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.

Splenic monocytes drive pathogenic subretinal inflammation in age-related macular degeneration.

Age-related macular degeneration (AMD) is invariably associated with the chronic accumulation of activated mononuclear phagocytes in the subretinal space. The mononuclear phagocytes are composed of microglial cells but also of monocyte-derived cells, which promote photoreceptor degeneration and choroidal neovascularization. Infiltrating blood monocytes can originate directly from bone marrow, but also from a splenic reservoir, where bone marrow monocytes develop into angiotensin II receptor (ATR1)+ splenic monocytes. The involvement of splenic monocytes in neurodegenerative diseases such as AMD is not well understood. Using acute inflammatory and well-phenotyped AMD models, we demonstrate that angiotensin II mobilizes ATR1+ splenic monocytes, which we show are defined by a transcriptional signature using single-cell RNA sequencing and differ functionally from bone marrow monocytes. Splenic monocytes participate in the chorio-retinal infiltration and their inhibition by ATR1 antagonist and splenectomy reduces the subretinal mononuclear phagocyte accumulation and pathological choroidal neovascularization formation. In aged AMD-risk ApoE2-expressing mice, a chronic AMD model, ATR1 antagonist and splenectomy also inhibit the chronic retinal inflammation and associated cone degeneration that characterizes these mice. Our observation of elevated levels of plasma angiotensin II in AMD patients, suggests that similar events take place in clinical disease and argue for the therapeutic potential of ATR1 antagonists to inhibit splenic monocytes for the treatment of blinding AMD.

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.

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.

Anti-Inflammatory Role of Netrin-4 in Diabetic Retinopathy.

Diabetic retinopathy is characterized by dysfunction of the retinal vascular network, combined with a persistent low-grade inflammation that leads to vision-threatening complications. Netrin-4 (NTN4) is a laminin-related secreted protein and guidance cue molecule present in the vascular basal membrane and highly expressed in the retina. A number of studies inferred that the angiogenic abilities of NTN4 could contribute to stabilize vascular networks and modulate inflammation. Analyzing human specimens, we show that NTN4 and netrin receptors are upregulated in the diabetic retina. We further evaluated a knock-out model for NTN4 undergoing experimental diabetes induced by streptozotocin. We investigated retina function and immune cells in vivo and demonstrated that NTN4 provides a protective milieu against inflammation in the diabetic retina and prevents cytokine production.

Correction to: Lack of netrin-4 alters vascular remodeling in the retina.

The article "Lack of netrin-4 alters vascular remodeling in the retina".

Lack of netrin-4 alters vascular remodeling in the retina.

PURPOSE: Netrin-4 (NTN4) is a protein that plays an important role in the regulation of angiogenesis in the pathological retina. Some evidences show that it can also have a role in inflammation and vascular stability. We will explore these questions in vivo in the mature mouse retina. METHODS: We created a NTN4 knockout that expresses EGFP in mononuclear phagocytes (CSFR1-positive cells) to track inflammation in vivo in the retina by scanning laser ophthalmoscopy (SLO). Fundus angiography permitted to study blood vessels. Retinal function was assessed with electroretinography (ERG). RESULTS: Lack of NTN4 leads to an increased amount of amoeboid mononuclear phagocytes in the adult retina, and blood vessels displayed increased tortuosity when compared with the wildtype. Inner retina function also seemed affected in NTN4 null. Lack of NTN4 resulted in a higher persistence of hyaloid artery and spontaneous leakage in the adult retina. No differences were found regarding vessel bifurcation, vessel width, or vein/artery ratio. CONCLUSIONS: These in vivo data show for the first time that lack of NTN4 induces changes in the retinal vascular phenotype in a non-pathological scenario. This evidence widens the role of NTN4 as a guidance cue in vascular remodeling.

Myeloid cells contribute indirectly to VEGF expression upon hypoxia via activation of Müller cells.

