Abstract Title

Homer Mediates Vascular Store-Operated CA2+ Entry and is Required for Neointima Formation after Vascular Injury

RAD Assignment Number

305

Presenter Name

Miguel Rodriguez

Abstract

Occlusive arterial disease (OAD) refers to the pathological obstruction of arteries that become progressively narrowed over time and are eventually blocked due to various risk factors, such as hypertension, diabetes, and atherosclerosis. This chronic arterial damage results from vascular wall remodeling, leading to neointima formation. Store-operated Ca2+ channels (SOCs) and entry (SOCE) play a central role in the vascular smooth muscle cell (VSMC) phenotypic change from contractile to migratory and proliferative states. In the present work, we ask if Homer is a critical molecular component of VSMC SOCE and does Homer mediate VSMC migration/proliferation and neointima formation. Homer binds to transient receptor potential canonical (TRPC) channels and is required for gating of TRPCs, while stromal interacting molecule1 (STIM1) binds to and regulates TRPC and Orai channels as SOCs. We cultured rat aortic VSMCs to increase their SOCE and migration/proliferation, as seen in OAD. Studies were done using small-interfering RNA (siRNA) targeting Homer1, STIM1, and TRPCs. Scratch wound migration assays were performed, and VSMC proliferation was assessed by cell count. In our in vivo OAD model (rat carotid artery balloon injury), the arteries were treated with adeno-associated virus (AAV) encoding short-hairpin RNA (shRNA) targeting Homer1. We found that Homer1 protein expression levels increase in balloon-injured vs. intact VSMCs, similar to known increases in protein expression levels of STIM1, Orai1, and TRPCs. Furthermore, we show that Homer1 binds to Orai1 and that interactions between Homer1 & Orai1/TRPCs and between STIM1 & Orai1/TRPCs markedly increase in injured vs. intact VSMCs. Cultured VSMCs treated with siHomer1 exhibit significant reduction in SOCE (56 ± 4.0%) vs. control (scrambled siRNA), similar to the SOCE reduction seen in siSTIM1-/siTRPC-treated cells. SiHomer1-treated cells also migrate significantly less over the wound surface area (73.3 ± 5.9%), and proliferate significantly less (73.3 ± 4.2%) vs. control, similar to observations seen in siSTIM1-/siTRPC-treated cells. Finally, immunofluorescence staining shows that the increased Homer1, STIM1, and Orai1 protein expression levels are localized in the neointima of the injured carotid artery. Knockdown of Homer1 using AAV-shHomer1 reduces this neointima. These studies provide evidence that Homer is a critical component of VSMC SOCE and neointima formation.

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Homer Mediates Vascular Store-Operated CA2+ Entry and is Required for Neointima Formation after Vascular Injury

Occlusive arterial disease (OAD) refers to the pathological obstruction of arteries that become progressively narrowed over time and are eventually blocked due to various risk factors, such as hypertension, diabetes, and atherosclerosis. This chronic arterial damage results from vascular wall remodeling, leading to neointima formation. Store-operated Ca2+ channels (SOCs) and entry (SOCE) play a central role in the vascular smooth muscle cell (VSMC) phenotypic change from contractile to migratory and proliferative states. In the present work, we ask if Homer is a critical molecular component of VSMC SOCE and does Homer mediate VSMC migration/proliferation and neointima formation. Homer binds to transient receptor potential canonical (TRPC) channels and is required for gating of TRPCs, while stromal interacting molecule1 (STIM1) binds to and regulates TRPC and Orai channels as SOCs. We cultured rat aortic VSMCs to increase their SOCE and migration/proliferation, as seen in OAD. Studies were done using small-interfering RNA (siRNA) targeting Homer1, STIM1, and TRPCs. Scratch wound migration assays were performed, and VSMC proliferation was assessed by cell count. In our in vivo OAD model (rat carotid artery balloon injury), the arteries were treated with adeno-associated virus (AAV) encoding short-hairpin RNA (shRNA) targeting Homer1. We found that Homer1 protein expression levels increase in balloon-injured vs. intact VSMCs, similar to known increases in protein expression levels of STIM1, Orai1, and TRPCs. Furthermore, we show that Homer1 binds to Orai1 and that interactions between Homer1 & Orai1/TRPCs and between STIM1 & Orai1/TRPCs markedly increase in injured vs. intact VSMCs. Cultured VSMCs treated with siHomer1 exhibit significant reduction in SOCE (56 ± 4.0%) vs. control (scrambled siRNA), similar to the SOCE reduction seen in siSTIM1-/siTRPC-treated cells. SiHomer1-treated cells also migrate significantly less over the wound surface area (73.3 ± 5.9%), and proliferate significantly less (73.3 ± 4.2%) vs. control, similar to observations seen in siSTIM1-/siTRPC-treated cells. Finally, immunofluorescence staining shows that the increased Homer1, STIM1, and Orai1 protein expression levels are localized in the neointima of the injured carotid artery. Knockdown of Homer1 using AAV-shHomer1 reduces this neointima. These studies provide evidence that Homer is a critical component of VSMC SOCE and neointima formation.