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Introduction: Sleep tracker data have not been utilized routinely in sleep-related disorders and their management. Sleep-related disorders are common in primary care practice and incorporating sleep tracker data may help in improving patient care. We conducted a pilot study to assess the feasibility of a sleep program using the Fitbit Charge 2™ device and SleepLife ® application. The main aim of the study was to examine whether a program using a commercially available wearable sleep tracker device providing objective sleep data would improve communication in primary care settings between patients and their providers. Secondary aims included whether patient satisfaction with care would improve as result of the program., Methods: A prospective, randomized, parallel group, observational pilot study was conducted in 20 primary care clinics in Indianapolis, IN from June 2018 to February 2019. Inclusion criteria included patients over the age of 18, have a diagnosis of insomnia identified by electronic medical record and/or a validated questionnaire, and were on a prescription sleep aid. The study was not specific to any sleep aid prescription, branded or generic, and was not designed to evaluate a drug or drug class. Each primary care clinic was randomized to either the SleepLife ® intervention or the control arm. All patients were provided with a Fitbit Charge 2™ device. Only patients in the intervention arm were educated on how to use the SleepLife ® application. Physicians in the intervention arm were set up with the SleepLife® portal on their computers., Results: Forty-nine physicians and 75 patients were enrolled in the study. Patients had a mean age of 57 (SD 12.8) years and 61% were female. Mean age of physicians was 47 (SD 10.6) years. Patients showed high rates of involvement in the program with 83% completing all survey questions. Physician survey completion rate was 55%. Only one physician logged into the SleepLife portal to check their patients' sleep status. At the end of the 6-week intervention, patients' composite general satisfaction scores with sleep health management decreased significantly in the intervention arm when compared to controls (p = 0.03). Patients' satisfaction with communication also decreased significantly in the intervention group (p = 0.01). The sleep outcomes, which were calculated on the basis of study questionnaire answers, improved significantly in the intervention group as compared to the control group (p = 0.04). Physician communication satisfaction scores remained unchanged (p = 0.12)., Conclusions: SleepLife ® and its related physician portal can facilitate physician-patient communication, and it captures patient sleep outcomes including behaviors and habits. Patients were highly engaged with the program, while physicians did not demonstrate engagement. The study design and questionnaires do not specifically address the reasons behind the decreased patient satisfaction with care and communication, but it was perceived to be a result of physician non-responsiveness. Sleep quality scores on the other hand showed an improvement among SleepLife ® users, suggesting that patients may have implemented good sleep practices on their own. Given that it was a feasibility study, and the sample size was small, we were not able to make major inferences regarding the difference between sleep disorder types. Additionally, we excluded patients with a history of alcohol use, substance abuse, or depression because of concerns that they may affect sleep independently. To promote the growth of technology in primary care, further research incorporating results from this study and physician engagement techniques should be included., (© 2022. The Author(s).)

Background: More efficient screening methods are needed to improve the ability to identify and follow genetic cohorts in Parkinson's disease (PD)., Objective: To explore the use of the electronic medical records (EMRs) to identify participants with PD., Methods: Using an algorithm previously developed in collaboration with Maccabi Healthcare Services (MHS), approximately 5,200 participants with PD were identified, more than 3,200 were screened, and 837 participants were enrolled and genotyped for leucine-rich repeat kinase 2 (LRRK2) and beta-glucocerebrosidase (GBA) variants. Questionnaires were completed to ascertain Ashkenazi Jewish (AJ) ancestry and family history of PD., Results: Among 837 participants with PD, 82% were 65 years and older and 72% had a family history of AJ ancestry. Among those with AJ ancestry, 15.6% reported having relatives with PD. The frequency of observed mutations for LRRK2 and GBA genes combined was approximately 15.4%. The frequency of observed LRRK2 mutation was 6.1% overall and 7.2% from those with AJ ancestry; and for GBA mutation was 9.3% overall and 11.2% from those with AJ ancestry., Conclusion: Although the frequency of observed mutations in this study was lower than anticipated, mutation carriers were enriched among those with a family history of AJ ancestry increasing nearly 2-3-fold, from 3% -7% (LRRK2) and 4% -11% (GBA). The identification (and selection) of PD patients through EMRs prior to genotyping is a viable approach, to establish a genetically defined cohort of patients with PD for clinical research.

Aims: To assess demographic and clinical characteristics associated with clinical inertia in a real-world cohort of type 2 diabetes mellitus patients not at hemoglobin A1c goal (<7%) on metformin monotherapy. Methods: Adult (≥18 years) type 2 diabetes mellitus patients who received care at Massachusetts General Hospital/Brigham and Women's Hospital and received a new metformin prescription between 1992 and 2010 were included in the analysis. Clinical inertia was defined as two consecutive hemoglobin A1c measures ≥7% ≥3 months apart while remaining on metformin monotherapy (i.e. without add-on therapy). The association between clinical inertia and demographic and clinical characteristics was examined via logistic regression. Results: Of 2848 eligible patients, 43% did not achieve a hemoglobin A1c goal of <7% 3 months after metformin monotherapy initiation. A sub-group of 1533 patients was included in the clinical inertia analysis, of which 36% experienced clinical inertia. Asian race was associated with an increased likelihood of clinical inertia (OR = 2.43; 95% CI = 1.48-3.96), while congestive heart failure had a decreased likelihood (OR = 0.58; 95% CI = 0.32-0.98). Chronic kidney disease and cardiovascular/cerebrovascular disease had weaker associations but were directionally similar to congestive heart failure. Conclusions: Asian patients were at an increased risk of clinical inertia, whereas patients with comorbidities appeared to have their treatment more appropriately intensified. A better understanding of these factors may inform efforts to decrease the likelihood for clinical inertia.

We developed an insomnia classification algorithm by interrogating an electronic medical records (EMR) database of 314,292 patients. The patients received care at Massachusetts General Hospital (MGH), Brigham and Women's Hospital (BWH), or both, between 1992 and 2010. Our algorithm combined structured variables (such as International Classification of Diseases 9th Revision [ICD-9] codes, prescriptions, laboratory observations) and unstructured variables (such as text mentions of sleep and psychiatric disorders in clinical narrative notes). The highest classification performance of our algorithm was achieved when it included a combination of structured variables (billing codes for insomnia, common psychiatric conditions, and joint disorders) and unstructured variables (sleep disorders and psychiatric disorders). Our algorithm had superior performance in identifying insomnia patients compared to billing codes alone (area under the receiver operating characteristic curve [AUROC] = 0.83 vs. 0.55 with 95% confidence intervals [CI] of 0.76-0.90 and 0.51-0.58, respectively). When applied to the 314,292-patient population, our algorithm classified 36,810 of the patients with insomnia, of which less than 17% had a billing code for insomnia. In conclusion, an insomnia classification algorithm that incorporates clinical notes is superior to one based solely on billing codes. Compared to traditional methods, our study demonstrates that a classification algorithm that incorporates physician notes can more accurately, comprehensively, and quickly identify large cohorts of insomnia patients.

