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Introduction Recent transcriptome analysis has demonstrated increased expression of Vascular Endothelial Growth Factor receptor-1 (VEGFR-1/FLT1) and in AD brain. Increased expression of VEGFR1 and its ligand VEGFB were associated with a more rapid rate of cognitive decline, providing evidence of a potential link between increased VEGFR-1 expression in AD pathogenesis. In this study, we explored the potential role of VEGFR-1 expression in neurons on AD pathology. Methods To confirm VEGFR1 expression in AD brains, we first performed immunostaining in AD brain sections (AD - Braak stage V-VI, and normal controls - Braak 0-II). And to determine a potential detrimental role of neuronal VEGFR1 expression on AD associated pathologies, we exposed SH-SY5Y human neuroblastoma cells and mouse primary neurons to either hypoxia conditions (1%O 2 ) or 5 μ Aβ1-42 oligomers or fibrils for 24, 28 and 72hrs. Results In this study, we found preferential staining of VEGFR-1 in the neuropil and neuronal cell bodies both in AD and Control hippocampus and increased VEGFR-1 immunoreactivity in dystrophic neuritic processes in the vicinity of Thio-S positive amyloid plaques in AD brains. And treatment of SH-SY5Y human neuroblastoma cell line and mouse primary neurons, with either hypoxia conditions or Aβ1-42 oligomers, resulted in increased VEGFR-1 expression and cleaved caspase 3 activation, leading to neuronal toxicities/cell death. Similarly, treatment with Aβ1-42 fibrils also increased VEGFR-1 and cleaved caspase 3 protein levels in the SH-SY5Y cells whereas treatment with Aβ1-42 monomers had no effect on VEGFR-1 expression. In addition, we show that over-expression of VEGFR-1 intracellular domains in SH-SY5Y cells directly induced neuronal toxicities and importantly, siRNA-mediated knockdown of VEGFR-1 in neurons prevented the hypoxia, Aβ1-42 oligomer and Aβ1-42 fibril-induced toxicities and cell death phenotypes. Treatment with either hypoxia or Aβ1-42 oligomers also reduced expression of cell survival genes including VEGFR-2 and Hippo pathway YAP1 and siRNA-mediated VEGFR-1 knockdown in the neurons normalized expression of both VEGFR-2 and YAP1. Using differential gene expression analysis, we demonstrated upregulation of several inflammatory/interferon-stimulated genes (ISGs) as well as increased expression of genes involved in activation of oxidative stress and cell death pathways in response to Aβ1-42 oligomers treatment in mouse primary neurons. And siRNA-mediated VEGFR-1 knockdown in the mouse primary neurons, reduced gene expression of both the ISGs and oxidative stress/cell death pathways in response to Aβ1-42 oligomer treatment. Discussion In summary, these results show that siRNA-mediated knockdown of VEGFR-1 in neurons significantly prevented hypoxia, Aβ1-42 oligomer and Aβ1-42 fibril-induced cellular toxicities and cell death phenotypes, indicating a potential detrimental role of aberrant VEGFR-1 expression and signaling in response to AD associated pathologies.

Myocardial infarction (MI) remains a leading cause of mortality worldwide, yet effective cardioprotective strategies remain limited in clinical settings. Vascular endothelial growth factor B (VEGFB) has emerged as a promising therapeutic candidate in MI, but the role of its co-receptor, Neuropilin-1 (NRP1), in cardiomyocyte (CM) survival under ischemic stress remains poorly understood. Here, we investigated VEGFB-NRP1 signaling using an in vivo zebrafish model of cardiac injury as well as in vitro hypoxia models in CMs. We demonstrated that VEGFB overexpression conferred protection against ischemic injury and enhanced cardiac regeneration in the zebrafish heart. Mechanistically, we showed that VEGFB treatment enhances CM viability through reducing reactive oxygen species (ROS), ferroptosis activation, and preserving mitochondrial integrity. We also demonstrated that NRP1 knockdown in the CMs abolished the VEGFB-mediated protective effects, indicating the significant role of NRP1 signaling in VEGFB-induced cardioprotective effects in MI. Lastly, using transcriptome analysis, we confirmed that VEGFB induces anti-apoptotic and anti-ferroptosis gene programs in CMs in response to hypoxic stress. Collectively, our findings provide mechanistic insight into cell death activation pathways, including ferroptosis, in response to ischemic stress and further validate the therapeutic potential of VEGFB in promoting CM survival in ischemic heart disease.

