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Browsing by Author "Gentile, Christopher L., committee member"

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    Cardiovascular-protective effects of blueberry consumption in postmenopausal women with above-normal blood pressure
    (Colorado State University. Libraries, 2023) Woolf, Emily K., author; Johnson, Sarah A., advisor; Gentile, Christopher L., committee member; Weir, Tiffany L., committee member; Rao, Sangeeta, committee member
    Endothelial dysfunction is the first step in atherosclerosis and contributes to its progression, and thus, is central to cardiovascular disease (CVD). It is driven by excessive oxidative stress and inflammation and characterized by impaired endothelium-dependent dilation. Estrogen-deficient postmenopausal women have oxidative stress-mediated suppression of endothelial function that is worsened by high blood pressure. Chronic blueberry consumption may be a beneficial dietary intervention for this population as it has shown to improve vascular function and blood pressure, though some studies have not demonstrated efficacy possibly due to the observed high interindividual variability in response to the intervention. Evidence indicates blueberries improve endothelial function, but studies have not been performed in postmenopausal women. Furthermore, ex vivo research has shown that blueberry (poly)phenols and their metabolites can decrease endothelial oxidative stress and inflammation, but whether these mechanisms translate to humans is unclear. The objectives of this dissertation were to 1) examine the efficacy of chronic blueberry consumption to improve endothelial function and blood pressure in estrogen-deficient postmenopausal women with above-normal blood pressure, with a specific focus on identifying mechanisms for improving endothelial function, 2) identify factors that contributed to the efficacy of blueberries as a dietary intervention for improving endothelial function, and 3) explore cellular mechanisms responsible for endothelial function improvements and the anti-atherogenic potential of blueberries. To investigate the aforementioned, we conducted a randomized, double-blind, placebo-controlled clinical trial and assessed endothelial function (measured through flow-mediated dilation (FMD)) and supine brachial blood pressure before and after daily consumption of 22 g of freeze-dried highbush blueberry powder or isocaloric placebo powder for 12 weeks. To examine mechanisms for improved endothelial function, FMD was assessed before and after infusing a supraphysiological dose of the antioxidant ascorbic acid (i.e. vitamin C) and normalized to shear rate area under the curve (FMD/SRAUC). To investigate factors impacting the interindividual variability in the endothelial function responses after the 12 weeks of blueberry consumption, we grouped the blueberry treatment group into responders (≥ +1% unit Δ FMD) and non-responders (< +1% unit Δ FMD) and performed secondary statistical analyses using data produced from the clinical trial. Lastly, to investigate mechanisms for improvements in endothelial function, we used a reverse translational human-to-cell approach leveraging human blood serum collected from participants in the clinical trial to perform ex vivo cell culture experiments. Results from the clinical trial showed that daily blueberry consumption significantly improved FMD/SRAUC compared to baseline by 96%. FMD not normalized for shear rate increased by 1.34% though the effects were not statistically significant (but were clinically significant). Improvements in FMD/SRAUC after blueberry consumption were due to reductions in oxidative stress as responses to ascorbic acid infusion were significantly reduced at 12 weeks in the blueberry group compared to baseline, with no changes in the placebo group. There were no major effects on blood pressure, arterial stiffness, endothelial cell protein expression, or other blood biomarkers of cardiovascular health. It was determined that the blueberry intervention was ~50% effective for improving FMD to clinically relevant levels of ≥ +1%, and that responders had decreased cardiovascular health and higher levels of circulating estrogen at baseline compared to non-responders. After 12 weeks of blueberry consumption, responders had reductions in oxidative stress, lower plasma nitrate levels, and higher phosphorylated endothelial nitric oxide synthase protein expression compared to non-responders. Lastly, we cultured HAECs with 15% serum (blueberry and placebo) for 1 h followed by 200 µM hydrogen peroxide (H2O2) for 24 h to induce endothelial dysfunction and evaluated the effects of blueberry (poly)phenol-rich serum on endothelial cell dysfunction and atherosclerosis progression. There were no statistically significant differences on monocyte binding, insulin-stimulated nitric oxide production, or peroxynitrite concentrations between dysfunctional HAECs treated with blueberry and placebo serum from the clinical trial. Collectively, results from these studies indicate that daily blueberry consumption for 12 weeks improves endothelial function in postmenopausal women with above-normal blood pressure through reductions in oxidative stress, and that efficacy (i.e. degree to which postmenopausal women responded to treatment in endothelial function) seems to be dependent on participant characteristics including cardiovascular risk factors and estradiol at baseline. Due to the inconclusive results regarding the ex vivo experiment, cellular mechanisms by which blueberry (poly)phenol metabolites impact endothelial function and atherosclerosis progression cannot be determined.