Aim: We assessed the longitudinal association between tooth loss and peripheral arterial disease (PAD) within the Nurses' Health Study., Materials and Methods: After excluding participants with prior cardiovascular diseases, 277 of 79,663 women were confirmed as PAD cases during 16 years of follow-up. Number of teeth and recent tooth loss were reported initially in 1992. Subsequent tooth loss was recorded in 1996 and in 2000. We evaluated the associations of baseline number of teeth and recent tooth loss with risk of PAD, adjusting for age, smoking, diabetes, hypertension, high cholesterol, aspirin use, family history of myocardial infarction, BMI, alcohol consumption, physical activity, postmenopausal hormone use, and use of vitamin E, vitamin D, multivitamin and calcium., Results: Incident tooth loss during follow-up was significantly associated with higher hazard of PAD (HR = 1.31 95% CI: 1.00-1.71). However, the association appeared inverse among never smokers. There was no dose-response relationship between baseline number of teeth and PAD., Conclusions: Tooth loss showed a modest association with PAD, but no dose-response relationship was observed., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Insomnia remains under-diagnosed and poorly treated despite its high economic and social costs. Though previous work has examined how patient characteristics affect sleep medication prescriptions, the role of physician characteristics that influence this clinical decision remains unclear. We sought to understand patient and physician factors that influence sleep medication prescribing patterns by analyzing Electronic Medical Records (EMRs) including the narrative clinical notes as well as codified data. Zolpidem and trazodone were the most widely prescribed initial sleep medication in a cohort of 1,105 patients. Some providers showed a historical preference for one medication, which was highly predictive of their future prescribing behavior. Using a predictive model (AUC = 0.77), physician preference largely determined which medication a patient received (OR = 3.13; p = 3 × 10 -37 ). In addition to the dominant effect of empirically determined physician preference, discussion of depression in a patient's note was found to have a statistically significant association with receiving a prescription for trazodone (OR = 1.38, p = 0.04). EMR data can yield insights into physician prescribing behavior based on real-world physician-patient interactions., Competing Interests: The authors declare no competing financial interests.

Background: Research targeting glycosylated hemoglobin A1c (HbA1c) to <6.5% to prevent coronary heart disease (CHD) events has conflicting results. We previously observed the haptoglobin (Hp) Hp2-2 genotype is associated with a ∼10-fold increased CHD risk among individuals with HbA1c ≥6.5%, and thus might be useful in identifying those at high risk of CHD who would benefit from maintaining HbA1c <6.5%., Objectives: This study sought to model whether HbA1c ≥ 6.5% in the Hp2-2 genotype is associated with CHD in a prospective case-control study nested within the Health Professionals Follow-Up Study (HPFS)., Methods: HbA1c concentration and Hp genotype were determined for 695 incident cases of CHD from 1994 to 2010 and matched control participants. Logistic regression models calculated relative risk (RR) and 95% CI, for the first and second halves of follow-up, adjusting for confounding variables. A dataset from the Nurses' Health Study served as a replication cohort., Results: The prevalence of the Hp2-2 genotype in HPFS was 39%. Compared with HbA1c <6.5%, the RR of CHD for HbA1c ≥6.5% for the Hp2-2 genotype over full follow-up was 3.07 (95% CI: 1.37 to 6.86) to 3.88 (95% CI: 1.31 to 11.52) during the first half of follow-up and 2.16 (95% CI: 0.61 to 7.61) in the second half. The corresponding RRs for the Hp1-1 + Hp2-1 genotypes were: full follow-up, 2.19 (95% CI: 1.14 to 4.24); first half, 1.60 (95% CI: 0.73 to 3.53); and second half, 4.72 (95% CI: 1.26 to 17.65)., Conclusions: In 2 independent cohorts, the risk of CHD associated with HbA1c ≥6.5% is pronounced in the Hp2-2 genotype, particularly in early cases. The Hp2-2 genotype may identify individuals at greatest CHD risk from hyperglycemia., (Copyright © 2015 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Background: Generalized allelic heterozygosity has been proposed to improve reproductive fitness and has been associated with higher blood pressure, but its association with chronic disease is not well characterized., Methods: Using the Affymetrix Genome-Wide Human 6.0 array, we performed whole genome scans in parallel case-control studies of coronary heart disease (CHD) nested in the Health Professionals Follow-up Study and Nurses' Health Study. We examined ~700,000 single nucleotide polymorphisms (SNPs) in 435 men with incident CHD and 878 matched controls and 435 women with incident CHD with 931 matched controls. We examined the relationship of genome-wide heterozygosity with risk of incident of CHD and with baseline levels of cardiovascular risk factors., Results: In both cohorts, approximately 227650 (SD 2000) SNPs were heterozygous. The number of heterozygous SNPs was not related to risk of CHD in either men or women (adjusted odds ratios per 2000 heterozygous SNPs 1.01 [95% confidence interval, 0.91-1.13] in women and 0.94 [0.84-1.06] in men). We also found no consistent associations of genome-wide heterozygosity with levels of lipids, inflammatory markers, adhesion molecules, homocysteine, adiponectin, or body-mass index., Conclusions: In these parallel nested case-control studies, we found no relationship of multilocus heterozygosity with risk of CHD or its major risk factors. Studies in other populations are needed to rule out associations with lower levels of heterozygosity.

Background: The healthiest dietary pattern for myocardial infarction (MI) survivors is not known. Specific long-term benefits of a low-carbohydrate diet (LCD) are unknown, whether from animal or vegetable sources. There is a need to examine the associations between post-MI adherence to an LCD and all-cause and cardiovascular mortality., Methods and Results: We included 2258 women from the Nurses' Health Study and 1840 men from the Health Professional Follow-Up Study who had survived a first MI during follow-up and provided a pre-MI and at least 1 post-MI food frequency questionnaire. Adherence to an LCD high in animal sources of protein and fat was associated with higher all-cause and cardiovascular mortality (hazard ratios of 1.33 [95% CI: 1.06 to 1.65] for all-cause mortality and 1.51 [95% CI: 1.09 to 2.07] for cardiovascular mortality comparing extreme quintiles). An increase in adherence to an animal-based LCD prospectively assessed from the pre- to post-MI period was associated with higher all-cause mortality and cardiovascular mortality (hazard ratios of 1.30 [95% CI: 1.03 to 1.65] for all-cause mortality and 1.53 [95% CI: 1.10 to 2.13] for cardiovascular mortality comparing extreme quintiles). An increase in adherence to a plant-based LCD was not associated with lower all-cause or cardiovascular mortality., Conclusions: Greater adherence to an LCD high in animal sources of fat and protein was associated with higher all-cause and cardiovascular mortality post-MI. We did not find a health benefit from greater adherence to an LCD overall after MI., (© 2014 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

Objective: Few studies have examined the roles of homocysteine and related nutrients in the development of peripheral artery disease (PAD). We examined the associations between plasma homocysteine, dietary B vitamins, betaine, choline, and supplemental folic acid use and incidence of PAD., Methods: We used two cohort studies of 72,348 women in the Nurses' Health Study (NHS, 1990-2010) and 44,504 men in the Health Professionals Follow-up Study (HPFS, 1986-2010). We measured plasma homocysteine in nested matched case-control studies of clinically recognized PAD within both cohorts, including 143 PAD cases and 424 controls within the NHS (1990-2010) and 143 PAD cases and 428 controls within the HPFS (1994-2008). We examined the association between diet and risk of incident PAD in the cohorts using a food frequency questionnaire and 790 cases of PAD over 3.1 million person-years of follow-up., Results: Higher homocysteine levels were positively associated with risk of PAD in men (adjusted IRR 2.17; 95% CI, 1.08-4.38 for tertile 3 vs. 1). There was no evidence of an association in women (adjusted IRR 1.14; 95% CI, 0.61-2.12). Similarly, higher folate intake, including supplements, was inversely associated with risk of PAD in men (adjusted HR 0.90; 95% CI, 0.82-0.98 for each 250 μg increase) but not women (HR 1.01, 95% CI, 0.88-1.15). Intakes of the other B vitamins, betaine, and choline were not consistently associated with risk of PAD in men or women., Conclusion: Homocysteine levels were positively associated and dietary folate intake was inversely associated with risk of PAD in men but not in women., (Copyright © 2014. Published by Elsevier Ireland Ltd.)

