Browsing by Subject "Cardiovascular"

Hutchison-Gilford Progeria Syndrome (HGPS) is a rare, accelerated aging disorder caused by nuclear accumulation of progerin, an altered form of the Lamin A gene. The primary causes of death are stroke and cardiovascular disease at an average age of 14 years. It is known that loss or malfunction of smooth muscle cells (SMCs) in the vasculature leads to cardiovascular defects, however, the exact mechanisms are still not understood. The contribution of other vascular cell types, such as endothelial cells, is still not known due to the current limitations of studying such a rare disorder. Due to limitations of 2D cell culture, mouse models, and the limited HGPS patient pool, there is a need to develop improved models of HGPS to better understand the development of the disease and discover novel therapeutics.

To address these limitations, we produced a functional, three-dimensional tissue model of HGPS that replicates an arteriole-scale tissue engineered blood vessel (TEBV) using induced pluripotent stem cell (iPSC)-derived cell sources from HGPS patients. To isolate the specific effects of HGPS SMCs, we initially used human cord blood-derived endothelial progenitor cells (hCB-EPCs) from a separate, healthy donor and iPSC-derived SMCs (iSMCs). TEBVs fabricated from HGPS patient iSMCs and hCB-EPCs (HGPS iSMC TEBVs) showed disease attributes such as reduced vasoactivity, increased medial wall thickness, increased calcification, excessive extracellular matrix protein deposition, and cell apoptosis relative to TEBVs fabricated from primary mesenchymal stem cells (MSCs) and hCB-EPCs or normal patient iSMCs with hCB-EPCs. Treatment of HGPS iSMC TEBVs for one week with the rapamycin analog Everolimus (RAD001), increased HGPS iSMC TEBV vasoactivity and iSMC differentiation in TEBVs.

To improve the sensitivity of our HGPS TEBV model and study the effects of endothelial cells on the HGPS cardiovascular phenotype, we adopted a modified differentiation protocol to produce iPSC-derived vascular smooth muscle cells (viSMCs) and endothelial cells (viECs) from normal and Progeria patient iPSC lines to create iPSC-derived vascular TEBVs (viTEBVs). Normal viSMCs and viECs showed structural and functional characteristics of vascular SMCs and ECs in 2D culture, while HGPS viSMCs and viECs showed various disease characteristics and reduced function compared to healthy controls. Normal viTEBVs had comparable structure and vasoactivity to MSC TEBVs, while HGPS viTEBVs showed reduced vasoactivity, increased vessel wall thickness, calcification, apoptosis and excess ECM deposition. In addition, HGPS viTEBVs showed markers of cardiovascular disease associated with the endothelium such as decreased response to acetylcholine, increased inflammation, and altered expression of flow-associated genes.

The treatment of viTEBVs with multiple Progeria therapeutics was evaluated to determine the potential of the HGPS viTEBV model to serve as a platform for drug efficacy and toxicity testing as well as to further elucidate the mechanisms behind each drugs mode of action. Treatment of viTEBVs with therapeutic levels of the farnesyl-transferase inhibitor (FTI), Lonafarnib, or Everolimus improved different aspects of HGPS viTEBV structure and function. Treatment with Everolimus alone increased response to phenylephrine, improved SMC differentiation and cleared progerin through autophagy. Lonafarnib improved acetylcholine response, decreased ECM deposition, decreased calcification and improved nitric oxide production. Most significantly, combined therapeutic treatment with both drugs showed an additive effect by improving overall vasoactivity, increasing cell density, increasing viSMC and viEC differentiation, and decreasing calcification and apoptosis in treated HGPS viTEBVs. On the other hand, toxic doses of both drugs combined resulted in significantly diminished HGPS viTEBV function through increased cell death. In summary, this work shows the ability of a tissue engineered vascular model to serve as an in vitro personalized medicine platform to study HGPS and potentially other rare diseases of the vasculature using iPSC-derived cell sources. It has also further identified a potential role of the endothelium in HGPS. Finally, this HGPS viTEBV model has proven effective as a drug testing platform to determine therapeutic and toxic doses of proposed therapeutics based on their specific therapeutic effects on HGPS viTEBV structure and function.

There is a scarcity of knowledge regarding the cardiovascular effects of low dose ionizing radiation (IR) such as the one experienced during medical tests, radiation therapy or space travel. This is becoming more of a pressing problem given the enormous increase in radiation exposure by the average American today, and the renewed interest in deep space travel. Multiple epidemiologic studies suggest a higher rate of delayed cardiovascular related morbidity and mortality after low dose acute radiation exposure. These studies are significantly limited by a number of confounders such as cancer comorbidity, poor follow up, and largely estimated radiation doses that might not be accurate. These limitations are also seen in studies on the effect of space radiation on long term cardiovascular mortality and accelerated atherosclerosis. Animal studies have been used to simulate the effect of terrestrial and space radiation scenarios on cardiac function. These studies have led to conflicting conclusions, and had important challenges related to methods of assessment of cardiac injury. Furthermore, available studies to date had limited follow up times, and no study has evaluated the effects of more complex radiation scenarios that are likely to be experienced in space such as Galactic Cosmic Rays (GCRs). Our overall hypothesis is that IR is associated with early damage to healthy cardiomyocytes and vascular cells that eventually leads to long term cardiovascular dysfunction.

