Vascular Stability Research Articles

Pericyte Insulin Receptors Modulate Retinal Vascular Remodeling and Endothelial Angiopoietin Signaling.

Pericytes regulate vascular development, stability, and quiescence; their dysfunction contributes to diabetic retinopathy. To explore the role of insulin receptors in pericyte biology, we created pericyte insulin receptor knockout mice (PIRKO) by crossing PDGFRβ-Cre mice with insulin receptor (Insr) floxed mice. Their neonatal retinal vasculature exhibited perivenous hypervascularity with venular dilatation, plus increased angiogenic sprouting in superficial and deep layers. Pericyte coverage of capillaries was unaltered in perivenous and periarterial plexi, and no differences in vascular regression or endothelial proliferation were apparent. Isolated brain pericytes from PIRKO had decreased angiopoietin-1 mRNA, whereas retinal and lung angiopoietin-2 mRNA was increased. Endothelial phospho-Tie2 staining was diminished and FoxO1 was more frequently nuclear localized in the perivenous plexus of PIRKO, in keeping with reduced angiopoietin-Tie2 signaling. Silencing of Insr in human brain pericytes led to reduced insulin-stimulated angiopoietin-1 secretion, and conditioned media from these cells was less able to induce Tie2 phosphorylation in human endothelial cells. Hence, insulin signaling in pericytes promotes angiopoietin-1 secretion and endothelial Tie2 signaling and perturbation of this leads to excessive vascular sprouting and venous plexus abnormalities. This phenotype mimics elements of diabetic retinopathy, and future work should evaluate pericyte insulin signaling in this disease.

A systems biology model of junctional localization and downstream signaling of the Ang\u2013Tie signaling pathway

The Ang–Tie signaling pathway is an important vascular signaling pathway regulating vascular growth and stability. Dysregulation in the pathway is associated with vascular dysfunction and numerous diseases that involve abnormal vascular permeability and endothelial cell inflammation. The understanding of the molecular mechanisms of the Ang–Tie pathway has been limited due to the complex reaction network formed by the ligands, receptors, and molecular regulatory mechanisms. In this study, we developed a mechanistic computational model of the Ang–Tie signaling pathway validated against experimental data. The model captures and reproduces the experimentally observed junctional localization and downstream signaling of the Ang–Tie signaling axis, as well as the time-dependent role of receptor Tie1. The model predicts that Tie1 modulates Tie2’s response to the context-dependent agonist Ang2 by junctional interactions. Furthermore, modulation of Tie1’s junctional localization, inhibition of Tie2 extracellular domain cleavage, and inhibition of VE-PTP are identified as potential molecular strategies for potentiating Ang2’s agonistic activity and rescuing Tie2 signaling in inflammatory endothelial cells.

New mechanism-based approaches to treating and evaluating the vasculopathy of scleroderma.

Utilizing recent insight into the vasculopathy of scleroderma (SSc), the review will highlight new opportunities for evaluating and treating the disease by promoting stabilization and protection of the microvasculature. Endothelial junctional signaling initiated by vascular endothelial-cadherin (VE-cadherin) and Tie2 receptors, which are fundamental to promoting vascular health and stability, are disrupted in SSc. This would be expected to not only diminish their protective activity, but also increase pathological processes that are normally restrained by these signaling mediators, resulting in pathological changes in vascular function and structure. Indeed, key features of SSc vasculopathy, from the earliest signs of edema and puffy fingers to pathological disruption of hemodynamics, nutritional blood flow, capillary structure and angiogenesis are all consistent with this altered endothelial signaling. It also likely contributes to further progression of the disease including tissue fibrosis, and organ and tissue injury. Restoring protective endothelial junctional signaling should combat the vasculopathy of SSc and prevent further deterioration in vascular and organ function. Indeed, this type of targeted approach has achieved remarkable results in preclinical models for other diseases. Furthermore, tracking this endothelial junctional signaling, for example by assessing vascular permeability, should facilitate insight into disease progression and its response to therapy.

