Remodeling senescent blood vessels
The retina is a thin layer of nervous tissue at the back of the eye that transforms light into neuronal signals. The retina is essential for vision and is supported ...by networks of blood vessels. In diabetic retinopathy, a common cause of vision loss, these microvessels degenerate and regrow in an aberrant manner. Such degeneration and regrowth can compromise the functioning of retinal nerve cells. Binet
et al.
observed that, after rapid proliferation, vascular endothelial cells in diseased blood vessels engaged molecular pathways linked to cellular senescence (see the Perspective by Podrez and Byzova). Senescent vascular units summoned an inflammatory response in which neutrophils extruded neutrophil extracellular traps onto diseased vessels to remodel them. This endogenous repair mechanism promoted the elimination of senescent blood vessels and could lead to beneficial vascular remodeling.
Science
, this issue p.
eaay5356
; see also p.
919
Remodeling of senescent vascular endothelial cells in the retina is mediated through neutrophil extracellular traps.
INTRODUCTION
Vision provides a critical survival advantage but requires a tight coupling between neuronal energy demands and their vascular supply. The degeneration and consequent aberrant regrowth of retinal vasculature is the hallmark of diseases such as diabetic retinopathy, retinopathy of prematurity, and age-related macular degeneration, which collectively are the most common causes of loss of sight in industrialized countries. Although considerable effort has been devoted to understanding how diseased blood vessels form, relatively little is known of the processes at play during late stages of pathological angiogenesis when blood vessels remodel and subsets of diseased vasculature regress.
RATIONALE
The retina is part of the central nervous system and thus has limited regenerative capacity. A relative exception to this rule are retinal blood vessels, which have a greater propensity to remodel depending on metabolic demand. We investigated the cellular mechanisms activated during the remodeling and regression of pathological blood vessels in retinopathy. We focused on a mouse model of oxygen-induced retinopathy, which has distinct and timed phases of vascular degeneration, neovascularization, and vascular regression. Our findings were verified in human patients with proliferative diabetic retinopathy. Understanding how diseased blood vessels remodel and yield functional networks has the potential to lead to strategies that enhance vascular normalization and helps to explain why retinas in certain patients have the propensity to repair themselves more readily than others.
RESULTS
We found that vascular remodeling in retinopathy is associated with bouts of sterile inflammation and tardy recruitment of neutrophils, an immune population typically associated with a first wave of invading leukocytes. We observed that, after rapid proliferation, vascular endothelial cells in diseased blood vessels engaged molecular pathways shared with aging and cellular damage that lead to cellular senescence. Senescent vascular units then released a secretome of cytokines and factors that attracted neutrophils and triggered the production of neutrophil extracellular traps (NETs). Through extrusion of NETs, neutrophils eliminated diseased senescent vasculature by promoting its apoptosis. By crippling the ability of neutrophils to produce NETs by genetically removing the peptidyl arginine deiminase type IV (PAD4) enzyme, clearance of senescent cells was impaired and regression of pathological angiogenesis compromised. Similar effects were observed with the neutrophil-depleting antibody anti-Ly6G or by pharmacological inhibition of the neutrophil receptor CXCR2.
CONCLUSION
We conclude that neutrophils, through the release of NETs, targeted pathological senescent vasculature for clearance and thus prepare the ischemic retina for reparative vascular regeneration. These findings imply that elimination of senescent blood vessels leads to beneficial vascular remodeling. Although cellular senescence is not necessarily synonymous with aging, our study may provide insight into a general mechanism in which senescent endothelial cells trigger NETosis and predispose to thrombotic events such as myocardial infarction, atherosclerosis, and stroke, which are typically seen in older populations.
Senescent blood vessels trigger neutrophil extracellular traps in retinopathy.
(
A
) Human samples and a mouse model were used to elucidate mechanisms of vascular remodeling in retinopathy. (
B
) Upon rapid proliferation, vascular cells in pathological tufts triggered pathways of cellular senescence, leading to cytokine secretion and the recruitment of neutrophils. (
C
) Factors secreted by senescent cells triggered NETosis. (
D
) NETs promoted the removal of senescent endothelial cells, ultimately leading to regression of pathological angiogenesis and promoting the regeneration of functional vessels.
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.
Attenuating pathological angiogenesis in diseases characterized by neovascularization such as diabetic retinopathy has transformed standards of care. Yet little is known about the molecular ...signatures discriminating physiological blood vessels from their diseased counterparts, leading to off-target effects of therapy. We demonstrate that in contrast to healthy blood vessels, pathological vessels engage pathways of cellular senescence. Senescent (p16INK4A-expressing) cells accumulate in retinas of patients with diabetic retinopathy and during peak destructive neovascularization in a mouse model of retinopathy. Using either genetic approaches that clear p16INK4A-expressing cells or small molecule inhibitors of the anti-apoptotic protein BCL-xL, we show that senolysis suppresses pathological angiogenesis. Single-cell analysis revealed that subsets of endothelial cells with senescence signatures and expressing Col1a1 are no longer detected in BCL-xL-inhibitor-treated retinas, yielding a retina conducive to physiological vascular repair. These findings provide mechanistic evidence supporting the development of BCL-xL inhibitors as potential treatments for neovascular retinal disease.
