The Role of Occludin in Vascular Endothelial Protection

Endothelial tight junction proteins play an important role in maintaining the integrity of vascular endothelial structure and physiological function. In recent years, studies have found that alterations in the expression, distribution, and structure of endothelial tight junction proteins may lead to many related vascular diseases and pathologies (such as diabetes, atherosclerosis, neurodegenerative diseases, and hypertension). Therefore, related strategies to prevent and/or tight junction proteins dysfunction may be an important therapeutic target. Occludin, as the most representative one among tight junction proteins, is mainly responsible for sealing intercellular junctions, maintaining cell permeability and the integrity of vascular endothelium. Here, we review the published biological information of occludin. We highlight the relationship between occludin and vascular endothelial injury-related disease. At the same time, we show our current knowledge of how vascular endothelial occludin exerts the protective effect and possible clinical applications in the future

The Dual Role of Hypoxia-Inducible Factor-1 in Ischemic Stroke: Neuroprotection and Blood-Brain Barrier Disruption

Stroke is a major cause of death and the leading cause of long-term disability in industrialized countries. Ischemic stroke-induced brain injury results from the interaction of complex pathophysiological processes, including energy failure, calcium overload, excitotoxicity, oxidative stress, disruption of blood-brain barrier (BBB) and inflammation. Despite the wealth of knowledge regarding the cellular and molecular mechanisms underlying neuronal death after stroke, research for several decades has failed to develop an effective and safe neuroprotective treatment. One complicating factor in the development of neuroprotective strategies is the dual nature of many of the processes that occur in the brain during stroke. Hypoxia-inducible factor 1 (HIF-1) is a master regulator of cellular and tissue adaption to hypoxia. It plays both protective and detrimental roles in ischemic stroke by inducing a wide array of target genes involved in angiogenesis, erythropoiesis, cell survival/death, and energy metabolism. The dual face of HIF-1 in the pathophysiology of cerebral ischemia is postulated to partially depend on thedifferent functions of its target proteins in specific type of brain cells. In the current studies, we hypothesize that neuronal HIF-1 accumulation is protective whereas endothelial HIF-1 induction is implicated in BBB disruption. We first evaluated HIF-1's role in the antioxidant N-acetylcysteine (NAC)-mediated neuroprotection in a transient cerebral ischemia animal model. The study demonstrated that pre-treatment of NAC increased the neuronal expression of HIF-1α, the regulatable subunit of HIF-1, and its target proteins erythropoietin (EPO) and glucose transporter (GLUT)-3 in the ischemic brain of rodents subjected to 90 min middle cerebral artery occlusion (MCAO) and 24 h reperfusion. Suppressing HIF-1 activity by pharmacological inhibitorsor by specific knock-out neuronal HIF-1α abolished NAC's neuroprotective effects. Furthermore, we observed that NAC increased HIF-1α stability through enhancingits interaction with heat-shock protein 90 (Hsp90) in ischemic brains. Increased BBB permeability and associated cerebral edema formation are potentially lethal complications of ischemic stroke. Accumulating evidence has shown that admission hyperglycemia in conjunction with ischemia/reperfusion causes exacerbated cerebrovascular endothelial cell dysfunction and increased BBB permeability, which leads to augmented brain edema and hemorrhagic transformation in ischemic stroke.The hypothesis of the second part of this dissertation is that endothelial HIF-1 is implicated in hyperglycemia-exacerbated BBB disruption after ischemia. Both in vitro and in vivo studies were undertaken to investigate the effect of hyperglycemia on (1) HIF-1α and its target genes expression; (2) ischemia-induced BBB permeability change; and (3) the effect of HIF-1α inhibition on BBB permeability after ischemia. The in vitrostudy showed that high glucose enhanced HIF-1αand its downstream factors expression in the endothelial cell culture after oxygen glucose deprivation (OGD)/reoxygenation. This was correlated withan increased paracellular permeability as well as diminished expression and disrupted continuity of tight junction (TJ) proteins. Suppressing HIF-1 activity by HIF-1α inhibitors ameliorated the alterations in paracellular permeability and expression and distribution pattern of TJ proteins induced by high glucose exposure. In in vivo studies, diabetic mice subjected to 90 min MCAOfollowed by reperfusiondemonstrated higher expression of HIF-1α and its target gene vascular endothelial growth factor (VEGF) in the ischemic brain microvessels than non-diabetic control mice. Diabetic mice also showed exacerbated BBB damage and TJ disruption, increased infarct volume, and worsened neurological deficits. SuppressingHIF-1 activity by specific knock-out endothelial HIF-1α ameliorated BBB leakage and brain infarction in diabetic animals. Taken together, these present studies provide new information concerning HIF-1's function in experimental models of acute ischemic stroke. Neuronal HIF-1α is an important mediator of antioxidant NAC's neuroprotective effect in ischemic stroke, whereas endothelial HIF-1α is involved in hyperglycemia-induced BBB breakdown after cerebral ischemia. The results suggest that developing therapeutic strategies by targeting HIF-1 needs to consider its multifunctional roles and differential effects on different cell types