Interferon-Tau regulates a plethora of functions in the corpus luteum

The corpus luteum (CL) plays a vital role in regulating the reproductive cycle, fertility, and in maintaining pregnancy. Interferon-tau (IFNT) is the maternal recognition of a pregnancy signal in domestic ruminants; its uterine, paracrine actions, which extend the CL lifespan, are widely established. However, considerable evidence also suggests a direct, endocrine role for IFNT. The purpose of this review is to highlight the importance of IFNT in CL maintenance, acting directly and in a cell-specific manner. A transcriptomic study revealed a distinct molecular profile of IFNT-exposed day 18, pregnant bovine CL, compared to the non-pregnant gland. A substantial fraction of the differentially expressed genes was downregulated, many of which are known to be elevated by prostaglandin F2A (PGF2A). In vitro, IFNT was found to mimic changes observed in the luteal transcriptome of early pregnancy. Key luteolytic genes such as endothelin-1 (EDN1), transforming growth factor-B1 (TGFB1), thrombospondins (THBSs) 1&2 and serpine-1 (SERPINE1) were downregulated in luteal endothelial cells. Luteal steroidogenic large cells (LGCs) were also found to be a target for the antilutelotytic actions of IFNT. IFNT-treated LGCs showed a significant reduction in the expression of the proapoptotic, antiangiogenic THBS1&2, as well as TGFBR1 and 2. Furthermore, IFNT was shown to be a potent survival factor for luteal cells in vivo and in vitro, activating diverse pathways to promote cell survival while suppressing cell death signals. Pentraxin 3 (PTX3), robustly upregulated by IFNT in various luteal cell types, mediated many of the prosurvival effects of IFNT in LGCs. A novel reciprocal inhibitory crosstalk between PTX3 and THBS1 lends further support to their respective survival and apoptotic actions in the CL. Even though IFNT did not directly regulate progesterone synthesis, it could maintain its concentrations, by increasing luteal cell survival and by supporting vascular stabilization. The direct effects of IFNT in the CL, enhancing cell survival and vasculature stabilization while curbing luteolytic activities, may constitute an important complementary branch leading to the extension of the luteal lifespan during early pregnancy.

RNF213 and GUCY1A3 in Moyamoya Disease: Key Regulators of Metabolism, Inflammation, and Vascular Stability.

Moyamoya disease is an idiopathic chronically progressive cerebrovascular disease, which causes both ischemic and hemorrhagic stroke. Genetic studies identified RNF213/Mysterin and GUCY1A3 as disease-causing genes. They were also known to be associated with non-moyamoya intracranial large artery disease, coronary artery disease and pulmonary artery hypertension. This review focused on these two molecules and their strong linker, calcineurin/NFAT signaling and caveolin to understand the pathophysiology of moyamoya disease and related vascular diseases. They are important regulators of lipid metabolism especially lipotoxicity, NF-κB mediated inflammation, and nitric oxide-mediated vascular protection. Although intimal thickening with fibrosis and damaged vascular smooth muscle cells are the distinguishing features of moyamoya disease, origin of the fibrous tissue and the mechanism of smooth muscle cell damages remains not fully elucidated. Endothelial cells and smooth muscle cells have long been a focus of interest, but other vascular components such as immune cells and extracellular matrix also need to be investigated in future studies. Molecular research on moyamoya disease would give us a clue to understand the mechanism preserving vascular stability.

An engineered tetra-valent antibody fully activates the Tie2 receptor with comparable potency to its natural ligand angiopoietin-1

Activation of the tyrosine kinase with Ig and epidermal growth factor homology domain 2 (Tie2) receptor by angiopoietin-1 (Ang1) is critical for vascular stabilization: it promotes survival signal transduction via auto-phosphorylation and reduces vascular permeability by strengthening tight junctions between endothelial cells. Thus, Tie2/Ang1 signaling is a promising therapeutic target for vascular diseases. However, in vivo use of existing Tie2 signaling modulators, such as recombinant Ang1, is restricted by limitations in manufacturability and stability. Here, we present a novel engineered tetra-valent agonistic antibody, ASP4021, which can specifically and fully activate the Tie2 receptor in an equivalent manner to Ang1. ASP4021 induced Tie2 self-phosphorylation and inhibited apoptosis in a human primary endothelial cell line. Additionally, single administration of ASP4021 significantly suppressed mustard-oil-induced vascular permeability in rats. ASP4021 may thus be a potential therapeutic candidate for diseases associated with vascular weakness such as diabetic retinopathy, diabetic macular edema and critical limb ischemia.

