Retinoic acid-inducible gene-I (RIG-I) is a putative RNA helicase and recently identified as a cytosolic RNA receptor in mammalian cells. The role of RIG-I in the regulation of vascular function under physiological and pathological conditions is unknown. Recent studies have shown that the inflammasome serves as a crucial initiator of cytokine-mediated inflammation mediating the pathogenesis of cardiovascular disease. The present study tested whether RIG-I activation triggers inflammasome formation and subsequent cytokine-mediated inflammation in the endothelium of mice coronary arteries. Using both genetic and pharmacological interventions of the RIG-I inflammasome, we first characterized whether specific activation of RIG-I via 3pRNA transfection induced the formation of an ASC-containing inflammasome in mouse vascular endothelial cells (MVECs). We confirmed that 3pRNA dose-dependently increased RIG-I protein levels and release of of type I IFNβ and IL-1b (a prototype cytokine from inflammasome activation), confirming RIG-I activation. We found that MVECs transfected with 3pRNA exhibited increased colocalization of RIG-I with apoptosis-associated speck-like protein (ASC) or caspase-1 and elevated active caspase-1 and IL-1β levels, indicating the formation and activation of the RIG-I inflammasome. This RIG-I inflammasome activation was accompanied by endothelial barrier dysfunction. In the presence of 3pRNA, ZO-1 and ZO-1 and VE-Cadherin expression diminished, and inhibiting caspase-1 and silencing inflammasome components attenuated this effect. To test the functional role of RIG-I and its affect on the permeability of mouse ECs, we performed a transwell permeability assay. Results confirmed that 3pRNA induced increased permeabilization of these mouse ECs, which was attenuated when inhibiting and silencing inflammasome components. These data indicate that increased expression and activity of RIG-I activate IL-1b producing inflammasomes in ECs, which may represent an early molecular mechanism mediating vascular inflammation and endothelial dysfunction independent of Nlrp3. Furthermore, we investigated the role of anti-aging gene Klotho in the regulation of RIG-I inflammasome activation. The Klotho protein has been shown to directly interact with RIG-I in senescent cells to block RIG-I multimerization and downstream production of pro-inflammatory cytokines. Administration of D-saccharic acid 1,4-lactone (saccharolactone) in vitro, a pharmacological inhibitor of Klotho activity, substantially increased inflammasome activation. In addition, mice injected with saccharolactone exhibited increased RIG-I inflammasome formation and activation within the coronary artery endothelium. These results suggest that decreased Klotho activity may activate RIG-I and thereby increase inflammasome activity. Therefore, the present study defines a novel role for RIG-I inflammasome activation in vascular dysfunction.

Cardiovascular disease is currently the foremost cause of death within the United States. Heart failure (HF) is a syndrome defined by the inability of the heart to adequately execute requisite pump function in order to deliver nutrients and oxygen to peripheral tissues, irrespective of etiology. One of the most common causes of HF is chronic pressure overload due to hypertension. Ischemic heart disease is also a common driver of HF, often in conjunction with hypertension. Pressure overload initially causes compensatory metabolic changes. Structural changes follow shortly thereafter typically resulting in left ventricular hypertrophy. Eventually, the heart loses the ability to compensate for the aberrant hemodynamic load and begins failing. The failing heart is unable to supply adequate adenosine triphosphate (ATP) for contractile function as evidenced by falling phosphocreatine (PCr) levels. This energy deficit occurs concurrently with a metabolic re-programming that results in a fuel utilization pattern resembling the fetal heart. Notably, enzymes involved in catabolism of fatty acids, the chief fuel substrate for ATP generation in the normal adult heart, are downregulated in the failing heart. However, the extent to which alternative fuels compensate for decreased fatty acid oxidation (FAO) is not well-known. Furthermore, consequences of the fuel substrate switches that occur in heart failure are not well established. In this work, we discover a new paradigm for alternate fuel utilization in the failing heart and define consequences of altered fuel metabolism in HF. We discovered a post-translational modification resultant from an accumulation of acetyl groups (C2) present in a mouse model of early-stage HF and human HF. Mitochondrial proteins were found to be hyperacetylated in the failing heart, and at least some of these alterations result in diminished electron-transport chain (ETC) capacity as shown by mutagenesis studies on succinate dehydrogenase A (SDHA). We also found an accumulation of C4-OH carnitine, a by-product of ketone oxidation in HF. This metabolite aggregation occurred alongside an increase in b-hydroxybutyrate dehydrogenase 1 (BDH1) transcript and protein levels. This signature suggested that the failing heart shifted to ketone bodies as a fuel. Subsequent experiments confirmed increased capacity for myocardial ketone oxidation in compensated cardiac hypertrophy and in HF. The consequences of increased ketone oxidation were then assessed using a cardiac-specific BDH1 knockout (BDH1 KO) mouse. Despite not having any apparent defect at baseline, we found BDH1 KO mouse hearts are completely unable to oxidize 3-hydroxybutyrate. The deficit for ketone oxidation capacity became consequential upon subjugation to transverse aortic constriction with a small apical myocardial infarction (TAC/MI). The BDH1 KO mice exhibit altered pathological cardiac remodeling compared to wild-type controls. These latter data suggest the increased reliance on ketone oxidation in HF, mediated by BDH1, is an adaptive response. Together the results of these studies provide important information regarding the consequences of altered fuel metabolism in HF. Recent reports of reduced HF mortality and elevated circulating ketone levels in patients prescribed Empagliflozin make cardiac ketone metabolism research in this dissertation particularly apropos.