Background: β2-Microglobulin and cystatin C may have advantages over creatinine in assessing risk associated with kidney function. We therefore investigated whether emerging filtration markers, β2-microglobulin and cystatin C, are prospectively associated with risk of the development of peripheral artery disease (PAD)., Methods and Results: We conducted nested case-control studies among women within the Nurses' Health Study (1990-2010) and among men within the Health Professionals Follow-up Study (1994-2008) with the use of archived blood samples collected before PAD diagnosis. During follow-up, symptomatic PAD was confirmed in 144 women and 143 men. Controls were matched 3:1 based on age, race, smoking status, fasting status, and date of blood sampling. Conditional logistic regression models were used to estimate relative risks (RRs) and were adjusted for plasma creatinine and cardiovascular risk factors. In women, the RRs (95% CI) per 1-SD) increment were 1.16 (0.85 to 1.58) for β2-microglobulin and 0.94 (0.69 to 1.28) for cystatin C. Corresponding RRs in men were 1.50 (1.08 to 2.09) for β2-microglobulin and 1.54 (1.07 to 2.22) for cystatin C. There was no association between creatinine and PAD risk in women, whereas the association in men (RR 1.41, 95% CI 1.10 to 1.81) disappeared after adjustment for either β2-microglobulin or cystatin C. In pooled analyses of men and women, only β2-microglobulin was associated with PAD risk (RR 1.31, 95% CI 1.04 to 1.64)., Conclusions: In pooled analyses, β2-microglobulin was associated with an increased risk of symptomatic PAD; a similar association with cystatin C was observed only in men. The findings suggest that β2-microglobulin may capture the atherosclerosis-promoting or atherosclerosis-related elements of kidney dysfunction better than creatinine., (© 2014 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

Objective: To evaluate the associations of dietary fiber after myocardial infarction (MI) and changes in dietary fiber intake from before to after MI with all cause and cardiovascular mortality., Design: Prospective cohort study., Setting: Two large prospective cohort studies of US women and men with repeated dietary measurements: the Nurses' Health Study and the Health Professionals Follow-Up Study., Participants: 2258 women and 1840 men who were free of cardiovascular disease, stroke, or cancer at enrollment, survived a first MI during follow-up, were free of stroke at the time of initial onset of MI, and provided food frequency questionnaires pre-MI and at least one post-MI., Main Outcome Measures: Associations of dietary fiber post-MI and changes from before to after MI with all cause and cardiovascular mortality using Cox proportional hazards models, adjusting for drug use, medical history, and lifestyle factors., Results: Higher post-MI fiber intake was significantly associated with lower all cause mortality (comparing extreme fifths, pooled hazard ratio 0.75, 95% confidence interval 0.58 to 0.97). Greater intake of cereal fiber was more strongly associated with all cause mortality (pooled hazard ratio 0.73, 0.58 to 0.91) than were other sources of dietary fiber. Increased fiber intake from before to after MI was significantly associated with lower all cause mortality (pooled hazard ratio 0.69, 0.55 to 0.87)., Conclusions: In this prospective study of patients who survived MI, a greater intake of dietary fiber after MI, especially cereal fiber, was inversely associated with all cause mortality. In addition, increasing consumption of fiber from before to after MI was significantly associated with lower all cause and cardiovascular mortality.

Importance: Information about diet after myocardial infarction (MI) and mortality is limited, despite the growing number of MI survivors in the United States., Objective: To examine the association of post-MI dietary quality and changes from pre- to post-MI with all-cause and cardiovascular mortality among MI survivors., Design, Setting, and Participants: We included 2258 women from the Nurses' Health Study and 1840 men from the Health Professionals Follow-up Study. Participants had survived an initial MI during the study follow-up period and completed the pre- and post-MI food frequency questionnaire. Diet quality was measured using Alternative Healthy Eating Index 2010 (AHEI2010), which consists of food and nutrients associated with the risk of chronic disease reported in the literature. We adjusted for medication use, medical history, and lifestyle risk factors using Cox proportional hazards regression models., Main Outcomes and Measures: All-cause and cardiovascular mortality., Results: During follow-up, we confirmed 682 all-cause deaths for women and 451 for men. The median survival time after the initial MI onset was 8.7 years for women and 9.0 years for men. When the results were pooled, the adjusted hazard ratio (HR) was 0.76 (95% CI, 0.60-0.96) for all-cause mortality and 0.73 (95% CI, 0.51-1.04) for cardiovascular mortality, comparing the extreme quintiles of post-MI AHEI2010. A greater increase in the AHEI2010 score from pre- to post-MI was significantly associated with lower all-cause mortality (pooled HR, 0.71; 95% CI, 0.56-0.91) and cardiovascular mortality (pooled HR, 0.60; 95% CI, 0.41- 0.86), comparing the extreme quintiles. The adjusted HRs associated with post-MI AHEI2010 were 0.73 (95% CI, 0.58-0.93) for all-cause mortality and 0.81 (95% CI, 0.64-1.04) for cardiovascular mortality when the alcohol component was excluded., Conclusions and Relevance: Myocardial infarction survivors who consume a higher-quality diet, which has been associated with a lower risk of coronary heart disease in primary prevention, have lower subsequent all-cause mortality.

