Connexin43 (Cx43) is one of the most abundant gap junction proteins in the central nervous system. Abnormal opening of Cx43 hemichannels after ischemic insults causes apoptotic cell death. In this study, we found persistently increased expression of Cx43 8 h to 7 d after hypoxia/ischemia (HI) injury in neonatal rats. Pre-treatment with Gap26 and Gap27, two Cx43 mimetic peptides, significantly reduced cerebral infarct volume. Gap26 treatment at 24 h after ischemia improved functional recovery on muscle strength, motor coordination, and spatial memory abilities. Further, Gap26 inhibited Cx43 expression and reduced active astrogliosis. Gap26 interacted and co-localized with Cx43 together in brain tissues and cultured astrocytes. After oxygen glucose deprivation, Gap26 treatment reduced the total Cx43 level in cultured astrocytes; but Cx43 level in the plasma membrane was increased. Degradation of Cx43 in the cytoplasm was mainly via the ubiquitin proteasome pathway. Concurrently, phosphorylated Akt, which phosphorylates Cx43 on Serine373 and facilitates the forward transport of Cx43 to the plasma membrane, was increased by Gap26 treatment. Microdialysis showed that increased membranous Cx43 causes glutamate release by opening Cx43 hemichannels. Extracellular glutamate concentration was significantly decreased by Gap26 treatment in vivo. Finally, we found that cleaved caspase-3, an apoptosis marker, was attenuated after HI injury by Gap26 treatment. Effects of Gap27 were analogous to those of Gap26. In summary, our findings demonstrate that modulation of Cx43 expression and astroglial function is a potential therapeutic strategy for ischemic brain injury.

Tim-3 has been linked to several inflammatory diseases by regulation on both adaptive and innate immunities. Here, we assessed the augmented expression of Tim-3 in brain tissue of ischemia-reperfusion mice and PBMCs of ischemic stroke (IS) patients. The augmented expression of Tim-3 significantly correlated with abnormal lipid levels. In vitro studies showed that plasma from ischemic stroke patients induced Tim-3 expression in THP- 1 cells. More importantly, our results revealed a significant correlation of Tim-3 expression on CD4 + T cells with systemic IL-17 in patients with ischemic stroke. Consistently, we also found a positive correlation of Tim-3 expression on CD14 + monocytes and serum TNF-a in IS patients. Collectively, augmented expression of Tim-3 may play an important role in the pathogenesis of ischemic stroke by regulation of proinflammatory cytokines. Further studies will give us new insights on the pathogenesis of ischemic stroke and potentially provide a new target at the medical therapy.

Background: Patients who have suffered an acute ischemic stroke (AIS) and are smokers may have a better outcome following thrombolytic therapy when compared with non-smokers. While this finding is controversial, data on baseline clinical risk factors to predict treatment efficacy of thrombolytic therapy using ambulatory status in patients who suffered AIS and are smokers is not common. Methods: Between 2010 and 2016, retrospective data on patients who have suffered an AIS and received recombinant tissue plasminogen activator (rtPA) were obtained from Greenville health system registry. Assessment of clinical risk factors and the likelihood of an improvement in post-stroke ambulation among smokers and non-smokers was carried out using multivariate logistic regression. Results: Of 1001 patients, 70.8% were smokers and 29.2% non-smokers. Among the smokers and non-smokers, 74.6% and 84.6% improvement in ambulation respectively at discharge. The odds of improved ambulation decrease among smokers as age group increases compared to those below 50 [(60–69 years, aOR, 0.30, 95% C.I, 0.108–0.850, p < 0.05), (70–79 years aOR, 0.27, 95% C.I, 0.096–0.734, p < 0.05), (80+ years aOR, 0.16, 95% C.I, 0.057–0.430, P < 0.01). Patients with National Institute of Health Stroke Scale Score (NIHSS) score > 7 (reference <7) were 91% less likely to have improved ambulation among smokers and non-smokers (aOR, 0.09, 95% C.I, 0.055–0.155, P = 0.01), and (aOR, 0.08, 95% C.I, 0.027–0.214, P = 0.01) respectively. Atrial fibrillation was an independent predictor of decreased improvement in ambulation only among smokers (aOR, 0.58, 95% C.I, 0.356–0.928 P < 0.05). Conclusion: Our findings suggest that elderly smokers with atrial fibrillation would benefit more from aggressive management of atrial fibrillation than non-smokers.

