Browsing by Author "Shrestha, Pawan"
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Item Evaluating the properties of extracellular vesicles that are affected by diabetic keratopathy.(2023) Hefley, Brenna; Shrestha, Pawan; Ma, Jian-Xing; Karamichos, DimitriosPurpose: Extracellular Vesicles (EVs) are small-sphere like structures that are released from cells. They play a role in cell-cell communication by passing genetic information from one cell to another. EVs are thought to be critical in future diagnostic targets for various diseases and conditions. Diabetic Keratopathy (DK) can often lead to different manifestations such as scarring and corneal erosions. One of the receptors that plays a role in DK is Peroxisome Proliferator-Activated Receptor alpha (PPARα), but its functions in the cornea is unknown, including its impact on the EVs formation. Our goal was to investigate the influence of PPARα in the production of EVs and its role in cell-cell interaction. Methods: Healthy Corneal Fibroblasts (HCFs), Type 1 Diabetes Mellitus (T1DM), and Type 2 Diabetes Mellitus (T2DM) corneal stromal cells were cultured on polycarbonate membranes for 4 weeks at a density of 1x106 cell/well in culture medium + 0.5mM stable Vitamin C. The culture media were processed and analyzed with the EV-TETRA-C chips paired with the ExoView R100. Results: The results showed that total particle counts were upregulated in HCF compared to T1DM and T2DM, but were downregulated in T1DM when compared with T2DM, during weeks 2 and 4. The total particle count for HCFs were downregulated during week 4 when compared to weeks 1 and 3. When comparing week 1 to weeks 2, 3, and 4, week 1 revealed the most significance for changes of CD63+, CD63+/CD81+, CD81+/CD9+, and CD63+/CD81+/CD9+ in HCFs and T1DMs. CD9+ showed significance during weeks 2, 3, and 4 in HCFs and T1DMs. During week 1, T2DMs had significant downregulation of CD63+, whereas CD9+, CD63+/CD9+, and CD63+/CD81+/CD9+ showed significant upregulation. During week 4, T2DMs showed significant downregulation in CD63+/CD81+ and significant upregulation of CD81+/CD9+. T1DMs were the only cell type to show significance in CD81+, which was during week 4 when compared to week 2. CD63+, CD81+, and CD9+ showed significance during week 1 and 2 in HCF, T1DM, and T2DM. The co/triple colocalizations showed significance in weeks 1, 2, and 4 in HCF, T1DM, and T2DM. Conclusion: EV formation (based on CD63+), immune system regulation (CD81+ and CD9+), and EV particle counts showed distinct phenotypes between HCF, T1DM, and T2DM. These distinctions could potentially serve as a diagnostic tool in the future and ultimately help individuals suffering from DK.Item Impact of sex and hypoxia on brain region-specific expression of androgen receptor AR45 and G protein Gαq in young adult rats(2024-03-21) Wilson, Elizabeth; Bradshaw, Jessica; Mabry, Steve; Shrestha, Pawan; Gardner, Jennifer; Cunningham, RebeccaPurpose: Sex differences in oxidative stress-associated cognitive decline are influenced by sex hormone levels. However, little is known regarding the expression of hormone receptors in brain regions associated with cognitive function. Notably, oxidative stress-associated neuronal cell death is exacerbated through testosterone signaling via membrane-associated androgen receptor AR45 and G protein Gαq. The objective of this study was to elucidate the expression of AR45 and Gαq in brain regions associated with cognitive function. Additionally, we investigated whether chronic intermittent hypoxia (CIH), an oxidative stressor with sex-specific effects, would modulate AR45 and Gαq expression. Methods: Adult male and female Sprague-Dawley rats were exposed to CIH or normoxia for 14 days. We quantified AR45 and Gαq protein expression in various cognition-associated brain regions [dorsal hippocampal CA1, CA3, DG, and entorhinal cortex (ETC)] via western blotting. For comparisons, AR45 and Gαq protein expression were also assessed in brain regions outside the hippocampal-ETC circuit [thalamus (TH) and striatum (STR)]. Results: The highest AR45 levels were expressed in the CA1 while the lowest expression was observed in the STR. The highest Gαq levels were expressed in the DG and ETC while the lowest expression was observed in the TH. We observed no effect of sex on AR45 or Gαq expression regardless of brain region assessed. Similarly, there was no effect of CIH on AR45 expression in any of the brain regions examined. However, CIH exposure increased Gαq expression only in the CA3 regardless of sex. Conclusions: Our findings reveal enrichment of AR45 and Gαq protein expression within the hippocampal-ETC circuit, which is vulnerable to oxidative stress and neurodegeneration during cognitive decline. Moreover, our data suggest the CA3 is the most vulnerable region to CIH-mediated oxidative stress. Overall, these findings were observed in both sexes, indicating that there are no observed sex differences in AR45 and Gαq expression or their modulation by CIH.Item Relationship of Down Syndrome with Keratoconus and Gonadotropins(2023) Shrestha, Pawan; Escandon, Paulina; Petersen, Melissa; Hjortdal, Jesper; Ramirez, Lito; Karamichos, DimitriosPurpose: Down syndrome (DS), also known as trisomy 21, is a common genetic disorder of chromosomal nondisjunction. DS has been strongly associated with Keratoconus (KC); however, the exact pathobiology remains unexplored. KC is one of the most significant corneal disorders which is characterized by thinning, cone-shaped protrusion, and steepening of the cornea leading to a significant reduction of visual acuity and even blindness. The aim of this study was to investigate the relationship of DS with KC in the context of gonadotrophic hormones. Methods: This study adhered to the declaration of Helsinki. Fifty-eight healthy controls (29 male, 29 female), one hundred and forty-nine KC (112 male, 37 female), and eighty DS (44 male, 36 female) patients were recruited for this study. Plasma samples were collected from all participants. We investigated the expression of Gonadotrophin-releasing hormone (GnRH) and Follicle stimulating hormone (FSH) using enzyme-linked immunosorbent assay (ELISA). Results: Significant downregulation of GnRH expression was observed in KCs when compared with healthy (p = 0.0006) and DSs (p = 0.00249). GnRH was significantly downregulated in KC and DS males, compared to their healthy counterparts, while no significance was observed in females across all diseases. Significant upregulation of FSH levels was observed in DSs compared to both healthy and KCs (p < 0.0001). FSH expression was also significantly elevated in both DS males and females when compared to healthy and KCs. Conclusions: Our results revealed downregulation of GnRH, but upregulation of FSH in DS participants as compared to KCs. These findings provide new insights into the potential association between DS and KC, and substantiate the role of gonadotropins. Further studies are warranted to further understand the underlying mechanisms and potential implications for the treatment and management of these conditions.