Mechanisms for Extracellular Matrix-Dependent Blood-Brain Barrier Dysfunction

Dysfunction of the blood vessels that form the blood-brain barrier (BBB) is observed across various neurological disorders, including multiple sclerosis (MS). As barrier loss culminates in neuronal dysfunction and degeneration, a better understanding of the mechanisms underlying BBB dysfunction is needed. Tight junctions are multiprotein complexes maintained by the endothelial cells lining the inner blood vessel wall to seal the intercellular space, and their disruption impairs BBB function. In my first chapter, I focus on how tight junctions are altered in CNS inflammatory demyelinating diseases (CNS-IDDs) like MS as BBB dysfunction is one of the earliest known stages in their disease progression. Literature searches were conducted for relevant studies involving three prominent tight junction protein families, namely the claudins, tight junction-associated MARVEL proteins (TAMPs), and angulins. As few studies analyzed patient tissues, additional literature searches were conducted for relevant cell culture and animal models. Particular attention is paid to studies involving pharmacological interventions or genetic manipulations as tight junctions are increasingly being recognized as possible therapeutic targets for preserving or restoring BBB function and, in turn, CNS homeostasis. In my second chapter, I explore how the molecular composition of the vascular basement membrane (BM) can influence barrier function. Under healthy conditions, the vascular BM surrounding the endothelium fosters BBB function through cell-extracellular matrix (ECM) protein interactions that promote the tight junction protein claudin-5. During inflammation, however, the molecular composition of the BM is perturbed. Recently, the ECM proteins collagen type I and decorin, both of which are typically absent from the BM, were observed surrounding vessels in the lesions of MS patients. As their roles in inflammation are poorly understood, I investigated whether they can influence barrier function or claudin-5 expression. Using a mouse model of inflammatory encephalomyelitis, I found that decorin is present within the BM during early disease when the onset of BBB dysfunction occurs. In complement, I conducted cell culture studies with mouse BBB endothelial cells, overall finding an inverse relationship between decorin and barrier function or claudin-5. Similar cell culture studies using collagen type I revealed a similar inverse relationship between it and barrier function or claudin-5. Overall, this work suggests that 1) tight junction proteins may be a viable therapeutic target in restoring BBB dysfunction and 2) inflammation-associated alterations to the vascular BM may contribute to BBB dysfunction by suppressing the tight junction protein claudin-5

Towards adipose tissue-derived stromal cells-based therapy for diabetic retinopathy