Morphological characterization of Etv2 vascular explants using fractal analysis and atomic force microscopy

The rapid engraftment of vascular networks is critical for functional incorporation of tissue explants. However, existing methods for inducing angiogenesis utilize approaches that yield vasculature with poor temporal stability or inadequate mechanical integrity, which reduce their robustness in vivo. The transcription factor Ets variant 2 (Etv2) specifies embryonic hematopoietic and vascular endothelial cell (EC) development, and is transiently reactivated during postnatal vascular regeneration and tumor angiogenesis. This study investigates the role for Etv2 upregulation in forming stable vascular beds both in vitro and in vivo. Control and Etv2+ prototypical fetal-derived human umbilical vein ECs (HUVECs) and adult ECs were angiogenically grown into vascular beds. These vessel beds were characterized using fractal dimension and lacunarity, to quantify their branching complexity and space-filling homogeneity, respectively. Atomic force microscopy (AFM) was used to explore whether greater complexity and homogeneity lead to more mechanically stable vessels. Additionally, markers of EC integrity were used to probe for mechanistic clues. Etv2+ HUVECs exhibit greater branching, vessel density, and structural homogeneity, and decreased stiffness in vitro and in vivo, indicating a greater propensity for stable vessel formation. When co-cultured with colon tumor organoid tissue, Etv2+ HUVECs had decreased fractal dimension and lacunarity compared to Etv2+ HUVECs cultured alone, indicating that vessel density and homogeneity of vessel spacing increased due to the presence of Etv2. This study sets forth the novel concept that fractal dimension, lacunarity, and AFM are as informative as conventional angiogenic measurements, including vessel branching and density, to assess vascular perfusion and stability.

Circulating progenitor cells are associated with plaque progression and long-term outcomes in heart transplant patients.

Circulating progenitor cells (CPCs) play a role in vascular repair and plaque stability, while osteocalcin (OC) expressing CPCs have been linked to unstable plaque and adverse cardiovascular outcomes. However, their role in cardiac allograft vasculopathy (CAV) has not been elucidated. This cohort study aimed to investigate the contribution of CPCs on CAV progression and cardiovascular events after heart transplantation. A total of 80 heart transplant patients (mean age 55 ± 14 years, 72% male) undergoing annual intravascular ultrasound (IVUS) had fresh CPCs marked by CD34, CD133, and OC counted in peripheral blood using flow cytometry, on the same day as baseline IVUS. CAV progression was assessed by IVUS as the change (Δ) in plaque volume divided by segment length (PV/SL), adjusted for the time between IVUS measurements [median 3.0, interquartile range (2.8-3.1) years] and was defined as ΔPV/SL that is above the median ΔPV/SL of study population. Major adverse cardiac events (MACEs) were defined as any incident of revascularization, myocardial infarction, heart failure admission, re-transplantation, stroke, and death. Patients with higher CD34+CD133+ CPCs had a decreased risk of CAV progression [odds ratio 0.58, 95% confidence interval (CI) (0.37-0.92), P = 0.01] and MACE [hazard ratio (HR) 0.79, 95% CI (0.66-0.99), P = 0.05] during a median (interquartile range) follow-up of 8.0 years (7.2-8.3). Contrarily, higher OC+ cell counts were associated with an increased risk of MACE [HR 1.26, 95% CI (1.03-1.57), P = 0.02]. Lower levels of CD34+CD133+ CPCs are associated with plaque progression and adverse long-term outcomes in patients who underwent allograft heart transplantation. In contrast, higher circulating OC+ levels are associated with adverse long-term outcomes. Thus, CPCs might play a role in amelioration of transplant vasculopathy, while OC expression by these cells might play a role in progression.

The Emerging Roles of Pericytes in Modulating Tumor Microenvironment.

