Cardiovascular
Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/21705
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Browsing Cardiovascular by Author "Cunningham, Joseph"
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Item Changes In TRPV4 Channel Function In Vasopressin Neurons Of Rats With Hepatic Cirrhosis(2016-03-23) Knapp, Blayne; Saxena, Ashwini; Bachelor, Martha; Cunningham, Joseph; Cunningham, Thomas IIPurpose: Dilutional hyponatremia associated with cardiac and hepatic failure negatively impacts morbidity and mortality of both diseases. Hyponatremia is a consequence of the dysregulation of vasopressin release but is not completely understood. In an animal model of liver failure, the membrane expression of transient receptor potential vanilloid 4 (TRPV4) channels increased in vasopressin-releasing neurons of the hypothalamus. Our hypothesis was that activation of TRPV4 channels with the specific agonist GSK 1019790A produces greater calcium influx in supraoptic nucleus (SON) vasopressin neurons collected from hyponatremic rats with liver failure induced by bile duct ligation surgery or to sham operated controls. Methods: Adult male Sprague-Dawley rats received bile duct ligation surgery or sham ligation surgery. After two weeks, all rats were anesthetized and injected in the SON with an adeno-associated viral 2 (AAV2) vector containing a construct for green fluorescent protein (GFP) driven by a vasopressin-specific promoter. Two weeks later, the rats were anesthetized and the SONs isolated and cultured for Fura-2 ratiometric calcium imaging. Initial experiments had the cultured cells sit overnight prior to imaging; subsequent experiments imaged cells on the same day as their isolation and culturing. SON cells were tested for changes in intracellular calcium produced by the specific TRPV4 agonist GSK 1019790A (20-200 nM). Cells were also tested for its response to a calcium ionophore. In some experiments, data were collected GFP positive (vasopressin) cells. Normalized ratio responses were tested by ANOVA. Results: In these studies using cells cultured for 24-48 hours, 1.5 nM GSK significantly increased calcium influx in GFP-positive cells from sham rats but not from BDL rats (Sham 1.53 ± 0.1, BDL 1.14 ± 0.1, P Conclusion: Our results demonstrate that hyponatremia produces gain in function changes consistent with increased TRPV4 expression. However, these results were strongly influenced by how long the cells were kept in culture.Item Homer Mediates Vascular Store-Operated CA2+ Entry and is Required for Neointima Formation after Vascular Injury(2016-03-23) Jia, Shuping; Wu, Qiong; Williams, Arthur Jr.; Little, Joel; Cunningham, Joseph; Mifflin, Steve; Ma, Rong; Yuan, Joseph; Rodriguez, MiguelOcclusive 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.