Abstract Background Myocardial infarction (MI) remains a leading cause of mortality worldwide. Recent studies suggest a cardioprotective role for vascular endothelial growth factor-B (VEGF-B) in MI. However, the molecular mechanisms of VEGF-B-mediated signaling via its co-receptor Neuropilin-1 (NRP1) in MI are poorly understood. In this study, we investigated the intricate signaling mechanisms involving VEGF-B and NRP1 in cardiomyocytes (CMs) using ischemic injury as a model of MI. And secondly, we further validated the protective role of VEFG-B in ischemic heart disease, shedding light on their roles not only in cardiac function but also in its therapeutic potential. Methods In this study, we utilized both in vitro and in vivo approaches to elucidate the role of VEGF-B and NRP1 signaling in MI and how it manifests a protective role in mitochondrial functions and cardiac regeneration following ischemic injury. We used two different cardiomyocyte cell lines, H9c2 (rat ventricular cardiomyocytes) and HL-1 (mouse ventricular cardiomyocytes), and induced hypoxia conditions, using either 1% oxygen or 200µM cobalt chloride (CoCl 2 ) to mimic the myocardial infarction-induced ischemic injury in the heart. In addition, we developed a novel heat shock inducible zebrafish model of a cardiomyocyte-specific VEGF-B overexpression system to further examine the protective role of VEGF-B in vivo . Results Our findings indicate that both VEGF-B and NRP1 are predominantly expressed in heart tissue compared to other tissues, and their expression is altered in response to hypoxia/ischemic injury. Our results demonstrate that VEGF-B treatment prior to hypoxia enhances cardiomyocyte survival, while NRP1 knockdown abolishes this protective effect, highlighting a prominent role of NRP1 signaling in VEGF-B-mediated cardio protection. Furthermore, we found that VEGF-B promotes cardiomyocyte survival by improving mitochondrial function, as evidenced by reduced oxidative stress and ROS accumulation, decreased oxidative stress, preserved mitochondrial membrane potential, and increased ATP levels. Lastly, using our VEGF-B transgenic zebrafish model, we demonstrated that VEGF-B overexpression in zebrafish cardiomyocytes protects the heart from ischemic injury and enhances cardiac regeneration in an NRP1-dependent manner. Conclusion Our study has uncovered an important role of VEGF-B-NRP1 signaling axis in VEGF-B mediated cell survival and in beneficial mitochondrial functions in the CMs. Importantly, we also demonstrated that VEGF-B is protective against ischemic injury in vivo using a novel zebrafish model Novelty and Significance What Is Known? Myocardial infarction (MI) results in mitochondrial dysfunction, cardiomyocyte death, and adverse ventricular remodeling. Vascular Endothelial Growth Factor B (VEGF-B) is traditionally associated with cardiac metabolism, survival, vascular biology, particularly in angiogenesis and lipid metabolism. The neurophilin-1 (NRP1) receptor, a co-receptor for VEGF-B, is expressed in cardiomyocytes and implicated in mitochondrial signaling pathways. Emerging evidence suggests that VEGF-B may influence mitochondrial integrity and function under stress conditions. What New Information Does This Article Contribute? VEGF-B exerts a protective effect on cardiomyocyte mitocondria following ischemic injury by preserving mitochondrial function and reducing oxidative stress. NRP1 receptor as a key mediator of VEGF-B modulates mitochondrial homeostasis in cardiomyocytes. VEGF-B signaling can attenuate apoptosis and enhance mitochondrial bioenergetics post-MI, suggesting a novel cardioprotective mechanism. Clinical Implication: These findings suggest that therapeutic strategies aimed at enhancing VEGF-B/NRP1 signaling may improve mitochondrial function and cardiac recovery after MI, offering a novel avenue for limiting heart failure progression in ischemic heart disease.