Pericytes (PCs), known as mural cells, play an important blood vessel (BV) supporting role in regulating vascular stabilization, permeability and blood flow in microcirculation as well as blood brain barrier. In carcinogenesis, defective interaction between PCs and endothelial cells (ECs) contributes to the formation of leaky, chaotic and dysfunctional vasculature in tumors. However, recent works from other laboratories and our own demonstrate that the direct interaction between PCs and other stromal cells/cancer cells can modulate tumor microenvironment (TME) to favor cancer growth and progression, independent of its BV supporting role. Furthermore, accumulating evidence suggests that PCs have an immunomodulatory role. In the current review, we focus on recent advancement in understanding PC’s regulatory role in the TME by communicating with ECs, immune cells, and tumor cells, and discuss how we can target PC’s functions to re-model TME for an improved cancer treatment strategy.

Vascular Endothelial Growth Factor Antagonists: Promising Players in the Treatment of Neovascular Age-Related Macular Degeneration.

Neovascular age-related macular degeneration (nAMD) treatment has been revolutionized by the introduction of vascular endothelial growth factor antagonists (anti-VEGF), but the need for frequent intravitreal injections poses a heavy burden to patients and physicians. Evolving anti-VEGF therapies include longer duration agents, approaches that target multiple pathways, topical anti-VEGF agents, sustained-release, and genetic therapies. Abicipar pegol, a designed ankyrin repeat protein (DARPin), demonstrated the ability to maintain stable visual acuity with 12-week dosing, but was not approved by the FDA due to higher than usual rates of intraocular inflammation. Conbercept, a recombinant anti-VEGF fusion protein, has been approved in China, and is in Phase 3 trials globally. KSI-301 is an anti-VEGF antibody biopolymer conjugate that allowed 66% of nAMD patients to maintain at least a 6-month treatment-free interval in Phase 1b studies. OPT-302, an inhibitor of VEGF-C/D, will be tested in phase 3 studies that compare anti-VEGF-A monotherapy against combination therapy with OPT-302. Faricimab is a bispecific anti-VEGF/Ang-2 antibody that upregulates the Tie-2 signaling pathway and promotes vascular stability; it is undergoing phase 3 trials with potential for 12- or 16-week dosing. PAN-90806 is a topical anti-VEGF agent that showed the ability to reduce injection frequency by 79% compared to ranibizumab monotherapy in a phase 1/2a trial. Sustained-release anti-VEGF therapies include the ranibizumab Port Delivery System (in phase 3 studies), GB-102 (Phase 2b), OTX-TKI (phase 1), and Durasert (preclinical). Suprachoroidal delivery of the tyrosine kinase inhibitor, axitinib, is in preclinical studies. Genetic therapies in phase 1 studies include RGX-314 and ADVM-022, which introduce a viral vector that modifies the retina’s cellular apparatus to create an anti-VEGF biofactory, potentially serving as a one-time treatment. Further investigation is warranted for drugs and delivery systems that hope to advance visual outcomes and reduce treatment burden of nAMD.

A twin herbal formula has gained sufficient clinical and laboratory evidences to be accepted as an effective cardio-vascular protective supplement

A two herbs formula has been successfully used as a cardiovascular supplement (tonic) to maintain a healthy vascular endothelial state in four groups of patients presenting with different symptom manifestations: coronary obstruction; hypertension; menopausal cardiovascular instability; and peripheral arterial disease. In parallel with the four clinical trials carried out separately in over ten years, laboratory platform studies to find out the mechanisms of action of the herbal formula have been carried out. The bioactivity tests demonstrated the general efficacy of the formula as being anti-inflammatory and anti-oxidative; while in addition, specific influences on vascular relaxation, cardiac protection, endothelial stability and blood lipid cholesterol adjustments were demonstrated.

Thymosin β4 protects against aortic aneurysm via endocytic regulation of growth factor signaling.

