Tsai, Michael Y.

Introduction: Hepatocyte growth factor (HGF) is released in response to endothelial injury, and higher levels of circulating HGF are associated with atherosclerosis. However, the association of HGF with atherosclerosis progression is unknown. Hypothesis: Circulating HGF is positively associated with atherosclerosis progression, measured as progression of 1) carotid artery plaque and 2) coronary artery calcium (CAC). Methods: The Multi-Ethnic Study of Atherosclerosis is a large, population-based cohort study of adults aged 45-84 and free of clinical cardiovascular disease. Participants had HGF measured in serum at baseline (2000-02), and were followed for progression of carotid plaque and CAC through 2012. We defined CAC and carotid plaque progression as dichotomous variables (progression, no progression). Carotid plaque progression is an increase in the number of carotid plaques. We used the Hokanson method to define CAC progression as an increase of ≥ 2.5 mm 3 in the square root-transformed calcium volume score. Relative risk regression quantified the association between HGF and carotid plaque progression. Cox regression models using inverse-probability-of-attrition weights estimated the association of HGF with progression of CAC. All effect estimates are adjusted for potential confounding variables. Results: The cohort included 6714 Americans with HGF measurements: 2593 non-Hispanic white, 797 Chinese, 1848 black, and 1476 Hispanic Americans. In 3363 participants with measurements for both carotid plaque and HGF, each standard deviation (259 pg/ml) increase in HGF was associated with a 4% greater risk (95% CI: 1.01-1.07) of carotid plaque progression (n=1875), which did not differ by race/ethnicity (interaction p = 0.3). Out of 6714 participants with measurements for both CAC and HGF, 2994 had CAC progression during follow-up. The association of HGF with CAC progression differed by race/ethnicity (interaction p = 0.01). Each standard deviation increase of HGF was associated with a 10% greater risk of CAC progression in non-Hispanic white (95% CI: 1.05-1.16) and black Americans (95% CI: 1.03-1.17), while HGF was not associated with CAC progression in Chinese (p = 0.5) nor Hispanic (p = 0.7) American participants. Conclusions: Circulating HGF was associated with a modestly increased risk of atherosclerosis progression in this large and diverse population, with the strongest associations between HGF and CAC progression in non-Hispanic white and black Americans. Our results suggest that HGF may have utility as a marker of atherosclerosis progression in some populations.

P-selectin is a cellular adhesion molecule that enhances procoagulant activity and activation of leukocyte integrins. P-selectin has been shown to be crucial in the development of atherosclerosis. Using a stratified random sample of 2406 participants in the Multi-Ethnic Study of Atherosclerosis (MESA) at Exam 2, we investigated the association of soluble P-selectin and cardiovascular disease. The Tobit model was used to investigate the association of P-selectin levels and coronary artery calcium (CAC) and Cox proportional hazards regression was used to assess the association between protein level and incident cardiovascular disease (CVD) events (myocardial infarction, stroke, angina, or other atherosclerotic death). Mean levels of soluble P-selectin differed significantly by ethnicity; 46±18 ng/mL African Americans, 58±20 ng/mL Caucasians, 43±15 ng/mL Chinese, and 61±20 ng/mL Hispanics (p<0.0001). CAC was present in 46% of African Americans, 61% of Caucasians, 52% of Chinese, and 53% of Hispanics. P-selectin levels were significantly associated with CAC in Caucasians (p < 0.001) and African Americans (p = 0.025), see Table. This association was robust to adjustment for traditional risk factors and a significant interaction of P-selectin and ethnicity was observed (p= 0.02). Over an average of 6.5 years of follow-up there were 111 incident CVD events after MESA exam 2, 26 in African Americans, 33 in Caucasians, 16 in Chinese, and 36 in Hispanics. The risk of CVD events increased 34% per standard deviation increase in P-selectin levels only in Caucasians (p = 0.007) and this finding was consistent after adjustment for CVD risk factors (HR = 1.30, p = 0.017). We have shown in this large cohort that mean circulating P-selectin levels differed by ethnicity. Despite similar percentages of subjects with CAC in each ethnic group, P-selectin levels were significantly associated with CAC only in African Americans and Caucasians. Furthermore, in Caucasians only, P-selectin levels were associated with incident CVD events. Association of P-selectin, per SD (20 ng/mL) for the population and cardiovascular disease Outcome African Americans n = 579 Caucasians n = 620 Chinese n = 602 Hispanics n = 605 CAC, Agatston Score Beta (S.E.) P-value Beta (S.E.) P-value Beta (S.E.) P-value Beta (S.E.) P-value Model 1 85.9 (38.4) 0.025 99.4 (28.7) <0.001 8.2 (33.3) 0.81 32.1 (27.3) 0.24 Model 2 79.3 (39.8) 0.046 94.7 (29.1) 0.001 −19.5 (33.6) 0.56 27.8 (27.6) 0.31 CVD Events HR (CI) HR (CI) HR (CI) HR (CI) Model 1 1.08 (0.75, 1.57) 0.67 1.34 (1.08, 1.65) 0.007 1.09 (0.57, 2.08) 0.80 0.99 (0.71, 1.39) 0.97 Model 2 1.10 (0.76, 1.58) 0.61 1.30 (1.05, 1.62) 0.017 0.82 (0.38, 1.78) 0.62 1.00 (0.71, 1.43) 0.98 Model 1 = age and sex ; Model 2 = age, sex, BMI, smoking status, LDL, HDL, triglycerides, hypertension, diabetes