Cardiovascular disease remains one of the most prevalent diseases in the United States and has remained as the leading cause of death. Large Mendelian randomization studies have found significant correlations between high blood pressure and cardiovascular disease (CVD). In fact, high blood pressure is the single most important independent risk factor for CVD. The purpose of this study was to determine the effect of L-citrulline on blood pressure to determine whether it could be advised as an effective treatment for high blood pressure. L-citrulline is a naturally occurring amino acid that readily converts to L-arginine within the human body. L-arginine has shown promise in decreasing both systolic blood pressure (SBP) and diastolic blood pressure (DBP) significantly by potently increasing levels of nitric oxide (NO) in the body. L-arginine, however, displays poor oral bioavailability compared to L-citrulline. Thus, L-citrulline may be a more effective method in raising plasma arginine levels, increasing NO, and decreasing SBP and DBP. A thorough systematic review and meta-analysis was conducted to extrapolate this effect. Using online databases, hundreds of articles were screened, and ultimately 11 studies were chosen, encompassing 224 total participants. Results showed an overall significant effect of L-citrulline on both resting SBP (MD: -3.74; 95% CI [-6.74, -0.74]; p=0.01) and DBP (MD: -2.00; 95% CI [-3.93, -0.06]; p=0.04). Further analysis of funnel plots was used to determine publication biases and subgroup analysis was performed to determine specific trial moderators that could have affected the overall outcome. In most cases, L-citrulline displayed a significant effect on blood pressure, and more research is warranted to investigate its potential therapeutic effect on cardiovascular health.

AIMS: The aim of the current project was to determine if lycopene could modulate T-cell function and activity. METHODS: 1) Peripheral blood mononuclear cells (PBMC) were isolated from healthy adults and incubated with lycopene-enriched liposomes (0.0-1.2 μg/ml) then activated with or without the mitogens Con A, anti-CD3 and LPS and cultured for various lengths of time before measuring CD3, CD4, CD8, CD69, CD11a and CD25 expression.  the production of IL-1β, IL-2, IL-4, IL-6, IL-10, IL-17, IFN-γ, TGF-β and TNF-α were also measured along with the percentage of FoxP3 and IL-10 positive T-regulatory cells.  2) <i>Ex-vivo</i> expression of CD11a, CD49d, CD54, CD3 and CD69, as well as Con A stimulated proliferation were measured in subjects before and after a 12 week tomato-food and lycopene intervention study. RESULTS: Lycopene reduced PBMC proliferation and CD69 expression irrespective of the type of T-cell considered.  Lycopene reduced IL-2, IL-10, IL-17 and IFN-γ but increased IL-1β and TNF-α production.  lycopene also reduced the number of CD4⁺/CD25⁺ cells present as well as those positive for IL-10. Other parameters were not affected. CONCLUSION: Lycopene reduces T-cell activity by mechanisms dependent on early cell activation but this modulation is not specific to T-cell types.  the data support a potential beneficial effect of lycopene in the reduction of atherosclerosis through the modulation of T-cell function.

