Treatment Strategies for Anti-VEGF Resistance in Neovascular Age-Related Macular Degeneration by Targeting Arteriolar Choroidal Neovascularization.

Despite extensive use of intravitreal anti-vascular endothelial growth factor (anti-VEGF) biologics for over a decade, neovascular age-related macular degeneration (nAMD) or choroidal neovascularization (CNV) continues to be a major cause of irreversible vision loss in developed countries. Many nAMD patients demonstrate persistent disease activity or experience declining responses over time despite anti-VEGF treatment. The underlying mechanisms of anti-VEGF resistance are poorly understood, and no effective treatment strategies are available to date. Here we review evidence from animal models and clinical studies that supports the roles of neovascular remodeling and arteriolar CNV formation in anti-VEGF resistance. Cholesterol dysregulation, inflammation, and ensuing macrophage activation are critically involved in arteriolar CNV formation and anti-VEGF resistance. Combination therapy by neutralizing VEGF and enhancing cholesterol removal from macrophages is a promising strategy to combat anti-VEGF resistance in CNV.

Growth hormone-releasing hormone receptor antagonist MIA-602 attenuates cardiopulmonary injury induced by BSL-2 rVSV-SARS-CoV-2 in hACE2 mice.

COVID-19 pneumonia causes acute lung injury and acute respiratory distress syndrome (ALI/ARDS) characterized by early pulmonary endothelial and epithelial injuries with altered pulmonary diffusing capacity and obstructive or restrictive physiology. Growth hormone-releasing hormone receptor (GHRH-R) is expressed in the lung and heart. GHRH-R antagonist, MIA-602, has been reported to modulate immune responses to bleomycin lung injury and inflammation in granulomatous sarcoidosis. We hypothesized that MIA-602 would attenuate rVSV-SARS-CoV-2-induced pulmonary dysfunction and heart injury in a BSL-2 mouse model. Male and female K18-hACE2tg mice were inoculated with SARS-CoV-2/USA-WA1/2020, BSL-2-compliant recombinant VSV-eGFP-SARS-CoV-2-Spike (rVSV-SARS-CoV-2), or PBS, and lung viral load, weight loss, histopathology, and gene expression were compared. K18-hACE2tg mice infected with rVSV-SARS-CoV-2 were treated daily with subcutaneous MIA-602 or vehicle and conscious, unrestrained plethysmography performed on days 0, 3, and 5 (n = 7 to 8). Five days after infection mice were killed, and blood and tissues collected for histopathology and protein/gene expression. Both native SARS-CoV-2 and rVSV-SARS-CoV-2 presented similar patterns of weight loss, infectivity (~60%), and histopathologic changes. Daily treatment with MIA-602 conferred weight recovery, reduced lung perivascular inflammation/pneumonia, and decreased lung/heart ICAM-1 expression compared to vehicle. MIA-602 rescued altered respiratory rate, increased expiratory parameters (Te, PEF, EEP), and normalized airflow parameters (Penh and Rpef) compared to vehicle, consistent with decreased airway inflammation. RNASeq followed by protein analysis revealed heightened levels of inflammation and end-stage necroptosis markers, including ZBP1 and pMLKL induced by rVSV-SARS-CoV-2, that were normalized by MIA-602 treatment, consistent with an anti-inflammatory and pro-survival mechanism of action in this preclinical model of COVID-19 pneumonia.

Ascending aortic aneurysm and histopathology in Alport syndrome: a case report.

BACKGROUND: Alport syndrome (AS) is caused by mutations in type IV collagen genes that typically target and compromise the integrity of basement membranes in kidney, ocular, and sensorineural cochlear tissues. Type IV and V collagens are also integral components of arterial walls, and whereas collagenopathies including AS are implicated in aortic disease, the incidence of aortic aneurysm in AS is unknown probably because of underreporting. Consequently, AS is not presently considered an independent risk factor for aortic aneurysm and more detailed case studies including histological evidence of basement membrane abnormalities are needed to determine such a possible linkage. CASE PRESENTATION: Here, we present unique histopathological findings of an ascending aortic aneurysm collected at the time of surgery from an AS patient wherein hypertension was the only other known risk factor. CONCLUSIONS: The studies reveal classical histological features of aortic aneurysm, including atheroma, lymphocytic infiltration, elastin disruption, and myxoid degeneration with probable AS association.

