Doctor of Philosophy, The Ohio State University, 1984, Graduate School

Committee: Not Provided (Other) Subjects: Health Sciences

PHD, Kent State University, 2025, College of Arts and Sciences / School of Biomedical Sciences

Cardiovascular disorders remain the leading cause of morbidity and mortality worldwide, with atherosclerosis as the predominant underlying pathology. In individuals with Metabolic Syndrome (MetS), which encompasses conditions such as obesity, diabetes, dyslipidemia, and hypertension, the development of atherosclerosis is accelerated. Vascular smooth muscle cells (VSMCs) play a pivotal role in atherosclerotic plaque formation through phenotypic switching—a process influenced by various extracellular factors, including TSP1. TSP1, a matricellular glycoprotein, has emerged as a key mediator of vascular inflammation, remodeling, and cell behavior. However, its precise role in VSMC differentiation and atherosclerosis in the context of MetS remains unclear. The significance of my dissertation lies in addressing the critical knowledge gap surrounding TSP1's role in VSMC phenotypic modulation within atherosclerotic lesions, particularly under the compounded metabolic stresses of MetS. The goal of this study was to elucidate the effect of TSP1 on VSMC behavior and plaque development. We specifically investigated the link between TSP1 expression, atherosclerosis, and SMC differentiation in MetS and the regulatory role of smooth muscle-specific TSP1 in SMC phenotypic changes in diabetes and the molecular pathways involved. We found that agouti KKAy+/-ApoE-/- mice (MetS) exhibited pronounced metabolic disturbances, including significant increases in body weight, non-fasted blood glucose levels (>250 mg/dL), and plasma lipid levels (cholesterol and triglycerides) compared to non-agouti KKAy-/-ApoE-/- controls (non-MetS). Male MetS mice demonstrated severe glucose intolerance, although insulin sensitivity was unaffected across groups. Male MetS mice exhibited significantly greater lipid burden and plaque area in the aortic root associated with upregulation of TSP1 and lesser testosterone levels compared to non-MetS males. However, female MetS mice did not display the same les (open full item for complete abstract)

Committee: Priya Raman (Advisor); Mohammad Yunus Ansari (Committee Member); Jessica Ferrell (Committee Member); Vahagn Ohanyan (Committee Member) Subjects: Biomedical Research; Pharmacology

PhD, University of Cincinnati, 2024, Medicine: Molecular, Cellular and Biochemical Pharmacology

Cardiovascular disease is the leading cause of death in the United States. Members of the low-density lipoprotein receptor (LDLR) family play integral roles in maintaining cardiovascular and metabolic homeostasis, and defects in these receptors are known to precede vascular occlusive diseases through their various roles. Apolipoprotein E Receptor-2 (ApoER2) is a transmembrane receptor in the LDLR family with a unique tissue expression limited to the brain, testis, and vascular and circulatory cells such as endothelial cells, smooth muscle cells, monocytes/macrophages, and platelets. Studies have revealed roles for ApoER2 in regulating atherosclerotic disease development, smooth muscle cell growth, tissue inflammation, and diet-induced obesity and diabetes. A single nucleotide polymorphism that encodes the R952Q sequence variant of ApoER2 has been associated with elevated plasma cholesterol levels and increased myocardial infarction risk in humans. The objective of this study was to delineate the mechanism underlying the association between the ApoER2-R952Q variant and increased atherosclerosis risk. Using a hyperlipidemic ApoER2-R952Q mouse model generated via a CRISPR/Cas9 strategy and intercrossing with LDLR knockout mice, this study investigated the development of atherosclerotic cardiovascular disease by feeding a Western-type, high-fat, high-cholesterol diet. Immunohistological analysis of atherosclerotic lesions revealed an acceleration of disease progression in ApoER2-R952Q mice. Plasma lipids and lipid distributions among the various lipoprotein classes were analyzed by colorimetric assay and revealed that hypercholesterolemia is exacerbated in ApoER2-R952Q mice. Tissue-specific effects of the R952Q sequence variant on atherosclerosis were analyzed by bone marrow transplant studies and showed that the ApoER2-R952Q mutation in bone marrow-derived cells instead of non-bone marrow-derived cells is responsible for the increase in hypercholesterolemia and atheros (open full item for complete abstract)

Committee: David Hui Ph.D. (Committee Chair); Guo-Chang Fan Ph.D. (Committee Member); Konstantinos Drosatos Ph.D. (Committee Member); Evangelia Kranias Ph.D. (Committee Member); Moises Huaman (Committee Member) Subjects: Pathology