Background: Given the global burden of peripheral arterial disease (PAD) and the associated morbidity, continuing investigation of potential biomarkers as therapeutic targets for preventing PAD is warranted. The association of P-selectin, an endothelial cell adhesion molecule, with the ankle brachial index (ABI) and PAD is not well established. Thus, we examined the association of circulating P-selectin with prevalent and incident PAD, the ABI, and change in the ABI. Methods: The Multi-Ethnic Study of Atherosclerosis (MESA) is a prospective population-based cohort including 6814 non-Hispanic white, African American, Hispanic and Chinese men and women aged 45-84 at baseline (2000-02). For this analysis, 5700 participants with both P-selectin at Exam 2 (2002-04) and the ABI at Exam 3 (2004-05) were available, with change in ABI from Exam 3 to Exam 5 (2010-12) available on 4276 participants. P-selectin was modeled per standard deviation (SD), while prevalent and incident PAD were defined as ABI ≤ 0.90 after excluding those with ABI>1.4. Linear regression was used for ABI, growth curve models for change in ABI, and logistic regression for prevalent and incident PAD. Results: In models adjusted for demographics, lifestyle factors, comorbidities, blood pressure, lipids, fasting glucose, c-reactive protein (CRP), interleukin-6 (IL-6), D-dimer, and fibrinogen, each SD (13 ng/mL) higher P-selectin was significantly associated with 0.007 lower ABI (95% CI ((-0.011, -0.004)), p<0.001), and an average change in the ABI of -0.006 ((-0.010, -0.003, p<0.001). P-selectin was also significantly associated with a 1.14-fold greater odds of prevalent PAD ((1.00, 1.28), p=0.04), and a 1.30-fold greater odds of incident PAD ((1.10, 1.55), p<0.001). There were no significant interactions of p-selectin by race/ethnicity or gender. Addition of P-selectin to models containing traditional PAD risk factors as well as CRP, IL-6, D-dimer and fibrinogen did not improve the c-statistic or net reclassification for incident PAD. Conclusions: P-selectin appears to be strongly associated with the development of PAD. However, further research is needed in population-based studies to confirm prospective associations of p-selectin with incident PAD and change in the ABI.

Incident hypertension varies across races/ethnicities. Mechanisms implicated in the pathogenesis are endothelial dysfunction with reduction of nitric oxide and alterations of the Renin-Angiotensin-Aldosterone System (RAAS). Stromal Derived Factor 1 (SDF1) and Transforming Growth Factor β1 (TGFβ1) are essential in these processes. SDF1 is a chemotactic protein that regulates migration of endothelial progenitor cells from the bone marrow to the arterial wall to repair endothelial damage; TGFβ1 promotes extra-cellular matrix deposition within arterial vessels, inhibits nitric oxide production and stimulates renin release. In order to investigate the association of circulating levels of these proteins with incident hypertension across different ethnicities, an ethnic stratified random sample of 2,880 participants from the Multi-Ethnic Study of Atherosclerosis (MESA) was used. Of those, 988 participants had available plasma samples and were without hypertension, defined as blood pressure ≥ 140/90 mmHg, or current use of antihypertensive medications, at baseline. During a median follow up of 8.5 years, 388 new cases of hypertension (101 in non-Hispanic whites, 94 in Chinese, 101 in African Americans and 92 in Hispanics) were observed. Time to hypertension incidence was estimated using Cox proportional hazards regression. Hazard Ratios were adjusted for age, sex, diabetes, LDL, HDL, triglycerides, BMI, smoking status and current alcohol use. The race/ethnicity by protein interaction p-values were 0.055 and 0.066 for TGFβ1 and SDF1 respectively, suggesting heterogeneity across races/ethnicities. In Chinese, a difference of one standard deviation of plasma TGFβ1 was associated with a 22% increase in incident hypertension (HR (95% CI) 1.22 (1.02, 1.47), p-value=0.03); and a difference of one standard deviation of plasma SDF1 was associated with a 31% decrease in incident hypertension (HR (95% CI) 0.69 (0.52, 0.91), p-value 0.008). No significant association was found between these proteins and incident hypertension among non-Hispanic whites, African or Hispanic Americans. In conclusion, soluble markers of endothelial integrity and of RAAS activation were associated with the incidence of hypertension among Chinese Americans. These results suggest heterogeneity in the pathophysiology of hypertension according to race/ethnicity.