Objectives: The goal of this study was to examine the prospective association between oxidation-specific biomarkers, primarily oxidized phospholipids (OxPL) on apolipoprotein B-100-containing lipoproteins (OxPL/apoB) and lipoprotein (a) [Lp(a)], and risk of peripheral artery disease (PAD). We examined, as secondary analyses, indirect measures of oxidized lipoproteins, including autoantibodies to malondialdehyde-modified low-density lipoprotein (MDA-LDL) and apolipoprotein B-100 immune complexes (ApoB-IC)., Background: Biomarkers to predict the development of PAD are lacking. OxPL circulate in plasma, are transported by Lp(a), and deposit in the vascular wall and induce local inflammation., Methods: The study population included 2 parallel nested case-control studies of 143 men within the Health Professionals Follow-up Study (1994 to 2008) and 144 women within the Nurses' Health Study (1990 to 2010) with incident confirmed cases of clinically significant PAD, matched 1:3 to control subjects., Results: Levels of OxPL/apoB were positively associated with risk of PAD in men and women: pooled relative risk: 1.37, 95% confidence interval: 1.19 to 1.58 for each 1-SD increase after adjusting age, smoking, fasting status, month of blood draw, lipids, body mass index, and other cardiovascular disease risk factors. Lp(a) was similarly associated with risk of PAD (pooled adjusted relative risk: 1.36; 95% confidence interval: 1.18 to 1.57 for each 1-SD increase). Autoantibodies to MDA-LDL and ApoB-IC were not consistently associated with risk of PAD., Conclusions: OxPL/apoB were positively associated with risk of PAD in men and women. The major lipoprotein carrier of OxPL, Lp(a), was also associated with risk of PAD, reinforcing the key role of OxPL in the pathophysiology of atherosclerosis mediated by Lp(a)., (Copyright © 2013 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Objective: Lower concentrations of adiponectin have been linked to subsequent risk of coronary heart disease in healthy individuals. Whether similar relationships exist for the development of systemic atherosclerosis, such as peripheral artery disease (PAD), is uncertain. We investigated the association between total adiponectin and risk of lower extremity PAD., Approach and Results: We performed a prospective, nested case-control study among 18,225 male participants of the Health Professionals Follow-up Study who were free of diagnosed cardiovascular disease at the time of blood draw (1993-1995). During 14 years of follow-up, 143 men developed PAD. Using risk set sampling, controls were selected in a 3:1 ratio and matched on age, smoking status, fasting status, and date of blood draw (n=429). Median (interquartile range) adiponectin concentrations at baseline were lower among cases compared with controls (4.1 [3.2-5.5] versus 5.4 [3.8-7.5] µg/mL; P<0.001). A log-linear inverse association was evident over the full spectrum of adiponectin concentrations with PAD risk after controlling for baseline cardiovascular risk factors using restricted spline conditional logistic regression. Adiponectin was associated with a 42% lower risk of PAD per SD increase in natural log-transformed adiponectin (relative risk, 0.58; 95% confidence interval, 0.45-0.74) after adjustment for cardiovascular risk factors. The relative risk was attenuated (relative risk, 0.68; 95% confidence interval, 0.51-0.92) after further accounting for high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, C-reactive protein, and cystatin C. Additional adjustment for hemoglobin A(1c), triglycerides, and γ-glutamyltransferase had little impact on this association (relative risk, 0.68; 95% confidence interval, 0.50-0.92)., Conclusions: Total adiponectin is inversely associated with risk of symptomatic lower extremity PAD in men.

Background: Hemoglobin A1c (HbA1c), a time-integrated marker of glycemic control, predicts risk of coronary heart disease (CHD) among diabetics. Few studies have examined HbA1c and risk of CHD among women and men without clinically elevated levels or previously diagnosed diabetes., Methods and Results: We conducted parallel nested case-control studies among women (Nurses' Health Study) and men (Health Professionals Follow-up Study). During 14 and 10 years of follow-up, 468 women and 454 men developed incident nonfatal myocardial infarction (MI) and fatal CHD. Controls were matched 2:1 based on age, smoking, and date of blood draw. For these analyses, participants with a history of diabetes or HbA1c levels ≥6.5% at baseline were excluded. Compared with HbA1c of 5.0% to <5.5%, those with an HbA1c of 6.0% to <6.5% had a multivariable-adjusted relative risk (RR) of CHD of 1.90 (95% CI 1.11 to 3.25) in women and 1.81 (95% CI 1.09 to 3.03) in men. The pooled RR of CHD was 1.29 (95% CI 1.11 to 1.50) for every 0.5%-increment increase in HbA1c levels and 1.67 (95% CI 1.23 to 2.25) for every 1%-increment increase, with the risk plateauing around 5.0%. Furthermore, participants with HbA1c levels between 6.0% and <6.5% and C-reactive protein levels >3.0 mg/L had a 2.5-fold higher risk of CHD compared with participants in the lowest categories of both biomarkers., Conclusions: Our findings suggest that HbA1c is associated with CHD risk among apparently healthy, nondiabetic women and men and may be an important early clinical marker of disease risk.

Objectives: This study sought to investigate into the biologically plausible interaction between the common haptoglobin (Hp) polymorphism rs#72294371 and glycosylated hemoglobin (HbA(1c)) on risk of coronary heart disease (CHD)., Background: Studies of the association between the Hp polymorphism and CHD report inconsistent results. Individuals with the Hp2-2 genotype produce Hp proteins with an impaired ability to prevent oxidative injury caused by elevated HbA(1c)., Methods: HbA(1c) concentration and Hp genotype were determined for 407 CHD cases matched 1:1 to controls (from the NHS [Nurses' Health Study]) and in a replication cohort of 2,070 individuals who served as the nontreatment group in the ICARE (Prevention of Cardiovascular Complications in Diabetic Patients With Vitamin E Treatment) study, with 29 CHD events during follow-up. Multivariate models were adjusted for lifestyle and CHD risk factors as appropriate. A pooled analysis was conducted of NHS, ICARE, and the 1 previously published analysis (a cardiovascular disease case-control sample from the Strong Heart Study)., Results: In the NHS, Hp2-2 genotype (39% frequency) was strongly related to CHD risk only among individuals with elevated HbA(1c) (≥ 6.5%), an association that was similar in the ICARE trial and the Strong Heart Study. In a pooled analysis, participants with both the Hp2-2 genotype and elevated HbA(1c) had a relative risk of 7.90 (95% confidence interval: 4.43 to 14.10) for CHD compared with participants with both an Hp1 allele and HbA(1c) <6.5% (p for interaction = 0.004), whereas the Hp2-2 genotype with HbA(1c) <6.5% was not associated with risk (relative risk: 1.34 [95% confidence interval: 0.73 to 2.46])., Conclusions: Hp genotype was a significant predictor of CHD among individuals with elevated HbA(1c)., (Copyright © 2013 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Context: Previous studies have examined the associations of individual clinical risk factors with risk of peripheral artery disease (PAD), but the combined effects of these risk factors are largely unknown., Objective: To estimate the degree to which the 4 conventional cardiovascular risk factors of smoking, hypertension, hypercholesterolemia, and type 2 diabetes are associated with the risk of PAD among men., Design, Setting, and Participants: Prospective study of 44,985 men in the United States without a history of cardiovascular disease at baseline in 1986; participants in the Health Professionals Follow-up Study were followed up for 25 years until January 2011. The presence of risk factors was updated biennially during follow-up., Main Outcome Measure: Clinically significant PAD defined as limb amputation or revascularization, angiogram reporting vascular obstruction of 50% or greater, ankle-brachial index of less than 0.90, or physician-diagnosed PAD., Results: During a median follow-up of 24.2 years (interquartile range, 20.8-24.7 years), there were 537 cases of incident PAD. Each risk factor was significantly and independently associated with a higher risk of PAD after adjustment for the other 3 risk factors and confounders. The age-adjusted incidence rates were 9 (95% CI, 6-14) cases/100,000 person-years (n = 19 incident cases) for 0 risk factors, 23 (95% CI, 18-28) cases/100,000 person-years (n = 99 incident cases) for 1 risk factor, 47 (95% CI, 39-56) cases/100,000 person-years (n = 176 incident cases) for 2 risk factors, 92 (95% CI, 76-111) cases/100,000 person-years (n = 180 incident cases) for 3 risk factors, and 186 (95% CI, 141-246) cases/100,000 person-years (n = 63 incident cases) for 4 risk factors. The multivariable-adjusted hazard ratio for each additional risk factor was 2.06 (95% CI, 1.88-2.26). Men without any of the 4 risk factors had a hazard ratio of PAD of 0.23 (95% CI, 0.14-0.36) compared with all other men in the cohort. In 96% of PAD cases (95% CI, 94%-98%), at least 1 of the 4 risk factors was present at the time of PAD diagnosis. The population-attributable risk associated with these 4 risk factors was 75% (95% CI, 64%-87%). The absolute incidence of PAD among men with all 4 risk factors was 3.5/1000 person-years., Conclusion: Among men in this cohort, smoking, hypertension, hypercholesterolemia, and type 2 diabetes account for the majority of risk associated with development of clinically significant PAD.