Doctor of Philosophy, The Ohio State University, 2024, Biomedical Sciences

Nearly 50% of the US population is predicted to be obese by 2030. Chronic inflammation is a central feature of obesity, which leads to cardiovascular disease, insulin resistance, cancer, and several other life-threatening complications. A healthy, lean individual is protected from adipose tissue inflammation and associated comorbidities due to well-coordinated interactions between adipocytes and immune cells in adipose tissue that maintain normal systemic metabolism. In obesity, pro-inflammatory changes occur in nearly all adipose immune cells. These proinflammatory changes include increased proinflammatory (T helper1) Th1 cells that produce interferon-gamma, which reduces immunoregulatory T cells (Tregs). T helper type 2 cells (Th2), innate lymphoid type 2 cells (ILC2), and anti-inflammatory M2-type macrophages also decrease, whereas proinflammatory neutrophils, M1-type macrophages, and cytotoxic CD8 T cells increase. The timeline of these events is established in several mouse models of obesity. However, these events in human adipose tissue remain elusive. Over the years, Hsueh laboratory has established a more comprehensive profile of the human AT microenvironment and has identified various mechanisms by which immune cells within the adipose tissue contribute to an obesity-related pro-inflammatory state that raises several metabolic diseases. One of the emerging mechanisms for AT inflammation is the transfer of gut microbes to adipose tissue due to increased gut dysbiosis in obesity. Microbes in AT can cause inflammation. We found that a mere two weeks of high-caloric diet consumption in healthy human subjects has shown signs of leaky gut, which overlapped with the recruitment of neutrophils to the adipose tissue. Interestingly, within the same cohort, we observed a remarkable decline in adipose tissue Tregs, a central player in regulating AT immune homeostasis. Therefore, we explored if a leaky gut resulted in neutrophil recruitment and subsequent proinflammato (open full item for complete abstract)

Committee: Willa Hsueh (Advisor); Daniel Spakowicz (Committee Member); Amy Lovett-Racke (Committee Member); Hazem Ghoneim (Committee Member) Subjects: Biomedical Research

MS, University of Cincinnati, 2023, Medicine: Molecular and Developmental Biology

Elevated cholesterol is a significant risk factor for cardiovascular diseases, particularly in women of peri-/post-menopausal age. The glucocorticoid receptor (GR) is a nuclear transcription factor that regulates the metabolism of virtually all major nutrients. However, its role in cholesterol metabolism remains unknown. rs6190, a coding single nucleotide polymorphism (SNP) in the GR protein (R23K), has been associated with changes in metabolic health, but the mechanisms remain unelucidated. We probed the large cohort of the UK Biobank (N=485,895), where this low-frequency coding variant associated with cholesterol (beta=0.055, P=0.0087) independently from known cholesterol-regulating variants. The SNP associated with increased levels of total, LDL-, and HDL-cholesterol, particularly in women of putative peri-/post-menopausal age. The effect was additive according to the number of SNP alleles (homo>hetero>reference). SNP homozygosity associated with increased odds ratio for hypercholesterolemia and death by cardiovascular disease. To understand the underlying mechanisms, we generated mice and human induced pluripotent stem cells (hiPSCs) genocopying the SNP in the GR gene locus using CRISPR editing. In SNP-bearing littermate mice, the SNP was sufficient to increase total, LDL-, and HDL- cholesterol levels on regular, high-fat, and high-cholesterol diets according to SNP zygosity. Liver RNA-seq and ChIP-seq data revealed that the SNP increased GR transactivation of Pcsk9 and Bhlhe40, negative regulators of LDL receptor and HDL receptor in liver. The molecular SNP effects were replicated in hiPSC-derived hepatocytes. Taken together, our data leverage a non-rare human variant to discover unanticipated mechanisms through which the GR regulates cardiovascular health in vulnerable population like peri-and post-menopausal women.

Committee: Mattia Quattrocelli Ph.D. (Committee Chair); Takanori Takebe M.D. (Committee Member); Kenneth Kaufman Ph.D. (Committee Member) Subjects: Cellular Biology

PHD, Kent State University, 2022, College of Arts and Sciences / School of Biomedical Sciences

The liver is one of the major organs for lipid metabolism. Disruption of lipid homeostasis in the liver may cause the development of many metabolic diseases, including nonalcoholic fatty liver disease (NAFLD). Histone deacetylase sirtuin 6 (SIRT6) regulates multiple biological processes via its deacetylation, deacylation, and mono-ADP-ribosylation activities. The role of SIRT6 in lipid metabolism is not fully understood. In this project, we aim to determine the impact of hepatic SIRT6 on the progression of diet-induced metabolic disorders in wild-type or Ldlr-deficient mice. We fed the control mice and mice lacking or overexpressing hepatic SIRT6 a diet enriched in fats and cholesterol, with or without fructose, to induce metabolic disorders. We investigated the effect of SIRT6 on the development of non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), atherosclerosis, and obesity. Our results show that adeno-associated virus serotype 8 (AAV8)-mediated overexpression of human SIRT6 in the liver ameliorates diet-induced NAFLD, atherosclerosis and obesity, whereas loss of hepatocyte SIRT6 has opposite effects. Further studies show that SIRT6 inhibits lipid droplet formation, apoptosis, bile acid synthesis, and intestinal fat and cholesterol absorption. Our data indicate that hepatic SIRT6 may be a promising therapeutic target for the treatment of metabolic disorders.

