Objective—: Use of alternative tobacco products including electronic cigarettes is rapidly rising. The wide variety of flavored tobacco products available is of great appeal to smokers and youth. The flavorings added to tobacco products have been deemed safe for ingestion, but the cardiovascular health effects are unknown. The purpose of this study was to examine the effect of 9 flavors on vascular endothelial cell function. Approach and Results—: Freshly isolated endothelial cells from participants who use nonmenthol- or menthol-flavored tobacco cigarettes showed impaired A23187-stimulated nitric oxide production compared with endothelial cells from nonsmoking participants. Treatment of endothelial cells isolated from nonsmoking participants with either menthol (0.01 mmol/L) or eugenol (0.01 mmol/L) decreased A23187-stimulated nitric oxide production. To further evaluate the effects of flavoring compounds on endothelial cell phenotype, commercially available human aortic endothelial cells were incubated with vanillin, menthol, cinnamaldehyde, eugenol, dimethylpyrazine, diacetyl, isoamyl acetate, eucalyptol, and acetylpyrazine (0.1–100 mmol/L) for 90 minutes. Cell death, reactive oxygen species production, expression of the proinflammatory marker IL-6 (interleukin-6), and nitric oxide production were measured. Cell death and reactive oxygen species production were induced only at high concentrations unlikely to be achieved in vivo. Lower concentrations of selected flavors (vanillin, menthol, cinnamaldehyde, eugenol, and acetylpyridine) induced both inflammation and impaired A23187-stimulated nitric oxide production consistent with endothelial dysfunction. Conclusions—: Our data suggest that short-term exposure of endothelial cells to flavoring compounds used in tobacco products have adverse effects on endothelial cell phenotype that may have relevance to cardiovascular toxicity.

Prior studies demonstrate mitochondrial dysfunction with increased reactive oxygen species generation in peripheral blood mononuclear cells in diabetes mellitus. Oxidative stress-mediated damage to mitochondrial DNA promotes atherosclerosis in animal models. Thus, we evaluated the relation of mitochondrial DNA damage in peripheral blood mononuclear cells s with vascular function in patients with diabetes mellitus and with atherosclerotic cardiovascular disease. We assessed non-invasive vascular function and mitochondrial DNA damage in 275 patients (age 57 ± 9 years, 60 % women) with atherosclerotic cardiovascular disease alone (N = 55), diabetes mellitus alone (N = 74), combined atherosclerotic cardiovascular disease and diabetes mellitus (N = 48), and controls age >45 without diabetes mellitus or atherosclerotic cardiovascular disease (N = 98). Mitochondrial DNA damage measured by quantitative PCR in peripheral blood mononuclear cells was higher with clinical atherosclerosis alone (0.55 ± 0.65), diabetes mellitus alone (0.65 ± 1.0), and combined clinical atherosclerosis and diabetes mellitus (0.89 ± 1.32) as compared to control subjects (0.23 ± 0.64, P < 0.0001). In multivariable models adjusting for age, sex, and relevant cardiovascular risk factors, clinical atherosclerosis and diabetes mellitus remained associated with higher mitochondrial DNA damage levels (β = 0.14 ± 0.13, P = 0.04 and β = 0.21 ± 0.13, P = 0.002, respectively). Higher mitochondrial DNA damage was associated with higher baseline pulse amplitude, a measure of arterial pulsatility, but not with flow-mediated dilation or hyperemic response, measures of vasodilator function. We found greater mitochondrial DNA damage in patients with diabetes mellitus and clinical atherosclerosis. The association of mitochondrial DNA damage and baseline pulse amplitude may suggest a link between mitochondrial dysfunction and excessive small artery pulsatility with potentially adverse microvascular impact.

