Kelly J. Shields

Cardiovascular Fat, Menopause, and Sex Hormones in Women: The SWAN Cardiovascular Fat Ancillary Study

CONTEXT Cardiovascular risk increases in women after menopause. Mounting evidence demonstrates a role of cardiovascular fat (CF) in the pathogenesis of coronary heart disease, but no research has examined CF in relation to sex hormones or menopausal status in women. OBJECTIVE The objective was to determine the relationship between CF depots, menopausal status, and endogenous sex hormones. DESIGN Cross-sectional and longitudinal study designs were used. SETTING The setting included the Study of Womens Health Across the Nation (SWAN) Heart and Cardiovascular Fat Ancillary Study. PARTICIPANTS A total of 456 women (mean age, 50.75 y); 62% premenopausal/early perimenopausal, and 38% late peri-/postmenopausal. INTERVENTION Menopausal status, endogenous sex hormones measured simultaneously with CF volumes, and circulating estradiol available 4.80 years (median) before CF measures. MAIN OUTCOME MEASURES Volumes of CF (epicardial adipose tissue [EAT], paracardial adipose tissue [PAT], total heart adipose tissue [TAT = EAT + PAT], and aortic perivascular adipose tissue [PVAT]). RESULTS In final models, late peri-/postmenopausal women had 9.88% more EAT, 20.72% more PAT, and 11.69% more TAT volumes than pre-/early perimenopausal women (P < .05). PVAT was not associated with menopausal status. In final models, lower estradiol concentrations were associated with greater volumes of PAT and TAT (P < .05). Women with the greatest reduction in estradiol since baseline had greater volumes of PAT compared to women with the least reduction (P = .02). CONCLUSIONS Late peri-/postmenopausal women have greater volumes of heart fat compared with pre-/early perimenopausal women independent of age, obesity, and other covariates. Endogenous sex hormones are associated with CF. Perhaps CF plays a role in the higher risk of coronary heart disease reported in women after menopause.

Postmenopausal Women With Greater Paracardial Fat Have More Coronary Artery Calcification Than Premenopausal Women: The Study of Women's Health Across the Nation (SWAN) Cardiovascular Fat Ancillary Study

Background Volumes of paracardial adipose tissue (PAT) and epicardial adipose tissue (EAT) are greater after menopause. Interestingly, PAT but not EAT is associated with estradiol decline, suggesting a potential role of menopause in PAT accumulation. We assessed whether volumes of heart fat depot (EAT and PAT) were associated with coronary artery calcification (CAC) in women at midlife and whether these associations were modified by menopausal status and estradiol levels. Methods and Results EAT and PAT volumes and CAC were measured using electron beam computed tomography scans. CAC was evaluated as (1) the presence of CAC (CAC Agatston score ≥10) and (2) the extent of any CAC (log CAC Agatston score >0). The study included 478 women aged 50.9 years (58% pre‐ or early perimenopausal, 10% late perimenopausal, and 32% postmenopausal). EAT was significantly associated with CAC measures, and these associations were not modified by menopausal status or estradiol. In contrast, associations between PAT and CAC measures were modified by menopausal status (interaction‐P≤0.01). Independent of study covariates including other adiposity measures, each 1‐SD unit increase in log PAT was associated with 102% higher risk of CAC presence (P=0.04) and an 80% increase in CAC extent (P=0.008) in postmenopausal women compared with pre‐ or early perimenopausal women. Additional adjustment for estradiol and hormone therapy attenuated these differences. Moreover, the association between PAT and CAC extent was stronger in women with lower estradiol levels (interaction P=0.004). Conclusions The findings suggest that PAT is a potential menopause‐specific coronary artery disease risk marker, supporting the need to monitor and target this fat depot for intervention in women at midlife.