Anti-Scg3 Gene Therapy to Treat Choroidal Neovascularization in Mice.

Neovascular age-related macular degeneration (nAMD) with choroidal neovascularization (CNV) is a leading cause of blindness in the elderly in developed countries. The disease is currently treated with anti-angiogenic biologics, including aflibercept, against vascular endothelial growth factor (VEGF) but with limited efficacy, treatment resistance and requirement for frequent intravitreal injections. Although anti-VEGF gene therapy may provide sustained therapy that obviates multiple injections, the efficacy and side effects related to VEGF pathway targeting remain, and alternative strategies to block angiogenesis independently of VEGF are needed. We recently reported that secretogranin III (Scg3) induces only pathological angiogenesis through VEGF-independent pathways, and Scg3-neutralizing antibodies selectively inhibit pathological but not physiological angiogenesis in mouse proliferative retinopathy models. Anti-Scg3 antibodies synergize dose-dependently with VEGF inhibitors in a CNV model. Here, we report that an adeno-associated virus-8 (AAV8) vector expressing anti-Scg3 Fab ameliorated CNV with an efficacy similar to that of AAV-aflibercept in a mouse model. This study is the first to test an anti-angiogenic gene therapy protocol that selectively targets pathological angiogenesis via a VEGF-independent mechanism. The findings support further safety/efficacy studies of anti-Scg3 gene therapy as monotherapy or combined with anti-VEGF to treat nAMD.

Mouse Model of Heart Failure With Preserved Ejection Fraction Driven by Hyperlipidemia and Enhanced Cardiac Low-Density Lipoprotein Receptor Expression.

Background The pathways of diastolic dysfunction and heart failure with preserved ejection fraction driven by lipotoxicity with metabolic syndrome are incompletely understood. Thus, there is an urgent need for animal models that accurately mimic the metabolic and cardiovascular phenotypes of this phenogroup for mechanistic studies. Methods and Results Hyperlipidemia was induced in WT-129 mice by 4 weeks of biweekly poloxamer-407 intraperitoneal injections with or without a single intravenous injection of adeno-associatedvirus 9-cardiac troponin T-low-density lipoprotein receptor (n=31), or single intravenous injection with adeno-associatedvirus 9-cardiac troponin T-low-density lipoprotein receptor alone (n=10). Treatment groups were compared with untreated or placebo controls (n=37). Echocardiography, blood pressure, whole-body plethysmography, ECG telemetry, activity wheel monitoring, and biochemical and histological changes were assessed at 4 to 8 weeks. At 4 weeks, double treatment conferred diastolic dysfunction, preserved ejection fraction, and increased left ventricular wall thickness. Blood pressure and whole-body plethysmography results were normal, but respiration decreased at 8 weeks (P<0.01). ECG and activity wheel monitoring, respectively, indicated heart block and decreased exercise activity (P<0.001). Double treatment promoted elevated myocardial lipids including total cholesterol, fibrosis, increased wet/dry lung (P<0.001) and heart weight/body weight (P<0.05). Xanthelasma, ascites, and cardiac ischemia were evident in double and single (p407) groups. Sudden death occurred between 6 and 12 weeks in double and single (p407) treatment groups. Conclusions We present a novel model of heart failure with preserved ejection fraction driven by dyslipidemia where mice acquire diastolic dysfunction, arrhythmia, cardiac hypertrophy, fibrosis, pulmonary congestion, exercise intolerance, and preserved ejection fraction in the absence of obesity, hypertension, kidney disease, or diabetes. The model can be applied to dissect pathways of metabolic syndrome that drive diastolic dysfunction in this lipotoxicity-mediated heart failure with preserved ejection fraction phenogroup mimic.

Safety and Efficacy of Systemic Anti-Scg3 Therapy to Treat Oxygen-Induced Retinopathy.