In the first part of this work, we hypothesize that IR is associated with a delayed cardiovascular derangement phenotype late after initial exposure. To test this hypothesis, we use a mouse animal model to study the effect of different radiation scenarios on cardiovascular function. Animals were exposed to one of the following (a) Gamma Rays (50-200 cGy), (b) 56Fe (15-50 cGy), (c) 16O (15-50cGy) heavy ions, and (d) 150cGy Galactic Cosmic Rays all using the particle accelerator at Brookhaven National Labs (BNL). They were then followed up for 9-12 months, and underwent cardiac MRI, pressure volume loop assessments, transthoracic echocardiograms and other histological evaluations. These studies revealed that GCR exposed animals had a clinically meaningful decline in their cardiac function, with a significant change in their arterial elastance. These findings were further confirmed on histology with their aortas demonstrating elastic fiber destruction and disorganization.

These animal studies however could not fully differentiate between a primary cardiac injury or a secondary cardiac response to a primary vascular injury to the aorta. Our hypothesis for the second part of this work, was that IR is associated with a unique and differentiated injury to cardiomyocytes that contributes to the previously seen cardiovascular phenotype. We therefore conducted additional experiments on isolated rat ventricular cardiomyocyte in collaboration with the Bursac lab. These studies used both 2D cultured cells, as well as a novel cardiac patch system both exposed to Gamma rays and X-rays (0.1-2 Gy). These cells underwent proteomics analysis, as well as a number of different biological and functional assays. While the acute functional effect of these radiation doses on cardiomyocytes was small, these irradiated cells produced a significant amount of reactive oxygen species and exhibited a large effect of radiation on mitochondrial related proteins, including elements of oxidative phosphorylation. We also noted a number of different pathways involved at different doses of radiation. This was an important finding, given that despite no changes in early cell death, the effect of these important proteomics changes on long term cardiac function maybe important.

Finally, for the last part of this dissertation, our hypothesis was that IR uniquely affects endothelial cells (ECs) and smooth muscle cells (SMCs) by inducing early senescence that is primarily due to over production of mitochondrial specific reactive oxygen species. To test this hypothesis, we use primary coronary artery endothelial cells, primary human aortic endothelial cells and primary coronary artery smooth muscle cells. These relevant cell types were then examined for their response to a single dose of radiation exposure. Given the previous findings of important mitochondria involvement even at low radiation doses, we used a novel mitochondrial specific ROS scavenger, that blocks the release of mROS, mito-TEMPO. Cells treated with mito-TEMPO had a significant decrease in observed cellular senescence an important hallmark indicator of cellular dysfunction. This strategy might have a potential therapeutic role in the prolonged cardiovascular effects of radiation exposure.

In summary, data generated in this dissertation supports the overall hypothesis that IR is associated with long term cardiovascular dysfunction that can be explained by early injury to cardiac, endothelial and smooth muscle cells. Galactic Cosmic Rays appear to significantly effect long term cardiovascular function, which has important implications on deep space travel. This effect is likely multifactorial, involving a number of organs, including the aorta. Cardiomyocytes, despite being resilient to death from radiation as compared to other cells types, appear to undergo a number of proteomics alterations after low dose radiation exposure, with significant involvement of the mitochondrial machinery. Finally, human vascular ECs and SMCs are highly sensitive to radiation exposure, and strategies that target mitochondrial specific ROS production might play an important role in mitigating the long-term vascular effects after radiation exposure.

Abstract

BACKGROUND: Cardiovascular disease (CVD) is the greatest killer of mankind. In Nepal, a small, landlocked country in South Asia and one of the poorest countries in the world, the burden of cardiovascular disease is high; national CVD monitoring has substantial gaps; and CVD risk factors are poorly controlled. Primary healthcare (PHC) is often at the front lines of prevention and control of CVD. Thus, strengthening local and national primary healthcare systems is an essential step toward prevention and management of CVD in low- and middle-income countries (LMICs). To inform further research and policy development, we conducted an exploratory, mixed-methods study to understand patient-side utilization of CVD services in rural and urban areas in Nepal.