Vascular stability and tone are maintained by contractile smooth muscle cells (VSMCs). However, injury-induced growth factors stimulate a contractile-synthetic phenotypic modulation which increases susceptibility to abdominal aortic aneurysm (AAA). As a regulator of embryonic VSMC differentiation, we hypothesized that Thymosin β4 (Tβ4) may function to maintain healthy vasculature throughout postnatal life. This was supported by the identification of an interaction with low density lipoprotein receptor related protein 1 (LRP1), an endocytic regulator of platelet-derived growth factor BB (PDGF-BB) signaling and VSMC proliferation. LRP1 variants have been implicated by genome-wide association studies with risk of AAA and other arterial diseases. Tβ4-null mice displayed aortic VSMC and elastin defects that phenocopy those of LRP1 mutants, and their compromised vascular integrity predisposed them to Angiotensin II–induced aneurysm formation. Aneurysmal vessels were characterized by enhanced VSMC phenotypic modulation and augmented PDGFR-β signaling. In vitro, enhanced sensitivity to PDGF-BB upon loss of Tβ4 was associated with dysregulated endocytosis, with increased recycling and reduced lysosomal targeting of LRP1–PDGFR-β. Accordingly, the exacerbated aneurysmal phenotype in Tβ4-null mice was rescued upon treatment with the PDGFR-β antagonist Imatinib. Our study identifies Tβ4 as a key regulator of LRP1 for maintaining vascular health, and provides insights into the mechanisms of growth factor–controlled VSMC phenotypic modulation underlying aortic disease progression.

115 An Audit on Dexamethasone Prescribing Practice on Surgically Managed Metastatic Spinal Cord Compression Patients in the Manchester Centre for Clinical Neurosciences: Lessons Learned in Closing the Loop

Abstract Metastatic Spinal Cord Compression is a surgical emergency owing to its propensity to cause significant morbidity and mortality. Dexamethasone is prescribed to reduce oedema and promote vascular membrane stabilization. The is done so until a more definitive management has been performed. This is a retrospective study evaluating the steroid-prescribing practice for MSCC patients in 2018 at a tertiary spinal unit in Greater Manchester. Thirty-three patients who underwent stabilisation were evaluated in terms of both steroid prescribing and weaning practices. Standards used against for both were the NICE guidelines and the Edinburgh/Christie’s guidelines. Of the 33 patients evaluated, only 1 patient received the correct loading dose of 16mg, whilst 27 went straight to being given 8mg BD. 5 did not receive dexamethasone at all. In terms of discontinuation/weaning, 26 were weaned accordingly and 2 were not weaned to continue as adjuvant treatment. Dexamethasone prescribing practises has since improved over the past two years. 93% of the patients have been correctly weaned as compared to 38% in the past. In terms of prescription, much needs to be done as almost all the patients evaluated did not get the correct loading dose despite receiving the appropriate maintenance.

Computational Systems Biology Modeling of the Angiopoietin‐Tie Signaling Pathway and its Crosstalk with α5β1 Integrin in Endothelial Cells

The Angiopoietin (Ang)-Tie signaling pathway is a major signaling pathway regulating vascular growth (angiogenesis), stability, and permeability. Dysregulation in the pathway is known to lead to vascular dysfunction related to many diseases, including cardiovascular diseases, systemic inflammation, diabetic macular edema, and cancer. Integrin α5β1 has been demonstrated to play an important role in regulating Ang-Tie and its downstream signaling. The understanding of the molecular mechanisms of the α5β1/Ang-Tie crosstalk has been limited due to the complexity of the reaction network formed by the ligands, receptors, integrin interactions, and the molecular regulatory mechanisms. The present study uses a mechanistic computational model of the Ang-Tie signaling pathway and its crosstalk with α5β1 to demonstrate the ability of integrin α5β1 to modulate Tie2 activation by affecting the junctional localization of the co-receptor Tie1. The model provides a mechanistic explanation of how AXT107, an α5β1-modulating peptide, promotes the Tie2-activation function of Ang2 and predicts that simultaneously targeting α5β1 and VE-PTP can synergistically enhance the activation of Tie2 in endothelial cells in pathological states. The model is also used to make quantitative prediction of the effects of simultaneously targeting various aspects of the α5β1/Ang-Tie crosstalk, including the integrin, VE-PTP, and Ang2.