BACKGROUND: To circumvent possible systemic side effects, anti-angiogenic drugs targeting vascular endothelial growth factor (VEGF) for ocular neovascular diseases in adults are approved only for intravitreal administration. However, intravitreal injection itself can elicit injection-related adverse effects, and premature eyes of infants with retinopathy of prematurity (ROP) may be particularly susceptible to intravitreal injection. Therefore, an unmet clinical need is to develop safe systemic anti-angiogenic therapies for ROP. We recently reported that secretogranin III (Scg3) is a disease-restricted angiogenic factor and that systemic anti-Scg3 mAb alleviates ROP in animal models with minimal side effects on developing eyes and organs. The aim of this study is to investigate the safety and efficacy of a humanized anti-Scg3 antibody via systemic administration. METHODS: We analyzed the safety and efficacy of a humanized anti-Scg3 antibody Fab fragment (hFab) delivered by intraperitoneal injection in oxygen-induced retinopathy (OIR) mice, a surrogate model of ROP. RESULTS: The results showed that systemic anti-Scg3 hFab effectively alleviated pathological retinal neovascularization in OIR mice with similar efficacy to the anti-VEGF drug aflibercept. Systemic aflibercept conferred significant adverse side effects in neonatal mice, including reduced body weight, abnormalities in retinal and renal development, and retarded physiological neovascularization, whereas systemic anti-Scg3 hFab elicited no such side effects. CONCLUSIONS: The findings suggest that systemic anti-Scg3 hFab is a safe and effective therapy for OIR and support further development for ROP treatment.

Secretogranin III stringently regulates pathological but not physiological angiogenesis in oxygen-induced retinopathy.

Conventional angiogenic factors, such as vascular endothelial growth factor (VEGF), regulate both pathological and physiological angiogenesis indiscriminately, and their inhibitors may elicit adverse side effects. Secretogranin III (Scg3) was recently reported to be a diabetes-restricted VEGF-independent angiogenic factor, but the disease selectivity of Scg3 in retinopathy of prematurity (ROP), a retinal disease in preterm infants with concurrent pathological and physiological angiogenesis, was not defined. Here, using oxygen-induced retinopathy (OIR) mice, a surrogate model of ROP, we quantified an exclusive binding of Scg3 to diseased versus healthy developing neovessels that contrasted sharply with the ubiquitous binding of VEGF. Functional immunohistochemistry visualized Scg3 binding exclusively to disease-related disorganized retinal neovessels and neovascular tufts, whereas VEGF bound to both disorganized and well-organized neovessels. Homozygous deletion of the Scg3 gene showed undetectable effects on physiological retinal neovascularization but markedly reduced the severity of OIR-induced pathological angiogenesis. Furthermore, anti-Scg3 humanized antibody Fab (hFab) inhibited pathological angiogenesis with similar efficacy to anti-VEGF aflibercept. Aflibercept dose-dependently blocked physiological angiogenesis in neonatal retinas, whereas anti-Scg3 hFab was without adverse effects at any dose and supported a therapeutic window at least 10X wider than that of aflibercept. Therefore, Scg3 stringently regulates pathological but not physiological angiogenesis, and anti-Scg3 hFab satisfies essential criteria for development as a safe and effective disease-targeted anti-angiogenic therapy for ROP.

Selectively targeting disease-restricted secretogranin III to alleviate choroidal neovascularization.

Choroidal neovascularization (CNV), a leading cause of blindness in the elderly, is routinely treated with vascular endothelial growth factor (VEGF) inhibitors that have limited efficacy and potentially adverse side effects. An unmet clinical need is to develop novel therapies against other angiogenic factors for alternative or combination treatment to improve efficacy and safety. We recently described secretogranin III (Scg3) as a disease-selective angiogenic factor, causally linked to diabetic retinopathy and acting independently of the VEGF pathway. An important question is whether such a disease-selective Scg3 pathway contributes to other states of pathological angiogenesis beyond diabetic retinopathy. By applying a novel in vivo endothelial ligand binding assay, we found that the binding of Scg3 to CNV vessels in live mice was markedly increased over background binding to healthy choriocapillaris and blocked by an Scg3-neutralizing antibody, whereas VEGF showed no such differential binding. Intravitreal injection of anti-Scg3 humanized antibody Fab (hFab) inhibited Matrigel-induced CNV with similar efficacy to the anti-VEGF drug aflibercept. Importantly, a combination of anti-Scg3 hFab and aflibercept synergistically alleviated CNV. Homozygous deletion of the Scg3 gene markedly reduced CNV severity and abolished the therapeutic activity of anti-Scg3 hFab, but not aflibercept, suggesting a role for Scg3 in VEGF-independent CNV pathogenesis and therapy. Our work demonstrates the stringent disease selectivity of Scg3 binding and positions anti-Scg3 hFab as a next-generation disease-targeted anti-angiogenic therapy for CNV.