METHODS: We surveyed 114 CVD patients from 10 PHC facilities across two regions of Nepal. Survey contents included sociodemographic information, disease history, and data on accessibility, affordability, availability, and utilization of PHC services. We further completed 20 in-depth interviews within our sample to understand patient-side perceptions of CVD-related PHC care.

RESULTS: In the final cohort, 23% had experienced a stroke, 26% had some form of existing heart disease, 76% presented with hypertension, and 67% had diabetes. For all conditions, hospitals were a more common place of diagnosis than PHC facilities. The mean visitation frequency to PHC facilities in the past year was 10.7 times, with healthier patients having higher visitation rates than those with poorer health. 69% of patients reported difficulty obtaining CVD medicine from their local PHC facility. Qualitative data revealed that PHC facilities lacked resources for diagnosing and managing CVD conditions. Additionally, behavior of physicians and affordability were both considered important components of PHC satisfaction. Patient recommendations included increased diagnostic equipment in PHC facilities, free essential medicines, and community awareness initiatives for cardiovascular disease.

CONCLUSION: There are clear shortcomings between national aims and objectives – such as free essential medicines and universal PHC services – and the reality faced by CVD patients in resource-limited settings in Nepal. Despite PHC facilities generally being close to patients, participants reported inadequate resources when seeking care for CVD and expressed a strong desire that such services could be provided locally. Based on the challenges articulated by CVD patients in our study, mhealth may be a relevant direction of future research for connecting hospital-based specialists to CVD patients in rural areas, improving follow-up, and decreasing expensive visits to far away tertiary care centers. Overall, we recommend increased national CVD monitoring, prioritization of NCDs and CVDs in national policymaking and strategizing, and continued provision of PHC facilities close to patients in their communities.

Keywords: primary care, cardiovascular, utilization, Nepal, public healthcare facilities

DEDICATION

It has been commonly accepted until recently that particulate matter (PM) is responsible for the cardiovascular toxicity of air pollution mixtures, while ozone (O3) mainly adversely affects respiratory health. However, there is increasing evidence that O3, independent of PM, is also associated with cardiovascular hospitalizations and mortality, even at levels below current regulatory standards. The mechanisms underlying these epidemiological associations between O3 and cardiovascular disease remain poorly understood. The goal of this dissertation research is to use human biomarker outcomes in real-world exposure scenarios to elucidate plausible mechanisms by which O3 affects cardiovascular health.

The findings of this dissertation research are primarily based on a single longitudinal cohort study designed to assess biomarker associations with time-activity-adjusted air pollutant exposures and with indoor air purification interventions, specifically different combinations of a high efficiency particulate air (HEPA) filter and a particle-removing and O3-producing electrostatic precipitator (ESP). Eighty-nine healthy participants living on a work campus in Changsha City, China were recruited for this study conducted from December 1st, 2014 - January 31st, 2015. The unique quasi-experimental setting of participants all living and working together on a work campus allowed for better characterization of air pollutant exposure effects due to minimal variation in potential confounders. At baseline, workers had a combination of an ESP and a HEPA in the central air handling units (AHUs) of their work and living spaces. During a five-week intervention period from December 6th, 2014 to January 13th, 2015, subjects were split into two groups, both of which had the ESPs turned off and one of which also had the HEPAs removed, and after this intervention original conditions were restored. Biomarkers indicative of inflammation and oxidative stress, arterial stiffness, myocardial function, blood pressure, thrombotic factors, and spirometry were measured at four sessions, one at baseline, two at two and four weeks into the intervention period, and one two weeks after restoring baseline conditions post-intervention. Indoor and outdoor O3 and PM of less than or equal to 2.5 µm in diameter (PM2.5), along with ambient co-pollutants NO2 and SO2, were monitored throughout the study period and combined with time-activity information and filtration conditions of each residence and office. These data were used to estimate 24-hour and 2-week combined indoor and outdoor average exposure concentrations, in addition to exposures in filtered and unfiltered environments.

