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Preeclampsia (PE), a hypertensive disorder of pregnancy, is a major contributor to morbidity and mortality for pregnant women and their offspring. Severe maternal hypertension is a risk factor for cerebrovascular accident and a leading indication for preterm delivery, which can have serious and lifelong consequences. Dysfunction of small resistance arteries, which demonstrate increased vasoconstrictor sensitivity and impaired endothelium-dependent relaxation, is a key contributor to maternal systemic vascular resistance and hypertension in PE. Kynurenine (Kyn) is an endogenous vasorelaxant derived from tryptophan catabolism via the Kyn pathway. Activation of the Kyn pathway by infection and inflammation contributes to the development of systemic hypotension. Kyn has direct vasorelaxant effects on arteries from several species and reduces systemic blood pressure in rats. This thesis tested the hypothesis that Kyn can ameliorate dysfunction in arteries from women with PE. The effects of Kyn in maternal omental and myometrial resistance arteries were investigated using isometric myography. In arteries from both maternal vascular beds, Kyn reduced sensitivity to a thromboxane mimetic (U46619) and induced substantial and reproducible relaxation of preconstricted arteries. Vasorelaxant effects were independent of the endothelium and wholly mediated via large-conductance calcium-activated potassium (BKCa) channels, a previously unrecognised mechanism for Kyn activity. Effects of Kyn were replicated, albeit slightly reduced, in omental arteries from women with PE, suggesting the potential for therapeutic application of Kyn to cause vasorelaxation in PE. A pilot study in normotensive Sprague Dawley rats determined treatment with Kyn or its precursor tryptophan (100 mg/kg/day) in drinking water is feasible, tolerable, and without any immediate maternal or fetal harm. Increased [Kyn] in fetal plasma following maternal Kyn treatment indicates efficacy of treatment for increasing Kyn bioavailability. In mesenteric arteries isolated from pregnant rats, Kyn caused a large reduction in U46619 sensitivity and induced BKCa mediated relaxation of preconstricted arteries; thus, Sprague Dawley rats recapitulate the vascular effects of Kyn observed in human arteries and are a suitable animal model for future investigations. This thesis provides compelling evidence indicating the vasorelaxant effects of Kyn may offer potential to develop a new clinical intervention for the treatment of hypertension in PE. As the next step towards clinical translation, future work will investigate the effects of Kyn pathway manipulation on maternal hypertension in an in vivo model of PE.

Inhalation of nano-TiO2 is linked to adverse clinical outcomes that affect people worldwide, particularly those who work in manufacturing. For example, cardiovascular consequences and changes in microvascular responses are, in part, associated with exposure to engineered nanomaterials such as nano-TiO2; however, the mechanism(s) driving this process is unknown. Nano-TiO2 is an engineered nanomaterial (ENM) that is reported to, upon inhalation, impair endothelium-dependent vasodilation in several vascular beds via enhanced local oxidant generation and diminished nitric oxide bioavailability. This dissertation aims to address the health concerns generated by occupational exposure to nano-TiO2 and identify a link between the oxidant producing enzyme, xanthine oxidoreductase (XOR) and nano-TiO2-mediated vascular dysfunction. The overarching hypothesis is that XOR plays a critical role in nano-TiO2-mediated vascular dysfunction and that altering XOR product identity from oxidants to NO can provide a novel treatment strategy. This hypothesis was tested using two aims: 1) define XOR-mediated contributions to vascular dysfunction allied to inhalation of nano-TiO2 and establish the liver as the source of amplified circulating XOR and 2) Assess the impact of manipulating XOR product identity from oxidants to nitric oxide on nano-TiO2-mediated vascular dysfunction. The results demonstrate that inhalation of nano-TiO2 mediates systemic vascular dysfunction as assessed in the mesenteric system concomitant with an elevation of circulating XO which is likely released by hepatocytes. In addition, treatment with nitrite can serve to switch XOR-derived products from oxidants to NO and thus protect vasculature, the results in this dissertation have helped to gain an insight to the mechanisms driving nano-TiO2-mediated vascular dysfunction and potential clinical intervention via nitrite supplementation.