L-selectin is an adhesion molecule constitutively expressed on leukocytes and mediating their interaction with the endothelium. As a member of the selectin family, it plays a crucial role in leukocyte adhesion to and migration through the endothelial wall during inflammation. A protective role of L-selectin in early atherosclerosis was demonstrated in animal models. Previous studies assessing the relationship of this protein with cardiovascular disease were inconsistent. Lower serum L-selectin levels were found in patients with coronary artery disease (CHD) and peripheral artery disease (PAD) compared to controls. In contrast, plasma levels of L-selectin were increased in patients with CHD and PAD. These inconsistencies might be due to an interaction of soluble L-selectin with clotting factors or anticoagulants present in plasma but not in serum. Also, the small number and Caucasian predominance of patients included in previous studies represent limitations that need to be addressed using a rigorous, population-based approach. Herein, we seek to investigate the association of serum L-selectin with subclinical outcomes in a large, ethnically heterogeneous population. In a randomly selected subset of 720 subjects for each of the four ethnicities included in the Multi-Ethnic Study of Atherosclerosis (MESA), serum samples were available for 2403 individuals (579 African Americans, 620 Caucasians, 600 Chinese and 604 Hispanics). Serum L-selectin levels and intima-media thickness (IMT) were measured at exam 2 and CAC was measured at exam 2 or 3 during the cohort follow up. Regression analysis and the Tobit model were used to assess the association of L-selectin levels with IMT and CAC respectively. The mean age was 63±10 years and 47% were men. The mean±SD protein levels were significantly different across ethnicities (867±192 ng/mL in African Americans, 956±215 ng/mL in Caucasians, 834±174 ng/mL in Chinese and 904±195 ng/mL in Hispanics; p<0.0001). In the univariate analysis, age was inversely correlated to the protein levels; in addition, levels were lower in males, regardless of ethnicity and in smokers, except among Hispanics. L-selectin was associated with modestly lower CAC only among Chinese (p=0.030), the association persisted after adjustment for traditional risk factors (p=0.046). No significant association was demontrated between the protein levels and IMT. In conclusion, in this large diverse population, we found that serum L-selectin was modestly associated to measures of subclinical atherosclerosis in Chinese but not in African, European or Hispanic Americans.

Pulse pressure (PP), defined as the difference between systolic and diastolic blood pressure, is considered a marker of large artery stiffness and has been shown to independently predict cardiovascular disease. Differences among ethnicities with regard to PP have been previously described, yet the underlying biological mechanisms are poorly understood. In addition, biomarkers of inflammation and matrix remodeling have been shown to be involved in the development of arterial stiffness. The aim of this study was to investigate the role of adhesion pathway components in the development of arterial stiffness measured by PP among four ethnicities. E-cadherin, HGF, L-selectin, P-selectin, 6-Ckine, MMP1, MMP2, RANTES, SDF1, SLPI, TGFβ1, TIMP2 and VCAM were measured in a random sample of 2402 participants (579 African Americans, 619 Caucasians, 600 Chinese and 604 Hispanics) in the Multi-Ethnic Study of Atherosclerosis (MESA) study at Exam 2. PP was measured concurrently and its association with each adhesion protein level was assessed via linear regression in each ethnicity. All analyses were adjusted for age, sex, and traditional cardiovascular risk factors. Mean PP was significantly different among ethnicities: 55.9±18.0 mmHg in African Americans, 52.1± 16.1 mmHg in Caucasians, 51.4±16.4 mmHg in Chinese, and 55.0±17.5 in Hispanics, p<0.001. MMP2 was positively associated with PP among African Americans (β=2.3 (0.6), p<0.001) and Caucasians (β=1.7 (0.5), p=0.001); TIMP2 among Caucasians (β=1.5 (0.5), p=0.002) and to a lesser extent among African Americans (β=1.2 (0.6) p=0.050) and Chinese (β=1.3 (0.6), p=0.034). SDF1 was associated to PP in African Americans (β=1.4 (0.6), p=0.017) and in Chinese (β=1.1 (0.5), p=0.042); finally an association existed between TGFβ1 and PP in C (β=1.1 (0.5), p=0.025). None of the studied proteins was associated with PP in Hispanics. In conclusion, components of the adhesion pathway were correlated to PP in this large multi-ethnic cohort, possibly giving new insight on the pathophysiology of arterial stiffness. In addition, heterogeneity by ethnicity suggests different mechanisms in the development of arterial stiffness.