Cardiovascular disease biomarkers across autoimmune diseases

Cardiovascular disease is increasingly recognized as a major cause of premature mortality among those with autoimmune disorders. There is an urgent need to identify those patients with autoimmune disease who are at risk for CVD so as to optimize therapeutic intervention and ultimately prevention. Accurate identification, monitoring and stratification of such patients will depend upon a panel of biomarkers of cardiovascular disease. This review will discuss some of the most recent biomarkers of cardiovascular diseases in autoimmune disease, including lipid oxidation, imaging biomarkers to characterize coronary calcium, plaque, and intima media thickness, biomarkers of inflammation and activated complement, genetic markers, endothelial biomarkers, and antiphospholipid antibodies. Clinical implementation of these biomarkers will not only enhance patient care but also likely accelerate the pharmaceutical pipeline for targeted intervention to reduce or eliminate cardiovascular disease in the setting of autoimmunity.

Harvest of pulmonary artery endothelial cells from patients undergoing right heart catheterization.

Endothelial dysfunction is a hallmark of pulmonaryarterial hypertension (PAH). Although the exact roleendothelial dysfunction plays in the initiation or progressionof PAH is not yet clear, impaired apoptosis and aberrantsignaling in endothelial cells is observed in most forms ofPAH. A variety of approaches have been utilized to studyendothelial cell biology in the context of PAH. Most studieshave focused on in situ visualization or purification ofpulmonary artery endothelial cells (PAECs) from explantedlungs of PAH patients, using untransplanted human donorlungs as controls. Although very useful for comparingendothelial cell biology in PAH versus normal pulmonaryvasculature, this approach is unable to provide informationon dynamic changes in PAEC biology over time within aliving patient.The definitive diagnosis of PAH requires measurement ofmean pulmonary artery (PA) pressure via right heartcatheterization (RHC), and thus RHC serves as a “goldstandard” for diagnosis and as a gateway for the manage-ment of PAH patients. We hypothesized that PAECs mightbe dislodged from the PA vessel wall by the balloon tip ofthe flow-directed pulmonary artery (Swan–Ganz) catheterduring RHC and would remain adherent to the balloon whenit is collapsed and the catheter extracted. Herein we reportour successful efforts to isolate, characterize and expandPAECs recovered from Swan–Ganz catheter balloons afterroutine RHC for the diagnosis and management of PAH.After obtaining institutional review board approval, 24patients undergoing RHC consented to participate in ourstudy. After the RHC procedure, the last 2 inches of theSwan–Ganz catheter, including the inflatable balloon, wascut and placed in a tube containing endothelial cell media(Cell Applications, Inc., San Diego, CA) and placed on iceuntil the samples were further processed. In the laboratory,catheter tips were washed vigorously 5 with phosphate-buffered saline (PBS)/trypsin and centrifuged at 1,500 rpmfor 10 minutes. The resulting pellets were re-suspended inammonium–chloride–potassium (ACK)/PBS solution tolyse the erythrocytes. These suspensions were centrifugedat 2,500 rpm for 10 minutes to pellet the cells. An aliquot ofthis final cell harvest was analyzed by fluorescence-activated cell sorting (FACS) with antibodies for endothelialantigens and the balance transferred to primary culture.In these studies, we relied on the classic marker profile forendothelial cells as reported in 2001 by Mancuso et al,

Three-dimensional micro computed tomography analysis of the lung vasculature and differential adipose proteomics in the Sugen/hypoxia rat model of pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a rare disease characterized by significant vascular remodeling. The obesity epidemic has produced great interest in the relationship between small visceral adipose tissue depots producing localized inflammatory conditions, which may link metabolism, innate immunity, and vascular remodeling. This study used novel micro computed tomography (microCT) three-dimensional modeling to investigate the degree of remodeling of the lung vasculature and differential proteomics to determine small visceral adipose dysfunction in rats with severe PAH. Sprague-Dawley rats were subjected to a subcutaneous injection of vascular endothelial growth factor receptor blocker (Sugen 5416) with subsequent hypoxia exposure for 3 weeks (SU/hyp). At 12 weeks after hypoxia, microCT analysis showed a decrease in the ratio of vascular to total tissue volume within the SU/hyp group (mean ± standard deviation: 0.27 ± 0.066; P = 0.02) with increased vascular separation (0.37 ± 0.062 mm; P = 0.02) when compared with the control (0.34 ± 0.084 and 0.30 ± 0.072 mm). Differential proteomics detected an up-regulation of complement protein 3 (C3; SU/hyp: control ratio = 2.86) and the adipose tissue–specific fatty acid binding protein-4 (FABP4, 2.66) in the heart adipose of the SU/hyp. Significant remodeling of the lung vasculature validates the efficacy of the SU/hyp rat for modeling human PAH. The upregulation of C3 and FABP4 within the heart adipose implicates small visceral adipose dysfunction. C3 has been associated with vascular stiffness, and FABP4 suppresses peroxisome proliferator–activated receptor, which is a major regulator of adipose function and known to be downregulated in PAH. These findings reveal that small visceral adipose tissue within the SU/hyp model provides mechanistic links for vascular remodeling and adipose dysfunction in the pathophysiology of PAH.