AbstractBackgroundRecent transcriptome analysis has demonstrated increased expression of Vascular Endothelial Growth Factor receptor‐1 (VEGFR‐1/FLT1) and decreased expression of VEGFR‐2/KDR in AD brain. Increased expression of VEGFR‐1 and its ligand VEGFB were associated with a more rapid rate of cognitive decline, providing evidence of a potential link between aberrant VEGFR‐1 expression in AD pathogenesis. In this study, we explored the potential role of aberrant VEGFR‐1 expression in neurons on AD pathology.MethodTo confirm VEGFR‐1 expression in AD brains, we performed immunostaining in AD brain sections (AD n = 5 (Braak stage V‐VI, and normal controls n = 5 Braak 0‐II) in AD brains. To determine a potential detrimental role of neuronal VEGFR‐1 expression on AD associated pathologies, we exposed SH‐SY5Y human neuroblastoma cells and mouse primary neurons to either hypoxia conditions (1%O2) or 5uM Aß 1‐42 oligomers for 72hrs.ResultWe found increased VEGFR‐1 immunoreactivity in dystrophic neurites in the vicinity of Thio‐S positive “cored” amyloid plaques, whereas VEGFR‐2 antibody staining showed minimal neuronal staining in AD samples. And treatment with hypoxia or Aß oligomers both in SH‐SY5Y cells and mouse primary neurons showed increased VEGFR‐1 expression and cleaved caspase 3 activation, leading to neuronal cell death. Significantly, siRNA mediated knockout of VEGFR‐1 in the neurons prevented both hypoxia and Aß oligomer induced cell death. We also show that treatment with either hypoxia or Aß oligomers reduced mRNA expression of cell survival genes including Hippo pathway YAP1 and VEGFR‐2, and siRNA mediated VEGFR‐1 knockdown normalized both YAP1 and VEGFR‐2 levels. Significantly, over‐expression of the VEGFR‐1 by itself reduced mRNA expression of YAP1 and VEGFR‐2 and readily induced neuronal cell death, implicating the VEGFR‐1/VEGFR‐2/YAP1 signaling axis in hypoxia and Aß oligomer induced neuronal cell death, which is a novel finding.ConclusionThese results demonstrate that VEGFR‐1 may play an important transcriptional regulatory role in neuronal cell survival responses and suggest that modulating VEGFR‐1 expression and signaling could have beneficial effects in AD pathogenesis.

Abstract We have generated transgenic (tg) mice expressing HLA-DQ8αβ (DQA1*0301/DQB*0302) or HLA-DQ6αβ (DQA1*0103/DQB1*0601) molecules lacking endogenous murine class II expression (Aβ0) to investigate the ability of these HLA class II to present type II collagen (CII) and induce collagen-induced arthritis. The DQ8αβ tg mice responded strongly to CII, developing severe arthritis, while DQ6αβ tg mice were nonresponsive to CII. The addition of the mixed haplotype DQ8α6β molecule did not significantly influence CII reactivity. To examine the interaction of DQ6αβ and DQ8αβ molecules in vivo, we generated double tg DQ6αβ/8αβ (Aβ0) mice expressing both the α- and β-chains of DQ6 and DQ8 molecules by mating DQ6αβ (Aβ0) and DQ8αβ (Aβ0) tg mice. CII-immunized DQ6αβ/8αβ tg mice developed severe experimental polychondritis, exhibiting both polyarthritis and auricular chondritis. The clinical, serologic, and histologic manifestations of experimental polychondritis are similar to those symptoms in human relapsing polychondritis. The susceptibility of DQ6αβ/8αβ tg mice compared with resistance in the parental strains suggests that expression of both the DQ6αβ and DQ8αβ tgs, unique to the DQ6αβ8αβ tg strain, is important in susceptibility to experimental polychondritis. The DQ6αβ/8αβ tg mice provide a model to investigate putative autoantigens and the mechanisms of pathogenesis involved in relapsing polychondritis as well as the influence of the expression of multiple HLA class II molecules on the disease process.

Objectives- Oxidative stress and endothelial dysfunction precedes cerebral β-amyloid deposits in human Alzheimer’s disease as well as in transgenic mouse models expressing mutations in amyloid precursor protein (APP). In the present study, we hypothesized that uncoupling of endothelial nitric oxide synthase (eNOS) caused by loss of its essential co-factor, tetrahydrobiopterin (BH 4 ), in cerebral arteries and microvessels contributes, in part, to oxidative stress and cerebral vascular dysfunction in a APP transgenic mouse (Tg2576) that express the Swedish double mutation of human APP. In addition, we examined whether treatment with Peroxisome Proliferator Activated Receptor-δ (PPARδ) activator - GW501516 restores bioavailability of BH 4 and reverses oxidative stress in APP Tg2576 mice. Methods- APP Tg2576 mice (4-5 months old) were treated with GW501516 , a selective PPARδ activator (2 mg/kg/day, po, 14 days). Following treatment, cerebral arteries and microvessels were obtained. Biopterin levels, enzymatic activity of GTP cyclohydrolase I (GTPCH I) and superoxide production were measured by HPLC and protein expression was studied by Western blotting. Results- Cerebral arteries and microvessels demonstrated increased expression of eNOS, while the bioavailability of its essential co-factor BH 4 was significantly reduced, suggestive of eNOS uncoupling in APP Tg2576 mice. Furthermore, expressions of catalase and manganese superoxide dismutase (MnSOD) were decreased, while superoxide production was increased (P<0.01, n=6) in cerebral microvessels of APP Tg2576 mice. Treatment with GW501516 restored the BH 4 /BH 2 ratio in cerebral arteries and microvessels of APP Tg2576 mice (P<0.05, n-7-8), while the enzymatic activity of GTPCH-I remained unchanged (P>0.05, n=6). PPARδ activation also prevented the attenuation in expressions of MnSOD and catalase, and inhibited the increased superoxide production (P<0.05, n=6) in cerebral arteries and microvessels of APP Tg2576 mice. Interestingly, PPARδ activation significantly inhibited the over-expression of APP in cerebral microvessels obtained from APP Tg2576 mice (P<0.001, n=4). Conclusion- Our results suggest that endothelial dysfunction in APP Tg2576 mice may be caused, in part, by reduced bioavailability of BH 4 and uncoupling of eNOS. Treatment of APP Tg2576 mice with PPARδ agonist GW501516 exerted cerebral vascular protection by multiple mechanisms: (a) by inhibiting eNOS uncoupling via increased expressions of MnSOD and catalase, as well as, (b) by attenuating the increased expression of APP in the cerebral microvessels of APP Tg2576 mice.

