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Our lab has recently shown that IL-19 is expressed in angiogenic ECs, opening the possibility for its use as a medicine to increase perfusion in patients with PAD. The first aim of the current study is to show IL-19’s ability to increase perfusion in vivo using C57BL/6 wild type and IL-19 KO mice in the hindlimb ischemia (HLI) model. Wild-type mice injected with 10ng/g/day of rmIL-19 after being subject to hindlimb ischemia showed significantly greater levels of perfusion than PBS injected littermates. Immunohistochemistry of harvested gastrocnemius muscle showed a greater level of capillary density in IL-19 injected mice as well. IL-19-/- mice also showed a slower recovery of perfusion in a ligated limb in addition to less CD31 positive cells in gastrocnemius muscle when compared to C57BL/6 wild type mice. IL-19 -/- mice also showed increased perfusion when injected with rmIL-19. The second aim of the study is to show more precisely if IL-19 increases angiogenesis by increasing angiogenic cytokine production, polarizing macrophage phenotype, or by influencing angiogenic and anti-angiogenic factors. Spleen, serum, and bone marrow derived macrophage (BMDM) from mouse models used in Aim 1 showed increased levels of angiogenic cytokines, decreased anti-angiogenic cytokines, and markers of M2 macrophage polarization when IL-19 was injected i.p. or present genetically. The third aim of the study examines whether or not IL-19 can increase perfusion within an atherosclerotic background. It also addresses whether IL-19 can both simultaneously reduce atherosclerosis and increase perfusion. This aim also uses mice lacking LDLR-/- genes to further evaluate these questions. LDLR-/- mice fed a high fat diet for 12 weeks underwent HLI and had perfusion levels measured using Doppler imaging in addition to four weeks of 10ng/g/day of IL-19 or PBS injections. Upon sacrifice mice also had their aortas harvested and stained for plaque measurement. This experiment seeks to demonstrate if IL-19 can increase perfusion on an atherosclerotic background. Additionally, a second set of experiments addresses if LDLR-/- mice injected with recombinant mouse IL-19 (rmIL-19) or PBS for 16 weeks on a HFD in addition to HLI being performed at week 12 showed decreased levels of plaque and increased levels of hindlimb perfusion. These experiments seek to demonstrate if IL-19 can simultaneously reduce atherosclerosis while increasing perfusion. A third set of experiments attempts to evaluate the hypothesis that double knock out mice (DKO) lacking both LDLR and IL-19 genes will have increased plaque after being fed a HFD for 16 weeks. These aims all support the overall hypothesis that IL-19 can increase angiogenesis while additionally proving to be anti-inflammatory and anti-atherogenic in vivo / Physiology

The global incidence of overweight and obese individuals has skyrocketed in the past few decades resulting in a new health epidemic. In 1980, 5% of males and 8% of females were categorized as obese; by 2008 these values doubled equating to half a billion adults worldwide. This surge of overweight and obese individuals has driven a dramatic increase in people afflicted with metabolic disorders. As such, the term "metabolic syndrome" (MetS) has been coined to describe several interrelated metabolic risk factors which often present in concert. Specifically, metabolic syndrome refers to the presence of at least three of the following five conditions: central obesity, elevated triglycerides, diminished high density lipoprotein (HDL) cholesterol, hypertension, and insulin resistance (IR). MetS is a major health concern due to its ability to increase the likelihood of cardiovascular disease (CVD), diabetes, and other life-threatening ailments. In light of this growing medical epidemic, we have concentrated our efforts in evaluating the role of microRNA-155 (miR-155) in MetS development. MicroRNAs are a newly defined class of small, non-coding RNA which contain the unique ability to regulate gene expression through RNA interference. As a result of this ability, microRNAs can mediate a wide variety of cellular processes. In order to evaluate the function of miR-155 in MetS, we established a novel miR-155-/-/ApoE-/- (DKO) mouse model. Coupling this model with the use of normal rodent or high fat diets allowed us to investigate how states of caloric balance and surplus affected the manifestation of the individual MetS components. We found that male and female DKO mice fed a high fat diet had significantly augmented body masses of 18% and 10% respectively, when compared to ApoE-/- counterparts on the same