Advisor(s)

Mark Olah, PhD
Ohio Northern University
Pharmaceutical & Biomedical Sciences
m-olah@onu.edu

Document Type

Poster

Location

ONU McIntosh Center; Activities Room

Start Date

22-4-2022 12:00 PM

End Date

22-4-2022 1:00 PM

Abstract

Millions of people are affected by diseases involving inflammatory states such as sepsis. Sepsis is associated with elevated tumor necrosis factor alpha (TNF-⍺) which mediates inflammation. Excessive TNF-⍺ contributes to death of endothelial cells (EC) with subsequent disruption of vascular endothelial function. Exchange Factor Directly Activated by cAMP (EPAC1) is a cyclic-AMP (cAMP) sensor that regulates multiple EC functions. We hypothesized that EPAC1 activation affects the response of EC to TNF-a. Microvascular EC were pretreated for 45 minutes with 100 mM 8-pCPT-2′-O-Me-cAMP (8-CPT, specific EPAC1 activator) or 10 mM forskolin (adenylyl cyclase activator that increases cAMP), then exposed to 5 ng/ml TNF-⍺ for 24 hours. MTT assays demonstrated that TNF-⍺ decreased cell viability to 76.3±2.3% of control and that 8CPT and forskolin protected the cells (92.4±4.8% and 98.7±3.3% of control, respectively). Western blotting for cleaved caspase-3 indicated TNF-⍺-induced death occurred via apoptosis and was inhibited by 8CPT and forskolin. To examine the role of EPAC1 in protection from TNF-⍺, EPAC1 expression was decreased via RNAi and MTT assays performed. In mock-transfected cells, 8CPT and forskolin inhibited TNF-a induced death. However, in EPAC1 siRNA-transfected cells, the protective effect of 8CPT was abolished while the response to forskolin was unaffected. This supports EPAC1 involvement in the effect of 8CPT but suggests an alternative pathway in response to a global increase in cAMP. Current studies are examining the role of the cAMP effector Protein Kinase A as well as further confirming the anti-apoptotic effect of EPAC1 in endothelial cells.

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Apr 22nd, 12:00 PM Apr 22nd, 1:00 PM

Exchange Protein Directly Activated by cAMP (EPAC1) Protects Human Endothelial Cells from Tumor Necrosis Factor-⍺ (TNF-⍺) Induced Cell Death

ONU McIntosh Center; Activities Room

Millions of people are affected by diseases involving inflammatory states such as sepsis. Sepsis is associated with elevated tumor necrosis factor alpha (TNF-⍺) which mediates inflammation. Excessive TNF-⍺ contributes to death of endothelial cells (EC) with subsequent disruption of vascular endothelial function. Exchange Factor Directly Activated by cAMP (EPAC1) is a cyclic-AMP (cAMP) sensor that regulates multiple EC functions. We hypothesized that EPAC1 activation affects the response of EC to TNF-a. Microvascular EC were pretreated for 45 minutes with 100 mM 8-pCPT-2′-O-Me-cAMP (8-CPT, specific EPAC1 activator) or 10 mM forskolin (adenylyl cyclase activator that increases cAMP), then exposed to 5 ng/ml TNF-⍺ for 24 hours. MTT assays demonstrated that TNF-⍺ decreased cell viability to 76.3±2.3% of control and that 8CPT and forskolin protected the cells (92.4±4.8% and 98.7±3.3% of control, respectively). Western blotting for cleaved caspase-3 indicated TNF-⍺-induced death occurred via apoptosis and was inhibited by 8CPT and forskolin. To examine the role of EPAC1 in protection from TNF-⍺, EPAC1 expression was decreased via RNAi and MTT assays performed. In mock-transfected cells, 8CPT and forskolin inhibited TNF-a induced death. However, in EPAC1 siRNA-transfected cells, the protective effect of 8CPT was abolished while the response to forskolin was unaffected. This supports EPAC1 involvement in the effect of 8CPT but suggests an alternative pathway in response to a global increase in cAMP. Current studies are examining the role of the cAMP effector Protein Kinase A as well as further confirming the anti-apoptotic effect of EPAC1 in endothelial cells.