MiR-126-HMGB1-HIF-1 Axis Regulates Endothelial Cell Inflammation during Exposure to Hypoxia-Acidosis.

Crosstalk between molecular regulators miR-126, hypoxia-inducible factor 1-alpha (HIF-1-α), and high-mobility group box-1 (HMGB1) contributes to the regulation of inflammation and angiogenesis in multiple physiological and pathophysiological settings. Here, we present evidence of an overriding role for miR-126 in the regulation of HMGB1 and its downstream proinflammatory effectors in endothelial cells subjected to hypoxia with concurrent acidosis (H/A). Methods. Primary mouse endothelial cells (PMEC) were exposed to hypoxia or H/A to simulate short or chronic low-flow ischemia, respectively. RT-qPCR quantified mRNA transcripts, and proteins were measured by western blot. ROS were quantified by fluorogenic ELISA and luciferase reporter assays employed to confirm an active miR-126 target in the HMGB1 3'UTR. Results. Enhanced expression of miR-126 in PMECs cultured under neutral hypoxia was suppressed under H/A, whereas the HMGB1 expression increased sequentially under both conditions. Enhanced expression of HMGB1 and downstream inflammation markers was blocked by the premiR-126 overexpression and optimized by antagomiR. Compared with neutral hypoxia, H/A suppressed the HIF-1α expression independently of miR-126. The results show that HMGB1 and downstream effectors are optimally induced by H/A relative to neutral hypoxia via crosstalk between hypoxia signaling, miR-126, and HIF-1α, whereas B-cell lymphoma 2(Bcl2), a HIF-1α, and miR-126 regulated gene expressed optimally under neutral hypoxia. Conclusion. Inflammatory responses of ECs to H/A are dynamically regulated by the combined actions of hypoxia, miR-126, and HIF-1α on the master regulator HMGB1. The findings may be relevant to vascular diseases including atherosclerotic occlusion and interiors of plaque where coexisting hypoxia and acidosis promote inflammation as a defining etiology.

Neurovascular regulation in diabetic retinopathy and emerging therapies.

Diabetic retinopathy (DR) is the leading cause of vision loss in working adults in developed countries. The disease traditionally classified as a microvascular complication of diabetes is now widely recognized as a neurovascular disorder resulting from disruption of the retinal neurovascular unit (NVU). The NVU comprising retinal neurons, glia and vascular cells coordinately regulates blood flow, vascular density and permeability to maintain homeostasis. Disturbance of the NVU during DR can lead to vision-threatening clinical manifestations. A limited number of signaling pathways have been identified for intercellular communication within the NVU, including vascular endothelial growth factor (VEGF), the master switch for angiogenesis. VEGF inhibitors are now widely used to treat DR, but their limited efficacy implies that other signaling molecules are involved in the pathogenesis of DR. By applying a novel screening technology called comparative ligandomics, we recently discovered secretogranin III (Scg3) as a unique DR-selective angiogenic and vascular leakage factor with therapeutic potential for DR. This review proposes neuron-derived Scg3 as the first diabetes-selective neurovascular regulator and discusses important features of Scg3 inhibition for next-generation disease-targeted anti-angiogenic therapies of DR.

Concurrent Physiological and Pathological Angiogenesis in Retinopathy of Prematurity and Emerging Therapies.

