Therapeutic targeting of cellular senescence in diabetic macular edema: preclinical and phase 1 trial results.

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 .

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.

Past history of obesity triggers persistent epigenetic changes in innate immunity and exacerbates neuroinflammation.

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 triggers tumoral senescence and renders poorly immunogenic malignancies amenable to senolysis.

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.

Myeloid-resident neuropilin-1 influences brown adipose tissue in obesity.

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.

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.

Pathological angiogenesis in retinopathy engages cellular senescence and is amenable to therapeutic elimination via BCL-xL inhibition.

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.

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.

RELi protocol: Optimization for protein extraction from white, brown and beige adipose tissues.

Global obesity rates have reached pandemic proportions, increasing the risk of metabolic complications for hundreds of millions of individuals worldwide. Gaining insight on adipose tissue biology and understanding how fat pads behave during obesity is critical to investigate metabolic syndromes. Elucidation of cellular signaling pathways engaged by adipose tissue both in health and disease requires standardized protocols for protein extraction that yield consistently pure samples. A recurrent problem of currently available protocols is lipid or detergent contamination in extracted protein samples, which renders protein quantification inaccurate and, as a consequence, consistency and reproducibility of protein loading become unreliable. To overcome this problem, we improved the process of adipose tissue protein extraction by improving tissue lysis and decreasing lipid contamination. Here we describe the Removal of Excess Lipids (RELi) protocol to obtain increased yields of total proteins extracted from adipose tissue. The RELi protocol allows accurate and reproducible adipose tissue sample preparation for Western blot analysis and other investigative techniques requiring adipose tissue-derived proteins.

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.

In Vivo Laser-Mediated Retinal Ganglion Cell Optoporation Using KV1.1 Conjugated Gold Nanoparticles.

Vision loss caused by retinal diseases affects hundreds of millions of individuals worldwide. The retina is a delicate central nervous system tissue stratified into layers of cells with distinct roles. Currently, there is a void in treatments that selectively target diseased retinal cells, and current therapeutic paradigms present complications associated with off-target effects. Herein, as a proof of concept, we introduce an in vivo method using a femtosecond laser to locally optoporate retinal ganglion cells (RGCs) targeted with functionalized gold nanoparticles (AuNPs). We provide evidence that AuNPs functionalized with an antibody toward the cell-surface voltage-gated K+ channel subunit KV1.1 can selectively deliver fluorescently tagged siRNAs or fluorescein isothiocyanate-dextran dye into retinal cells when irradiated with an 800 nm 100 fs laser. Importantly, neither AuNP administration nor irradiation resulted in RGC death. This system provides a novel, non-viral-based approach that has the potential to selectively target retinal cells in diseased regions while sparing healthy areas and may be harnessed in future cell-specific therapies for retinal degenerative diseases.

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.

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.

MicroRNA signatures in vitreous humour and plasma of patients with exudative AMD.

Age-related macular degeneration (AMD) is a leading cause of blindness worldwide affecting individuals over the age of 50. The neovascular form (NV AMD) is characterized by choroidal neovascularization (CNV) and responsible for the majority of central vision impairment. Using non-biased microRNA arrays and individual TaqMan qPCRs, we profiled miRNAs in the vitreous humour and plasma of patients with NV AMD. We identified a disease-associated increase in miR-146a and a decrease in miR-106b and miR-152 in the vitreous humour which was reproducible in plasma. Moreover, miR-146a/miR-106b ratios discriminated patients with NV AMD with an area under the Receiver Operating Characteristic curve (ROC AUC) of 0,977 in vitreous humour and 0,915 in plasma suggesting potential for a blood-based diagnostic. Furthermore, using the AMD Gene Consortium (AGC) we mapped a NV AMD-associated SNP (rs1063320) in a binding site for miR-152-3p in the HLA-G gene. The relationship between our detected miRNAs and NV AMD related genes was also investigated using gene sets derived from the Ingenuity Pathway Analysis (IPA). To our knowledge, our study is the first to correlate vitreal and plasma miRNA signatures with NV AMD, highlighting potential future worth as biomarkers and providing insight on NV AMD pathogenesis.

Treatment Recruitment and Retention of Geriatric Participants With Hoarding Disorder.

This study describes the initial contact of 255 potential participants, recruitment of 63 participants, and retention of 57 participants from three geriatric Hoarding Disorder (HD) studies. Patients with HD were easily recruited from the community, primarily through the use of clinician referrals and posted flyers, as evidenced by steady patient flow despite lack of compensation for participation. Contrary to treatment outcomes of late life mood and anxiety disorders, geriatric HD patients are largely retained in clinical research treatment studies. Results demonstrate that older adults with HD can be engaged in treatment. Participants often needed the study staff to provide substantial support, including informal motivational interviewing and problem solving, in scheduling and attending the initial visit.