To test the hypothesis that air pollutant exposures observed during this study would be associated with biomarker outcomes, associations between each exposure measure and biomarker were analyzed with single- and two-pollutant linear mixed models. The 24-hour mean O3 exposure concentrations during the study ranged from 1.4 to 19.4 ppb, corresponding with daily 8-hour maximum outdoor concentrations ranging from 3.6 to 60.5 ppb, with all but six days during the study period falling below the WHO 8-hour mean O3 guideline of 50 ppb6. Within this range, in models controlling for a second co-pollutant and other potential confounders, a 10 ppb increase in 24-hour O3 was associated with mean percent increases (95% CIs) of 36.3% (29.9%, 43.0%) in the platelet activation marker soluble P-selectin (sCD62P), 2.8% (0.6%, 5.1%) in diastolic blood pressure (DBP), and 18.1% (4.5%, 33.5%) and 31.0% (0.2%, 71.1%) in the pulmonary inflammation markers fractional exhaled nitric oxide (FeNO) and exhaled breath condensate nitrite and nitrate (EBCNN), respectively, as well as a -9.5% (-17.7%, -1.4%) decrease in arterial stiffness marker augmentation index (AI) and a -15.5% (-23.8%, -6.2%) decrease in the systemic oxidative stress marker urinary malondialdehyde (UMDA). A 10 ppb increase in 2-week O3 was associated with increases of 61.1% (37.8%, 88.2%) in sCD62P and 126.2% (12.1%, 356.2%) in EBCNN. In contrast, PM2.5, NO2, and SO2 exposure measures were variably and weakly associated with markers indicating increased arterial stiffness and endothelial cell dysfunction. Only the O3 associations with sCD62P are robust in two-pollutant models and multiple testing p-value correction. These results suggest that O3 exposure enhances cardiovascular disease risk through platelet activation and blood pressure increases at levels lower than those capable of affecting lung function.

To examine if the removal of HEPA filtration and ESP in the indoor air purification systems were associated with changes in biomarker outcomes, Bayesian hierarchical generalized ridge regression (GRR) models accounting for subject-specific intercept random effects were used to assess associations between categorical intervention variables while controlling for cumulative pollutant exposures in unfiltered microenvironments, namely outdoors and places other than the offices and dorms. The GRR models allowed for more stable maximized likelihood estimates when model predictors were highly correlated. When factoring in time-activity patterns, subjects without HEPA filtration had total 24-hour PM2.5 exposures on average 37.9 µg/m3 (88.3%) higher than subjects with HEPA filtration, and the removal of the ESPs resulted in a small average reduction of 2.2 ppb (a 32.8% decrease as compared to the overall mean 24-hour O3 exposure) in each subject’s total 24-hour O3 exposure. Despite this large change in PM2.5 exposure, no biomarkers were associated with HEPA removal in any models, but ESP removal was associated with decreases of -17.1% (-23.1%, -11.3%) in sCD62P, -3.6% (-5.5%, -1.4%) in systolic blood pressure (SBP), and -3.3% (-5.9%, -0.7%) in DBP. In addition, though subjects spent an average of 64.5% of their time in filtered locations during each two-week period between sampling visits, cumulative air pollutant exposure in unfiltered environments was associated with increased sCD62P for O3, increased FeNO for PM2.5, and increased EBC MDA and decreased subendocardial viability ratio (SEVR, a marker of myocardial oxygen supply and demand) for SO2. This study suggests that ESP use may result in O3-associated adverse health effects, biomarkers traditionally associated with PM exposure may not show a response weeks into an intervention, and time spent in environments filtered by particulate air filters, though perhaps not ESPs, should be maximized to avoid the health effects of cumulative high exposures in unfiltered locations.

These O3 associations with platelet activation and blood pressure are consistent with related results in some studies but not others, and so I hypothesized that age and sex may influence each individual’s response to O3 and account for some of this variability. I tested this hypothesis by assessing pollutant exposure by age or by sex interaction term estimates in association with biomarker outcomes in the GRR models. This statistical analysis was applied not only to the main study conducted in Changsha, but also to a subsequent study conducted in Shanghai with similar exposure and biomarker measurements that had younger study participants with more balanced sex ratio. In addition, the exposure and biomarker data between these two studies were pooled for an additional analysis checking the results from the individual study findings. In the main Changsha Study, significant age by pollutant exposure interaction terms were observed for the associations between 24-hour and 2-week O3 and sCD62P, 2-week O3 and SBP, and 2-week SO2 and PWV. In addition, the association between PWV and 2-week SO2 was significantly higher in men, and the association between PWV and 24h O3 was significantly higher in women, though the latter interaction term became nonsignificant in a sensitivity analysis assessing the independent interaction effect. No interaction terms were significant in the Shanghai Study analysis. In the pooled analysis, the 24-hour O3 exposure by age interaction term was significant for both sCD62P and SBP. Also, the pooled analysis showed that women had a significantly higher association between 24-hour O3 exposure and PWV as had been seen in the Changsha Study, but as in that case this association was not robust to the sensitivity analyses. These results indicate that older individuals are more susceptible to O3-associated effects on platelet activation and blood pressure, which is supported by literature examining age-associated changes in platelets and vascular tone.

Taken together these results and the findings in previous research examining cardiovascular pathophysiologic mechanisms, a coherent, plausible mechanistic pathway emerges. In this pathway, O3-associated reaction products in the airway lead to the propagation of signals that activate platelets, which in turn enhance blood pressure and induce a procoagulant state. The findings of this dissertation contribute to the mechanistic understanding of how O3 exposure affects cardiovascular health outcomes.