Committee: Yianqiao Zhang (Advisor); James Hardwick (Committee Member); Yoon-Kwang Lee (Committee Member); Feng Dong (Committee Member); Rafaela Takeshita (Committee Member) Subjects: Biomedical Research

PHD, Kent State University, 2022, College of Arts and Sciences / School of Biomedical Sciences

A growing body of research points to protein O-GlcNAcylation as a potential pathogenic factor in diabetes and its associated vascular problems. Diabetes patients show an increased propensity for vascular smooth muscle cell (VSMC) migration and proliferation, characteristic of VSMC de-differentiation from "quiescent" contractile to "synthetic" proliferative phenotypes. Previously, we reported that the development of atherosclerotic lesions and high glucose-induced VSMC proliferation is correlated with increased protein O-GlcNAcylation. However, the role of O-GlcNAc transferase (OGT), a key regulator of O-GlcNAc signaling, in the etiology of diabetic atherosclerosis remains elusive. The objective of this study was to investigate whether OGT directly contributes to SMC de-differentiation and diabetes atherosclerosis. Briefly, female OGTfl/fl mice crossed with tamoxifen-inducible male Myh11-CreERT2 (Cretg) were developed on an ApoE-/- background. Cre recombination was initiated in 6-wks-old male Cretg/OGTfl/y/ApoE-/- and age-matched Cretg/OGT+/y/ApoE-/- littermates via once daily i.p. injection of tamoxifen (Tmx, 60mg/Kg/day) or vehicle (peanut oil) for 5 days. Two weeks post-Tmx, mice were fed Western diet for 8 wks. In a parallel study, hyperglycemia was induced in 8-wks-old male Tmx-treated Cretg/OGTfl/y and age-matched Cretg/OGT+/y littermates via STZ (50 mg/Kg/day, i.p.) or citrate buffer (vehicle) for 5 days; mice were kept on regular chow diet for this study. Animals were harvested at 14-16-wks-age; plasma, heart and aortic vessels utilized for biochemical and molecular studies. Immunoblotting of aortic lysates confirmed loss of OGT expression in Tmx-treated Cretg/OGTfl/y/ApoE-/- (smOGTKO;ApoE-/-) and Cretg/OGTfl/y (smOGTKO) mice vs. Tmx-treated Cretg/OGT+/y/ApoE-/- (smOGTWT;ApoE-/-) and Cretg/OGT+/y (smOGTWT) littermates. VSMC-restricted OGT deletion attenuated atherosclerotic lesion lipid burden (Oil red O), plaque area (H&E) and leukocyte infiltration (CD (open full item for complete abstract)

Committee: Priya Raman (Advisor) Subjects: Biomedical Research; Molecular Biology

Doctor of Philosophy, The Ohio State University, 2022, Biomedical Engineering

Endothelial cells (ECs) line all blood vessels and vasculature in the human body and are exposed to mechanical shear stress from blood flow. EC dysfunction has been shown to be one of the root causes of cardiovascular atherosclerotic disease. Atherosclerosis is a site-specific phenomenon and occurs in vessels at areas of disturbed blood flow (atheroprone regions). It is known that hemodynamic forces and mechanochemical environments alter the intracellular signal responses in ECs and these alterations are known to contribute to EC dysfunction. Calcium (Ca2+) is a key intracellular signalling ion and is a ubiquitous secondary messenger that is responsible for many intracellular signalling mechanisms. Intracellular calcium concentration ([Ca2+]i) oscillations are an important signaling response to hemodynamic forces and fluid shear stress. Oscillatory changes in [Ca2+]i occur due to inositol 1,4,5 trisphosphate (IP3)-regulated Ca2+ release from the endoplasmic reticulum (ER), and require Ca2+ transport between the ER and mitochondria. Ca2+ uptake by mitochondria is a major determinant of bioenergetics, adenosine triphosphate (ATP) production and EC fate. Mitochondrial Ca2+ uptake occurs via the mitochondrial Ca2+ uniporter (MCU) complex, an inner mitochondrial membrane (IMM) protein assembly consisting of a core MCU Ca2+ channel and MCU complex regulatory/auxiliary subunit proteins. The MCU is a Ca2+-selective channel that is responsible for the influx of cytosolic Ca2+ into the mitochondria. In vivo and in vitro evidence showed upregulated MCU expression in vascular ECs under conditions associated with cardiovascular disease (CVD). However, the critical role of MCU in regulating the mitochondrial Ca2+ concentration ([Ca2+]m) in ECs exposed to fluid shear stress and the regulation of the MCU complex expression by hemodynamic forces are currently unknown. This work examined the role of MCU in cultured human ECs exposed to hemodynamic shear stresses. This included mon (open full item for complete abstract)

Committee: Jonathan Song (Advisor); Yi Zhao (Committee Member); Shaurya Prakash (Committee Member) Subjects: Biomedical Engineering; Cellular Biology

PHD, Kent State University, 2022, College of Arts and Sciences / School of Biomedical Sciences