Aims: The aim of this study was to examine the association between long-term alcohol consumption, alcohol consumption before and after myocardial infarction (MI), and all-cause and cardiovascular mortality among survivors of MI., Methods and Results: The Health Professionals Follow-up Study (HPFS) is a prospective cohort study of 51 529 US male health professionals. From 1986 to 2006, 1818 men were confirmed with incident non-fatal MI. Among MI survivors, 468 deaths were documented during up to 20 years of follow-up. Long-term average alcohol consumption was calculated beginning from the time period immediately before the first MI and updated every 4 years afterward. Cox proportional hazards were used to estimate the multivariable-adjusted hazard ratios (HR) and 95% confidence intervals (CI). Compared with non-drinkers, the multivariable-adjusted HRs for all-cause mortality were 0.78 (95% CI: 0.62-0.97) for 0.1-9.9 g/day, 0.66 (95% CI: 0.51-0.86) for 10.0-29.9 g/day, and 0.87 (95% CI: 0.61-1.25) for ≥30 g/day (P(quadratic)= 0.006). For cardiovascular mortality, the corresponding HRs were 0.74 (95% CI: 0.54-1.02), 0.58 (95% CI: 0.39-0.84), and 0.98 (95% CI: 0.60-1.60), P(quadratic)= 0.003. These findings were consistent when restricted to pre- and post-MI alcohol assessments. In subgroup analyses, moderate alcohol consumption was inversely associated with mortality among men with non-anterior infarcts, and among men with mildly diminished left ventricular function., Conclusion: Long-term moderate alcohol consumption is inversely associated with all-cause and cardiovascular mortality among men who survived a first MI. This U-shaped association may be strongest among individuals with less impaired cardiac function after MI and should be examined further.

Background and Objective: CRP has several potentially antibacterial effects, and variation in the CRP gene is known to influence CRP levels. Whether this variation influences risk of infection, and hence whether CRP has anti-infective activity in humans, is uncertain., Methods: We evaluated a series of haplotype-tagging single nucleotide polymorphisms among 5374 individuals in the Cardiovascular Health Study, a cohort of older adults from four communities, who were followed for community-acquired pneumonia for 12-13 years. Secondarily, we evaluated whether these polymorphisms varied among men in the Health Professionals Follow-up Study who self-reported pneumonia on biennial questionnaires., Results: There were 581 (507 white and 74 black) Cardiovascular Health Study participants with incident hospitalizations for pneumonia. No single nucleotide polymorphism or haplotypes were associated with risk among white Cardiovascular Health Study participants. Among black participants, the haplotype tagged by A790T was associated with lower risk of incident pneumonia (hazard ratio 0.5; 95% confidence interval: 0.3-0.9) and with higher CRP levels. In Health Professionals Follow-up Study, a separate haplotype was associated with less frequent self-reported pneumonia but not with circulating CRP levels., Conclusions: Some genetic variants in CRP may be associated with risk of pneumonia, but haplotypes associated with risk are variably associated with baseline CRP levels. If CRP is a relevant component of innate immunity in humans, the inducibility or tissue-specificity of expression may be at least as important as chronic circulating levels.

Background: Previous studies of genetic variants of paraoxonase 1 (PON1) and coronary heart disease (CHD) have been conflicting and the modifying effects of lifestyle factors that affect PON1 activity are uncertain., Methods and Results: In parallel nested case-control studies, the prospective associations between PON1 polymorphisms Q192R and L55M and incident CHD were examined among participants in the Nurses' Health and Health Professionals Follow-up Studies. Women were followed for 8 years and men for 6 years, and 249 women and 266 men were documented with incident CHD. Neither polymorphism was associated with risk of CHD in either sex, and neither monounsaturated fat intake nor smoking interacted with genotype. Among women, there was a possible interaction of Q192R with alcohol intake (P interaction 0.06) and a suggestion of a similar interaction with the L55M genotype (P interaction 0.11). In analyses of both polymorphisms, alcohol intake > or =2.5 g/day was associated with lower risk among all women (odds ratio 0.45), except those with the Q192Q/L55M genotype (OR 1.33; P 3-way interaction 0.07)., Conclusions: PON1 polymorphisms are not associated with the risk of CHD nor do they interact with smoking or monounsaturated fat intake. A possible gene-alcohol interaction should be considered in future studies of PON1 and CHD.

Background: C-reactive protein (CRP), an acute phase reactant and marker of inflammation, has been shown to predict risk of incident cardiovascular events. However, few studies have comprehensively examined six common single-nucleotide polymorphisms (SNPs) in the CRP gene, haplotypes, and plasma CRP levels with risk of coronary heart disease (CHD)., Methods and Findings: We conducted parallel nested case-control studies within two ongoing, prospective cohort studies of U.S. women (Nurses' Health Study) and men (Health Professionals Follow-up Study). Blood samples were available in a subset of 32,826 women and 18,225 men for biomarker and DNA analyses. During 8 and 6 years of follow-up, 249 women and 266 men developed incident nonfatal myocardial infarction or fatal CHD, and controls (498 women, 531 men) were matched 2:1 on age, smoking, and date of blood draw from participants free of cardiovascular disease at the time the case was diagnosed. Among both women and men, minor alleles were significantly associated with higher CRP levels for SNPs 1919A>T and 4741G>C, but associated with lower CRP levels for SNPs 2667G>C and 3872C>T. SNP 2667G>C was individually associated with increased risk of CHD in both women [OR 1.57 (95% CI 1.01-2.44); p = 0.047] and men [1.93 (95% CI 1.30-2.88); p = 0.001]. Two of the five common haplotypes were associated with lower CRP levels, and Haplotype 4 which included minor alleles for 2667 and 3872 was associated with significantly lower CRP levels and an elevated risk of CHD. The remaining SNPs or haplotypes were not associated with CHD in both populations., Conclusions: Common variation in the CRP gene was significantly associated with plasma CRP levels; however, the association between common SNPs and CRP levels did not correspond to a predicted change in CHD risk. The underlying inflammatory processes which predict coronary events cannot be captured solely by variation in the CRP gene.

The ATP-binding cassette transporter A-1 (ABCA1) regulates cholesterol efflux from cells and is involved in high-density lipoprotein (HDL) metabolism and atherogenesis. We investigated whether common ABCA1 variants, previously reported to have phenotypic effects in humans, were associated with plasma lipids and CHD in a prospective study of coronary heart disease (CHD) in healthy women. Three polymorphisms in the promoter region (-565C/T, -191G/C, and -17C/G) and two in the coding region (I883M and R1587K) were genotyped in the Nurses' Health Study. During 8 years of follow-up, 249 incident cases of CHD were identified and matched to controls (1:2) on age and smoking. The I883M variant was associated with higher HDL-cholesterol levels among younger women. Nearly complete linkage disequilibrium was observed between -565C/T and -191G/C and their less common alleles predicted a lower risk of CHD (odds ratio of CHD per -191C allele: 0.8; 95% CI, 0.6-1.0). Neither the -17C/G SNP nor the 2 the coding polymorphisms were associated with risk of CHD. The -565C/T and the -191G/C variants were inversely associated with risk of CHD among healthy women, without pronounced effects on plasma lipids.