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    Implications of diet-induced obesity on metabolic and immune homeostasis: the role of the mesenteric lymph nodes
    (Colorado State University. Libraries, 2020) Hill, Jessica Lynn, author; Michelle, Foster T., advisor; Weir, Tiffany L., committee member; Gentile, Christopher L., committee member; Schenkel, Alan, committee member
    Obesity is a major public health crisis among adolescents and adults. The development of obesity is associated with several comorbidities as a result of underlying systemic chronic inflammation, the culmination of which increases one’s risk for chronic and infectious disease. Excessive accumulation of visceral adipose tissue is shown to confer the greatest disease risk. This is primarily due to inherent depot differences, namely proximity to and a shared blood supply with the liver and gastrointestinal (GI) tract. Recent work demonstrates the considerable influence gut physiology has over both local and systemic homeostasis, as GI diseases such as inflammatory bowel disease are associated with metabolic derangements characteristic of obesity. While the mechanisms that mediate this inter-organ crosstalk continue to be elucidated, several studies suggest that inflammation originating from the gut triggers these broad metabolic and immunologic changes found in obesity. Previous work from our lab has demonstrated that high-fat diet (HFD) induced obesity results in mesenteric lymph node (MLN) fibrosis, which was associated with a localized impairment in immune function. MLNs, located within mesenteric adipose tissue (MAT) surrounding the GI tract, constitutively monitor the mesenteric adipose depot and draining sections of the small and large intestines, serving as critical inductive sites for adaptive immune responses. Subsequently, they are essential for overall tissue maintenance and protection. Hence, further study into the role of the MLNs in obesity-associated pathology is an important area of research. The goals of this dissertation research were to 1) examine the relationship between MLNs and GI inflammation on metabolic outcomes, and 2) characterize immunologic changes associated with models of chronic inflammation. To investigate the above-mentioned, we conducted four separate preclinical studies utilizing mouse models of diet-induced obesity, MLN cauterization, and dextran sulfate sodium (DSS) induced GI inflammation. In the first study (Chapter 2), we examined the contribution of the MLNs on disease pathology associated with HFD-induced obesity. We found that MLN dysfunction, either as a result of surgical manipulation or obesity-induced fibrosis, led to metabolic dysfunction. Furthermore, that functional MLNs are needed for the full restorative effects of Pirfenidone treatment. In the second study (Chapter 3), we examined the effect of chronic low-dose DSS induced GI inflammation, independent of diet and obesity, on metabolic and immune function. We found that non-obese mice treated with DSS had a modest reduction in total body weight and MAT mass yet showed substantial alterations in tissue immune cell populations and frequencies. These adaptations occurred without a concurrent change in glucose homeostasis. Finally, in the third study (Chapter 4) we characterized immunologic parameters within a normal weight and obese human population, free of disease, through the ex vivo challenge of peripheral blood mononuclear cells (PBMCs) with the T lymphocyte mitogen Concanavalin A (ConA). We found that PBMCs isolated from obese adults had a modest increase in cell proliferation and IFNγ secretion upon stimulation within ConA relative to their normal weight controls. Additionally, we found a distinct expansion of CD4+CD8+ T cells, CD16+ monocytes, and NK cells within ConA stimulated PBMCs from obese donors. Collectively, these studies provide evidence that 1) the MLNs are critical for metabolic homeostasis as their dysfunction exacerbates features of HFD-induced obesity; 2) chronic GI iv inflammation, independent of diet and obesity, can reshape the immune milieu without altering glucose homeostasis; and 3) obesity distinctly alters the PBMC response to acute ex vivo challenge as compared to that of normal weight individuals. Future studies should further elucidate mechanisms of crosstalk between the immune system, MLNs, and GI tract on metabolic homeostasis in models of obesity.