Retinoic Acid Receptor Alpha (RARα) is a nuclear hormone receptor activated by All-trans Retinoic Acid (AtRA) which is a metabolite of fat-soluble vitamin A. When activated, RARα promotes transcription of downstream target genes. Even though AtRA has been studied in different metabolic conditions, the role of RARα in regulating lipid metabolism and associated metabolic diseases is not completely known. Metabolic diseases including non-alcoholic fatty liver diseases (NAFLD), atherosclerosis, obesity, and hyperlipidemia are caused partly by dysregulated triglyceride and cholesterol metabolism. In this project, we used loss of function approaches to address the cell- specific role of RARα in regulating triglyceride and cholesterol homeostasis. For the loss of function studies, we crossed cell-specific cre recombinase-expressing transgenic mice including Albumin Cre, Lyz-M Cre, Adipoq Cre, and Vilin Cre with RARα floxed (RARαfl/fl) mice to knockout RARα in hepatocytes, macrophages, adipocytes, and intestinal epithelial cells (IEC), respectively. Our data show that loss of hepatocyte RARα exacerbates hepatosteatosis by increasing hepatic triglyceride levels in aged chow diet-fed mice, high fat diet (HF)-fed mice and high fat/cholesterol/fructose diet (HFCF)-fed mice. Mechanistic studies show that AtRA protects from hepatosteatosis by regulating fatty acid uptake and lipid droplet formation in a hepatocyte RARα-dependent manner. In atherosclerotic study, loss of macrophage RARα increases lipid accumulation in macrophages and induces atherosclerosis in high fat/high cholesterol diet (HFHC)-fed mice by inhibiting cholesterol efflux and increasing inflammation. AtRA induces cholesterol efflux and cholesterol efflux transporters dependent on macrophage RARα. In addition, our data demonstrates that the loss of adipocyte RARα increases adiposity, reduces overall energy expenditure, and decreases stimulated lipolysis. Loss of adipocyte RARα induces steatohepatitis in aged chow-f (open full item for complete abstract)

Committee: Yanqiao Zhang (Advisor); James Hardwick (Committee Member); Takhar Kasumov (Committee Member); Yoon-Kwang Lee (Committee Member); Min-Ho Kim (Committee Member) Subjects: Biomedical Research; Molecular Biology; Pharmacology

Doctor of Philosophy, University of Akron, 2021, Chemistry

Cysteinyl leukotrienes (cys-LTs) - leukotriene C4 (LTC4), leukotriene D4 (LTD4) and leukotriene E4 (LTE4) are potent inflammatory lipid mediators that act via two different G-protein coupled receptors: CysLT1R and CysLT2R. These metabolites are implicated in pathophysiology of various inflammatory diseases like asthma, cancer, cardiovascular diseases and monocyte recruitment to the inflammation site. However, the molecular mechanism by which cys-LTs modulate their function is still elusive. Macrophages use receptor-mediated phagocytosis to identify foreign particles and cellular debris and maintain tissue homeostasis during inflammatory processes. It was shown in Chapter III of the dissertation that cys-LTs increases phagocytosis of zymosan bioparticles and ox-LDL in macrophages. Furthermore, the underlying mechanism revealed that cys-LT-mediated activation of scavenger receptors CD36 and OLR1, as well as an increase in the cytokine MCP-1, improves macrophage phagocytic abilities. Macrophages tend to alter their function dynamically in response to local micro environmental cues. To understand the role of cys-LT/CysLTR signaling in modulating macrophage plasticity, BMDMs were polarized into M1 and M2 phenotypes which were then validated with the literature. In chapter IV of the dissertation, murine bone marrow cells polarization into M1 phenotype was shown to be NFkB, STAT1, and p38 dependent whereas M2 polarization was shown to require KLF4 and PPARγ. Using CysLT1R and CysLT2R null mice, it was shown for the first time that BMDMs and peritoneal macrophages require CysLT1R to attain inflammatory as well as resolution phenotype. Additionally, the study revealed protection form LPS induced septic shock in absence of CysLT1R which was supported by attenuated systemic response compared to WT and CysLT2R null mice. Further, in chapter VI of the dissertation it was observed that wound healing process requires CysLT1R signaling which could be a result of impaired macr (open full item for complete abstract)

Committee: Sailaja Paruchuri (Advisor); Leah Shriver (Committee Member); Nic Leipzig (Committee Member); Yi Pang (Committee Member); Adam Smith (Committee Member) Subjects: Biochemistry; Chemistry; Immunology

PhD, University of Cincinnati, 2021, Medicine: Molecular, Cellular and Biochemical Pharmacology

