Upregulated STIM2 And Orai2 Are Involved In The Phenotypic Transition Of Pulmonary Arterial Smooth Muscle Cells From Contractile To Proliferative Phenotype Ruby A. Fernandez, Jun Wan, Qiang Guo, and Jason X.-J. Yuan Department of Medicine, Division of Pulmonary; Critical Care, Sleep and Allergy Medicine; Department of Pharmacology; Center for Cardiovascular Research, University of Illinois at Chicago, Chicago, IL Pulmonary hypertension (PH) is a fatal disease attributed to an increased pulmonary vascular resistance (PVR). Elevated PVR is partially due to sustained pulmonary vasoconstriction and excessive pulmonary vascular remodeling. In adult patients with idiopathic PAH, only a minority of the patients respond to vasodilators, and can decrease PAP and PVR to the normal level in approximately 15% patients. Thus indicating that, at early stage, sustained pulmonary vasoconstriction plays an important role in the initiation and development of pulmonary hypertension in patients with PAH; while at late stage, a gradual transition from sustained vasoconstriction to vascular remodeling may play a critical role in the development and progression of pulmonary hypertension in the patients. We hypothesize that STIM2 upregulation may play an important role in the early transition of the disease (PAH) pathogenesis from the sustained vasoconstrictive stage to highly proliferative stage due to the transition of PASMC from contractile phenotype to highly proliferative phenotype and results in increased PAP, leading to PH. Introduction ResultsTranslational Summary Contractile markers are downregulated and STIM2 and TRPC6 are upregulated in cultured PASMC in comparison to isolated PA STIM2, TRPC6, and Orai2 are upregulated in proliferating PASMC as compared to contractile PASMC SOCE is enhanced while voltage-dependent Ca 2+ influx through VDCC is attenuated in primary cultured rPASMC SOCE is attenuated with knockdown of Orai2 by siRNA in proliferative PASMC SOCE is enhanced with overexpression of STIM2 in proliferative PASMC STIM2, TRPC6, and Orai2 are upregulated in proliferative PASMC (10% FBS) compared to quiescent differentiated PASMC (1% FBS) TGF  decreases the proliferation rate of rPASMC Differentiation of rPASMC with TGF  increases contractile marker expression and decreases SOC/ROC channels and STIM expression Differentiation of rPASMC with heparin increases contractile marker expression and decreases SOC/ROC channels and STIM2 expression Conclusion STIM2 upregulation may play an important role in the early transition of the disease (PAH) pathogenesis from the sustained vasoconstrictive stage to highly proliferative stage due to the transition of PASMC from contractile phenotype to highly proliferative phenotype and results in increased PAP, leading to PH. I would like to thank my mentor, Dr. Jason X.-J. Yuan, and my fellow lab members for their valuable guidance and help. The project described was supported by NIH grants: HL and HL In addition, this project was supported by the National Center for Advancing Translational Sciences, National Institutes of Health, through Grant UL1TR The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. Acknowledgements Cell Preparation: Freshly dissociated, primary, and cultured PASMC were isolated from male Sprague Dawley rats and used for experiments. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 10% FBS. Western blot: PASMC were lysed and supernatants were used as a sample protein. Samples were separated through a SDS-polyacrylamide gel and transferred to PVDF membranes. Blots were incubated with a primary Ab, anti-STIM2, Orai1, Orai2, TRPC6, MYH, and SM-22 . Band intensity was quantified with ImageJ, normalized to β-actin, and expressed as arbitrary units. Tension measurements: Pulmonary arteries were isolated and cut into rings and mounted to an isometric force transducer in a profusion chamber. Isometric tension was continuously monitored and recorded using DATAQ data acquisition software. Measurement of [Ca 2+ ] cyt : [Ca 2+ ] cyt was measured in PASMC using fura- 2 and a Nikon digital fluorescent imaging system. Cells were loaded with 4 μmol/L fura-2 acetoxymethyl ester (fura-2/AM) for 60 minutes at 25°C and [Ca 2+ ] cyt was measured using a ratiometric method at 32°C. Proliferation Assay: Proliferation of PASMC was determined using an automated cell counter (TC10; Bio-Rad Laboratories, Hercules, CA). rPASMC were counted and replated (0 hours) into 8-well multidishes with 1×10 5 cells/well. Materials & Methods Figure 1. STIM and Orai are the molecular components that mediate SOCE. Depletion of the ER/SR Ca 2+ store, through activation of the IP 3 R, leads to STIM oligomerization, translocation to puncta near the plasma membrane junction leading to formation and activation of Orai hexameric channels. Recent data also states STIM’s ability to inhibit VDCC. Figure 2. Differentiated SMC have increased contractile marker expression and decreased STIM2, TRPC6, and Orai2 expression compared to dedifferentiated proliferative SMC. Figure 3. SOCE is enhanced while voltage-dependent Ca 2+ influx through VDCC is attenuated in proliferative PASMC. Also, STIM2, TRPC6, and Orai2 are upregulated in proliferative PASMC as compared to contractile PASMC. Figure 4. Cell differentiation with 1% and 3% FBS significantly attenuates rPASMC growth rate and decreases STIM2, TRPC6, and Orai2 expression. Figure 5. Cell differentiation with TFG  attenuates rPASMC growth rate and decreases TRPC6, STIM1, and Orai2 expression. Figure 6. Cell differentiation with heparin increases the contractile markers and decreases TRPC6, STIM1, STIM2, Orai1, Orai2, and Orai3 expression. Figure 7. Knockdown of Orai2 attenuates SOCE in rPASMC and decreases proliferating cellular nuclear antigen. Figure 8. Overexpression of STIM2 enhances SOCE in rPASMC. Figure 9. Diagram illustrating the proposed role of increased STIM2 on phenotypic switching from a contractile phenotype to a proliferative phenotype, as seen in cultured rPASMC. This activation results in a rise in [Ca 2+ ] cyt. Increased [Ca 2+ ] cyt is may be major trigger for sustained vasoconstriction and excessive vascular remodeling in PASMC from PAH.