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    Influence of FADS2 expression on cardiovascular risk: role of mitochondrial arachidonic acid
    (Colorado State University. Libraries, 2020) Li Puma, Lance Christopher, author; Chicco, Adam J., advisor; Bouma, Gerrit J., committee member; Amberg, Gregory C., committee member; Gentile, Christopher L., committee member; Legare, Marie E., committee member
    Long-chain polyunsaturated fatty acids (LC-PUFA) are widely believed to influence cardiovascular health and disease in humans and can be supplied through the diet or endogenously synthesized from the essential PUFAs linoleic acid (LA; n6) and alpha-linolenic acid (ALA; n3). Redistribution of PUFAs in serum and tissue phospholipids has been associated with various pathologies, manifesting primarily as a proportional loss of the essential PUFA LA paralleled by reciprocal increases in its long-chain product arachidonic acid (AA; n6). Epidemiological studies have linked greater AA/LA ratios in serum phospholipids to multiple parameters of cardiometabolic disease (CMD), such as obesity, insulin resistance, hypertension, atherosclerosis, and coronary artery disease. Single nucleotide polymorphisms in the FADS2 gene are associated with haplotypes of greater expression of FADS2, which may increase the production of AA from LA and increase serum AA/LA ratios. FADS2 encodes delta-6 desaturase, the rate-limiting enzyme in endogenous LC-PUFA biosynthesis, which is believed to participate in the development of CMD by interacting with the high dietary LA content found in the modern Western diet to disproportionally produce AA over other LC-PUFAs. The overarching hypothesis of this dissertation is that greater expression of FADS2 promotes the development of cardiovascular risk parameters. To investigate this, we generated mice with global (CMV promoter) transgenic overexpression of Fads2 (Fads2TG); these mice exhibit classic serum shifts in PUFA distribution characteristic of human FADS2 polymorphisms. A series of three projects were undertaken: 1) Investigate the interaction between dietary essential fatty acid intakes and Fads2 expression on cardiovascular risk; 2) Establish methodology for simultaneous measurement of mitochondrial respiration and ROS release in vitro; 3) Investigate effects of Fads2 expression on cardiac mitochondrial responses to Ca++-overload. These studies discovered that greater Fads2 expression is sufficient to increase several aspects of cardiovascular risk that were independent of the dietary ratio of n6:n3 essential PUFAs. Further investigation demonstrated that cardiac cardiolipin AA content predicted ischemia-reperfusion (IR) injury, suggesting a mechanistic role of mitochondria in this phenotype. Gain- and loss-of-function approaches in mice established that greater Fads2 expression lowers mitochondrial tolerance to Ca++-overload demonstrated by loss of OXPHOS-linked respiration, greater mitochondrial ROS release, and increased mitochondrial permeability transition. Furthermore, mitochondrial permeability transition by Ca++-overload could be attenuated by inhibition of AA release or metabolism. Collectively, the significance of these studies establish the influence of Fads2 on serum and tissue PUFA composition and the pathogenesis of IR injury through modulation of mitochondria membrane composition, thereby demonstrating Fads2 expression as an independent factor for cardiovascular risk.