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•Senescent cells accumulate during diabetic retinopathy•Pathological vasculature engages p16INK4A and BCL-xL•Clearance of p16INK4A-expressing cells suppresses pathological angiogenesis•BCL-xL inhibitor UBX1967 eliminates senescent cells and suppresses neovascularization
Pathological angiogenesis is a cardinal feature of retinopathies and cancer. Crespo-Garcia, Tsuruda, Dejda et al. demonstrate that diseased blood vessels can be discriminated by their propensity to engage pathways of cellular senescence. They show that targeting senescence effector/anti-apoptotic protein BCL-xL suppresses pathological angiogenesis, providing a target for elimination of deregulated neovascularization.
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.
Abstract
The beneficial effects of brown adipose tissue (BAT) on obesity and associated metabolic diseases are mediated through its capacity to dissipate energy as heat. While immune cells, such as ...tissue-resident macrophages, are known to influence adipose tissue homeostasis, relatively little is known about their contribution to BAT function. Here we report that neuropilin-1 (NRP1), a multiligand single-pass transmembrane receptor, is highly expressed in BAT-resident macrophages. During diet-induced obesity (DIO), myeloid-resident NRP1 influences interscapular BAT mass, and consequently vascular morphology, innervation density and ultimately core body temperature during cold exposure. Thus, NRP1-expressing myeloid cells contribute to the BAT homeostasis and potentially its thermogenic function in DIO.
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 IL1β, 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.
Age-related macular degeneration is a prevalent neuroinflammatory condition and a major cause of blindness driven by genetic and environmental factors such as obesity. In diseases of aging, ...modifiable factors can be compounded over the life span. We report that diet-induced obesity earlier in life triggers persistent reprogramming of the innate immune system, lasting long after normalization of metabolic abnormalities. Stearic acid, acting through Toll-like receptor 4 (TLR4), is sufficient to remodel chromatin landscapes and selectively enhance accessibility at binding sites for activator protein-1 (AP-1). Myeloid cells show less oxidative phosphorylation and shift to glycolysis, ultimately leading to proinflammatory cytokine transcription, aggravation of pathological retinal angiogenesis, and neuronal degeneration associated with loss of visual function. Thus, a past history of obesity reprograms mononuclear phagocytes and predisposes to neuroinflammation.
Obesity is a major risk factor for cancer. Conventional thought suggests that elevated adiposity predisposes to heightened inflammatory stress and potentiates tumor growth, yet underlying mechanisms ...remain ill-defined. Here, we show that tumors from patients with a body mass index >35 carry a high burden of senescent cells. In mouse syngeneic tumor models, we correlated a pronounced accretion of senescent cancer cells with poorly immunogenic tumors when mice were subjected to diet-induced obesity (DIO). Highly immunogenic tumors showed lesser senescence burden suggesting immune-mediated elimination of senescent cancer cells, likely targeted as a consequence of their senescence-associated secretory phenotype. Treatment with the senolytic BH3 mimetic small molecule inhibitor ABT-263 selectively stalled tumor growth in mice with DIO to rates comparable to regular diet-fed mice. Thus, consideration of body adiposity in the selection of cancer therapy may be a critical determinant for disease outcome in poorly immunogenic malignancies.
Compromised vascular endothelial barrier function is a salient feature of diabetic complications such as sight-threatening diabetic macular edema (DME). Current standards of care for DME manage ...aspects of the disease, but require frequent intravitreal administration and are poorly effective in large subsets of patients. Here we provide evidence that an elevated burden of senescent cells in the retina triggers cardinal features of DME pathology and conduct an initial test of senolytic therapy in patients with DME. In cell culture models, sustained hyperglycemia provoked cellular senescence in subsets of vascular endothelial cells displaying perturbed transendothelial junctions associated with poor barrier function and leading to micro-inflammation. Pharmacological elimination of senescent cells in a mouse model of DME reduces diabetes-induced retinal vascular leakage and preserves retinal function. We then conducted a phase 1 single ascending dose safety study of UBX1325 (foselutoclax), a senolytic small-molecule inhibitor of BCL-xL, in patients with advanced DME for whom anti-vascular endothelial growth factor therapy was no longer considered beneficial. The primary objective of assessment of safety and tolerability of UBX1325 was achieved. Collectively, our data suggest that therapeutic targeting of senescent cells in the diabetic retina with a BCL-xL inhibitor may provide a long-lasting, disease-modifying intervention for DME. This hypothesis will need to be verified in larger clinical trials. ClinicalTrials.gov identifier: NCT04537884 .
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)-1alpha 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 HIF1alpha is triggered in inflammation. Here, we demonstrate that cells of the innate immune system require activity of the inositol-requiring enzyme 1alpha (IRE1alpha/XBP1) axis in order to initiate HIF1alpha-dependent production of cytokines such as IL1beta, IL6 and VEGF-A. Knockout of either HIF1alpha or IRE1alpha 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 HIF1alpha, may co-regulate immune adaptation to low oxygen. Keywords: HIF1alpha, Retina, Angiogenesis, Inflammation, IRE1alpha, Myeloid, Mononuclear phagocytes, Microglia, Hypoxia, ER stress
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
and
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.