in neutrophil crawling and emigration to VEGF-A. Isolated CD49d(+) neutrophils featured increased chemokinesis but not chemotaxis compared with CD49d(-) neutrophils in the presence of VEGF-A. Finally, by targeting the integrin α4 subunit (CD49d) in a transplantation-based angiogenesis model that used avascular pancreatic islets transplanted to striated muscle, we demonstrated that inhibiting the recruitment of circulating proangiogenic neutrophils to hypoxic tissue impairs vessel neoformation. Thus, angiogenesis can be modulated by targeting cell-surface receptors specifically involved in VEGF-A-dependent recruitment of proangiogenic neutrophils without compromising recruitment of the neutrophil population involved in the immune response to pathogens.

Assessment of vascular regeneration in the CNS using the mouse retina.

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer's, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

Systemic inflammation perturbs developmental retinal angiogenesis and neuroretinal function.

PURPOSE: Perinatal inflammatory stress in preterm babies is associated with increased rates of severe retinopathy of prematurity (ROP) and adverse neurological dysfunction. In this study, we set out to determine the consequences of severe systemic inflammatory stress on developmental retinal vascularization and evaluate the subsequent outcome on retinal function in later life. METHODS: Systemic inflammatory stress was induced in C57BL/6J mouse pups by an intraperitoneal injection of lipopolysaccharide (LPS; 1 mg/kg) at postnatal day 4. In response to LPS, retinal inflammation was confirmed by quantitative RT-PCR analysis of diverse inflammatory markers. A detailed and systematic analysis of retinal microglial infiltration, retinal vascular morphology, density, and growth rate was performed at key time points throughout retinal vascularization. Retinal function in adult life was assessed by using electroretinography at 6 weeks postinjection. RESULTS: As early as 48 hours after intraperitoneal administration of LPS, a significant increase in retinal vascular density was noted throughout the retina. A pronounced increase in the number of activated microglial cell was observed in the retinal ganglion cell layer and in the outer plexiform layer just prior to their vascularization; direct physical contact between activated microglia and sprouting vessels suggested that microglia partake in promoting the aberrant retinal vascularization. With maturity, animals subjected to perinatal inflammatory stress displayed depleted retinal vascular beds and had significantly decreased retinal function as determined by electroretinography. CONCLUSIONS: Our data reveal that early severe postnatal inflammatory stress leads to abnormal retinal vascular development and increased vessel anastomosis and, ultimately, permanently compromises retinal function. The aberrant and initially exaggerated retinal vascularization observed is associated with microglial activation, providing a cellular mechanism by which perinatal sepsis predisposes to ROP.

Neuron-derived semaphorin 3A is an early inducer of vascular permeability in diabetic retinopathy via neuropilin-1.

The deterioration of the inner blood-retinal barrier and consequent macular edema is a cardinal manifestation of diabetic retinopathy (DR) and the clinical feature most closely associated with loss of sight. We provide evidence from both human and animal studies for the critical role of the classical neuronal guidance cue, semaphorin 3A, in instigating pathological vascular permeability in diabetic retinas via its cognate receptor neuropilin-1. We reveal that semaphorin 3A is induced in early hyperglycemic phases of diabetes within the neuronal retina and precipitates initial breakdown of endothelial barrier function. We demonstrate, by a series of orthogonal approaches, that neutralization of semaphorin 3A efficiently prevents diabetes-induced retinal vascular leakage in a stage of the disease when vascular endothelial growth factor neutralization is inefficient. These observations were corroborated in Tg(Cre-Esr1)/Nrp1(flox/flox) conditional knockout mice. Our findings identify a therapeutic target for macular edema and provide further evidence for neurovascular crosstalk in the pathogenesis 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.

Ischemic neurons prevent vascular regeneration of neural tissue by secreting semaphorin 3A.

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) is secreted by hypoxic neurons in the avascular retina in response to the proinflammatory cytokine IL-1ß. Sema3A contributes to vascular decay and later forms a chemical barrier that repels neo-vessels toward the vitreous. Conversely, silencing Sema3A expression enhances normal vascular regeneration within the ischemic retina, thereby diminishing aberrant neovascularization and preserving neuroretinal function. Overcoming the chemical barrier (Sema3A) released by ischemic neurons accelerates the vascular regeneration of neural tissues, which restores metabolic supply and improves retinal function. Our findings may be applicable to other neurovascular ischemic conditions such as stroke.

Interaction between arsenic trioxide (ATO) and human neutrophils.

The cytotoxic effect of arsenic trioxide (ATO) is known to be mediated by its ability to induce cell apoptosis in a variety of cells, including neutrophils. More recently, we demonstrated that ATO induced several parameters involved in endoplasmic reticulum (ER) stress-induced neutrophil apoptosis but that caspase-4 was not involved. The aim of this study was to better understand how neutrophils are activated by ATO and to further demonstrate that ATO is an ER stressor. Human neutrophils were isolated from healthy blood donors and incubated in vitro in the presence or absence of ATO and several parameters were investigated. We found that ATO induced the expression of the proapoptotic GADD153 protein, a key player involved in ER stress-induced apoptosis, activated nuclear nuclear factor κB (NF-κB) DNA binding activities, and increased prostaglandine E2 (PGE2) production. Using an antibody array approach, we found that ATO increased the production of several cytokines, with interleukin 8 (IL-8) being the predominant one. We confirmed that ATO increased the production of IL-8 by enzyme-linked-immunosorbent assay (ELISA). Treatment with a caspase-4 inhibitor did not inhibit IL-8 production. The results of the present study further support the notion that ATO is an ER stressor and that, although its toxic effect is mediated by induction of apoptosis, this chemical also induced, in parallel, NF-κB activation, the production of PGE2 and several cytokines probably involved in other cell functions. Also, we conclude that the production of IL-8 is not induced by a caspase-4-dependent mechanism, suggesting that ATO-induced caspase-4 activation is involved in other as yet unidentified functions in human neutrophils.

Arsenic trioxide induces endoplasmic reticulum stress-related events in neutrophils.

We recently reported that the endoplasmic reticulum (ER)-induced cell pathway of apoptosis is operational in human neutrophils and that some ER stressors can accelerate this process. Recent data suggest that arsenic trioxide (As(2)O(3) or ATO), may also act as an ER stressor. The aims of the present study were to elucidate if other ER stress-related events occur in ATO-induced neutrophils, and to determine the role of caspase-4 in the proapoptotic activity of ATO. We found that ATO induced ubiquitination of proteins, and increased calcium concentration and gene expression of calcineurin in neutrophils. In addition to caspase-4, activities of caspase-3, -8 and -9 were increased by ATO. The processing of caspase-4 was reversed by a caspase-8 inhibitor, indicating that caspase-4 activation requires the action of upstream initiator components, questioning on the role of caspase-4 in ATO-induced ER stress-mediated cell apoptosis. Using caspase-4 deficient THP-1 cells, we demonstrated that the proapoptotic effect of ATO was similar to that of control caspase-4-positive cells. We conclude that ATO is an ER stressor that can induce cell apoptosis by a mechanism which does not require caspase-4. In addition, we conclude that caspase-4 activation in ATO-induced neutrophils could be involved in functions other than apoptosis.