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    Novel role of acetylcholine in vascular control in humans
    (Colorado State University. Libraries, 2020) Terwoord, Janée D., author; Dinenno, Frank A., advisor; Amberg, Gregory C., committee member; Chicco, Adam J., committee member; Gentile, Christopher L., committee member
    The vascular endothelium is remarkably sensitive to the molecule acetylcholine (ACh), which binds to muscarinic receptors to initiate endothelium-dependent vasodilation. Although vasodilatory responsiveness to ACh is considered the gold standard index of endothelial function, an obligatory role for ACh in peripheral blood flow control has been challenging to elucidate. Thus, muscarinic ACh receptors on endothelial cells are widely considered to be evolutionary remnants with no real physiological function in humans. Administration of exogenous ACh amplifies endothelial sensitivity to other vasodilatory stimuli and blunts sympathetic vasoconstrictor signaling; therefore, we sought to determine whether endogenous ACh contributes to these processes in vivo. Accordingly, the overall goal of this dissertation research was to evaluate the role of ACh in modulating sympathetic α adrenergic vasoconstriction and eliciting vasodilation in healthy, young adults. The primary findings are that 1) ACh interacts with the endothelium-dependent vasodilator adenosine triphosphate (ATP) to augment vasodilation and limit α1 adrenergic vasoconstriction in the skeletal muscle resistance vasculature, 2) endogenous ACh blunts sympathetic vasoconstriction within active skeletal muscle and is an obligatory mechanism of functional sympatholysis during exercise at high intensities, and 3) ACh mediates flow-induced vasodilation of conduit arteries in response to sustained and transient increases in shear rate induced by handgrip exercise and reactive hyperemia, respectively. Collectively, these studies reveal a novel, physiological role of ACh in peripheral blood flow regulation in humans.
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    Sympathetic inhibition attenuates hypoxia induced insulin resistance in healthy adult humans
    (Colorado State University. Libraries, 2012) Peltonen, Garrett Lee, author; Bell, Christopher, advisor; Miller, Benjamin F., committee member; Hamilton, Karyn L., committee member; Gentile, Christopher L., committee member
    Acute and chronic exposure to hypoxia is known to decrease insulin sensitivity in healthy humans and animals, while simultaneously increasing the activity of the sympathetic nervous system (SNS). Likewise, obstructive sleep apnea (OSA) and chronic obstructive pulmonary disease (COPD) are clinical conditions characterized by hypoxia, elevated SNS activity, and a high prevalence of insulin resistance. In contrast to hypoxic exposure and hypoxic related diseases, hyperoxia (FIO2 = 1.00) has been shown to improve insulin sensitivity while concomitantly decreasing SNS activity. Consistent with this, continuous positive airway pressure (CPAP), a common OSA treatment, has proven effective in abolishing nocturnal bouts of intermittent hypoxia, enhancing insulin sensitivity and diminishing SNS activity. Although the underlying mechanism of hypoxia induced insulin resistance remains unclear, it appears that elevated SNS activity may be a mediating factor. Therefore, we hypothesized that inhibition of the SNS would attenuate hypoxia induced insulin resistance. METHODS: 10 males (23±1 years, body mass index 24.2±0.8 kg/m2 (mean±SE)) reported to our laboratory on 4 separate mornings, separated by a minimum of 7 days, after a 12-hour fast and 48-hour abstention from exercise. Insulin sensitivity was determined via the hyperinsulinemic euglycemic clamp technique under each of the following conditions: normoxia (FIO2=0.21), hypoxia (FIO2=0.11), normoxia and SNS inhibition (48-hour transdermal clonidine administration (Catapres-TTS; 0.2mg/day)), and hypoxia SNS inhibition. RESULTS: Oxyhemoglobin saturation was decreased (P<0.01) during hypoxia (63±2%) compared to normoxia (96±0%) and there was no significant effect of SNS inhibition on oxyhemoglobin saturation in either normoxia or hypoxia (P>0.25). Norepinephrine was elevated in hypoxia (137±13%; P=0.02), as determined by area under curve and expressed relative to normoxia. SNS inhibition prevented the hypoxia induced increase in norepinephrine (94±14%; P=0.43). The glucose infusion rate (adjusted for fat free mass and circulating insulin), required to maintain blood glucose at 90 mg/dl (5 mmol/L) during administration of insulin, was decreased during hypoxia (128±30 nmol/kg fat free mass/pmol/L/min; P=0.03) compared to normoxia (225±23) and remained unchanged during normoxia and SNS inhibition (219±19; P=0.86), and hypoxia and SNS inhibition (169±23; P=0.23). CONCLUSION: Inhibition of the SNS attenuates hypoxia induced insulin resistance.