Atherosclerosis is a chronic inflammatory disease that results from the accumulation of cholesterol-containing LDL particles in the vessel wall, and is the underlying pathology for many cardiovascular diseases. Current clinical guidelines focus on LDL-cholesterol lowering as a means of cardiovascular disease prevention. The immune system also plays a major role in the pathogenesis of this disease, and emerging evidence points to the therapeutic potential of targeting inflammation to combat cardiovascular diseases. Studies have identified dual roles of apolipoprotein E (apoE) and apoE receptors in modulating plasma lipids and inflammation, and have implicated these proteins as key players in atherogenesis. Moreover, genome-wide association studies have linked polymorphisms in the genes encoding these proteins with altered cardiovascular disease risk in humans. ApoE was first identified as a component of circulating lipoproteins, and the importance for ligand-receptor interactions between lipoprotein-associated apoE and hepatic endocytic receptors for maintaining healthy cholesterol levels has been since well established. However, the roles of apoE and apoE receptors expressed in other tissues and their involvement in the pathogenesis of atherosclerosis have not been fully elucidated. The goals of this dissertation research are to (1) investigate the impact of human APOE polymorphisms on macrophage-driven mechanisms of atherosclerosis, and (2) evaluate the effect of global and immune-cell specific expression of a novel mutation of the apoE receptor, LRP1, on development of metabolic disease and atherosclerosis. The functions of macrophage expression of the different human apoE isoforms – apoE2, apoE3, and apoE4 – in modulating atherogenesis was assessed by transplantation of donor bone marrow expressing the apoE isoforms into recipient ApoE-/- mice. Despite the similar plasma cholesterol levels between groups, we observed significant reduction in atherosclerosis (open full item for complete abstract)

Committee: David Hui Ph.D. (Committee Chair); Guo-Chang Fan Ph.D. (Committee Member); Marie-Dominique Filippi Ph.D. (Committee Member); Evangelia Kranias Ph.D. (Committee Member); Alex Lentsch Ph.D. (Committee Member); William Miller Ph.D. (Committee Member) Subjects: Pathology

Doctor of Philosophy (PhD), Ohio University, 2021, Chemical Engineering (Engineering and Technology)

The field of biomedical nanotechnology has witnessed an unprecedented growth over the past decades. However, developing non-invasive and highly targeted platforms remains a challenge. Different types of hard and soft vesicles have been developed to optimize nanoparticle targeting and internalization in diseased cells while minimizing toxicity to healthy cells. However, many challenges remain. First, there is still limited understanding of how physiologically relevant conditions, such as the hydrodynamic conditions of blood flow, can affect nanoparticle targeting to cells. In addition, new surface engineering and advanced coatings are needed to enhance particle targeting. On the other hand, while it is possible to incorporate one or few proteins in particle structures, mimicking the exact composition, distribution and orientation of proteins in the native structure of plasma membrane is difficult. Finally, nanoparticle toxicity still remains an issue with many drug delivery vehicles and there is a need to better understand the mechanisms of nanotoxicity. In my dissertation, I have first developed a new liposomal targeting system that is capable of targeting epithelial cells that overexpress vascular cell adhesion molecule-1 (VCAM1). I have shown that functionalized liposomes can target the cells of interest under static and flow conditions. I have then focused on developing a cell-derived liposomal targeting system, which includes all membrane proteins. Going forward, I utilized giant plasma membrane vesicles (GPMVs) to construct a safe delivery platform that prevents the time consuming steps of making protein-functionalized liposomes and can be used to deliver drugs or drug-loaded nanoparticles. Also, I used GPMVs as the membrane model to examine the toxicity of solid nanoparticles to the cell plasma membrane. Finally, I investigated the role of membrane proteins and lipids in nanoparticle-induced toxicity.

Committee: Monica Burdick (Advisor) Subjects: Biomedical Engineering; Cellular Biology; Chemical Engineering

Doctor of Philosophy, University of Akron, 2019, Biomedical Engineering

Peripheral arterial disease (PAD) is the result of an obstruction of arteries that limits blood flow to the extremities which impacts an estimated 8.5 million Americans with nearly 40% showing no symptoms of the disease. Of those with PAD, the femoral and popliteal arteries are affected in as many as 90% of people. Severe PAD can lead to ischemic ulceration, gangrene, and potentially major amputation of the affected limbs if left untreated. Arterial geometry is a known factor of treatment success for minimally invasive devices such as stents. Much of the geometry data used for describing the lower extremities has been leveraged from data presented in coronary literature although coronary vessels are known to differ from peripheral arteries. Recently, a 2019 FDA warning for SFA paclitaxel SFA stents noting mortality concerns has triggered a call by vascular researchers to for fundamental SFA geometry and plaque morphology characterization. This characterization is needed to better understand the environment SFA stents experience following implant. The main objective of this effort has identified and summarized one of the largest virtual histology IVUS datasets known to exist of the SFA. IVUS is one of the highest resolution imaging modalities clinically used to visualize arteries. Geometry and plaque morphology are summarized for a 79-subject cohort. Similarly, associations with demographic and medical comorbidities are presented. The second objective of this research developed 3D printing methodology to introduce patient specific mechanical properties to a geometrically accurate SFA model. These findings also allow the model to serve as an ultrasound phantom for medical imaging. The third objective of this effort has developed a software algorithm that allows the IVUS imaging of an SFA stent to be 3D reconstructed. This software allows for current and future stents to be imaged without ionizing radiation. The (open full item for complete abstract)