Interstitial Collagen IV: A template for pericyte recruitment

During an acute ischemic stroke, the integrity of the cerebrovascular wall is compromised (Hu et al. 2017). This specialized vessel barrier is formed through coordination between endothelial cells (ECs) and pericytes (PCs) and involves extracellular matrix (ECM) proteins comprising the vascular basement membrane (vBM). A component of the vBM, Type IV Collagen (Col-IV), modulates cell adhesion and migration with the Col4a1 subunit in particular regulating angiogenic remodeling. Aberrant Col4a1 expression in humans has been linked to diseases such as stroke (Alamowitch et al. 2009). Herein, we utilize a murine embryonic stem cell (ESC) model to investigate how perturbations in Col-IV deposition, and specifically with respect to Col4a1, impacts PC-EC interactions during vascular development. Col-IV was targeted by (i) administering α,α-dipyridyl (an iron chelator that disrupts collagen fibril formation, Ikeda et al. 1992) or (ii) plasmid transfection (pLKO.1) with shRNA insert targeting Col4a1 (pLKO-Col4a1; MISSION shRNA Plasmid DNA, Sigma). This study aims to inform rational drug design for promoting cerebrovascular stability post-acute ischemic stroke. In the current study, we administered α,α-dipyridyl on days 6 and 8 of ESC differentiation into primitive vascular structures and cultured these vessel networks for 10 days. We quantified confocal images of vessels and Col-IV content, finding a ~20% increase in Col-IV density, with a 5% increase in EC networks with the 25µM dose. Application of a 50µM dose, however, yielded a ~60% decrease in Col-IV and EC densities, when compared to time-matched, untreated controls. The 25µM dose did not appear to affect ESC-derived vessel formation but altered PC recruitment to the emerging endothelium. Additionally, we observed that a subset of cells in these cultures appeared to reactively overproduce Col-IV with the 25µM dose, presumably to prevent vascular degradation. The majority of Col-IV signal appeared to be associated with EC in all scenarios, with a reduction in the PC reporter signal (Ng2:DsRed) when α,α-dipyridyl was administered, suggesting that interstitial Col-IV (i.e. Col-IV beyond the vessel wall) may contribute to successful PC recruitment. In separate experiments, we transfected ESCs with a pLKO.1-Col4a1 plasmid 7 days after differentiation and cultured ESC-derived vessels for 8 days. Quantification of confocal images revealed a 19.3% reduction in Col-IV content and a 93.6% reduction in Col4a1 via qRT-PCR. The Col-IV reduction do not appear to be spatially localized to ECs, indicating that interstitial Col-IV is more likely to be reduced first following transfection. Furthermore, we observed a reduction in the PC reporter signal upon the pLKO.1-Col4a1 transfection, further suggesting that interstitial Col-IV may be involved in PC recruitment to vessel ECs. Next steps include testing lower α,α-dipyridyl doses (5, 10, 15, & 20µM) and Col4a1 targets (using different pLKO.1-Col4a1 plasmids) to establish conditions in which interstitial Col-IV is reduced at different levels and EC-associated Col-IV can also be reduced. We will also pursue similar experiments with a focus on Col3a1 as our lab and others have found that Col3a1 may be relevant to vascular stability (Frank et al. 2015) and PC recruitment mechanisms.

Characterization of p38 Inflammatory Response in Human Brain Vascular Pericyte Cells

G protein-coupled receptor (GPCR) mediated mitogen activated protein kinase (MAPK) p38 activation is a crucial regulator of pro-inflammatory and pro-angiogenic responses. Prior studies have revealed an atypical pathway for p38 activation that induces auto-phosphorylation of p38 through the direct binding of transforming growth factor β activated kinase 1 binding protein-1 (TAB1) in endothelial cells. During angiogenesis and inflammation, endothelial cells are known for their rapid proliferation, migration, and expression of cytokines. Pericyte cells serve to guide these endothelial cells, modulating vascular stability and orchestrating vascular inflammatory responses. During inflammation, pericytes migrate away from the vasculature leaving endothelial tubules exposed and fragile. Atypical p38 activation has not yet been examined in pericyte cells. We hypothesize that atypical p38 signaling regulates G protein-coupled receptor-mediated vascular function through control of pericyte signaling. Our preliminary data shows for the first time that a family of GPCR ligands, including thrombin and histamine, can activate p38 via the atypical p38 signaling pathway in primary human brain vascular pericytes. Chemical inhibition and siRNA depletion of TAB1-TAB2 abrogates inflammatory signaling by brain pericyte cells. As the role of GPCR induced p38 signaling in pericytes has not been examined, our current studies focus on defining this role, including establishing a co-cultured model of pericyte and endothelial cells. Using this model, we are the first to define atypical p38 signaling in pericyte cytokine expression, migration, and endothelial vascular formation. These studies represent an unexplored branch of neurovascular inflammatory signaling, which we predict is a critical driver of neurovascular disease.