Abstract: Background: Sepsis is a dysregulated response to infection that results in life-threatening organ dysfunction. The elderly are disproportionately affected by sepsis and suffer an alarmingly high mortality rate. We hypothesized that increased mortality in the aged is due to a more highly dysfunctional vasculature secondary to loss of soluble guanylate cyclase signalling. As sepsis mortality is high, we aimed to provide insight into septic vascular dysfunction and discover a novel sepsis treatment that would be effective in the young and aged. Methods: In all studies included herein, hemodynamics and vascular function were assessed in-vivo and ex-vivo. In Study 1, young (3 to 4 months) and aged (22 to 24 months) mice were anesthetized and instrumented for hemodynamic assessments. Mice received an intraperitoneal injection of fecal slurry or an equivalent volume of vehicle. Systolic blood pressure, diastolic blood pressure, and heart rate were continuously recorded for a maximum of 600 minutes. A subset of mice received norepinephrine treatment. All mice received intravenous methacholine every 30 minutes to measure vascular reactivity. Following euthanization, tissues and arteries were collected for analysis of cGMP production and wire myography. Study 2 examined the young septic murine response to the soluble guanylate cyclase activator cinaciguat using the same protocol as above while Study 3 examined aged mice. The cytokine Oncostatin M is released in sepsis and plays a role in hemodynamic stability. As such, its receptor deficiency in both young males and females was investigated using a model of Oncostatin M receptor deficiency combined with the above septic protocol (Study 4). Results: In study 1, aged mice had impaired non-septic and septic vasorelaxation responses in-vivo compared to the young. Non-septic aged mice were more responsive to methacholine, whereas septic aged mice were less responsive. Ultimately, aged mice demonstrated decreased sepsis survival. Conventional sepsis treatment with norepinephrine resulted in a similar sepsis survival time in both age groups. cGMP production in aged lung and kidney did not increase. Notable aged myography findings included increased sensitivity to methacholine in mesenteric arteries and increased vasorelaxation 120 minutes post-sepsis induction. Due to the mortality associated with sepsis, we aimed to discover a treatment that would be effective in young (Study 2) and aged (Study 3) septic mice. Mice were treated with saline or the soluble guanylate cyclase activator cinaciguat to increase cGMP production and improve vascular dysfunction in sepsis. Cinaciguat improved the survival of young septic mice, coinciding with improved hemodynamics. Cinaciguat increased cGMP production in young lung and kidney. Myography revealed an increased sensitivity of cinaciguat-treated young septic mesenteric vessels to methacholine and sodium nitroprusside. Hemodynamic improvements were also noted in septic aged mice treated with cinaciguat. Baseline aged systolic and diastolic blood pressure response to methacholine was increased while septic blood pressure was decreased. Aged sepsis survival was also increased. However, no change in cGMP production was noted in aged lung or kidney. Myography demonstrated minimal vascular effect of cinaciguat. In Study 4, both sexes of Oncostatin M receptor deficient mice displayed a distinct hemodynamic profile demonstrating systolic and diastolic hypertension as well as tachycardia. Septic response to methacholine was decreased in male but not female knockout mice (compared to controls). Oncostatin M deficiency did not improve survival for either sex. Oncostatin M receptor deficiency did demonstrate sex-specific myography effects associated with a decreased level of relaxation secondary to methacholine and sodium nitroprusside in both non-septic and septic male vessels. These changes were not seen in female vessels. Summary and Conclusion: This is one of the first studies to examine both in-vivo and ex-vivo vascular function in a young and aged septic murine model. The findings demonstrate that aged septic mice experience vascular dysfunction, which may contribute to mortality. The novel use of cinaciguat resulted in augmentation of vascular function in young mice and improvement in sepsis survival in both young and aged mice. Finally, Oncostatin M receptor deficiency did not improve sepsis survival but led to the development of distinct non-septic and septic hemodynamic phenotypes. Together, these results underscore the need to further delineate the mechanisms that underlie vascular dysfunction and septic circulatory collapse. This new information sets the stage for further testing of cinaciguat to understand the molecular mechanism(s) in improving septic outcomes.

Omega-3 polyunsaturated fatty acids (n-3 PUFAs) may lower cardiovascular disease risk by reducing blood pressure (BP). We hypothesized that fat-1 transgenic mice, which convert n-6 PUFAs to n-3 PUFAs, would have improved vascular function in BP regulation and following secondhand smoke (SHS) exposure, compared to wild type (WT) mice. BP was recorded in mice fed an n-6 PUFA, or standard chow diet. On the n-6 PUFA diet fat-1 mice had a lower mean arterial pressure and a greater decrease in diastolic BP when treated with an ACE inhibitor, compared to WT mice. No differences were observed on the chow diet. Next, fat-1 mice on the n-6 PUFA or chow diet were exposed to SHS. SHS enhanced vasoconstriction on the n-6 PUFA diet, but not on chow, but did not affect vasorelaxation. These data suggest n-3 PUFAs have a protective role in basal BP regulation and SHS-induced vascular dysfunction.