Retinopathy of prematurity (ROP) is an ocular vascular disease affecting premature infants, characterized by pathological retinal neovascularization (RNV), dilated and tortuous retinal blood vessels, and retinal or vitreous hemorrhages that may lead to retinal detachment, vision impairment and blindness. Compared with other neovascular diseases, ROP is unique because of ongoing and concurrent physiological and pathological angiogenesis in the developing retina. While the disease is currently treated by laser or cryotherapy, anti-vascular endothelial growth factor (VEGF) agents have been extensively investigated but are not approved in the U.S. because of safety concerns that they negatively interfere with physiological angiogenesis of the developing retina. An ideal therapeutic strategy would selectively inhibit pathological but not physiological angiogenesis. Our group recently described a novel strategy that selectively and safely alleviates pathological RNV in animal models of ROP by targeting secretogranin III (Scg3), a disease-restricted angiogenic factor. The preclinical profile of anti-Scg3 therapy presents a high potential for next-generation disease-targeted anti-angiogenic therapy for the ROP indication. This review focuses on retinal vessel development in neonates, the pathogenesis of ROP and its underlying molecular mechanisms, including different animal models, and provides a summary of current and emerging therapies.

Nuclear Osteopontin Is a Marker of Advanced Heart Failure and Cardiac Allograft Vasculopathy: Evidence From Transplant and Retransplant Hearts.

BACKGROUND: Heart transplant is the gold standard therapy for patients with advanced heart failure. Over 5,500 heart transplants are performed every year worldwide. Cardiac allograft vasculopathy (CAV) is a common complication post-heart transplant which reduces survival and often necessitates heart retransplantation. Post-transplant follow-up requires serial coronary angiography and endomyocardial biopsy (EMB) for CAV and allograft rejection screening, respectively; both of which are invasive procedures. This study aims to determine whether osteopontin (OPN) protein, a fibrosis marker often present in chronic heart disease, represents a novel biomarker for CAV. METHODS: Expression of OPN was analyzed in cardiac tissue obtained from patients undergoing heart retransplantation using immunofluorescence imaging (n = 20). Tissues from native explanted hearts and three serial follow-up EMB samples of transplanted hearts were also analyzed in five of these patients. RESULTS: Fifteen out of 20 patients undergoing retransplantation had CAV. 13/15 patients with CAV expressed nuclear OPN. 5/5 patients with multiple tissue samples expressed nuclear OPN in both 1 st and 2 nd explanted hearts, while 0/5 expressed nuclear OPN in any of the follow-up EMBs. 4/5 of these patients had an initial diagnosis of dilated cardiomyopathy (DCM). CONCLUSION: Nuclear localization of OPN in cardiomyocytes of patients with CAV was evident at the time of cardiac retransplant as well as in patients with DCM at the time of the 1 st transplant. The results implicate nuclear OPN as a novel biomarker for severe CAV and DCM.

c-Kit suppresses atherosclerosis in hyperlipidemic mice.

Atherosclerosis is the most common underlying cause of cardiovascular morbidity and mortality worldwide. c-Kit (CD117) is a member of the receptor tyrosine kinase family, which regulates differentiation, proliferation, and survival of multiple cell types. Recent studies have shown that c-Kit and its ligand stem cell factor (SCF) are present in arterial endothelial cells and smooth muscle cells (SMCs). The role of c-Kit in cardiovascular disease remains unclear. The aim of the current study is to determine the role of c-Kit in atherogenesis. For this purpose, atherosclerotic plaques were quantified in c-Kit-deficient mice (KitMut) after they were fed a high-fat diet (HFD) for 16 wk. KitMut mice demonstrated substantially greater atherosclerosis compared with control (KitWT) littermates (P < 0.01). Transplantation of c-Kit-positive bone marrow cells into KitMut mice failed to rescue the atherogenic phenotype, an indication that increased atherosclerosis was associated with reduced arterial c-Kit. To investigate the mechanism, SMC organization and morphology were analyzed in the aorta by histopathology and electron microscopy. SMCs were more abundant, disorganized, and vacuolated in aortas of c-Kit mutant mice compared with controls (P < 0.05). Markers of the "contractile" SMC phenotype (calponin, SM22α) were downregulated with pharmacological and genetic c-Kit inhibition (P < 0.05). The absence of c-Kit increased lipid accumulation and significantly reduced the expression of the ATP-binding cassette transporter G1 (ABCG1) necessary for lipid efflux in SMCs. Reconstitution of c-Kit in cultured KitMut SMCs resulted in increased spindle-shaped morphology, reduced proliferation, and elevated levels of contractile markers, all indicators of their restored contractile phenotype (P < 0.05).NEW & NOTEWORTHY This study describes the novel vasculoprotective role of c-Kit against atherosclerosis and its function in the preservation of the SMC contractile phenotype.