Patients with atrial fibrillation (AF) are at increased risk of stroke. Warfarin anticoagulation therapy reduces the incidence of stroke and increases the incidence of hemorrhagic events. This dissertation further informs the decision to use anticoagulation therapy in AF patients by examining outcomes in patients with major hemorrhages, further examination of stroke risk in diabetic patients with AF, and by evaluating the association between warfarin and stroke while accounting for competing risk events. These studies utilized data from the AnTicoagulation and Risk Factors In Atrial Fibrillation (ATRIA) and ATRIA-CVRN (Cardiovascular Research Network) (Study 1 only) studies which consist of patients from Kaiser Permanente Northern and Southern California. Study 1 examined short and long-term mortality in patients who experienced major gastrointestinal (GI) hemorrhages. In the ATRIA cohort, patients using and not using warfarin at the time of GI hemorrhage were equally likely to die within 30-days, while in ATRIA-CVRN, patients using warfarin were much less likely to die within 30-days (adjusted mortality rate ratio (aMRR): 0.33, 95% CI: 0.16-0.70). For longer-term mortality, both cohorts were consistent with a reduced mortality rate among patients whose GI hemorrhage occurred while using warfarin. Study 2 assessed the association between diabetes characteristics (duration of diabetes and glycemic control) and incidence of ischemic stroke among patients with AF and diabetes. Duration ≥ 3 years was associated with a large increase in rate of stroke (adjusted hazard ratio (aHR): 2.04, 95% CI: 1.27-3.26) compared to patients with duration < 3 years. Patients with the poorest glycemic control (hemoglobin A1c (HbA1c) values ≥ 9.0%) did not have an increased rate of ischemic stroke compared to patients with HbA1c < 7.0%. Study 3 evaluated the association between warfarin and thromboembolism in analyses that did and did not account for competing death events. In analyses not accounting for competing events, the adjusted HR was 0.61 (95% CI: 0.54-0.69), and after accounting for competing death events this association was attenuated (aHR: 0.87, 95% CI: 0.77-0.99). In summary, these studies add to the literature about the benefits of warfarin therapy and risk of stroke in patients with AF, findings that can improve decisions about use of anticoagulants in patients with AF.

PURPOSE: Ischemic retinopathies such as diabetic retinopathy (DR), retinal vein occlusions (RVO), and age-related macular degeneration (AMD) are ocular diseases caused by abnormal changes in the microvasculature that results in ischemia. This is often followed by a secondary phase characterized by pathological neovascularization and leakage of fluid, which contributes to a loss of visual acuity in affected patients. Anti-VEGF therapy, the current standard of treatment for ischemic retinopathies, is invasive, costly, and lacks a known treatment period. Supplemental oxygen provides the therapeutic potential of not only oxygenating hypoxic retinal cells, but also reducing the neovascularization and edema associated with many ischemic retinopathies through the downregulation of proangiogenic and pro-inflammatory cytokines.The objective of this study is to understand the biochemical underpinnings of treating ischemic retinopathies with hyperoxia. The elucidation of the effect hyperoxia on the molecular level may help guide the development of future studies regarding this novel treatment. METHODS: 68 undiluted vitreous samples were obtained during pars plana vitrectomy (PPV) and the concentration analysis of 34 proteins was analyzed using the Bio-Plex Pro Human Cancer Biomarker Assay. Vitreous samples were divided into three groups: (1) eyes of patients who underwent PPV for epiretinal membrane peeling (ERMP) and/or macular hole (MH) with no history of diabetes mellitus (non-DM group); (2) eyes of patients who underwent PPV for ERMP and/or MH with a history of diabetes or nonproliferative diabetic retinopathy (DM group); (3) eyes of patients who underwent PPV for proliferative diabetic retinopathy (PDR group). Mann-Whitney U tests were performed to compare the biomarker concentrations between the three groups. RESULTS: Numerous growth factors and inflammatory cytokines were significantly upregulated between the non-DM and PDR groups - Angiopoietin-2, EGF, Endoglin, G-CSF, HB-EGF, HGF, PDGF, PIGF, sHER2/neu, TIE-2, VEGF-A, VEGF-D, IL-18, IL-6, IL-8, PECAM-1, sCD40L, SCF, sFASL, sIL-6Ra, TNF-⍺, Leptin, PAI-1, and uPA. A literature search of these proteins revealed many to be directly activated by HIF-1 transcription factor, which is the "master switch" for genes transcribed during a hypoxic event. CONCLUSION: The abundance of proangiogenic and pro-inflammatory factors in PDR that are also upregulated by HIF-1 demonstrate the potential for using hypoxia to treat PDR (and other ischemic retinopathies) through the reduction of HIF-1. This study also shows the wide variability in the expression levels of these proteins which helps provide a better understanding of their degree of involvement in the pathogenesis of ischemic retinopathies. / 2021-06-14T00:00:00Z