Anti-VEGF-directed therapies have been a milestone for treating retinal vascular diseases. Depletion of monocyte lineage cells suppresses pathological neovascularization in the oxygen-induced retinopathy mouse model. However, the question whether myeloid-derived VEGF-A expression is responsible for the pathogenesis in oxygen-induced retinopathy remained unknown. We analyzed LysMCre-driven myeloid cell-specific VEGF-A knockout mice as well as mice with complete depletion of circulating macrophages through clodronate-liposome treatment in the oxygen-induced retinopathy model by immunohistochemistry, qPCR, and flow cytometry. Furthermore, we analyzed VEGF-A mRNA expression in MIO-M1 cells alone and in co-culture with BV-2 cells in vitro. The myeloid cell-specific VEGF-A knockout did not change relative retinal VEGF-A mRNA levels, the relative avascular area or macrophage/granulocyte numbers in oxygen-induced retinopathy and under normoxic conditions. We observed an insignificantly attenuated pathology in systemically clodronate-liposome treated knockouts but evident VEGF-A expression in activated Müller cells on immunohistochemically stained sections. MIO-M1 cells had significantly higher expression levels of VEGF-A in co-culture with BV-2 cells compared to cultivating MIO-M1 cells alone. Our data show that myeloid-derived cells contribute to pathological neovascularization in oxygen-induced retinopathy through activation of VEGF-A expression in Müller cells.

Spatial distribution of CD115+ and CD11b+ cells and their temporal activation during oxygen-induced retinopathy in mice.

PURPOSE: The model of oxygen-induced retinopathy (OIR) is widely used to analyze pathomechanisms in retinal neovascularization. Previous studies have shown that macrophages (MP) play a key role in vessel formation in OIR, the influence of microglia (MG) having been discussed. The aim of our study was to analyze the spatial and temporal distribution and activation of MP/MG expressing CD115 and CD11b during the process of neovascularization in OIR. METHODS: We used MacGreen mice expressing the green fluorescence protein (GFP) under the promoter for CD115. CD115+ cells were investigated in vivo by scanning laser ophthalmoscopy at postnatal days (P) 17 and 21 in MacGreen mice with OIR (75% oxygen from P7 to P12), and were compared to MacGreen room-air controls. In addition MP/MG were examined ex vivo using immunohistochemistry for CD11b+ detection on retinal flatmounts at P14, P17, and P21 of wild type mice with OIR. RESULTS: In-vivo imaging revealed the highest density of activated MP/MG in tuft areas at P17 of MacGreen mice with OIR. Tufts and regions with a high density of CD115+ cells were detected close to veins, rather to arteries. In peripheral, fully vascularized areas, the distribution of CD115+ cells in MacGreen mice with OIR was similar to MacGreen room-air controls. Correspondingly, immunohistochemical analyses of retinal flatmounts from wild type mice with OIR induction revealed that the number of CD11b+ cells significantly varies between vascular, avascular, and tuft areas as well as between the retinal layers. Activated CD11b+ cells were almost exclusively found in avascular areas and tufts of wild type mice with OIR induction; here, the proportion of activated cells related to the total number of CD11b+ cells remained stable over the course of time. CONCLUSIONS: Using two different approaches to monitor MP/MG cells, our findings demonstrated that MP/MG concentrate within pathologically vascularized areas during OIR. We were able to clarify that reactive changes of CD11b+ cell distribution to OIR primarily occur in the deep retinal layers. Furthermore, we found the highest proportion of activated CD11b+ cells in regions with pathologic neovascularization processes. Our findings support previous reports about activated MP/MG guiding revascularization in avascular areas and playing a key role in the formation and regression of neovascular tufts.

The TetO rat as a new translational model for type 2 diabetic retinopathy by inducible insulin receptor knockdown.

AIMS/HYPOTHESIS: Although the renin-angiotensin system plays an important role in the progression of diabetic retinopathy, its influence therein has not been systematically evaluated. Here we test the suitability of a new translational model of diabetic retinopathy, the TetO rat, for addressing the role of angiotensin-II receptor 1 (AT1) blockade in experimental diabetic retinopathy. METHODS: Diabetes was induced by tetracycline-inducible small hairpin RNA (shRNA) knockdown of the insulin receptor in rats, generating TetO rats. Systemic treatment consisted of an AT1 blocker (ARB) at the onset of diabetes, following which, 4-5 weeks later the retina was analysed in vivo and ex vivo. Retinal function was assessed by Ganzfeld electroretinography (ERG). RESULTS: Retinal vessels in TetO rats showed differences in vessel calibre, together with gliosis. The total number and the proportion of activated mononuclear phagocytes was increased. TetO rats presented with loss of retinal ganglion cells (RGC) and ERG indicated photoreceptor malfunction. Both the inner and outer blood-retina barriers were affected. The ARB treated group showed reduced gliosis and an overall amelioration of retinal function, alongside RGC recovery, whilst no statistically significant differences in vascular and inflammatory features were detected. CONCLUSIONS/INTERPRETATION: The TetO rat represents a promising translational model for the early neurovascular changes associated with type 2 diabetic retinopathy. ARB treatment had an effect on the neuronal component of the retina but not on the vasculature.