diet. Evaluation of this phenotype with body composition analysis revealed an 18% and 46% increase in body fat percentage among the male DKO mice on normal and high fat diets, respectively. This trend was also observed in female DKO mice, albeit to a lesser extent. This phenotype was further substantiated by the observation of augmented gonadal white adipose tissue pad mass within male and female DKO mice fed either chow. This equated to a 43% and 112% increase in male mice and a 45% and 57% augmentation in female mice for normal and high fat chow diets, respectively. In light of our findings, we also evaluated how miR-155 impacted glucose and insulin sensitivity. We found levels of insulin to be augmented by 181% and 148% in male DKO mice on normal and high fat diets, respectively. Furthermore, we found these mice to be euglycemic. These observations suggest that DKO mice are IR but capable of compensating for their insensitivity with elevated insulin production. Due to the tight association between MetS and the development of non-alcoholic fatty liver disease (NAFLD) as well as CVD, we felt it prudent to investigate the manifestation of these conditions. We found elevated hepatic mass of 40% and 13% in male and female DKO mice on high fat chow. Furthermore, hepatic discoloration was seen in these mice prompting us to perform in-depth histological evaluation which revealed widespread steatosis, a hallmark of NAFLD. Meanwhile, investigation of atherosclerosis, the key underlying cause of most CVDs, unexpectantly revealed diminished development. Due to the complex nature of atherosclerosis it is tough to explain the exact reason for this observation. Independent reports have shown that miR-155 plays a critical role in the development, maturation, or activation of B-cell, T-cells, macrophages, and dendritic cells. As a result, decreased immune cell infiltration may be the root cause for the observed decline in atherosclerosis. Taking into account our observations of obesity, IR, and NAFLD in conjunction with independent findings of blood pressure mitigation by miR-155, we feel confident in reporting that miR-155 is a vital factor in preventing MetS and NAFLD development. Despite this, we surprisingly found atherosclerosis development to be diminished in these mice suggesting a pro-inflammatory role in atherogenesis. This duality highlights the complex and ambiguous nature of miRNAs. In light of this, further evaluations should be conducted to gain additional insight into these pathologies and hopefully the development of novel therapeutics. / Pharmacology

Patients with chronic kidney disease (CKD) develop hyperhomocysteinemia (HHcy), have increased inflammatory monocytes (MC) and 10-times higher cardiovascular mortality than the general population. Here, we investigated HHcy-related MC differentiation in CKD. Twenty seven CKD and CVD, and 14 healthy subjects were recruited. CD40 was selected as a CKD-induced MC activation marker by mining for CKD-MC-mRNA screen database. We found that CD14++CD16+ MC, often denoted as inflammatory subset, soluble CD40 ligand (sCD40L), and TNFα/IL-6 levels were augmented in CVD and CKD subjects. CD40hiCD14++CD16+ MC, plasma homocysteine (Hcy) and S-adenosylhomocysteine (SAH) levels were increased in CVD and further elevated in CKD subjects. In cultured human peripheral blood mononuclear cells, CKD patient serum, Hcy, CD40L and TNFα/IL-6 induced CD40hiCD14++CD16+ MC differentiation, which was prevented by Hcy-lowering folic acid and neutralizing antibodies against TNFα and IL-6. Interestingly, CD14++CD16+ and CD40hiCD14++CD16+ MCs were negatively correlated with plasma S-adenosylmethionine/SAH (SAM/SAH) ratios, an indicator of methylation status, in CKD and CVD subjects. In white blood cells (WBC) isolated from CKD and CVD subjects with lower SAM/SAH ratios, hypomethylation was identified on the CG pair of NFκB consensus element in the core promoter located at the CpG island of CD40 gene by DNA methylation mapping using bisulfite converting pyrosequencing. Moreover, Hcy inhibited DNA methyltransferase-1 activity in cultured human blood MC. In conclusion, HHcy induces CD14++CD16+ and CD40hiCD14++CD16+ MC differentiation, at least in part, via sCD40L induction and CD40 DNA hypomethylation in CKD and CVD subjects. To study the role of CD40 in the development of kidney pathology and vascular disease, we then established mouse model of CKD-induced CVD (5/6 nephrectomy CKD model plus left carotid artery ligation) in CD40-/- mice. Bone marrow (BM)-derived cells were traced by the transplantation of BM cells from enhanced green fluorescent protein (EGFP) transgenic CD40+/+ mice after sublethal