VEGFA165 gene therapy ameliorates blood-labyrinth barrier breakdown and hearing loss.

Millions of people are affected by hearing loss. Hearing loss is frequently caused by noise or aging and often associated with loss of pericytes. Pericytes populate the small vessels in the adult cochlea. However, their role in different types of hearing loss is largely unknown. Using an inducible and conditional pericyte depletion mouse model and noise-exposed mouse model, we show that loss of pericytes leads to marked changes in vascular structure, in turn leading to vascular degeneration and hearing loss. In vitro, using advanced tissue explants from pericyte fluorescence reporter models combined with exogenous donor pericytes, we show that pericytes, signaled by VEGF isoform A165 (VEGFA165), vigorously drive new vessel growth in both adult and neonatal mouse inner ear tissue. In vivo, the delivery of an adeno-associated virus serotype 1–mediated (AAV1–mediated) VEGFA165 viral vector to pericyte-depleted or noise-exposed animals prevented and regenerated lost pericytes, improved blood supply, and attenuated hearing loss. These studies provide the first clear-cut evidence that pericytes are critical for vascular regeneration, vascular stability, and hearing in adults. The restoration of vascular function in the damaged cochlea, including in noise-exposed animals, suggests that VEGFA165 gene therapy could be a new strategy for ameliorating vascular associated hearing disorders.

Microvascular cells: A special focus on heterogeneity of pericytes in diabetes associated complications.

Pericytes (PC) are microvascular mural cells that make specific cell-to-cell contacts with the endothelial cells (EC). These cells are obligatory constituents of the microvessels including the retinal vasculature and they serve as regulators of vascular development, stabilization, maturation and remodeling. During early stages of diabetic retinopathy (DR), apoptotic loss of PC surrounding the retinal vasculature occurs. This may lead to reduced vessel stability, the onset of EC apoptosis, and subsequent retinal ischemia leading to angiogenesis and eventually, severe vision loss due to late proliferative diabetic retinopathy (PDR). Similarly, diabetic nephropathy (DN) is a chronic kidney disease due to hyperglycemia that particularly affects renal PC. Chronic high blood glucose level causes migration of peritubular PC away from the capillary into the interstitial space, which destabilizes the micro vessels, resulting in microvascular rarefaction. In both diabetes associated complications, the identification of specific biomarkers is necessary to stabilize the PC at an early stage. This review largely covers the importance of PC towards the pathogenesis of diabetes associated complications, and their heterogeneity in healthy and angiogenic vasculature.

Abstract P729: Glp-1 Receptor Nitration Contributes to Brain Pericytes Dysfunction in Diabetes

Brain pericytes are unique, multi-functional mural cells located at the center of the NVU. Pericytes maintain the blood-brain barrier (BBB), promote vascular stability, and control the capillary blood flow. We have previously shown that diabetes alters the functions of the pericytes causing BBB damage and increased cerebrovascular pathological neovascularization. GLP-1 agonists, used in the management of diabetes, showed vascular protective effects. However, the underlying mechanisms are unclear. We hypothesis that GLP-1 receptor nitration contributes to the loss of pericytes functions in diabetes. Methods: Expression of GLP-1R in pericytes was assessed in the brain of control and diabetic mice and human brain microvascular pericytes using IHC, RT-PCR, and Western blots. BBB was assessed using Western blot/IHC of Occludin, VEGF, and MMPs gelatinase. Vascular integrity was determined by measuring cerebrovascular neovascularization indices (vascular density, tortuosity, and branching density). GLP-1R nitration was determined by immunoprecipitation and slot blotting. Results: In the present study, we provide novel evidence that the human brain pericytes express the GLP-1R (P<0.05). Moreover, diabetes increased GLP-1R expression in pericytes (P<0.05). Diabetes caused BBB dysfunction, loss of cerebrovascular integrity, and increased all pathological neovascularization indices (P<0.05). Diabetes increased nitration of GLP-1R. Treatment of pericytes with Exendin-4, a GLP-1 agonist, under diabetic conditions, decreased diabetes-induced inflammation, oxidative stress, and pericytes migration, which ameliorated the integrity of the BBB, and prevented the pathological neovascularization (P<0.05). Conclusion: Our results provide novel evidence that pericytes express GLP-1R and that diabetes increased receptor expression and nitration. Treatment of pericytes with GLP-1R agonist exerts neurovascular protective effects, making it a promising strategy in preventing diabetes- mediated cerebrovascular microangiopathy.