Background: Electronic cigarettes (e‐cigarettes) have been proposed as a potential harm reduction tool for combustible cigarette smokers. The majority of adult e‐cigarette users continue to smoke combustible cigarettes and are considered dual users. The vascular impact of e‐cigarettes remains incompletely defined. Methods and Results: We examined the association of e‐cigarette use with measures of vascular function and tonometry, preclinical measures of cardiovascular injury. As part of the CITU (Cardiovascular Injury due to Tobacco Use) study, we performed noninvasive vascular function testing in individuals without known cardiovascular disease or cardiovascular disease risk factors who were nonsmokers (n=94), users of combustible cigarettes (n=285), users of e‐cigarettes (n=36), or dual users (n=52). In unadjusted analyses, measures of arterial stiffness including carotid‐femoral pulse wave velocity, augmentation index, carotid‐radial pulse wave velocity, and central blood pressures differed across the use groups. In multivariable models adjusted for age, sex, race, and study site, combustible cigarette smokers had higher augmentation index compared with nonusers (129.8±1.5 versus 118.8±2.7, P =0.003). The augmentation index was similar between combustible cigarette smokers compared with sole e‐cigarette users (129.8±1.5 versus 126.2±5.9, P =1.0) and dual users (129.8±1.5 versus 134.9±4.0, P =1.0). Endothelial cells from combustible cigarette smokers and sole e‐cigarette users produced less nitric oxide in response to A23187 stimulation compared with nonsmokers, suggestive of impaired endothelial nitric oxide synthase signaling. Conclusions: Our findings suggest that e‐cigarette use is not associated with a more favorable vascular profile. Future longitudinal studies are needed to evaluate the long‐term risks of sustained e‐cigarette use.

Nuclear-mitochondrial DNA segments (NUMTs) are mitochondrial DNA (mtDNA) fragments that have been inserted into the nuclear genome. Some NUMTs are common within the human population but most NUMTs are rare and specific to individuals. NUMTs range in size from 24 base pairs to encompassing nearly the entire mtDNA and are found throughout the nuclear genome. Emerging evidence suggests that the formation of NUMTs is an ongoing process in humans. NUMTs contaminate sequencing results of the mtDNA by introducing false positive variants, particularly heteroplasmic variants present at a low variant allele frequency (VAF). In our review, we discuss the prevalence of NUMTs in the human population, the potential mechanisms of de novo NUMT insertion via DNA repair mechanisms, and provide an overview of the existing approaches for minimizing NUMT contamination. Apart from filtering known NUMTs, both wet lab-based and computational methods can be used to minimize the contamination of NUMTs in analyses of human mtDNA. Current approaches include: (1) isolating mitochondria to enrich for mtDNA; (2) applying basic local alignment to identify NUMTs for subsequent filtering; (3) bioinformatic pipelines for NUMT detection; (4) k-mer-based NUMT detection; and (5) filtering candidate false positive variants by mtDNA copy number, VAF, or sequence quality score. Multiple approaches must be applied in order to effectively identify NUMTs in samples. Although next-generation sequencing is revolutionizing our understanding of heteroplasmic mtDNA, it also raises new challenges with the high prevalence and individual-specific NUMTs that need to be handled with care in studies of mitochondrial genetics.

Mitochondrial abnormalities have long been described in the setting of cardiomyopathies and heart failure (HF), yet the mechanisms of mitochondrial dysfunction in cardiac pathophysiology remain poorly understood. Many studies have described HF as an energy-deprived state characterized by a decline in adenosine triphosphate production, largely driven by impaired oxidative phosphorylation. However, impairments in oxidative phosphorylation extend beyond a simple decline in adenosine triphosphate production and, in fact, reflect pervasive metabolic aberrations that cannot be fully appreciated from the isolated, often siloed, interrogation of individual aspects of mitochondrial function. With the application of broader and deeper examinations into mitochondrial and metabolic systems, recent data suggest that HF with preserved ejection fraction is likely metabolically disparate from HF with reduced ejection fraction. In our review, we introduce the concept of the mitochondrial ecosystem, comprising intricate systems of metabolic pathways and dynamic changes in mitochondrial networks and subcellular locations. The mitochondrial ecosystem exists in a delicate balance, and perturbations in one component often have a ripple effect, influencing both upstream and downstream cellular pathways with effects enhanced by mitochondrial genetic variation. Expanding and deepening our vantage of the mitochondrial ecosystem in HF is critical to identifying consistent metabolic perturbations to develop therapeutics aimed at preventing and improving outcomes in HF.