Objective To determine if hepatocyte growth factor (HGF), a promising biomarker of coronary heart disease (CHD) given its release into circulation in response to endothelial damage, is associated with subclinical and clinical CHD in a racial/ethnic diverse population. Methods HGF was measured in 6738 participants of the Multi-Ethnic Study of Atherosclerosis (MESA). Highest mean HGF values (pg/mL) were observed in Hispanic, followed by African, non-Hispanic white, then Chinese Americans. Results In all races/ethnicities, HGF levels were associated with older age, higher systolic blood pressure (SBP) and body mass index, lower high-density lipoprotein, diabetes and current smoking. In fully adjusted models, each SD higher HGF was associated with an average increase in coronary artery calcium (CAC) of 55 Agatston units for non-Hispanic whites (p<0.001) and 51 Agatston units for African-Americans (p=0.007) but was not in the other race/ethnic groups (interaction p=0.02). There were 529 incident CHD events, and CHD risk was 41% higher in African (p<0.001), 17% in non-Hispanic white (p=0.026) and Chinese (p=0.36), and 6% in Hispanic Americans (p=0.56) per SD increase in HGF. Conclusion In a large and diverse population-based cohort, we report that HGF is associated with subclinical and incident CHD. We demonstrate evidence of racial/ethnic heterogeneity within these associations, as the results are most compelling in African-Americans and non-Hispanic white Americans. We provide evidence that HGF is a biomarker of atherosclerotic disease that is independent of traditional risk factors.

Background and Purpose— Hepatocyte growth factor (HGF) is positively associated with ischemic and hemorrhagic stroke risk factors. However, understanding the relation between HGF and stroke is in its infancy. Therefore, we sought to examine the association of circulating HGF with incident stroke using data from the MESA (Multi-Ethnic Study of Atherosclerosis). We hypothesized that circulating HGF would be positively associated with an increased risk of stroke. Methods— Participants aged 45 to 84 years (n=6711) had HGF measured between 2000 and 2002 and were followed for incident stroke through 2013 (n=233). Cox proportional hazards regression was used to calculate hazard ratios and 95% confidence intervals for incident stroke. A secondary analysis stratified results by adjudicated stroke type (n=183 ischemic; n=39 hemorrhagic; n=11 other). Results— After adjustment for potential confounding variables, risk of stroke was 17% higher with each standard deviation increase in HGF (hazard ratio, 1.17; 95% confidence interval, 1.03–1.34). This association was mainly driven by ischemic stroke and did not change on exclusion of cardioembolic strokes, although the number of excluded cases was small. The few hemorrhagic and other types of stroke were not associated with HGF. Conclusions— Circulating HGF was positively associated with the incidence of stroke in a diverse, population-based cohort of men and women from the United States. Our findings support the hypothesis that circulating HGF is a marker of endothelial damage and suggest that HGF may have utility as a prognostic marker of stroke risk.

Inflammation plays a pivotal role in peripheral artery disease (PAD). Cellular adhesion proteins mediate the interaction of leukocytes with endothelial cells during inflammation. To determine the association of cellular adhesion molecules with ankle–brachial index (ABI) and ABI category (≤1.0 vs >1.0) in a diverse population, 15 adhesion proteins were measured in the Multi-Ethnic Study of Atherosclerosis (MESA). To assess multivariable associations of each protein with ABI and ABI category, linear and logistic regression was used, respectively. Among 2364 participants, 23 presented with poorly compressible arteries (ABI > 1.4) and were excluded and 261 had ABI ≤ 1.0. Adjusting for traditional risk factors, elevated levels of soluble P-selectin, hepatocyte growth factor, and secretory leukocyte protease inhibitor were associated with lower ABI ( P = .0004, .001, and .002, respectively). Per each standard deviation of protein, we found 26%, 20%, and 19% greater odds of lower ABI category ( P = .001, .01, and .02, respectively). Further investigation into the adhesion pathway may shed new light on biological mechanisms implicated in PAD.