Regulation of Vascular Calcification by Growth Hormone-Releasing Hormone and Its Agonists.

RATIONALE: Vascular calcification (VC) is a marker of the severity of atherosclerotic disease. Hormones play important roles in regulating calcification; estrogen and parathyroid hormones exert opposing effects, the former alleviating VC and the latter exacerbating it. To date no treatment strategies have been developed to regulate clinical VC. OBJECTIVE: The objective of this study was to investigate the effect of growth hormone-releasing hormone (GHRH) and its agonist (GHRH-A) on the blocking of VC in a mouse model. METHODS AND RESULTS: Young adult osteoprotegerin-deficient mice were given daily subcutaneous injections of GHRH-A (MR409) for 4 weeks. Significant reductions in calcification of the aortas of MR409-treated mice were paralleled by markedly lower alkaline phosphatase activity and a dramatic reduction in the expression of transcription factors, including the osteogenic marker gene Runx2 and its downstream factors, osteonectin and osteocalcin. The mechanism of action of GHRH-A was dissected in smooth muscle cells isolated from human and mouse aortas. Calcification of smooth muscle cells induced by osteogenic medium was inhibited in the presence of GHRH or MR409, as evidenced by reduced alkaline phosphatase activity and Runx2 expression. Inhibition of calcification by MR409 was partially reversed by MIA602, a GHRH antagonist, or a GHRH receptor-selective small interfering RNA. Treatment with MR409 induced elevated cytosolic cAMP and its target, protein kinase A which in turn blocked nicotinamide adenine dinucleotide phosphate oxidase activity and reduced production of reactive oxygen species, thus blocking the phosphorylation of nuclear factor κB (p65), a key intermediate in the ligand of receptor activator for nuclear factor-κ B-Runx2/alkaline phosphatase osteogenesis program. A protein kinase A-selective small interfering RNA or the chemical inhibitor H89 abolished these beneficial effects of MR409. CONCLUSIONS: GHRH-A controls osteogenesis in smooth muscle cells by targeting cross talk between protein kinase A and nuclear factor κB (p65) and through the suppression of reactive oxygen species production that induces the Runx2 gene and alkaline phosphatase. Inflammation-mediated osteogenesis is thereby blocked. GHRH-A may represent a new pharmacological strategy to regulate VC.

Secretogranin III: a diabetic retinopathy-selective angiogenic factor.

Secretogranin III (Scg3) is a member of the granin protein family that regulates the biogenesis of secretory granules. Scg3 was recently discovered as an angiogenic factor, expanding its functional role to extrinsic regulation. Unlike many other known angiogenic factors, the pro-angiogenic actions of Scg3 are restricted to pathological conditions. Among thousands of quantified endothelial ligands, Scg3 has the highest binding activity ratio to diabetic vs. healthy mouse retinas and lowest background binding to normal vessels. In contrast, vascular endothelial growth factor binds to and stimulates angiogenesis of both diabetic and control vasculature. Consistent with its role in pathological angiogenesis, Scg3-neutralizing antibodies alleviate retinal vascular leakage in mouse models of diabetic retinopathy and retinal neovascularization in oxygen-induced retinopathy mice. This review summarizes our current knowledge of Scg3 as a regulatory protein of secretory granules, highlights its new role as a highly disease-selective angiogenic factor, and envisions Scg3 inhibitors as "selective angiogenesis blockers" for targeted therapy.

Loss of c-Kit function impairs arteriogenesis in a mouse model of hindlimb ischemia.