Objective: The objective was to examine the association of 5 common single nucleotide polymorphisms (SNPs) at the adiponectin locus with risk of coronary heart disease (CHD) in men and women., Methods and Procedures: We genotyped five common SNPs in the adiponectin gene (rs266729, -11365C>G; rs822395, -4034A>C; rs822396, -3964A>G; rs2241766, +45T>G; and rs1501299, +276G>T) in men (Health Professionals Follow-up Study) and women (Nurses' Health Study) in a nested case control setting. Among participants free of cardiovascular disease at baseline, 266 men and 249 women developed non-fatal myocardial infarction or fatal CHD during 6 and 8 years of follow-up, respectively. In addition, 564 men had coronary artery bypass graft surgery or percutaneous transluminal coronary angioplasty. Using risk set sampling, controls were selected 2:1 matched on age, smoking, and date of blood draw., Results: The -4034CC genotype was related to an increased risk of non-fatal myocardial infarction or fatal CHD compared with the AA genotype [relative risk (RR), men, 1.69; 95% confidence interval (CI), 0.99 to 2.89; women, 2.04; 95% CI, 1.20 to 3.49); however, this genotype was not related to risk of coronary artery bypass graft surgery or percutaneous transluminal coronary angioplasty or to plasma adiponectin levels. Other SNPs or haplotypes defined by the 5 SNPs were not consistently related to risk of CHD in men and women or to plasma adiponectin levels., Discussion: Our study does not support the hypothesis that these 5 common SNPs in the adiponectin gene play an important role in the development of CHD among men and women, although we cannot exclude an association between the -4034CC genotype and risk of CHD.

Aims: Complement factor H (CFH) Y402H polymorphism is located in a region that binds C-reactive protein and may affect inflammatory processes and risk of coronary heart disease (CHD). We assessed the association between Y402H and risk of CHD in nested case-control studies among two large prospective cohorts of US male health professionals and female nurses., Methods and Results: Among participants who were disease-free at baseline, we confirmed 266 (men) and 249 (women) incident CHD deaths and non-fatal myocardial infarctions (MIs) over 6 and 8 years of follow-up, respectively. Using risk-set sampling, controls were matched 2:1 on the basis of age, smoking, and date of blood draw. Comparing homozygous HH with YY, the relative risk (RR) of CHD was 0.94 [95% confidence interval (CI) 0.59-1.49] among men and 0.51 (95% CI 0.29-0.89) among women (pooled RR 0.73, 95% CI 0.51-1.04). The HH genotype was inversely associated with CHD among those <65 years at onset (men: RR 0.39, 95% CI 0.16-0.95; women: 0.21, 95% CI 0.07-0.65; pooled: 0.30, 95% CI 0.15-0.61), but not among those > or =65 years (pooled RR 1.09, 95% CI 0.71-1.68)., Conclusion: CFH Y402H was inversely associated with CHD among women, but not men. This inverse association was observed in both populations with earlier age of CHD.

The pro-inflammatory cytokine LTA (lymphotoxin-alpha) has multiple functions in regulating the immune system and may contribute to inflammatory processes leading to CHD (coronary heart disease). The aim of the present study was to investigate whether the common C804A (resulting in a Thr(26)-->Asp amino acid substitution) and A252G polymorphisms of the LTA gene and the C3279T polymorphism of the galectin-2 (LGALS2) gene, which affects LTA secretion, are associated with inflammatory parameters and cell adhesion molecules, and whether these polymorphisms are related to CHD in American women and men. We conducted a prospective nested case-control study within the Nurses' Health Study and Health Professionals Follow-Up Study. Among participants free of cardiovascular disease at baseline, 249 women and 266 men developed CHD during 8 and 6 years of follow-up respectively, and we matched controls 2:1 based on age and smoking. The LGALS2 gene variant was significantly associated with a decreased risk of CHD in women [odds ratio (95% confidence interval), 0.70 (0.50-0.97); P=0.03]. In addition, the LGALS2 polymorphism was directly associated with CRP (C-reactive protein) levels in cases from both studies (P<0.05). The LTA gene polymorphisms were directly associated with levels of sTNFRs (soluble tumour necrosis factor receptors) and VCAM-1 (vascular cell adhesion molecule-1) in both women and men with CHD (P<0.05). However, no overall effect was demonstrated between LTA gene polymorphisms and risk of CHD.

Objective: Genetic variation in CC-chemokine receptor-2 (CCR2) and -5 (CCR5), and their common haplotypes, acting through inflammatory responses, may affect atherosclerosis and risk of coronary heart disease (CHD)., Method and Results: We examined seven common variants in the CCR2 and CCR5 loci and risk of CHD among women in the Nurses' Health Study. During 8 years of follow-up, we documented 248 incident cases of nonfatal myocardial infarction and fatal CHD, and matched controls 2:1 based on age and smoking. The distribution of alleles was similar between cases and controls. The haplotype-specific odds ratios (ORs) were not statistically significant nor was the globally-adjusted p-value (p=0.61). However, there was a statistically significant association for CCR5-Delta32 and A58755G (rs2856758) between cases and controls comparing age of onset <55 and >or=55 years. For Delta32, the OR for having the variant was 0.12 (0.02-0.76) for age <55, and 1.14 (0.69-1.88) for age >or=55 years (p, interaction=0.04). The CCR5-Delta32 was in linkage disequilibrium with 58755G, and a similar association was observed for having the 58755G., Conclusions: In this population, CCR2-CCR5 haplotypes were not associated with risk of CHD. However, our data suggest a strong inverse association for certain CCR5 variants and early age of CHD onset.

Objective: Moderate alcohol consumption is associated with substantially lower risk of cardiovascular disease (CVD). We assessed the relationship between alcohol intake and inflammatory markers to partially explain this beneficial effect., Methods and Results: From two large prospective studies, we sampled 959 healthy male and 473 healthy female health professionals with reported alcohol intake. Markers of inflammation were soluble tumor necrosis factor-alpha receptors 1 and 2 (sTNF-R1 and sTNF-R2), C-reactive protein (CRP), and interleukin-6 (IL-6). We found significant inverse linear trends for sTNF-R1 (p-trend<0.001 men; 0.03 women) and sTNF-R2 (p-trend=0.002 men; 0.08 women) with increasing alcohol intake. Compared to non-drinkers, men who consumed on average 1-2 drinks/day had 26% lower CRP (-0.66 mg/L, p=0.13), and 36% lower IL-6 (-1.12 pg/ml, p=0.02) levels. Among women, a similar though stronger association was observed at half drink per day. Compared to non-drinkers, both men and women who consumed 1-2 drinks/drinking day had significantly lower sTNF-R1 (-9% in men, -6% in women) and sTNF-R2 (-7% in men, -6% in women) levels as well as lower CRP (-10% in men, -32% in women) and IL-6 (-45% in men, -27% in women) levels., Conclusions: Alcohol in moderation is associated with lower levels of inflammatory markers and may lower risk of CVD through these mechanisms.

