Profiling of the Differentially Expressed Genes of Inflammation and Angiogenesis of Human Brain Microvascular Endothelial Cells Evoked by Cell Injury In-Vitro to Mimic Acute Ischemic Stroke-Like Conditions
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Ischemic stroke (IS) is a leading cause of disability and death worldwide. IS arises from a blocked artery in the brain that causes a lack of oxygen and nutrient supply to other brain cells, causing cell injury and neuronal death. In-vitro Acute ischemic stroke models were developed in the lab, by a method of oxygen-glucose deprivation (OGD), to understand the mechanisms and pathophysiology of brain cell injury in IS. One important cell type of brain is the primary human brain microvascular endothelial cell (PHBMEC), which builds up the blood-brain barrier (BBB), and any disruption of it, could cause injury to other cells in the brain. Acute IS (AIS) injury of PHBMEC is not well characterized in terms of its consequences on cell functions using genomic function profiling. In our study, we aimed to establish a new model of an acute in-vitro ischemic stroke, within a time limit of 2 hrs., with depriving whole nutrients and not just glucose, under hypoxic condition, in a model we created as oxygen nutrients deprivation (OND). Control cells were exposed to normal conditions of glucose (5 mM) and other nutrients with 20% oxygen. In order to establish this model, different methods were used to assess cell injury such as viability, death, and apoptosis, cytoskeleton or F-actin stain, and biochemical changes. RNA extracted was analyzed using RT-PCR profiling of angiogenesis and inflammatory genes, differentially expressed genes were analyzed by Gene Globe analysis, and signaling pathways were identified using IPA software, Qiagen. Major findings of OND have decreased cell viability, increased cell death, increased F-actin stress fibers that indicated severe cell injury and BBB disruption, and increased acidity. Profiling of the gene expression revealed dysregulation of 20 genes (5 upregulated and 15 down regulated) of both inflammation and angiogenesis. Major signaling pathways identified were HIF1A and NOTCH3. These signaling pathways affected different functions, including cell survival, proliferation, angiogenesis, and cytoskeleton organization. In conclusion, the established OND model could cause severe and acute cell injury that could mimic AIS in humans. Cell injury was evidenced by the results of phenotype changes, differential gene expression, and the signaling pathways identified.
- Biomedical Sciences [54 items ]