MicroCT analysis of vascular morphometry: a comparison of right lung lobes in the SUGEN/hypoxic rat model of pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a rare disease characterized by significant vascular remodeling within the lung. Clinical computed tomography (CT) scans are routinely used to aid in PAH diagnosis. Animal models, including the Sugen-hypoxic rat model (SU/hyp), of PAH closely mimic human PAH development. We have previously used micro-computed tomography (microCT) to find extensive right lung vascular remodeling in the SU/hyp. We hypothesized that the individual right lung lobes may not contribute equally to overall lung vascular remodeling. Sprague-Dawley rats were subjected to a subcutaneous injection of vascular endothelial growth factor receptor blocker (Sugen 5416) and subsequently exposed to chronic hypoxic conditions (10% O2) for three weeks. Following perfusion of the lung vasculature with an opaque resin (Microfil), the right lung lobes were microCT-imaged with a 10-µm voxel resolution and 3D morphometry analysis was performed separately on each lobe. As expected, we found a significantly lower ratio of vascular volume to total lobe volume in the SU/hyp compared with the control, but only in the distal lobes (inferior: 0.23 [0.21–0.30] versus 0.35 [0.27–0.43], P = 0.02; accessory: 0.27 [0.25–0.33] versus 0.37 [0.29–0.43], P = 0.06). Overall, we observed significantly fewer continuous blood vessels and reduced vascular density while having greater vascular lumen diameters in the distal lobes of both groups (P < 0.05). In addition, the vascular separation within the SU/hyp lobes and the vascular surface area to volume ratio were significantly greater in the SU/hyp lobes compared with controls (P < 0.03). Results for the examined parameters support the overall extensive vascular remodeling in the SU/hyp model and suggest this may be lobe-dependent.

In situ expression of Bcl-2 in pulmonary artery endothelial cells associates with pulmonary arterial hypertension relative to heart failure with preserved ejection fraction

We have previously reported that pulmonary artery endothelial cells (PAECs) can be harvested from the tips of discarded Swan-Ganz catheters after right heart catheterization (RHC). In this study, we tested the hypothesis that the existence of an antiapoptotic phenotype in PAECs obtained during RHC is a distinctive feature of pulmonary arterial hypertension (PAH; World Health Organization group 1) and might be used to differentiate PAH from other etiologies of pulmonary hypertension. Specifically, we developed a flow cytometry-based measure of Bcl-2 activity, referred to as the normalized endothelial Bcl-2 index (NEBI). We report that higher NEBI values are associated with PAH to the exclusion of heart failure with preserved ejection fraction (HFpEF) and that this simple diagnostic measurement is capable of differentiating PAH from HFpEF without presenting addition risk to the patient. If validated in a larger, multicenter study, the NEBI has the potential to assist physicians in the selection of appropriate therapeutic interventions in the common and dangerous scenario wherein patients present a clinical and hemodynamic phenotype that makes it difficult to confidently differentiate between PAH and HFpEF.