Hypertensive retinopathy in a transgenic angiotensin-based model.

Severe hypertension destroys eyesight. The RAS (renin-angiotensin system) may contribute to this. This study relied on an established angiotensin, AngII (angiotensin II)-elevated dTGR (double-transgenic rat) model and same-background SD (Sprague-Dawley) rat controls. In dTGRs, plasma levels of AngII were increased. We determined the general retinal phenotype and observed degeneration of ganglion cells that we defined as vascular degeneration. We also inspected relevant gene expression and lastly observed alterations in the outer blood-retinal barrier. We found that both scotopic a-wave and b-wave as well as oscillatory potential amplitude were significantly decreased in dTGRs, compared with SD rat controls. However, the b/a-wave ratio remained unchanged. Fluorescence angiography of the peripheral retina indicated that exudates, or fluorescein leakage, from peripheral vessels were increased in dTGRs compared with controls. Immunohistological analysis of blood vessels in retina whole-mount preparations showed structural alterations in the retina of dTGRs. We then determined the general retinal phenotype. We observed the degeneration of ganglion cells, defined vascular degenerations and finally found differential expression of RAS-related genes and angiogenic genes. We found the expression of both human angiotensinogen and human renin in the hypertensive retina. Although the renin gene expression was not altered, the AngII levels in the retina were increased 4-fold in the dTGR retina compared with that in SD rats, a finding with mechanistic implications. We suggest that alterations in the outer blood-retinal barrier could foster an area of visual-related research based on our findings. Finally, we introduce the dTGR model of retinal disease.

In vivo analysis of the time and spatial activation pattern of microglia in the retina following laser-induced choroidal neovascularization.

Microglia play a major role in retinal neovascularization and degeneration and are thus potential targets for therapeutic intervention. In vivo assessment of microglia behavior in disease models can provide important information to understand patho-mechanisms and develop therapeutic strategies. Although scanning laser ophthalmoscope (SLO) permits the monitoring of microglia in transgenic mice with microglia-specific GFP expression, there are fundamental limitations in reliable identification and quantification of activated cells. Therefore, we aimed to improve the SLO-based analysis of microglia using enhanced image processing with subsequent testing in laser-induced neovascularization (CNV). CNV was induced by argon laser in MacGreen mice. Microglia was visualized in vivo by SLO in the fundus auto-fluorescence (FAF) mode and verified ex vivo using retinal preparations. Three image processing algorithms based on different analysis of sequences of images were tested. The amount of recorded frames was limiting the effectiveness of the different algorithms. Best results from short recordings were obtained with a pixel averaging algorithm, further used to quantify spatial and temporal distribution of activated microglia in CNV. Morphologically, different microglia populations were detected in the inner and outer retinal layers. In CNV, the peak of microglia activation occurred in the inner layer at day 4 after laser, lacking an acute reaction. Besides, the spatial distribution of the activation changed by the time over the inner retina. No significant time and spatial changes were observed in the outer layer. An increase in laser power did not increase number of activated microglia. The SLO, in conjunction with enhanced image processing, is suitable for in vivo quantification of microglia activation. This surprisingly revealed that laser damage at the outer retina led to more reactive microglia in the inner retina, shedding light upon a new perspective to approach the immune response in the retina in vivo.

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 .

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.

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 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.

An exploratory investigation of brain collateral circulation plasticity after cerebral ischemia in two experimental C57BL/6 mouse models.

Brain collateral circulation is an essential compensatory mechanism in response to acute brain ischemia. To study the temporal evolution of brain macro and microcollateral recruitment and their reciprocal interactions in response to different ischemic conditions, we applied a combination of complementary techniques (T2-weighted magnetic resonance imaging [MRI], time of flight [TOF] angiography [MRA], cerebral blood flow [CBF] imaging and histology) in two different mouse models. Hypoperfusion was either induced by permanent bilateral common carotid artery stenosis (BCCAS) or 60-min transient unilateral middle cerebral artery occlusion (MCAO). In both models, collateralization is a very dynamic phenomenon with a global effect affecting both hemispheres. Patency of ipsilateral posterior communicating artery (PcomA) represents the main variable survival mechanism and the main determinant of stroke lesion volume and recovery in MCAO, whereas the promptness of external carotid artery retrograde flow recruitment together with PcomA patency, critically influence survival, brain ischemic lesion volume and retinopathy in BCCAS mice. Finally, different ischemic gradients shape microcollateral density and size.

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.