BACKGROUND: Arteriogenesis is a process whereby collateral vessels remodel usually in response to increased blood flow and/or wall stress. Remodeling of collaterals can function as a natural bypass to alleviate ischemia during arterial occlusion. Here we used a genetic approach to investigate possible roles of tyrosine receptor c-Kit in arteriogenesis. METHODS: Mutant mice with loss of c-Kit function (KitW/W-v), and controls were subjected to hindlimb ischemia. Blood flow recovery was evaluated pre-, post-, and weekly after ischemia. Foot ischemic damage and function were assessed between days 1 to 14 post-ischemia while collaterals remodeling were measured 28 days post-ischemia. Both groups of mice also were subjected to wild type bone marrow cells transplantation 3 weeks before hindlimb ischemia to evaluate possible contributions of defective bone marrow c-Kit expression on vascular recovery. RESULTS: KitW/W-v mice displayed impaired blood flow recovery, greater ischemic damage and foot dysfunction after ischemia compared to controls. KitW/W-v mice also demonstrated impaired collateral remodeling consistent with flow recovery findings. Because arteriogenesis is a biological process that involves bone marrow-derived cells, we investigated which source of c-Kit signaling (bone marrow or vascular) plays a major role in arteriogenesis. KitW/W-v mice transplanted with bone marrow wild type cells exhibited similar phenotype of impaired blood flow recovery, greater tissue ischemic damage and foot dysfunction as nontransplanted KitW/W-v mice. CONCLUSION: This study provides evidence that c-Kit signaling is required during arteriogenesis. Also, it strongly suggests a vascular role for c-Kit signaling because rescue of systemic c-Kit activity by bone marrow transplantation did not augment the functional recovery of KitW/W-v mouse hindlimbs.

A Reliable Mouse Model of Hind limb Gangrene.

BACKGROUND: Lack of a reliable hind limb gangrene animal model limits preclinical studies of gangrene, a severe form of critical limb ischemia. We develop a novel mouse hind limb gangrene model to facilitate translational studies. METHODS: BALB/c, FVB, and C57BL/6 mice underwent femoral artery ligation (FAL) with or without administration of NG-nitro-L-arginine methyl ester (L-NAME), an endothelial nitric oxide synthase inhibitor. Gangrene was assessed using standardized ischemia scores ranging from 0 (no gangrene) to 12 (forefoot gangrene). Laser Doppler imaging (LDI) and DiI perfusion quantified hind limb reperfusion postoperatively. RESULTS: BALB/c develops gangrene with FAL-only (n = 11/11, 100% gangrene incidence), showing mean limb ischemia score of 12 on postoperative days (PODs) 7 and 14 with LDI ranging from 0.08 to 0.12 on respective PODs. Most FVB did not develop gangrene with FAL-only (n = 3/9, 33% gangrene incidence) but with FAL and L-NAME (n = 9/9, 100% gangrene incidence). Mean limb ischemia scores for FVB undergoing FAL with L-NAME were significantly higher than for FVB receiving FAL-only. LDI score and capillary density by POD 28 were significantly lower in FVB undergoing FAL with L-NAME. C57BL/6 did not develop gangrene with FAL-only or FAL and L-NAME. CONCLUSIONS: Reproducible murine gangrene models may elucidate molecular mechanisms for gangrene development, facilitating therapeutic intervention.

Myocardial reparative functions of exosomes from mesenchymal stem cells are enhanced by hypoxia treatment of the cells via transferring microRNA-210 in an nSMase2-dependent way.

Hypoxia treatment enhances paracrine effect of mesenchymal stem cells (MSCs). The aim of this study was to investigate whether exosomes from hypoxia-treated MSCs (ExoH) are superior to those from normoxia-treated MSCs (ExoN) for myocardial repair. Mouse bone marrow-derived MSCs were cultured under hypoxia or normoxia for 24 h, and exosomes from conditioned media were intramyocardially injected into infarcted heart of C57BL/6 mouse. ExoH resulted in significantly higher survival, smaller scar size and better cardiac functions recovery. ExoH conferred increased vascular density, lower cardiomyocytes (CMs) apoptosis, reduced fibrosis and increased recruitment of cardiac progenitor cells in the infarcted heart relative to ExoN. MicroRNA analysis revealed significantly higher levels of microRNA-210 (miR-210) in ExoH compared with ExoN. Transfection of a miR-210 mimic into endothelial cells (ECs) and CMs conferred similar biological effects as ExoH. Hypoxia treatment of MSCs increased the expression of neutral sphingomyelinase 2 (nSMase2) which is crucial for exosome secretion. Blocking the activity of nSMase2 resulted in reduced miR-210 secretion and abrogated the beneficial effects of ExoH. In conclusion, hypoxic culture augments miR-210 and nSMase2 activities in MSCs and their secreted exosomes, and this is responsible at least in part for the enhanced cardioprotective actions of exosomes derived from hypoxia-treated cells.