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    The efficacy of red beetroot juice supplementation to improve cardiometabolic health in middle-aged/older adults with overweight or obesity
    (Colorado State University. Libraries, 2019) Litwin, Nicole S., author; Johnson, Sarah A., advisor; Pagliassotti, Michael J., committee member; Seals, Douglas R., committee member; Gentile, Christopher L., committee member; Rao, Sangeeta, committee member
    Cardiovascular disease (CVD) is the leading cause of morbidity and mortality in developed societies worldwide. Advancing age is the primary risk factor for CVD, with lifestyle factors such as diet and nutrition also playing a role. Aging results in adverse changes to the arteries including vascular endothelial dysfunction which is characterized by a decline in nitric oxide (NO)-mediated endothelium-dependent dilation, and increased stiffening of large elastic arteries. This age-associated vascular dysfunction is predominantly driven by increased oxidative stress and chronic inflammation and contributes to the development of CVD through the development of atherosclerotic plaque and hypertension. Previous research suggests that a single high-fat meal may result in transient impairments in postprandial vascular endothelial function, which is thought to be driven by a postprandial pro-inflammatory and oxidative stress response to hypertriglyceridemia and/or hyperglycemia, resulting in a decline in NO bioavailability. This phenomenon may be exaggerated in aging individuals with overweight or obesity, though previous research findings have been inconclusive. Nonetheless, repeated high-fat meal consumption may increase CVD risk through impairments in postprandial vascular endothelial function, thus warranting further investigation. While the mechanisms of postprandial vascular endothelial dysfunction continue to be fully elucidated, an emerging area of research suggests that the oral microbiota may determine steady-state NO levels. Recent scientific discoveries indicate that the oral microbiota reduces dietary inorganic nitrate to nitrite and NO (known as the enterosalivary nitrate-nitrite-NO pathway), thus providing a new therapeutic target for CVD risk management. Red beetroot juice is a rich source of inorganic nitrate as well as other bioactive compounds such as betalains, flavonoids, carotenoids, and ascorbic acid, and previous research suggests that it may improve several parameters of cardiometabolic health including vascular endothelial function. The goals of this dissertation research were to 1) examine the clinical efficacy of acute and chronic red beetroot juice supplementation on postprandial vascular endothelial function after a high-fat meal challenge in middle-aged/older men and postmenopausal women with overweight or obesity, and 2) investigate the underlying mechanisms that contribute to vascular and metabolic responses to the meal challenge and supplementation, including the nitrate-dependent and -independent effects of red beetroot juice. To investigate the aforementioned, we conducted a randomized, double-blind, placebo-controlled, 4-period, crossover, clinical trial. To investigate the nitrate-dependent and -independent effects of red beetroot juice, we used 1) a placebo concentrate devoid of inorganic nitrate or polyphenols, 2) red beetroot juice concentrate, 2) nitrate-depleted red beetroot juice concentrate, and 4) a placebo concentrate with an equivalent dose of inorganic nitrate to that of red beetroot juice. We first examined the impact of acute and chronic red beetroot juice supplementation on postprandial vascular endothelial function and other cardiometabolic responses to a high-fat meal challenge. We found that the high-fat meal led to postprandial alterations in several cardiometabolic parameters but did not impair vascular endothelial function. Significant acute and chronic increases in saliva and plasma NO metabolites were observed following consumption of red beetroot juice and the placebo plus inorganic nitrate, but these increases were not paralleled by significant changes in vascular endothelial function. Although the meal and treatments altered several other parameters of cardiometabolic health, there were no consistent effects of the treatments on those parameters. Next, we examined the relationship between oral nitrate-reducing bacteria and NO metabolites following acute and chronic red beetroot juice supplementation to gain insight on the impact of the oral microbiota on dietary nitrate metabolism and vascular responses to the high-fat meal. We found that red beetroot juice and inorganic nitrate salt supplementation may alter the oral microbiome to favorably affect NO metabolism and vascular endothelial function in this population. Taken together, these results suggest that although red beetroot juice did not modulate postprandial vascular endothelial function, it may be a promising dietary intervention for targeting the enterosalivary nitrate-nitrite-NO pathway to increase NO bioavailability in middle-aged/older adults with overweight or obesity. Further research is needed to evaluate the potential of red beetroot juice as an oral microbiota targeted therapy for improving NO bioavailability and overall cardiovascular health. Additionally, further research is needed to better understand the impact of high-fat meal consumption on cardiometabolic health.