Objective: Activation of the peroxisome proliferator-activated receptor-gamma (PPARgamma) improves insulin sensitivity and exerts antiatherogenic effects. A common alanine for proline substitution at codon 12 in the PPARG2 gene is related to lower receptor activity. Studies suggest that the A12 allele is associated with reduced risk of type 2 diabetes; however, data on the risk of coronary heart disease (CHD) are scarce and controversial., Methods and Results: We examined the relationship between PPARG2 P12A and CHD risk in women (Nurses' Health Study) and men (Health Professionals Follow-Up Study) in nested case control settings. Among participants free of cardiovascular disease at baseline, 249 women and 266 men developed nonfatal myocardial infarction (MI) or fatal CHD during 8 and 6 years of follow-up, respectively. Using risk-set sampling, controls were selected 2:1 matched on age, smoking, and date of blood draw. The relative risk (RR) of nonfatal MI or fatal CHD of carriers compared with noncarriers of the A12 allele was 1.17 (95% CI, 0.82 to 1.68) among women and 1.44 (95% CI, 1.00 to 2.07) among men (pooled RR, 1.30 [95% CI, 1.00 to 1.67]). We found a significantly increased risk associated with the A12 allele among individuals with a body mass index > or =25 kg/m2 (women: RR, 1.88; 95% CI, 1.01 to 3.50; men: RR, 1.55; 95% CI, 0.92 to 2.60; pooled: RR, 1.68; 95% CI, 1.13 to 2.50) but not among those <25 kg/m2 (pooled RR, 0.86; 95% CI, 0.37 to 1.97; P heterogeneity overweight versus nonoverweight 0.16)., Conclusions: These data do not support the hypothesis that the A12 allele is associated with a decreased risk of CHD. The potential interaction between PPARG2 P12A, overweight, and increased CHD risk needs further evaluation.

Kidney dysfunction and high C-reactive protein (CRP) levels are independently associated with coronary events. However, it is unclear whether the risk of coronary events associated with decreased kidney function is at least partially mediated by inflammation and whether the association between inflammatory biomarkers and coronary events is influenced by level of kidney function. With the use of a prospective, nested, case-control study design, the association among kidney function, inflammatory biomarker levels, and coronary events was studied. A total of 244 women who were participants in the Nurses' Health Study and had no history of cardiovascular disease received a diagnosis of an incident coronary event (defined as nonfatal myocardial infarction or death as a result of coronary disease) during the follow-up period from 1990 to 1998 and were matched to 486 control subjects. Serum creatinine and inflammatory biomarker levels were measured in blood samples collected in 1989. Creatinine clearance (CrCl) was estimated using creatinine, age, weight, and height. In multivariate analyses, the odds ratio (OR) for a coronary event in women with an estimated CrCl <60 ml/min was 2.33 (95% confidence interval [CI], 1.01 to 5.38) compared with those with a CrCl > or =90 ml/min. When soluble tumor necrosis factor receptor (sTNFR) I and II levels were added into this model individually, the observed OR for women with CrCl <60 ml/min was attenuated. In analyses stratified by estimated CrCl, higher high-sensitivity CRP (hs-CRP), IL-6, and sTNFR I and II levels each were significantly associated with an increased odds of coronary events in women with an estimated CrCl < or =74 ml/min but not in women with an estimated CrCl > or =75 ml/min. The OR per 5-mg/L unit increase in hs-CRP was 1.68 (95% CI, 1.13 to 2.52) for women with an estimated CrCl < or =74 ml/min, compared with 1.23 (95% CI, 0.86 to 1.76) and 0.99 (95% CI, 0.76 to 1.29) for women with an estimated CrCl 75 to 89 and > or =90 ml/min, respectively (P = 0.004 for interaction). In conclusion, kidney dysfunction is associated with an increased odds of coronary events, and inflammation, as assessed by higher sTNFR I and II levels, may mediate some of this risk. Higher inflammatory biomarkers levels, specifically, hs-CRP, IL-6, and sTNFR I and II, were significantly associated with coronary events only in women with reduced kidney function. These findings warrant further investigation in other populations.

Efficacy and safety of a low-molecular-weight heparin (LMWH) were studied in 33 stable maintenance hemodialysis patients who had a bleeding tendency on unfractionated heparin. The optimal dose of LMWH for each patient was titrated before the study; the mean total LMWH dosage was 1,152 +/- 574 IU. No major bleeding or clot formation was noted in a total of 2,470 hemodialysis sessions during 6 months of LMWH administration. The mean value of plasma anti-factor Xa (anti-Xa) activity increased from 0.05 +/- 0.03 IU/ml before dialysis to 0.34 +/- 0.28 IU/ml after 2 h of dialysis and returned to 0.15 +/- 0.09 IU/ml after 4 h of dialysis; the mean activated partial thromboplastin time was 26.1 +/- 4.4 s before dialysis, 30.7 +/- 9.5 s (an 18% increase) after 2 h of dialysis, and 26.2 +/- 4.4 s after 4 h of dialysis. No significant change in serum antithrombin levels was noted throughout the whole study period. We conclude that a low dosage of LMWH is safe and effective in hemodialysis patients who have a risk of bleeding with unfractionated heparin. Serum anti-Xa activity is better than activated partial thromboplastin time and antithrombin in assessing the optimal dose of LMWH. A plasma anti-Xa activity of 0.37 IU/ml after 2 h of hemodialysis may represent an optimal dosage of LMWH for most patients.

The association of mutant forms of Ras protein with a variety of human cancers has stimulated intense interest in therapies based on inhibiting oncogenic Ras signaling. Attachment of Ras proteins to the plasma membrane is required for effective Ras signaling and is initiated by the enzyme farnesyl protein transferase. We found that in the presence of potent farnesyl protein transferase inhibitors, Ras proteins in the human colon carcinoma cell line DLD-1 were alternatively prenylated by geranylgeranyl transferase-1. When H-Ras, N-Ras, K-Ras4A, and K-Ras4B were expressed individually in COS cells, H-Ras prenylation and membrane association were found to be uniquely sensitive to farnesyl transferase inhibitors; N- and K-Ras proteins incorporated the geranylgeranyl isoprene group and remained associated with the membrane fraction. The alternative prenylation of N- and K-Ras has significant implications for our understanding of the mechanism of action of farnesyl protein transferase inhibitors as anti-cancer chemotherapeutics.

The mechanism of phospholipase D (PLD) activation by platelet-derived growth factor (PDGF) was examined using a NIH 3T3 fibroblast cell line (3T3-gamma 1) that stably overexpresses PLC-gamma 1 isozyme. Immunoblot analysis revealed that 3T3-gamma 1 cells contained about 10-fold more PLC-gamma 1 than a control cell line (3T3-C) transfected with expression vector lacking PLC-gamma 1 cDNA. PDGF-stimulated PLD activation was 10-fold greater in 3T3-gamma 1 cells than in 3T3-C cells, indicating that PLD activation is dependent upon the level of PLC-gamma 1. Phorbol 12-myristate 13-acetate (PMA) treatment increased PLD activity to a similar extent in both 3T3-gamma 1 cells and control cells. Pretreatment with tyrosine kinase inhibitors including staurosporine and genistein decreased PLD activity by 82.6% and 87.2%, respectively, and completely blocked tyrosine phosphorylation of PDGF receptor and PLC-gamma 1 in 3T3-gamma 1 cells stimulated with PDGF. Moreover, down-regulation of protein kinase C by pretreatment of PMA caused complete inhibition of PDGF- and PMA-stimulated PLD activation. Therefore, these results suggest that PDGF-induced PLD activation may be a consequence of primary stimulation of PLC-gamma 1 and that PLD may play a role downstream from PLC-gamma 1 in PDGF-triggered mitogenesis.