ABSTRACT Aggregated amyloid β (Aβ) peptides in the Alzheimer's disease (AD) brain are hypothesized to trigger several downstream pathologies, including cerebrovascular dysfunction. Previous studies have shown that Aβ peptides can have antiangiogenic properties, which may contribute to vascular dysfunction in the early stages of the disease process. We have generated data showing that brain endothelial cells (ECs) exposed to toxic Aβ1‐42 oligomers can readily enter a senescence phenotype. To determine the effect of Aβ oligomers on brain ECs, we treated early passaged human brain microvascular ECs and HUVECs with high MW Aβ1‐42 oligomers (5 µM, for 72 h). For controls, we used no peptide treatment, 5 µM Aβ1‐42 monomers, and 5 µM Aβ1‐42 fibrils, respectively. Brain ECs treated with Aβ1‐42 oligomers showed increased senescence‐associated β‐galactosidase staining and increased senescence‐associated p21/ p53 expression. Treatment with either Aβ1‐42 monomer or Aβ1‐42 fibrils did not induce senescence in this assay. We then measured vascular endothelial growth factor receptor (VEGFR) expression in the Aβ1‐42 oligomer‐treated ECs, and these cells showed significantly increased VEGFR‐1 expression and decreased VEGFR‐2 levels. Overexpression of VEGFR‐1 in brain ECs readily induced senescence, suggesting a direct role of VEGFR‐1 signaling events in this paradigm. More importantly, small interfering RNA‐mediated knockdown of VEGFR‐1 expression in brain ECs was able to prevent up‐regulation of p21 protein expression and significantly reduced induction of senescence following Aβ1‐42 oligomer treatment. Our studies show that exposure to Aβ1‐42 oligomers may impair vascular functions by altering VEGFR‐1 expression and causing ECs to enter a senescent phenotype. Altered VEGFR expression has been documented in brains of AD patients and suggests that this pathway may play a role in AD disease pathogenesis. These studies suggest that modulating VEGFR‐1 expression and signaling events could potentially prevent senescence and rejuvenate EC functions, and provides us with a novel target to pursue for prevention and treatment of cerebrovascular dysfunction in AD.—Angom, R. S., Wang, Y., Wang, E., Pal, K., Bhattacharya, S., Watzlawik, J. O., Rosenberry, T. L., Das, P., Mukhopadhyay, D. VEGF receptor‐1 modulates amyloid β 1–42 oligomer‐induced senescence in brain endothelial cells. FASEB J. 33, 4626–4637 (2019). www.fasebj.org

Drug development for Alzheimer’s disease has endeavored to lower amyloid β (Aβ) by either blocking production or promoting clearance. The benefit of combining these approaches has been examined in mouse models and shown to improve pathological measures of disease over single treatment; however, the impact on cellular and cognitive functions affected by Aβ has not been tested. We used a controllable APP transgenic mouse model to test whether combining genetic suppression of Aβ production with passive anti-Aβ immunization improved functional outcomes over either treatment alone. Compared with behavior before treatment, arresting further Aβ production (but not passive immunization) was sufficient to stop further decline in spatial learning, working memory, and associative memory, whereas combination treatment reversed each of these impairments. Cognitive improvement coincided with resolution of neuritic dystrophy, restoration of synaptic density surrounding deposits, and reduction of hyperactive mammalian target of rapamycin signaling. Computational modeling corroborated by in vivo microdialysis pointed to the reduction of soluble/exchangeable Aβ as the primary driver of cognitive recovery.