Committee: Francis Loth PhD (Advisor); Rouzbeh Amini PhD (Committee Member); James Keszenheimer PhD (Committee Member); Jiang Zhe PhD (Committee Member); Rolando J.J. Ramirez PhD (Committee Member) Subjects: Biomedical Engineering; Biomedical Research

Doctor of Philosophy (PhD), Ohio University, 2019, Translational Biomedical Sciences

Chronic diseases account for approximately 45% of all deaths in developed countries and are particularly prevalent in countries with the most sophisticated and robust public health systems. Chronic metabolic diseases, specifically lifestyle-related diseases pertaining to diet and exercise, continue to be difficult to treat clinically. One of the most prevalent chronic metabolic diseases is cardiovascular disease (atherosclerosis). WNT proteins are highly conserved glycoproteins best known for their role in development. However, expression of WNT proteins and dysfunctional WNT signaling have been reported in many chronic diseases. The following studies focus on the mechanisms of WNT signaling in macrophages and vascular smooth muscle cells in the context of atherosclerosis. The results presented here support the hypothesis that WNT signaling is an important regulatory pathway in atherosclerosis. Specifically, it is hypothesized that WNT5A promotes foam cell formation in both macrophages and vascular smooth muscle cells. Secondarily, it is hypothesized that WNT5A and WNT3A perform distinct functions in vascular smooth muscle cells to regulate phenotypic differentiation. First, our data demonstrate that WNT5A activates Planar Cell Polarity signaling components in macrophage derived foam cells and that this signaling pathway is present in human atherosclerotic plaques. Important in the progression of atherosclerosis is the accumulation of modified low-density-lipoproteins (e.g. oxidized LDL) within the artery wall. We demonstrate a novel autocrine signaling mechanism for oxLDL-induced WNT signaling and foam cell formation involving functional co-localization of the FZD5 and ROR2 receptors in vitro and in patients with atherosclerosis. Thus, WNT signaling is an important regulator of foam cell development. Second, our data demonstrate that archetypal canonical and non-canonical WNT proteins (WNT3A and WNT5A, respectively) are expressed in vascular smooth muscle cells in (open full item for complete abstract)

Committee: Ramiro Malgor (Advisor); Amir Farnoud (Advisor); Fabian Benencia (Committee Member); Leslie Consitt (Other); Darlene Berryman (Committee Chair) Subjects: Biology; Biomedical Research; Cellular Biology; Molecular Biology; Pathology

Doctor of Philosophy, The Ohio State University, 2019, Integrated Biomedical Science Graduate Program

The primary function of adipose tissue (AT) was previously thought to be a passive storage site for excess caloric intake. However, increasing evidence suggests that inflammatory processes taking place within AT play a fundamental role in the development of obesity-related complications. Within mouse models of obesity, there is an accumulation and shift in the inflammatory profile of the immune cells that traditionally constitute the lean AT from anti-inflammatory macrophages (M2-like), immunosuppressive regulatory T cells (Tregs), T helper type 2 cells (Th2) and innate lymphoid type 2 cells (ILC2) to proinflammatory M1-like macrophages, T helper type 1 cells (Th1), CD8+ cytotoxic T cells and proinflammatory neutrophils. This shift to a more pro-inflammatory milieu in states of chronic over-nutrition has been linked to the development of insulin resistance and fatty liver disease in mouse models. However, the inflammatory microenvironment in human AT remains largely unexplored. Thus, our laboratory has developed a more comprehensive profile of the human AT microenvironment and has identified multiple immune cells that contribute to an obesity-related pro-inflammatory state that fosters metabolic disease. Similar to mouse AT, T cells and macrophages comprise a significant proportion of the immune cells within human AT, and obesity leads to a pro-inflammatory shift within these immune cell populations. While the total number of T cells within the lean and obese state remains fairly constant, the number of the anti-inflammatory Tregs is reduced in obesity. They undergo rapid and acute depletion in lean patients who are placed on a hypercaloric diet with about a 50% reduction in subcutaneous AT T cells within 2 weeks. In addition to a change in specific T lymphocytes, AT macrophages are highly prevalent in obesity, contributing to ~10% of the AT stromal vascular fraction (SVF). We discovered that human AT macrophages are mainly comprised of two distinct populations: (open full item for complete abstract)

Committee: Willa Hsueh MD (Advisor); Martha Belury PhD (Committee Member); William Lafuse PhD (Committee Member); Peter Mohler PhD (Committee Member) Subjects: Biomedical Research

PhD, University of Cincinnati, 2018, Medicine: Pathobiology and Molecular Medicine