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Stroke is a leading cause of morbidity and long-term neurological disability in the U.S. Ischemic stroke, which accounts for approximately 90% of all strokes, is the result of an occlusion in the arteriole cerebrovascular network. No effective treatment options exist to provide neuroprotection from occlusion, and limited success has been seen clinically when attempting to restore blood flow to vulnerable neural tissue regions. Enhancement of pial collateral remodeling (Arteriogenesis) has recently been shown to improve blood flow and mitigate neural tissue damage following stroke (1-3). Arteriogenesis is the remodeling of pre-existing arteriole vessel which are able to re-route blood to blood-deprived regions of tissue. Arteriogenesis requires endothelial cell (EC) and smooth muscle cell proliferation, extracellular matrix degradation and recruitment of circulating bone marrow-derived cells (4-6). Unlike spouting angiogenesis, which requires weeks following occlusion to develop, arteriogenesis begins as early as 24-48hrs post-stroke (7, 8) and can expeditiously enhance blood flow to ischemic regions, making it an attractive target for therapeutic intervention. Our preliminary studies, in an EphA4 global knockout mouse model, indicated that EphA4 receptor tyrosine kinase severely limits pial arteriole collateral formation. The preliminary work also showed that activation of EC EphA4 receptor in vitro inhibited vascular formation. Additionally, ECs lining the collateral vessel have been shown to play a role in collateral remodeling (9). Taken together, the objective of this dissertation was to elucidate the cell autonomous role of the EphA4 receptor and given the central role of the EC in collateral remodeling, we postulated that EphA4 receptor on ECs the limits pial collateral formations. Using a cell-specific loss-of-function approach, we tested the hypothesis that EC-specific EphA4 plays an important role in pial collateral development and remodeling after induced stroke. The results from this dissertation show that (1) EphA4 expression on ECs suppress the formation of pial collaterals during development and limits EC growth via suppression of p-Akt in vitro (2) EC-specific EphA4 ablation leads to increased collateral remodeling, enhanced blood flow recovery, tissue protection and improved neurological behavioral outcomes after stroke and (3) Mechanistically, EphA4 limits pial collateral remodeling via attenuation of the Tie2/Angiopoietin-2 signaling pathway. The work presented in this dissertation demonstrate that EphA4 can be targeted therapeutically to increase pial collateral remodeling to alleviate neurological deficits after ischemic stroke. / Doctor of Philosophy / Stroke is the fifth leading cause of death in the United States. Ischemic stroke is the most common type of stroke and occurs when blood flow to part of the brain is impeded. Lack of blood results in cell death and tissue damage in the brain. In an effort to restore blood flow, specialized blood vessels in the brain called collaterals remodel and become larger to allow re-routed blood to the blood-deprived region of the brain. The duration it takes to remodel these remarkable blood vessels and re-route blood varies in humans, and sometimes is not able to prevent adequate tissue damage. The current work explores novel therapeutic targets to accelerate collateral remodeling in an effort to reduce tissue loss after stroke. We present studies which show that a protein called EphA4, found on endothelial cells restricts remodeling, and when inhibited in the brain can increase collateral remodeling and reduced adverse effects after ischemic stroke.