The functional significance of phospholipase D (PLD) could most easily be investigated using selective inhibitors. We have isolated a family of fungal metabolites, ketoepoxides, that inhibit chemotactic peptide (formyl-Met-Leu-Phe)-stimulated PLD activation and superoxide generation in granulocytes in the low micromolar range (SCH 49210 having an IC50 of 1.6 microM). Unlike receptor-mediated PLD activation, ketoepoxides were poor inhibitors of phorbol ester-induced PLD activity in granulocytes (IC50 = 43 microM for SCH 49210). Ketoepoxides did not inhibit platelet-derived growth factor-stimulated PLD activity in fibroblasts at up to 50 microM. We also tested the effect of ketoepoxides on in vitro epidermal growth factor receptor and neu tyrosine kinase activities. SCH 49210 (and 49209) did not inhibit the tyrosine kinases at up to 100 microM. These results suggest that ketoepoxides do not inhibit PLD activation due to effects on tyrosine kinase activity. fMLP-induced phospholipase A2 (PLA2) activation is also inhibited by ketoepoxides in the low micromolar range (SCH 49210 having an IC50 of 3.2 microM), but the ketoepoxides were poorer inhibitors of Ca2+ ionophore A23187-induced PLA2 (SCH 49210 having an IC50 of 83 microM). As a measure of phospholipase C (PLC) activity, the generation of inositol-1,4,5 triphosphate in thrombin-stimulated platelets was measured. The ketoepoxides did not inhibit PLC activation indicating that, unlike the aminosteroid U73122, ketoepoxides exhibit some selectivity among receptor-linked phospholipases. The ketoepoxides were also effective inhibitors of tumor cell invasion, as measured by penetration of HT1080 human fibrosarcoma cells into a reconstituted basement membrane matrix. Interestingly, both PLD inhibition and anti-tumor invasion activity correlate closely. These ketoepoxides are, therefore, potential anti-metastatic compounds and may be useful probes to study the role of PLD in cell function.

Rat 6 fibroblasts that stably overexpress cDNA for the beta 1 isozyme of protein kinase C (PKC3 cells) were used to determine the effect of protein kinase C (PKC) overexpression on hormonal stimulation of phospholipid hydrolysis. In control Rat 6 cells, inositol trisphosphate levels (InsP3) were increased 9-fold in 15 s in response to 10 nM alpha-thrombin, compared with only a 2-fold increase in PKC3 cells. PKC overexpression also inhibited thrombin-stimulated production of 1,2-diacylglycerol, the other product of phosphatidylinositol 4,5-bisphosphate hydrolysis, by 73% at 15 s. In permeabilized cells, PKC overexpression greatly reduced guanosine thiotriphosphate-stimulated InsP3 accumulation, but did not affect InsP3 stimulation by increased free calcium concentration. These data suggest that desensitization of thrombin-stimulated phosphoinositide-phospholipase C is enhanced by PKC-beta 1 overexpression and may involve modulation of G-protein/phospholipase C coupling. In contrast, thrombin was 4.5-fold more effective in stimulation of phosphatidylcholine-phospholipase D activity in PKC3 cells than in control cells, as determined by phosphatidylethanol formation. In permeabilized cells, guanosine thiotriphosphate also stimulated phospholipase D activity more effectively in PKC3 cells than in control cells, suggesting that upregulation of phospholipase D activity by PKC overexpression occurs distal to the thrombin receptor. These results suggest that PKC may act as a switch to up-regulate phosphatidylcholine-phospholipase D and down-regulate phosphoinositide-phospholipase C stimulations.

The binding of a variety of agonists to their receptors leads to the breakdown of membrane phospholipids and the formation of intracellular second messengers. Hydrolysis of inositol phospholipids by phospholipase C results in the formation of two second messengers, inositol-1,4,5-trisphosphate which mobilizes intracellular calcium and the neutral lipid diacylglycerol (DAG) which binds to and activates protein kinase C (PKC). PKC is actually a family of homologous serine/threonine protein kinases which play a central role in regulation of growth, differentiation and secretion reactions in a variety of cell types. In addition to these feedforward roles of PKC, it is thought to play an important feedback role, regulating early events in signal transduction. To explore these feedback functions we have examined the effect of PKC inhibitors on second messenger formation in thrombin-stimulated human platelets (a rapidly responding system) and the effect of PKC overexpression on second messenger formation and mitogenesis in rat fibroblasts (a system where sustained signaling occurs). Treatment of platelets with inhibitors of PKC potentiates DAG mass formation in response to thrombin while prior activation of PKC with phorbol esters blocks DAG mass formation, consistent with PKC playing a negative feedback role, inhibiting inositol phospholipid breakdown. DAG can also be formed by the sequential hydrolysis of phosphatidylcholine by phospholipase D and phosphatidic acid phosphohydrolase. This is a minor reaction in the rapidly responding platelet system, but may play a role in sustained signaling events. We have found that fibroblasts which overexpress the beta 1 isozyme of PKC display greatly enhanced DAG formation and phospholipase D activation in response to phorbol ester treatment. Upon stimulation of fibroblasts with thrombin, phospholipase D activation is also enhanced by PKC overexpression while formation of inositol phosphates is suppressed. These data suggest that PKC may act as a switch, terminating inositol phospholipid hydrolysis and activating the hydrolysis of phosphatidylcholine. Furthermore, we have observed a strong correlation between activation of phospholipase D and mitogenesis, suggesting an important role for this enzyme in long-term cellular responses to activation.

Endothelins (ETs) are a family of extremely potent vasoconstrictor peptides. In addition, ET-1 acts as a potent mitogen and activates phospholipase C in smooth muscle cells and fibroblasts. We examined the effects of ET-1 on phosphatidylcholine (PC) metabolism and thymidine incorporation in control Rat-6 fibroblasts and in cells that overexpress protein kinase C beta 1 (PKC). PC pools were labeled with [3H]myristic acid, and formation of phosphatidylethanol (PEt), an unambiguous marker of phospholipase D (PLD) activation, was monitored. ET-1 stimulated much greater PEt formation in the PKC overexpressing cells. ET-1 action was dose-dependent with a half-maximal effect at 1.0 x 10(-9) M. With increasing ethanol concentrations, [3H]PEt formation increased at the expense of [3H]phosphatidic acid (PA). Propranolol, an inhibitor of PA phosphohydrolase, increased [3H]PA accumulation and decreased [3H]diacylglycerol (DAG) formation. These data are consistent with the formation of [3H]DAG from PC by the sequential action of PLD and PA phosphohydrolase. Phorbol esters are known to stimulate thymidine incorporation and PLD activity to a greater extent in PKC overexpressing cells than in control cells. ET-1 also stimulates thymidine incorporation to a greater extent in the PKC overexpressing cells. The effect of ET-1 on thymidine incorporation into DNA in the overexpressing cells was also dose-dependent with a half-maximal effect at 0.3 x 10(-9) M. Enhanced PLD activity induced by ET-1 in the overexpressing cells may contribute to the mitogenic response, especially in light of a possible role of the PLD product, PA, in regulation of cell growth.