Background: Atherosclerosis is a multifactorial inflammatory disease that begins with the accumulation of lipid in arterial endothelium. Atherosclerosis is the leading cause of deaths attributable to cardiovascular disease in the United States. Epidemiological studies showing high-density lipoprotein (HDL) cholesterol is inversely correlated with atherosclerosis has made it a pharmacological target for preventing cardiovascular disease. However, outcomes from clinical trials have raised questions about HDL's protective properties. Investigating the molecular interactions of apolipoprotein (apo)A-I, which accounts for approximately 70% of total HDL protein, can help translate HDL structure to cardioprotective function. Lecithin: cholesterol acyl transferase (LCAT) is a critical HDL-modifying protein that performs a key function in reverse cholesterol transport by using apoA-I as a cofactor to esterify cholesterol. Data from our lab and others demonstrate that apoA-I molecules dimerize into an antiparallel stacked ring-structure that encapsulates lipid in reconstituted (r)HDL. Cross-linking analysis of rHDL implies that apoA-I molecules exist in at least two distinct organizations: one with helix 5 of an apoA-I molecule adjacent to helix 5 of its antiparallel partner (5/5 helical registry), and the other in a 5/2 registry. We hypothesized that the orientation of apoA-I molecules on rHDL modulates LCAT activity. Objective: Identify the mechanism by which apoA-I activates LCAT to determine how HDL interacts with its immense proteome. Linking HDL structure and function will allow for therapeutic development that targets HDL-associated inflammatory diseases. Major Findings: 1) Antiparallel apoA-I molecules adopt a thumbwheel mechanism to generate a discontinuous epitope for LCAT activation. Site-directed cysteine mutagenesis was used to “lock” two apoA-I molecules into an antiparallel 5/5, 5/2, and 5/1 helical registry on rHDL. The 5/5 mutant demonstrated higher LCAT acti (open full item for complete abstract)

Committee: William Sean Davidson Ph.D. (Committee Chair); Christopher A. Crutchfield Ph.D. (Committee Member); Philip Howles Ph.D. (Committee Member); Francis McCormack M.D. (Committee Member); Thomas Thompson Ph.D. (Committee Member); Laura Woollett Ph.D. (Committee Member) Subjects: Pathology

Doctor of Philosophy (PhD), University of Toledo, 2018, Biomedical Sciences (Molecular Medicine)

Insulin resistance has long been considered to play a crucial role in the pathophysiology of metabolic syndrome that is the leading cause of mortality and morbidity worldwide. Metabolic diseases consist of a group of metabolic abnormalities that increase the risk of health problems, such as type 2 diabetes (T2D) and cardiovascular disease. Nonalcoholic fatty liver disease (NAFLD) is associated with obesity and metabolic syndromes. It is the fastest growing cause of liver dysfunction. Its progressive form nonalcoholic steatohepatitis (NASH) is associated with hepatic fibrosis that can develop into cirrhosis. In addition, there is a growing body of evidence that among risk factors that promote atherosclerosis, metabolic syndrome is a potent predictor of cardiovascular events. Insulin resistance seems to play a major role in the pathophysiology of atherosclerosis in relation with metabolic syndrome. Given that patients with NAFLD/NASH are at a high risk to develop atherosclerosis; these two diseases may share some pathology. However, precise molecular mechanisms underlying the pathogenesis and progression of these diseases are not well understood. Thus studying the molecular link between them would pinpoint sites of more effective pharmacologic interventions. The carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), a protein that is markedly reduced in the liver of patients with NASH, promotes insulin clearance to regulate insulin action. Whether or not CEACAM1 links hyperinsulinemia to NAFLD/NASH and atherosclerosis still needs to be determined. CEACAM1 enhances the rate of uptake of the insulin-receptor complex into the clathrin-coated vesicles of hepatocytes. It also plays a major role in mediating the negative acute effect on fatty acid synthase (Fasn) activity. As such, CEACAM1 regulates insulin and lipid metabolism. Mice with global null mutation of CEACAM1 (Cc1–/–) display hyperinsulinemia resulting from impaired insulin clearance, insulin resist (open full item for complete abstract)

Committee: Sonia Najjar PhD (Committee Chair); Guillermo Vazquez PhD (Committee Member); Jennifer Hill PhD (Committee Member); Rajesh Gupta MD (Committee Member); David Kennedy PhD (Committee Member); Steven Haller PhD (Committee Member) Subjects: Biomedical Research

Doctor of Philosophy (PhD), Wright State University, 2017, Biomedical Sciences PhD

Phospholipase D (PLD) is a cell membrane remodeling and signaling protein implicated in the pathology of chronic inflammation. As PLD is also central to macrophage cell migration, we investigated the molecular basis of PLD's involvement and regulation in macrophage-initiated inflammation (atherosclerosis) and resolution. We have found that PLD is associated with signaling proteins and positively affects cell movement, phagocytosis and NADPH-initiated release of Reactive Oxygen Species (ROS). We found that PLD2 but not PLD1 is important for foam cell formation that causes atherogenesis. We have also found a novel way of inducing macrophage class-switch (polarization) by PLD overexpression. PLD induces a macrophage M1 to M2 class-switch that accelerates resolution of inflammation and limits damage to blood vessels and affected tissues during atherosclerosis and other inflammatory conditions. We also investigated a new molecular pathway for macrophage; class-switch (M1-to-M2) by overexpressed PLD resulting in inflammation by bacterial phagocytosis or resolution of inflammation by efferocytosis. In order to understand the physiological relevance of PLD's role in inflammation and resolution, we studied the effect of resolvins, a class of specialized proresolving lipid mediators (SPMs), on PLD expression and activity in the different macrophage populations taking into consideration the time course of inflammation and resolution. We found that RvD5 upregulates PLD activity and expression in M2 macrophages confirming a molecular mechanism for PLD's role in resolution of inflammation.