irradiation of the recipient CD40-/- mice. We demonstrated here that CKD accelerated carotid artery atherosclerosis, exacerbated metabolism, increased spleen weight and circulating CD40+ inflammatory MC, and further increased differentiation of mononuclear phagocytic cells (MPC); CD11b+F4/80- MC, CD11b+F4/80+ macrophage (Mϕ) and CD11c+CD11b+F4/80+ bone marrow-derived dendritic cell in the kidney and aorta, which were abolished by CD40-/- mice. We also found that CKD kidney elevated CD40 expression and induced MC chemotactic signals; CCL2, CCL12, and CCL5 chemokines, which were abolished in CD40-/- mice. In conclusion, our results suggest that CD40 induction in the chronic kidney disease mediates kidney chemokine production, which in turn contributes to acceleration of myeloid cell infiltration, MPC differentiation, and carotid artery atherosclerosis. / Pharmacology

The purpose of this study was to investigate time commitment, self-efficacy and social environment as it relates to physical activity in a selected sample of hypertensive African Americans. In addition, this study focused on identifying additional research areas in regards to hypertensive African Americans. This study utilized a quantitative method for data collection. The survey instrument utilized contained the following subtopics: (1) demographics;(2)hypertension risk factors;(3) prevention and treatment;(4)hypertension knowledge, and (5)physical activity participation. Data collected did not support the hypotheses or information contained in the review of literature. It was revealed from data collection that 69% of the respondents (n=90) disagreed with the survey statement that "exercise takes too much of my time (time commitment)." Fifty-two percent of the respondents (n=68) either disagreed or strongly disagreed with the statement "exercise tires me (self-efficacy belief)." Forty-three percent of the respondents (n=56) disagreed with the statement "my spouse (or significant other) does not encourage exercise." There is a need for future investigation to examine how additional barriers to physical effect activity African Americans individually, and is there a culmination of specific barriers to physical activity that work in conjunction to inhibit African Americans to engage in physical activity. / Ph. D.

Introduction: Garlic-derived organosulfur compounds are associated with physiological benefits, including the reduction of cardiovascular disease (CVD) risk, possibly by reducing the risk marker trimethylamine-N-oxide (TMAO). TMAO production in humans is largely influenced by the metabolic activity of the intestinal bacteria on dietary precursors including L-carnitine. Dietary supplementation of bioactive garlic phytochemical allicin has recently been suggested to reduce the formation of TMAO precursor molecule trimethylamine (TMA) from L-carnitine through impact on the intestinal bacteria, thereby limiting the formation of TMAO by the host. Purpose: The objective of this research was to evaluate and compare the efficacy of fresh and aged garlic extracts (rich in alliin and allicin, respectively) in the reduction of circulating TMAO levels produced from L-carnitine metabolism and identify shifts in the abundances of gastrointestinal bacterial genes that may contribute to reduction in circulating TMA levels, which may, in turn, influence the levels of circulating TMAO. Methods: Five-week old female C57BL/6 mice (n = 12) were challenged with L-carnitine to assess the animal's capacity for TMAO production. Animals were gavaged daily with fresh or aged garlic extract dissolved in L-carnitine for 13 days, then challenged with L-carnitine post-treatment to evaluate changes in TMAO production. Whole blood samples were evaluated for TMAO content using UPLC-MS/MS and compared to non-extract consuming control groups. Post-mortem hepatic tissues were collected and analyzed for TMA-oxidizing flavin monooxygenase 3 (Fmo3) gene abundance and protein expression using quantitative real-time PCR (qPCR) and ELISA. Fecal samples collected prior to and following treatment were analyzed using qPCR to quantify shifts in the abundance of L-carnitine metabolizing genes cntAB and grdH. Results: Postprandial and circulating TMAO levels were not significantly affected (p < 0.05) by inclusion of garlic extract in the diet. Dietary intervention with extracts significantly increased L-carnitine-derived proatherogenic CVD risk marker γ-butyrobetaine levels ~28% higher than the increased levels observed in the positive control group supplemented with L-carnitine only. Mice administered garlic extracts had significant increases of, γ-butyrobetaine, relative to negative control mice