Associations of cardiovascular fat radiodensity and vascular calcification in midlife women: The SWAN cardiovascular fat ancillary study

BACKGROUND AND AIMS Fat radiodensity, measured via CT Hounsfield units (HU), is a potential marker of fat quality. We sought to determine the cross-sectional associations of total heart fat (TAT) and aortic perivascular fat (PVAT) radiodensity with cardiovascular risk factors, coronary artery calcification (CAC), and aortic calcification (AC) in midlife women. METHODS Fat radiodensity, CAC, and AC were quantified using CT scans. A total of 528 women (mean age: 50.9 ± 2.9 years; 37% Black) were included in analyses. RESULTS Women in the lowest TAT radiodensity tertile were more likely to have adverse cardiovascular risk factors. Independent of cardiovascular risk factors, women in the middle and high TAT radiodensity tertiles were less likely to have CAC (OR (95% CI): 0.32 (0.18, 0.59); 0.43 (0.24, 0.78), respectively) compared with women in the lowest TAT radiodensity tertile. Although adjusting for BMI attenuated the overall association, women in the middle TAT radiodensity tertile remained at significantly lower odds of CAC when compared to the low radiodensity tertile, 0.47 (0.24, 0.93), p=0.03. No significant associations were found for PVAT radiodensity and calcification measures in multivariable analysis. CONCLUSIONS Lower TAT radiodensity was associated with a less favorable cardiometabolic profile. Women with mid-range TAT radiodensity values had a lower odds of CAC presence, independent of CVD risk factors and BMI. More research is necessary to understand radiodensity as a surrogate marker of fat quality in midlife women.

In vivo Endocrine Secretion of Prostacyclin Following Expression of a Cyclooxygenase-1/Prostacyclin Fusion Protein in the Salivary Glands of Rats Via Nonviral Gene Therapy

Pulmonary arterial hypertension (PAH) is a progressive disease that culminates in right heart failure and death. Prostacyclin (PGI2) and its derivatives are effective treatments for PAH when administered as continuous parenteral infusions. This treatment paradigm requires medical sophistication, and patients are at risk for complications from an indewelling catheter; drug interruptions may result in rebound pulmonary hypertension and death. We hypothesized that the salivary gland can be repurposed into an endogenous production site for circulating PGI2 through the expression of a fusion protein embodying cyclooxygenase-1 (Cox1) and prostacyclin synthase (PGIS) domains. We utilized ultrasound-assisted gene transfer, a nonviral gene transfer strategy that achieves robust gene transfer to the salivary gland. We initially found that Cox1-PGIS expression in livers of mice using an adenoviral vector dramatically increased circulating PGI2 relative to untreated rats or rats treated with PGIS alone. We then utilized ultrasound-assisted gene transfer to express Cox1-PGIS in the submandibular glands of rats and showed a significant elevation of circulating PGI2 that corresponded to approximately 30% of that seen in humans undergoing intravenous infusion therapy for PAH. These results suggest the feasibility of gene therapy to drive endogenous biosynthesis of PGI2 as a therapeutic strategy for the treatment of PAH.

3D MicroCT spatial and temporal characterization of thoracic aorta perivascular adipose tissue and plaque volumes in the ApoE-/- mouse model

ABSTRACT Perivascular adipose tissue (PVAT) influences vascular function and pathology. We present a protocol using micro-computed tomography (microCT), a novel imaging technique typically used for hard biological tissue, to characterize the temporal and spatial development of aorta PVAT and luminal plaque soft tissue. Apolipoprotein E deficient (ApoE) and C57Bl/6J (control) mice were fed a high fat western diet up to 30 weeks. 3D microCT reconstructions were used to quantify: 1) vascular wall volume, a surrogate measure of remodeling, was greater in ApoE, 2) aorta PVAT volume was reduced in ApoE, 3) plaque volumes increased over time in ApoE, 4) plaque development co-localized with luminal ostia, origins of branching arteries, which traveled through areas of greatest PVAT volume, 5) qualitatively, the same arteries showed evidence of increased tortuosity in ApoE. This study reflects the potential of microCT analyses to assess vascular wall, PVAT and arterial trajectory modifications in relevant animal models. Abbreviations: PVAT: perivascular adipose tissue; ApoE: apolipoprotein E deficient mouse strain; Control: C57Bl/6J mouse strain; PTA: 0.3% phosphotungstic acid; microCT: micro-computed tomography; CV: cardiovascular; CVD: cardiovascular disease; IQR: interquartile range; PPARγ: peroxisome proliferator activated receptor – gamma; VV: vasa vasorum; 3D: three dimensional