Abstract PS17-51: Angiogenesis in a bulk tumor is associated with cancer immunity and metastasis

Abstract Angiogenesis is included in one of the hallmarks of cancer because it not only provides conduit to supply oxygen and nutrients to the cancer cells, but also deliver molecules that confer immune resistance, cell adhesion proteins, and other various growth factors. We hypothesized that angiogenetic score that accumulate 200-related genes will accurately grasp angiogenesis in bulk tumor that enable as to assess clinical relevance of angiogenesis in breast cancer. Angiogenesis pathway score was defined using the molecular Signatures Database Hallmark angiogenesis gene set. Median was used to divide into high and low score groups within each cohort. High angiogenesis score is associated with high expression of Vascular endothelial growth factor (VEGF)- (VEGFA/B, VEGFR1/2/3; p = 0.094, < 0.001, < 0.001, < 0.001, < 0.001, respectively), Endothelial cell surface marker- (CD31 and VWF; all p < 0.001), Vascular stability- (TIE1/2, ANGPT1/2, VE-Cadherin, Claudin 5 and JAM2; all p < 0.001), Hypoxia- (HIF1A/1B; p < 0.001 and 0.004), and Shlingosine-1-phosphate (S1P)- (SphK1/2, S1PR1, SPNS2; all p < 0.001) related genes in TCGA cohort. These results were validated by METABRIC cohort. Even though none of the angiogenesis-related genes analyzed were included in the angiogenesis pathway score except for VEGFA. Although angiogenesis score was not associated with aggressive clinical features nor response to neoadjuvant chemotherapy, high angiogenesis score tumors were associated with both favorable and unfavorable immune cell infiltrations including CD4 memory (p < 0.001 and 0.03), T helper 1 (Th1) (p < 0.001 in both cohorts), B cell (p < 0.001 in both cohorts), T helper 2 (p < 0.001 in both cohorts) and regulatory T cell (p < 0.001 and 0.037) in both cohorts. On the other hand, Dendritic cell and M2 macrophage were highly infiltrated in high angiogenesis score tumors in both cohorts (p < 0.001 in both cohorts). TIL regional fraction was higher in the low angiogenesis score group (p < 0.001), while Leukocyte fraction and lymphocyte infiltration signature showed higher values in the high angiogenesis score tumors (both p < 0.001). High angiogenesis pathway score significantly enriched immune response-related Hallmark gene sets; interferon (IFN)-γ response, IL2-STAT5 signaling, and IFN-α response. Furthermore, high angiogenesis score significantly enriched unfavorable inflammation-related Hallmark gene sets; inflammatory response, IL6-JAK-STAT3 signaling, TNF-α signaling via NFkB, and TGF-β signaling and hypoxia. Furthermore, we found that metastasis-related genes sets; including epithelial mesenchymal transition (EMT), HEDGEHOG signaling, NOTCH signaling and WNT-β catenin signaling were also enriched in high angiogenesis score group in both cohorts (all FDR < 0.25). In particular, EMT pathway score was strongly correlated with angiogenesis pathway score in both cohorts (spearman r = 0.894 [p < 0.01] and 0.868 [p < 0.868] respectively). High angiogenesis pathway scores were significantly associated with site-specific metastasis-free survival especially brain and bone metastasis (p = 0.029 and 0.043 respectively). In conclusion, the angiogenesis score covers many of the angiogenesis-related genes, and it show the correlation between the amount of angiogenesis with inflammation and metastasis in breast cancer. Citation Format: Masanori Oshi, Newman Stephanie, Yoshihisa Tokumaru, Ryusei Matsuyama, Itaru Endo, Kazuaki Takabe. Angiogenesis in a bulk tumor is associated with cancer immunity and metastasis [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-51.