Purpose: The purpose of these studies was to determine if arterial angulations (through sitting or standing), shear stress (through local heating), or a combination of these factors affected vascular function. Moreover, this studied estimated the magnitude of the effect that biological sex has on these vascular responses. Methods: Twenty-six healthy, young (18-40 years old) males (n=13) and females (n=13) completed two experimental trials (2-h sitting and 2-h standing). In a randomized fashion, one leg was passively heated in order to increase shear rate. Following a 48-h washout, participants returned to the laboratory to complete the other trial. Flow-mediated dilation (FMD) at the superficial femoral artery (SFA), and central and peripheral pulse-wave velocity (PWV) were measured using vascular ultrasound. Endothelin-1 (ET-1) was analyzed using an enzyme-linked immunoassay (ELISA). Results: There was a non-significant decrease in FMD (1.45%; p = .06) during sitting and the decline in FMD was not different between biological sexes (p > .05). Further, standing resulted in a significant increase in peripheral PWV (50 cm/s; p < .05) but there was no change during sitting (p > .05). Overall, passive heating (1.42%; p < .05) and standing (1.42%; p < .05) both independently improved FMD in comparison to sitting. However, neither intervention attenuated the increases in peripheral PWV that occurred during standing (p > .05). ET-1 did not increase over the course of either trial and was not different between sexes (p > .05). Antegrade shear rate increased (p < .001) and retrograde stress decreased (p < .001) when passively heated. Further, there was vasoconstriction during standing with SFA diameter decreasing compared to sitting (0.28 mm; p < .001). Conclusions: Both interventions, passive heating and standing, appear to be equally effective at improving FMD. Further, we demonstrate that there is no significant impact of biological sex on sitting induced vascular dysfunction and the interventions intended to prevent this dysfunction. However, standing increased peripheral PWV and passive heating did not attenuate this response.

Excess plasma homocysteine (Hcy; hyperhomocysteinemia, HHcy) remains an independent risk factor for cardiovascular disease (CVD) and treatments remain elusive. The source of Hcy, methionine, is an essential amino acid acquired by ingestion of animal foods. Normal methionine metabolism effectively removes Hcy via recycling back into methionine or excretion via the kidney. However, in aberrant methionine metabolism, Hcy accumulates and causes damage to the vascular system by increasing oxidative stress; the exact mechanism of how this occurs is unknown. Importantly, the B vitamins B6, B9 and B12 are essential to proper methionine/Hcy metabolism and are often found in low levels in patients presenting with HHcy; this has provided a potentially viable treatment strategy in the clinical setting. Disappointingly, clinical trials administering B vitamins to reduce HHcy have been unsuccessful in reducing CVD and treatments continue to be sought. The NADPH oxidase (Nox) family of enzymes are expressed in a broad range of cell types throughout the body and are the primary source of superoxide (Nox1, Nox2) and hydrogen peroxide (Nox4) within the vasculature under both physiological and pathological conditions. Nox1, 2 and 4 are of primary interest in vascular disease, as there is evidence that Hcy can interfere with the proper function of Nox1, 2 and 4 signalling, potentially leading to an over-expression of pro-oxidants. Nox1, 2 and 4 have been implicated in vascular disease (endothelial dysfunction), hypertension, vascular inflammation, stroke, diabetes, and atherosclerosis, and putative inhibitors of these enzymes are now available. Additionally, nitric oxide (NO) is also measured as a marker of proper vascular function; indeed, the current gold standard of assessing NO availability is by indirectly measuring vascular responses to acetylcholine. Accordingly, a loss of NO bioavailability is linked to the development of many of the same vascular pathologies caused by HHcy and also potentially increased Nox1, 2, and 4 activity. Thus, this thesis examined if current putative Nox inhibitors could prevent vascular dysfunction caused by homocysteine (as indirectly measured by acetylcholine-mediated vasorelaxation). Using New Zealand white rabbits, C57BL/6 mice and a Nox2-/- (C57BL/6 background) mouse models, we observed that pharmacological intervention with single Nox1, 2 and 4 inhibitors reduced the effect of acetylcholine on vasorelaxation. In 1% methionine-fed Nox2-/- mice, we observed an improvement in function. We also assessed combinations of Nox1, 2 and 4 inhibitors and found that, although function was not restored to control levels, it was improved compared with single Nox inhibition. Due to these results, we performed a gp91ds-tat dose response in rabbit aorta. We found that in our models of vascular dysfunction, lower doses of gp91ds-tat significantly improved acetylcholine-mediated vasorelaxation. These results showed for the first time that in both pharmacological and diet-induced HHcy, high dose putative Nox inhibitors might not be effective. In conclusion, the observations made in this thesis highlight the important role that Hcy plays in the redox balance in the context of vascular function. Future work in this area should focus on low dose Nox inhibition in Hcy induced disease in in vivo models, in order to better determine which drug can be used to HHcy induced vascular damage.