and mice supplemented with broad-spectrum antibiotics. Mice supplemented fresh garlic extract saw a 25-fold increase in circulating γ-butyrobetaine levels after intervention; mice supplemented aged garlic extract saw a 23-fold increase in circulating γ-butyrobetaine levels after intervention. Furthermore, FMO3 protein expression levels in either extract treatment group were not significantly different (p < 0.05) from controls. Abundances of L-carnitine metabolizing genes in fecal samples of mice fed either garlic extract were not significantly higher than levels observed in positive or negative controls. Interestingly, treatment with broad-spectrum antibiotics significantly increased abundances of L-carnitine metabolizing genes cntAB and grdH when compared with controls. Abundances of hepatic Fmo3 mRNA transcript in mice supplemented garlic extracts were not significantly different from the positive control group when data were normalized to mg of liver used. Mice supplemented aged garlic extracts significantly lowered Fmo3 mRNA transcript levels relative to the negative control. Significance: This research suggests that garlic extract supplementation in conjunction with excess L-carnitine consumption may not be an appropriate dietary intervention strategy to reduce CVD risk. As it stands, garlic extract supplementation may increase CVD risk by promoting the biosynthesis of proatherogenic γ-butyrobetaine. The impact of garlic extract mediated increases in γ-butyrobetaine should be further investigated in tandem with CVD outcomes to confirm the findings presented in this study. / Doctor of Philosophy / Garlic compounds that contain sulfur are associated with many health benefits, including the reduction of heart disease risk, possibly by lowering the amount of risk marker trimethylamine-N-oxide (TMAO) in the body. TMAO is produced when the gut bacteria break down L-carnitine into trimethylamine (TMA), which is then absorbed and converted to TMAO in the liver. Garlic supplementation has recently been suggested to reduce TMAO formation, which may, in turn, reduce heart disease risk. The objective of this research was to evaluate the potential of fresh and aged garlic extracts (which have different sulfur compounds in them) to reduce TMAO levels and identify changes in the gut bacteria that may contribute to this lowering effect. Mice were fed daily with either fresh or aged garlic extract for 13 days, then given L-carnitine to evaluate changes in TMAO levels in the blood. These levels were then compared to mice that did not consume any garlic extract. Liver samples were tested for their ability to turn TMA into TMAO. Fecal samples were tested to determine if there were any changes to gut bacteria caused by the garlic extracts. TMAO levels in the mice were not significantly affected by consuming garlic extracts. Consuming garlic extracts did, however, increase another risk marker of heart disease known as γ-butyrobetaine. Feeding mice garlic extracts did not affect the ability of mice to turn TMA into TMAO, nor did it affect the gut bacteria. This research suggests that garlic extracts may not be an appropriate strategy to reduce heart disease risk. As it stands, garlic extract supplementation may increase heart disease risk by promoting the γ-butyrobetaine formation. The means that garlic extracts increase γ-butyrobetaine levels should be further investigated.

This experiment tested two hypotheses linking the right cerebral regulation of hostility and cardiovascular arousal. First, replication of previous research supporting heightened cardiovascular (systolic blood pressure, diastolic blood pressure, and heart rate) reactivity among high hostile participants was attempted. Second, dynamic variations in functional cerebral asymmetry in response to pain (cold pressor) and emotional linguistic processing was measured. Low- and high-hostile participants were identified using the Cook Medley Hostility Scale (CMHS). All participants completed either the negative affective verbal learning test (Experiment 1) or the cold pressor paradigm (Experiment 2). Cardiovascular measures (SBP, DBP, and HR) were recorded and either dichotic listening procedures (Experiment 1) or tachistoscopic lexical recognition procedures (Experiment 2) were administered before and after the stressor. The primary finding of this research was greater left cerebral activation (decreased cardiovascular reactivity) following the dichotic phoneme listening and the tachistoscopic lexical recognition tasks and greater right cerebral activation following pain (cold pressor) and emotionally linguistic (affective verbal learning) stressors. / Master of Science