Master of Science (MS), Wright State University, 2021, Biological Sciences

Atherosclerosis is responsible for 50% of all deaths in western society and is the main cause of cardiovascular diseases (Pahwa et al. 2020). Vascular smooth muscle cells (VSMC) play a vital role in the development of atherosclerotic plaques; this is due to their ability to proliferate and migrate in response to inflammation and damage to arteries' inner linings. To further understand what causes these cells to proliferate and migrate, our lab has previously found that VSMC express all three subunits of the IL-2 receptor (IL-2R). Out of these three subunits, IL-2Rα appeared to change the most depending on the phenotype of the cell. Our studies have shown that IL-2Rα could play a vital role in VSMC switching from a proliferative phenotype to a quiescent phenotype, in response to injury and inflammation. The present studies found that not only does the nuclear localization of IL-2Rα impact VSMC proliferation, but that commercially available knockout mice are not fully knocked out. In the first study, we utilized immunofluorescent staining, western blotting, and proliferation and migration assays to show that, when IL-2Rα localizes to the nucleus, VSMC proliferation is inhibited, therefore providing a possible means for regulation. In the second study, we used similar methodology in addition to PCR to show that IL-2Rα knock out mice are not completely devoid of IL-2Rα. Because the IL-2Rα knock out was generated using a neomycin resistance gene insert, we were able to eliminate the majority of IL-2Rα producing cells via treatment with G418, a neomycin analog. Treatment with G418 therefore allowed us to study VSMC nearly depleted of IL-2Rα. These findings are important in contributing to knowledge of the role of IL2-Rα in VSMC function, which could later aid in developing treatments for intimal hyperplasia and, in turn, atherosclerosis. In addition, knowing that the mice are only partially knocked out is beneficial to future investigators working with these mice.

Committee: Lucile E. Wrenshall M.D., Ph.D. (Advisor); Shulin Ju Ph.D. (Committee Member); Paula Bubulya Ph.D. (Committee Member) Subjects: Biology

Doctor of Philosophy, Case Western Reserve University, 2021, Molecular Medicine

Mechanism-based therapies that mitigate phenotypic modulation of vascular smooth muscle cells (SMC) represent appealing targets to improve outcomes of vascular intervention by limiting restenosis. Canonical transient receptor potential channel subtype-6 (TRPC6) is upregulated in arterial segments following balloon angioplasty, though its role in the pathogenesis of neointimal hyperplasia (IH) is unclear. We hypothesized that TRPC6 is required for maintenance of myocardin expression and stabilizes the contractile phenotype in SMC to reduce stenosis in a model of arterial intervention. In this study, histologic evaluation of TRPC6-/- common carotid arteries (CCA) demonstrated subtle structural deficits, including luminal dilation, medial thinning and decreased elastin lamella compared to WT CCA. These structural deficits were associated with reduced myocardin expression in TRPC6-/- CCA homogenates. To assess the effects of TRPC6 on arterial healing after vascular intervention, WT and TRPC6-/- mice were subjected to carotid wire injury. Evaluation at 28 days post injury demonstrated that luminal stenosis, negative arterial remodeling and neointimal hyperplasia were accentuated in TRPC6-/- mice compared to WT mice. TRPC6-/- carotid arteries showed increased medial and neointimal cell proliferation after injury. Immunohistochemistry suggested persistent attenuation of contractile biomarker expression in the media of TRPC6-/- CCA but complete restoration of contractile biomarker expression in in the media of WT CCA 28 days after wire injury. In vitro, cultured primary aortic TRPC6-/- SMC showed decreased expression of SM22, MYH11 and myocardin. Loss of contractile biomarker expression in TRPC6-/- SMC was accompanied by the emergence of IH-related behaviors including enhanced proliferation and migration. Acute siRNA-mediated knockdown of TRPC6 in immortalized arterial SMC was sufficient to induce myocardin downregulation and phenotypic modulation. In total, these results (open full item for complete abstract)

Committee: Linda Graham MD (Advisor); Jonathan Smith PhD (Committee Chair); Christine Moravec PhD (Committee Member); Mitchell Olman MD (Committee Member); Edward Maytin MD PhD (Committee Member) Subjects: Medicine; Molecular Biology; Surgery

Doctor of Philosophy, The Ohio State University, 2016, Biomedical Sciences

The Notch signaling pathway has long been intricately linked with the development and function of the vasculature. In vascular smooth muscle cells (VSMCs), Notch signaling has a great influence on phenotype and is a strong promoter of differentiation and expression of contractile genes necessary to produce a functional vessel wall. However, the role of Notch signaling in VSMC proliferation and survival is less well defined, and some cases contradictory reports are given. Also, the contributions of each individual Notch receptor have not been clearly described. Thus, to better understand Notch signaling in VSMC phenotype, we investigated the specific roles of the predominant Notch receptors in VSMCs as they relate to differentiation, proliferation, and survival. We found that Notch3 promotes Platelet-Derived Growth Factor (PDGF)-induced proliferation in VSMCs, while Notch2 inhibits it. We also found that Notch3 was able to promote cell survival in response to apoptosis cues, while Notch2 had no discernible effect. Interestingly, we also found the expression of Notch2 and Notch3 were changed in response to proliferation and apoptosis inducers. Notch2 mRNA was significantly decreased after PDGF-BB treatment, a proliferation inducer, and Notch3 protein was degraded rapidly in response to induction of apoptosis. Additionally, we demonstrated that Notch3's induction of cell survival genes required MEK/ERK signaling and Notch3 was capable of increasing levels of phosphorylated ERK. Altogether, these findings demonstrate that Notch2 and Notch3 have unique functions in regulating VSMC phenotype. In a mouse model devoid of Notch2 and Notch3 in smooth muscle cells, we were able to show that Notch2 and Notch3 are required for normal closure of the ductus arteriosus. Animals without Notch2 in VSMCs presented with patent ductus arteriosus with increasing incidence combined with the loss of Notch3. These mice died within one day of birth and also presented with aortic dilation. (open full item for complete abstract)

