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.
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 β-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.
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.
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.
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.
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