In this thesis, we investigated the impact of hyperglycemia on adipose tissue-derived stromal cells (ASC) as a prelude to their use in the future therapeutic treatment of diabetic retinopathy (DR). Ultrastructure analyses of co-cultured ASC-endothelial cells featured the pericytic role of ASC in the maintenance of the vascular architecture under normal and high glucose conditions. The ROS-induced mitochondrial dysfunction and hyperglycemia-induced apoptosis partially influenced the pericytic functions of ASC. Injected ASC into an angiogenic mouse models, were detected at pericytic positions on newly formed vessels. Our recommendation is to preculturing of ASC under ‘chronic’ hyperglycemia, before injecting into a hyperglycemic environment to reduce their expression of pro-inflammatory and pro-angiogenic genes. ASC-conditioned medium (ASC-Cme) delivered from ASC cultured in chronically HG, protected bovine retinal endothelial cells (BREC) from hyperglycemia-induced apoptosis and inflammatory activation. This effect on BREC was evoked by the ROS-neutralizing capacity of ASC-Cme in culture that was associated with a reduced NF-κB activation which showed as a downmodulation of HG-upregulated pro-inflammatory genes in BREC. Towards using ASC for treatment of DR, we present a new hypothermic storing technique of cells in their own culture medium to well-maintain and transport the prepared cells to their clinical destination. The new pharmacologic compound, (6-hydroxyl-2,5,7,8-tetramethylchroman-2-yl)(4-(2-hydroxyethyl)piperazin-1-yl) methanone (SUL-109) shields ASC during cell preservation from hypothermic cell death without influencing their multi-potency capacity and proliferation through maintenance of the mitochondrial membrane potential and promoting the activation of mitochondrial complexes I and IV, consequently sustaining ATP production and preventing the overproduction of ROS under hypothermic conditions

Age-related Alterations on Ultrastructure and Gene Expression Profile of the Female Blood-brain Barrier

Blood-Brain Barrier (BBB) breakdown occurs in ageing and neurodegenerative disorders and affects several brain regions including cortex and hippocampus. During ageing, structural and functional changes affecting the main BBB components (brain endothelial cells (BECs), pericytes and astrocytes), appear to be associated with altered expression of genes and microRNAs (miRNAs) potentially related to development, protein synthesis or longevity pathways. However, little is known about the age-related BBB dysfunction in females. In this study, we aimed to assess the relation between ultrastructural and transcriptional changes in the ageing female BBB. A combination of transmission electron microscopy (TEM) and 3D reconstruction was used to study microvessel ultrastructure in 6- and 24-month-old female C57BL/6J mice. According to our results, the ageing female BBB shows a significant increase in basement membrane (BM) thickness, volume and number of BEC pseudopods, pericyte mitochondrial volume, pericyte – BEC contact and tight junction (TJ) tortuosity. Also, cortical capillaries appeared to be prominently more affected during ageing than hippocampal capillaries. These results suggest a higher impact of ageing on the cortical BBB in females, promoting changes that lead to a pro-inflammatory state, among other processes. In addition, sequencing results showed that the majority of upregulated genes in the ageing female BBB were involved in inflammation and immune response pathways, whereas the downregulated genes were mostly related to metabolism and signalling pathways. Amongst the age-deregulated mRNAs and miRNAs, miR-144-3p (upregulated) and Dnmt3a (downregulated) were selected for functional analysis in a human BEC line (hCMEC/D3), where their inverse correlation was confirmed. However, DNMT3A, not miR-144-3p, was shown to influence BEC function when deregulated, thereby promoting higher leukocyte adhesion and mRNA levels of adhesion molecules (ICAM-1, VCAM-1) and the chemokine CCL5. Age-induced increase of miR-144-3p appears to modulate DNMT3A expression, but DNMT3A might be independently contributing more to switching BECs into a pro-inflammatory state

THE PREBIOTIC INULIN BENEFICIALLY MODULATES THE GUT-BRAIN AXIS BY ENHANCING METABOLISM IN AN APOE4 MOUSE MODEL

Alzheimer’s disease (AD) is the most common form of dementia and a growing disease burden that has seen pharmacological interventions primarily fail. Instead, it has been suggested that preventative measures such as a healthy diet may be the best way in preventing AD. Prebiotics are one such potential measure and are fermented into metabolites by the gut microbiota and acting as gut-brain axis components, beneficially impact the brain. However, the impact of prebiotics in AD prevention is unknown. Here we show that the prebiotic inulin increased multiple gut-brain axis components such as scyllo-inositol and short chain fatty acids in the gut, periphery, and in the case of scyllo-inositol, the brain. We found in E3FAD and E4FAD mice fed either a prebiotic or control diet for 4-months, that the consumption of the prebiotic inulin can beneficially alter the gut microbiota, modulate metabolic function, and dramatically increase scyllo-inositol in the brain. This suggests that the consumption of prebiotics can beneficially impact the brain by enhancing metabolism, helping to decrease AD risk factors