Pathogenic role and therapeutic potential of pleiotrophin in mouse models of ocular vascular disease.

Angiogenic factors play an important role in the pathogenesis of diabetic retinopathy (DR), neovascular age-related macular degeneration (nAMD) and retinopathy of prematurity (ROP). Pleiotrophin, a well-known angiogenic factor, was recently reported to be upregulated in the vitreous fluid of patients with proliferative DR (PDR). However, its pathogenic role and therapeutic potential in ocular vascular diseases have not been defined in vivo. Here using corneal pocket assays, we demonstrated that pleiotrophin induced angiogenesis in vivo. To investigate the pathological role of pleiotrophin we used neutralizing antibody to block its function in multiple in vivo models of ocular vascular diseases. In a mouse model of DR, intravitreal injection of pleiotrophin-neutralizing antibody alleviated diabetic retinal vascular leakage. In a mouse model of oxygen-induced retinopathy (OIR), which is a surrogate model of ROP and PDR, we demonstrated that intravitreal injection of anti-pleiotrophin antibody prevented OIR-induced pathological retinal neovascularization and aberrant vessel tufts. Finally, pleiotrophin-neutralizing antibody ameliorated laser-induced choroidal neovascularization, a mouse model of nAMD, suggesting that pleiotrophin is involved in choroidal vascular disease. These findings suggest that pleiotrophin plays an important role in the pathogenesis of DR with retinal vascular leakage, ROP with retinal neovascularization and nAMD with choroidal neovascularization. The results also support pleiotrophin as a promising target for anti-angiogenic therapy.

Enhanced Cardioprotection by Human Endometrium Mesenchymal Stem Cells Driven by Exosomal MicroRNA-21.

Our group recently reported positive therapeutic benefit of human endometrium-derived mesenchymal stem cells (EnMSCs) delivered to infarcted rat myocardium, an effect that correlated with enhanced secretion of protective cytokines and growth factors compared with parallel cultures of human bone marrow MSCs (BMMSCs). To define more precisely the molecular mechanisms of EnMSC therapy, in the present study, we assessed in parallel the paracrine and therapeutic properties of MSCs derived from endometrium, bone marrow, and adipose tissues in a rat model of myocardial infarction (MI). EnMSCs, BMMSCs, and adipose-derived MSCs (AdMSCs) were characterized by fluorescence-activated cell sorting (FACS). Paracrine and cytoprotective actions were assessed in vitro by coculture with neonatal cardiomyocytes and human umbilical vein endothelial cells. A rat MI model was used to compare cell therapy by intramyocardial injection of BMMSCs, AdMSCs, and EnMSCs. We found that EnMSCs conferred superior cardioprotection relative to BMMSCs or AdMSCs and supported enhanced microvessel density. Inhibitor studies indicated that the enhanced paracrine actions of EnMSCs were mediated by secreted exosomes. Analyses of exosomal microRNAs (miRs) by miR array and quantitative polymerase chain reaction revealed that miR-21 expression was selectively enhanced in exosomes derived from EnMSCs. Selective antagonism of miR-21 by anti-miR treatment abolished the antiapoptotic and angiogenic effects of EnMSCs with parallel effects on phosphatase and tensin homolog (PTEN), a miR-21 target and downstream Akt. The results of the present study confirm the superior cardioprotection by EnMSCs relative to BMMSCs or AdMSCs and implicates miR-21 as a potential mediator of EnMSC therapy by enhancing cell survival through the PTEN/Akt pathway. The endometrium might be a preferential source of MSCs for cardiovascular cell therapy. Stem Cells Translational Medicine 2017;6:209-222.