Indiana University-Purdue University Indianapolis (IUPUI) / Coronary artery disease (CAD) is a leading cause of death among Americans and is fueled by underlying metabolic syndrome (MetS). The prevalence and lethality of CAD necessitates rigorous investigations into its underlying mechanisms and to facilitate the development of effective treatment options. Coronary smooth muscle (CSM) phenotypic modulation from quiescent to synthetic, proliferative, and osteogenic phenotypes is a key area of investigation, with underlying mechanisms that remain poorly understood. Using a well-established pre-clinical model of CAD and MetS, the Ossabaw miniature swine, we established for the first time the time course of Ca2+ dysregulation during MetS-induced CAD progression. In particular, we used the fluorescent Ca2+ dye, fura-2, to examine alterations in CSM intracellular Ca2+ regulation during CAD progression, as perturbations in intracellular Ca2+ regulation are implicated in several cellular processes associated with CAD pathology, including CSM contractile responses and proliferative pathways. These studies revealed that the function of several CSM Ca2+ handling proteins is elevated in early CAD, followed by loss of function in severe atherosclerotic plaques. Decreased intracellular Ca2+ regulation occurred concurrently with reductions in CSM proliferation, measured with Ki-67 staining. In particular, alterations in sarcoplasmic reticulum (SR) Ca2+ store together with altered function of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) were associated with induction of proliferation. Organ culture of coronary arterial segments revealed that culture-induced medial thickening was prevented by SERCA inhibition with cyclopiazonic acid (CPA). Activation of SERCA with the small molecule activator, CDN1163, increased CSM proliferation, which was attenuated by treatment with CPA, thus establishing upregulated SERCA function as a proximal inducer of CSM proliferation. Further, we demonstrated that in vitro treatment of CSM from lean Ossabaw swine with the glucagon-like peptide-1 (GLP-1) receptor agonist, exenatide, increased SERCA function. However, in vivo treatment of Ossabaw swine with MetS with the GLP-1 receptor agonist, AC3174, had no effect on CAD progression and in vitro examination revealed resistance of SERCA to GLP-1 receptor agonism in MetS. These findings further implicate SERCA in CAD progression. Collectively, these are the first data directly linking SERCA dysfunction to CSM proliferation and CAD progression, providing a key mechanistic step in CAD progression.

Indiana University-Purdue University Indianapolis (IUPUI) / For years, the leading killer of Americans has been coronary artery disease (CAD), which has a strong correlation to the U.S. obesity epidemic. Obesity, along with the presence of other risk factors including hyperglycemia, hypercholesterolemia, dyslipidemia, and high blood pressure, comprise of the diagnosis of metabolic syndrome (MetS). The presentation of multiple MetS risk factors increases a patients risk for adverse cardiovascular events. CAD is a complex progressive disease. We utilized the superb model of CAD and MetS, the Ossabaw miniature swine, to investigate underlying mechanisms of CAD progression. We studied the influence of coronary epicardial adipose tissue (cEAT) and coronary smooth muscle cell (CSM) intracellular Ca2+ regulation on CAD progression. By surgical excision of cEAT from MetS Ossabaw, we observed an attenuation of CAD progression. This finding provides evidence for a link between local cEAT and CAD progression. Intracellular Ca2+ is a tightly regulated messenger in CSM that initiates contraction, translation, proliferation and migration. When regulation is lost, CSM dedifferentiate from their mature, contractile phenotype found in the healthy vascular wall to a synthetic, proliferative phenotype. Synthetic CSM are found in intimal plaque of CAD patients. We investigated the changes in intracellular Ca2+ signaling in enzymatically isolated CSM from Ossabaw swine with varying stages of CAD using the fluorescent Ca2+ indicator, fura-2. This time course study revealed heightened Ca2+ signaling in early CAD followed by a significant drop off in late stage calcified plaque. Coronary artery calcification (CAC) is a result of dedifferentiation into an osteogenic CSM that secretes hydroxyapatite in the extracellular matrix. CAC is clinically detected by computed tomography (CT). Microcalcifications have been linked to plaque instability/rupture and cannot be detected by CT. We used 18F-NaF positron emission tomography (PET) to detect CAC in Ossabaw swine with early stage CAD shown by mild neointimal thickening. This study validated 18F-NaF PET as a diagnostic tool for early, molecular CAC at a stage prior to lesions detectable by CT. This is the first report showing non-invasive PET resolution of CAC and CSMC Ca2+ dysfunction at an early stage previously only characterized by invasive cellular Ca2+ imaging.