The association between diabetes and coronary artery disease (CAD) has been recognized as a major public health problem in the developed world. While there is an increased prevalence of silent myocardial ischaemia among asymptomatic individuals with diabetes, diabetic individuals with CAD in their larger number are usually asymptomatic, and when they present with signs of disease, there is extensive and severe CAD. It should be noted that amongst black South African, ischemic heart disease (IHD) remains rare, and there is little data linking diabetes mellitus with IHD. However, contrary to early reports that have suggested a low prevalence of CAD in black population in Africa, many studies have indicated a rapid change on the spectrum of CAD in numerous parts of the African continent. Despite the emerging report of high prevalence of risk factors there are only limited data investigating prevalence of CAD in black African with diabetes. The purpose of this thesis was to use myocardial perfusion imaging (MPI) at rest and after stress testing to detect CAD in a group of asymptomatic black patients suffering from diabetes and therefore assess the prevalence of CAD; to assess the changes in myocardial perfusion in asymptomatic diabetic black individuals and compare the differences seen in myocardial perfusion changes between the asymptomatic diabetic black and, the asymptomatic diabetic white and Indian individuals; to include data from symptomatic diabetic patients who were referred for MPI as part of their routine clinical management for possible comparison Consecutive 94 asymptomatic diabetic black patients and 50 asymptomatic diabetic white and Indian patients were recruited from the outpatient diabetic clinic of the Johannesburg hospital. Data from 90 subjects forming a group of symptomatic diabetic patients, 45 blacks and 45 whites and Indians referred for MPI as part of their clinical management were also analyzed. A two-day protocol for SPECT MPI was used in all participants: on the first day the stress testing MPI while the rest MPI was consistently done on the second day. Both exercise and pharmacologic stress testing were used. Technetium-99m methoxy-isobutylisonitrile (MIBI) was used as the myocardial perfusion radiopharmaceutical. Myocardial perfusion was assessed by means of semi-quantitative scoring system to measure the extent and severity of perfusion abnormality. Visual inspection of the reconstructed SPECT MPI images was carried out to assess perfusion deficit where there was a doubt on the extent and severity of perfusion abnormality. The QPS/QGS software allows obtaining resting and post stress left ventricular ejection fraction (LVEF). The means and percentages on study variables were obtained. The Spearmen correlation coefficient was used to calculate correlations between variables. The Kruskal-Wallis test was used to assess differences between black diabetic and white or Indian diabetic patients and Wilcoxon scores (rank sum) two-sided were used to measure differences within these racial groups. There were 123 females (52.6%) and 111 males (47.4%) in total. From the recruited participants, 53 (56.4%) asymptomatic females and 41 (43.6%) asymptomatic males were blacks whereas 24 (48%) asymptomatic females and 26 (52%) asymptomatic males were whites or Indians. The symptomatic group was comprised of 26 (57.8%) female and 19 (42.2%) male black patients and 20 (44.5%) female and 25 (55.5%) male white or Indian patients. Asymptomatic diabetic black participants were younger than the participants from the asymptomatic diabetic white and Indian group with a mean age of 60 (SD±7.2) years Vs 64 (SD±7.7) [p=0.003]. Fourteen percent of asymptomatic black participants had evidence of ischaemia by showing improvement of perfusion on stress testing versus twenty eight percent of white and Indian asymptomatic participants (p=0.62). Perfusion defects that did not change from rest to post stress testing MPI (fixed defects) were also noted in 20% of asymptomatic black and 26% of asymptomatic white and Indian diabetic participants. These fixed perfusion defects are indicative of previous myocardial infarctions and therefore suggestive of CAD. No significant difference was noted on the changes of perfusion that could account either for ischaemia or infract between asymptomatic diabetic black participants and their white and Indian counterparts (p=0.47). The difference on the improvement of perfusion from rest to post-stress MPIs or reversibility of perfusion to suggest only the presence of ischaemia did not also show a significant difference between these two racial groups (p=0.62). Our data demonstrated a high prevalence of CAD in asymptomatic diabetic black participants similar to other racial groups. Our study has demonstrated evidence to recommend screening of asymptomatic diabetic black individuals in equal manner than other races for the detection of CAD. More importantly, stress MPI should be routinely used as a noninvasive investigation in our environment and be utilized more actively in the management of all asymptomatic diabetic patients.

Following myocardial infarction, as a result of thrombus formation, angiogenesis occurs and permits reperfusion of damaged myocardium. Sphingolipids, sphingosine 1-phosphate (S1P) and sphingosylphosphorylcholine (SPC) are naturally occurring lipid mediators released from platelets and found in high concentrations at sites of thrombosis. S1P and SPC may therefore be involved in regulating angiogenesis following myocardial infarction and may influence reperfusion. The aims of this study were to determine the effects of S1P and SPC in human coronary arterial cell angiogenesis and delineate the subsequent mechanisms. An in vitro model of angiogenesis was developed using a co-culture of human coronary artery endothelial cells, human coronary smooth muscle cells and human fibroblasts. In this model S1P and SPC inhibited angiogenesis and this was dependent on the presence of smooth muscle cells. The mechanism of the inhibitory effect was via S1P-/SPC-induced release of a soluble mediator from smooth muscle cells. This mediator was identified as tissue inhibitor of metalloproteinase-2 (TIMP- 2). TIMP-2 release was dependent on sphingolipid-induced activation of S1P2 receptor and Rho-kinase signalling and directly contributed to incomplete formation of endothelial cell adherens junctions. This was observed as a diffuse localization of VE-cadherin leading to decreased tubulogenesis. This effect may occur via both matrix metalloproteinase-dependent and/or matrix metalloproteinase-independent pathways. A similar inhibitory response to S1P was demonstrated in ex vivo human and mouse arterial models of angiogenesis. In summary, S1P- and SPC-induced inhibition of angiogenesis in human artery endothelial cells mediated by TIMP-2 from vascular smooth muscle cells. This reduces the integrity of intercellular junctions between nascent endothelial cells. S1P and SPC may therefore inhibit the angiogenic response following myocardial infarction.

Diss. (sammanfattning) Uppsala : Univ., 2000. / Härtill 6 uppsatser.

Diss. (sammanfattning) Uppsala : Univ., 2001. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 6 uppsatser.

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