Committee: Julian Gomez-Cambronero Ph.D. (Advisor); Nancy Bigley Ph.D. (Committee Chair); Juliusz A. Kozak Ph.D. (Committee Member); Yong-jie Xu M.D./Ph.D. (Committee Member); Michael Markey Ph.D. (Committee Member); Gerald Alter Ph.D. (Committee Member) Subjects: Biochemistry; Cellular Biology; Immunology; Molecular Biology

PhD, University of Cincinnati, 2017, Medicine: Molecular Genetics, Biochemistry, and Microbiology

Deposition, maturation, and regulation of the extracellular matrix (ECM) are key components of the overall fibrotic response, which plays a major pathological role in many forms of cardiovascular disease (CVD). Paradoxically, ECM remodeling is also required for preserving ventricular chamber integrity after injury and for maintaining vessel compliance and structure under physiological conditions. However, excessive and dysregulated ECM deposition occurs during disease, such as vessel wall remodeling during atherosclerosis or the replacement of dying cardiomyocytes by noncompliant scar tissue in the setting of myocardial infarction (MI). Although the molecular players that regulate pathological ECM remodeling are still poorly understood, one superfamily known as the matricellular proteins, a group of stress-induced ECM modulators, has received particular attention. Here we investigated the roles played by two matricellular protein family members, periostin and transforming growth factor beta induced (TGFBI), in mouse models of atherosclerosis, coronary artery disease (CAD) and hypertensive heart disease (HHD). Periostin and TGFBI are known to interact directly with a number of ECM proteins and have roles in cell adhesion and migration, two essential components of the fibrotic response. We first investigated the role periostin plays in atherosclerotic plaque development. Using a well-characterized model of atherosclerosis, the ApoE knockout mouse (ApoE-/-), we show that periostin is induced within the plaque and also in the circulating blood, suggesting a role for periostin in disease progression. Using a periostin-deficient global knockout mouse model crossed to the ApoE-/- mouse, we find that loss of periostin reduces plaque burden through a mechanism involving decreased collagen maturation within the plaque and impaired macrophage recruitment. We then investigated the role of TGFBI, a paralog of periostin, in ventricular remodeling in the heart after injury. B (open full item for complete abstract)

Committee: Jeffery Molkentin Ph.D. (Committee Chair); Burns Blaxall Ph.D. (Committee Member); William Miller Ph.D. (Committee Member); Thomas Thompson Ph.D. (Committee Member); David Wieczorek Ph.D. (Committee Member) Subjects: Molecular Biology

Master of Science in Biomedical Sciences (MSBS), University of Toledo, 2017, Biomedical Sciences (Bioinformatics and Proteomics/Genomics)

Macrophages are critical cells in the immune system implicated in various diseases with a chronic inflammatory component, such as atherosclerosis. The dominant macrophage types, M1 and M2, play key roles in progression and regression of atherosclerotic plaques. In this study, we profiled the transcriptomes of these macrophage subsets using RNA-Seq and microarray to examine molecular signatures and distinctive pathways for each subset. RNA-Seq analysis revealed a total of 2,127 differentially expressed genes (including coding and non-coding transcripts) between the M1 and M2 subsets. Validation by qRT-PCR of 10 top differentially expressed genes showed that 8 of the upregulated and 9 of the downregulated genes followed the expected trend based on the RNA-Seq analysis. Subsequently, pathway analysis of the upregulated and downregulated gene sets showed M1 and M2 macrophages to be enriched in pathways such as the Th1 and glioma signaling respectively. Microarray analysis revealed a total of 163 differentially expressed circRNAs between the two macrophage subsets and predictions of circRNA/miRNA interactions. From these circRNA/miRNA interactions, a number of the differentially expressed circRNAs were predicted to target athero-relevant miRNAs. As examples, circRNA_41878 (gene Ankrd42) was predicted to target miR-382-5p while circRNA_19794 (gene Lmbrd1) was predicted to target miR-124-3p, miRNAs of recognized effects in atherosclerosis. Overall, this work provides insight into distinctive molecular signatures and pathways enriched in bone-marrow derived M1 and M2 macrophages, and reveals the presence and contribution of non-protein-coding RNAs to their transcriptomes. In addition, our circRNA results provide the basis for future studies on the function of circRNAs in the context of macrophage functions in atherosclerosis.

Committee: Guillermo Vazquez (Committee Chair); Bina Joe (Committee Member); Beata Lecka-Czernik (Committee Member); Sivarajan Kumarasamy (Committee Member) Subjects: Bioinformatics; Biomedical Research