An In Vitro Bovine Cellular Model for Human Schlemm's Canal Endothelial Cells and Their Response to TGFβ Treatment.
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2020-06-25
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Purpose:Due to the limited availability of primary human Schlemm's canal (SC) endothelial cells, we aimed to develop an in vitro cellular model using the angular aqueous plexus (AAP) cells from bovine eyes. Methods:We harvested a mixture of cells from the trabecular meshwork region including AAP loops from multiple donors, followed by puromycin treatment and immunostaining of Von Willebrand factor and vascular endothelial (VE)-cadherin to confirm identity. Previously identified differentially expressed genes in glaucomatous SC cells were examined in non-glaucomatous SC cells (n = 3) under 0% or 15% equibiaxial strain for 24 hours using droplet digital polymerase chain reaction (ddPCR) and analyzed using the Ingenuity Pathway Analysis (IPA) software application to identify upstream regulators. To compare the cellular responses to candidate regulators of these mechanoresponsive genes, AAP and human SC cells (n = 3) were treated with 5 or 10 ng/mL transforming growth factor beta-2 (TGFβ2) for 24 or 48 hours, followed with expression profiling using real-time PCR or ddPCR. Results:We found that the isolated AAP cells displayed uniform cobblestone-like morphology and positive expression of two endothelial markers. In stretched SC cells, nine glaucoma-related genes were upregulated, and IPA implicated TGFβ as a potential upstream regulator. The effects of TGFβ2 treatment were similar for both AAP and SC cells in a dose- and time-dependent manner, activating TGFBR1 and SMAD2, inhibiting BMP4, and altering expression of three glaucoma-related genes (DCN, EZR, and CYP1B1). Conclusions:Bovine AAP cells may serve as an alternative cellular model of human SC cells. Translational Relevance:These AAP cells may be used to study the functional pathways related to the outflow facility.
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Cai, Jingwen, Kristin Perkumas, W Daniel Stamer and Yutao Liu (2020). An In Vitro Bovine Cellular Model for Human Schlemm's Canal Endothelial Cells and Their Response to TGFβ Treatment. Translational vision science & technology, 9(7). p. 32. 10.1167/tvst.9.7.32 Retrieved from https://hdl.handle.net/10161/21830.
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Scholars@Duke
W Daniel Stamer
My laboratory studies the disease of glaucoma, the second leading cause of blindness in the United States, affecting nearly 3 million people (70 million Worldwide). The primary risk factor for developing glaucoma is ocular hypertension (high intraocular pressure, IOP). IOP is a function of the regulated movement of aqueous humor into and out of the eye. Elevated IOP in glaucoma is a result of disease in the primary efflux route, the conventional outflow pathway, affecting proper homeostatic control of aqueous humor drainage.
Lowering IOP in glaucoma patients, whether or not they have ocular hypertension, is important because large clinical trials involving tens of thousands of patients repeatedly demonstrate that significant, sustained IOP reduction slows or halts vision loss. Unfortunately, current first-line medical treatments do not target the diseased conventional pathway and do not lower IOP sufficiently in most people with glaucoma. Therefore, finding new, more effective ways to medically control IOP by targeting the conventional pathway is a central goal the Stamer Laboratory.
Using molecular, cellular, organ and mouse model systems, my laboratory seeks to identify and validate novel drug targets in the human conventional outflow pathway to facilitate the development of the next generation of treatments for ocular hypertension and glaucoma.
Yutao Liu
Dr. Liu's research interest is to identify genetic risk factors related to age-related complex human diseases, including but not limited to primary open-angle glaucoma (POAG), keratoconus, exfoliation glaucoma (XFG), amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease), and Post-traumatic Stress Disorder (PTSD). Dr. Liu is also interested in the role of genomic structural variation (i.e., DNA copy number variants) in human disease. He was funded by the Duke Translational Medicine Institute and Glaucoma Research Foundation to study the effects of DNA copy number variants (CNV) in glaucoma. This research is currently funded by American Health Assistance Foundation (AHAF). The research for exfoliation glaucoma is currently supported by The Glaucoma Foundation (TGF). He is currently characterizing genetic risk variants identified through large scale genome-wide association studies (GWAS) using bioinformatics, molecular genetics, and animal models. He is also interested in applying next-generation sequencing, such as exome sequencing, to identify genetic mutations in multiplex families affected by ocular disorders, such as keratoconus.
Dr. Liu is also the Director of Duke Molecular Genomics Core (MGC) lab(http://wwwchg.duhs.duke.edu/research/molecular.html). The MGC is a state-of-the-art facility that offers a variety of experimental platforms to facilitate genomics research, including but not limited to gene expression profiling for mRNA and microRNA, DNA genotyping, next generation DNA/RNA sequencing, and DNA methylation profiling. For more information, please visit the MGC website (http://wwwchg.duhs.duke.edu/research/molecular.html).
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