Committee: Brenda Lilly PhD (Advisor); Joy Lincoln PhD (Committee Member); Andrea Doseff PhD (Committee Member); Aaron Trask PhD (Committee Member) Subjects: Biomedical Research; Cellular Biology; Developmental Biology; Molecular Biology

Doctor of Philosophy (PhD), Wright State University, 2014, Biomedical Sciences PhD

Atherosclerosis and high circulating levels of oxidized low density lipoproteins (oxLDL) are considered among the most important risk factors for the occurrence and development of cardiovascular disease (CVD). During the atherosclerotic lesion repair, phenotypic transition of vascular smooth muscle cells (VSMCs) from contractile to synthetic states plays a central role. In this process, enhanced proliferation/migration of VSMCs, from the tunica media to the intima, is required to sustain blood vessel endothelium integrity, and for inducing vessel wall remodeling in response to injury. At the molecular level, the activity of electroneutral potassium-chloride cotransporters or KCCs, is necessary to: a) allow changes in cell volume (key prerequisites to coordinate cell proliferation/migration) and b) sustain normal cardiovascular function. Stimulation of the adipokine apelin and its receptor APJ (apelin/APJ) signaling pathway has been shown to protect against atherosclerotic lesion formation by lowering blood pressure levels and promoting vasodilation. Upon binding to APJ, apelin promotes nitric oxide (NO)-mediated vasodilation, and cell proliferation via the PI3K/Akt and MAPK pathways. Apelin/APJ exert its action through the same signaling pathways regulating the KCCs. However, the mechanisms of KCC regulation by apelin/APJ remain to be determined. Thus, we hypothesized that KCC expression and activity play an important role during VSMCs' phenotypic transition and could be involved in the apelin/APJ cardioprotective effects. In addition, it is possible that the apelin-mediated effect on KCC activity could also be dependent on factors affecting the transporter, such as, serum, ionic strength, osmolality, cell proliferation and migration. This hypothesis will be tested using rat aortic VSMCs that were immunologically validated by specific markers. KCC activity was measured by atomic absorption spectrophotometry using rubidium as a potassium (K+) congener. KCC exp (open full item for complete abstract)

Committee: Norma Adragna Ph.D. (Advisor); Peter Lauf M.D. (Committee Member); Lawrence Prochaska Ph.D. (Committee Chair); David Cool Ph.D. (Committee Member); Mauricio Di Fulvio Ph.D. (Committee Member); Mill Miller Ph.D. (Other); Karen Luchin (Other) Subjects: Biomedical Research

PHD, Kent State University, 2005, School of Biomedical Sciences

Angiotensin (Ang) II is well known for its classic role in the renin-angiotensin system. However, it also plays a central role in the remodeling of the vascular wall associated with hypertension, atherosclerosis and restenosis via the activation of AT1 receptors on vascular smooth muscle cells (VSMC). Upon activation by Ang II, AT1 receptors stimulate the cytosolic phospholipase A2 (cPLA2)-dependant release of arachidonic acid (ArAc) in VSMC. ArAc release mediates reactive oxygen species (ROS) production and transactivation of the epidermal growth factor receptor, leading to the activation of downstream kinases resulting in VSMC growth. To determine the involvement of Akt in this mitogenic pathway, I used cultured rat VSMC to link Ang II-induced ArAc release to ROS production, Akt phosphorylation, Akt kinase activity, and VSMC growth. Using western analysis, I observed that Ang II (100nM), ArAc (20uM), or H2O2 (200uM) increased Akt phosphorylation by 45, 46 or 39%, respectively, while increasing Akt activity by 324, 250 or 249%, respectively. We also observed that Ang II, ArAc, or H2O2 increased 3H-thymidine incorporation into DNA by 210, 150 or 140%, respectively. The Akt inhibitor SH6 (10uM) effectively blocked Ang II-, ArAc-, or H2O2-induced Akt phosphorylation, Akt kinase activity, and VSMC growth. The inhibition of phosphoinositide 3-kinase (PI3K) by10uM LY294002 decreased Akt phosphorylation, Akt kinase activity, and VSMC growth by 95, 91, or 95%, respectively, indicating this pathway is PI3K-dependant. Inhibition of cPLA2 by 10uM AACOCF3 blocked Ang II-induced Akt phosphorylation, Akt kinase activity, and VSMC growth by 94, 76 or 100%, respectively. Finally, the ROS scavenger NaC (10mM) decreased Ang II-induced Akt phosphorylation and Akt kinase activity by 61 or 75%, respectively, and ArAc-induced Akt phosphorylation and Akt kinase activity by 91 or 60%, respectively. Thus it appears that AT1 receptor activation, subsequent ArAc release and ROS production is (open full item for complete abstract)

Committee: Ernest Freeman (Advisor) Subjects: Biology, Molecular