PreSERVE-AMI: A Randomized, Double-Blind, Placebo-Controlled Clinical Trial of Intracoronary Administration of Autologous CD34+ Cells in Patients With Left Ventricular Dysfunction Post STEMI.

RATIONALE: Despite direct immediate intervention and therapy, ST-segment-elevation myocardial infarction (STEMI) victims remain at risk for infarct expansion, heart failure, reinfarction, repeat revascularization, and death. OBJECTIVE: To evaluate the safety and bioactivity of autologous CD34+ cell (CLBS10) intracoronary infusion in patients with left ventricular dysfunction post STEMI. METHODS AND RESULTS: Patients who underwent successful stenting for STEMI and had left ventricular dysfunction (ejection fraction≤48%) ≥4 days poststent were eligible for enrollment. Subjects (N=161) underwent mini bone marrow harvest and were randomized 1:1 to receive (1) autologous CD34+ cells (minimum 10 mol/L±20% cells; N=78) or (2) diluent alone (N=83), via intracoronary infusion. The primary safety end point was adverse events, serious adverse events, and major adverse cardiac event. The primary efficacy end point was change in resting myocardial perfusion over 6 months. No differences in myocardial perfusion or adverse events were observed between the control and treatment groups, although increased perfusion was observed within each group from baseline to 6 months (P<0.001). In secondary analyses, when adjusted for time of ischemia, a consistently favorable cell dose-dependent effect was observed in the change in left ventricular ejection fraction and infarct size, and the duration of time subjects was alive and out of hospital (P=0.05). At 1 year, 3.6% (N=3) and 0% deaths were observed in the control and treatment group, respectively. CONCLUSIONS: This PreSERVE-AMI (Phase 2, randomized, double-blind, placebo-controlled trial) represents the largest study of cell-based therapy for STEMI completed in the United States and provides evidence supporting safety and potential efficacy in patients with left ventricular dysfunction post STEMI who are at risk for death and major morbidity. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01495364.

Nitric Oxide Contributes to Vasomotor Tone in Hypertensive African Americans Treated With Nebivolol and Metoprolol.

Endothelial dysfunction is more prevalent in African Americans (AAs) compared with whites. The authors hypothesized that nebivolol, a selective ß1 -antagonist that stimulates nitric oxide (NO), will improve endothelial function in AAs with hypertension when compared with metoprolol. In a double-blind, randomized, crossover study, 19 AA hypertensive patients were randomized to a 12-week treatment period with either nebivolol 10 mg or metoprolol succinate 100 mg daily. Forearm blood flow (FBF) was measured using plethysmography at rest and after intra-arterial infusion of acetylcholine and sodium nitroprusside to estimate endothelium-dependent and independent vasodilation, respectively. Physiologic vasodilation was assessed during hand-grip exercise. Measurements were repeated after NO blockade with L-N(G) -monomethylarginine (L-NMMA) and after inhibition of endothelium-derived hyperpolarizing factor (EDHF) with tetraethylammonium chloride (TEA). NO blockade with L-NMMA produced a trend toward greater vasoconstriction during nebivolol compared with metoprolol treatment (21% vs 12% reduction in FBF, P=.06, respectively). This difference was more significant after combined administration of L-NMMA and TEA (P<.001). Similarly, there was a contribution of NO to exercise-induced vasodilation during nebivolol but not during metoprolol treatment. There were significantly greater contributions of NO and EDHF to resting vasodilator tone and of NO to exercise-induced vasodilation with nebivolol compared with metoprolol in AAs with hypertension.

Subclinical Vascular Dysfunction Associated with Metabolic Syndrome in African Americans and Whites.

CONTEXT: The diagnosis of metabolic syndrome (MetS) identifies individuals at risk for developing diabetes and cardiovascular disease. African Americans (AAs) have high rates of cardiovascular disease and subclinical vascular disease including arterial stiffness and microvascular dysfunction but have relatively low rates of MetS. OBJECTIVE: The objective of the study was to evaluate the relationship between MetS and vascular function in a biracial cohort with the hypothesis that the diagnosis of MetS underestimates subclinical vascular disease in AAs. DESIGN: We measured components of MetS in a community-based cohort of 951 AAs and white subjects (aged 48.8 ± 11 y, 47% AA, 55% female). MAIN OUTCOME MEASURES: Using digital pulse amplitude tonometry, we estimated the reactive hyperemia index (RHI), a measure of microvascular endothelial function. Using applanation tonometry (Sphygmocor), central augmentation index (CAIx) and pulse wave velocity (PWV) were measured as indices of wave reflections and arterial stiffness, respectively. RESULTS: MetS was present in 24.0% of subjects and was associated with increased PWV (P < .001) and CAIx (P < .001) and a trend to lower RHI (P = .068) in both races. However, in subjects without MetS, AAs had lower RHI (P < .001) and higher PWV (P = .003) and CAIx (P = .002) compared with white subjects. Addition of an extra MetS criterion point for AAs with hypertension eliminated the racial differences in PWV and CAIx but not RHI. CONCLUSION: Although MetS is associated with microvascular dysfunction and increased arterial stiffness in both racial groups, AAs without MetS have greater vascular dysfunction compared with whites. Additional weighting for hypertension in AAs attenuated the racial differences in subclinical disease associated with MetS.

Differential effects of nebivolol and metoprolol on arterial stiffness, circulating progenitor cells, and oxidative stress.

Unlike traditional beta receptor antagonists, nebivolol activates nitric oxide. We hypothesized that therapy with nebivolol compared with metoprolol would improve arterial stiffness, increase levels of circulating progenitor cells (PC), and decrease oxidative stress (OS). In a randomized, double-blind, cross-over study, 30 hypertensive subjects received either once daily nebivolol or metoprolol succinate for 3 months each. Pulse wave velocity and augmentation index were measured using tonometry. Flow cytometry was used to measure circulating PC. OS was measured as plasma aminothiols. Measurements were performed at baseline, and repeated at 3 and 6 months. No significant differences were present between the levels of OS, arterial stiffness, and PC numbers during treatment with metoprolol compared with nebivolol. In subgroup analyses of beta-blocker naïve subjects (n = 19), nebivolol reduced pulse wave velocity significantly compared with metoprolol (-1.4 ± 1.9 vs. -0.1 ± 2.2; P = .005). Both nebivolol and metoprolol increased circulating levels of CD34+/CD133 + PC similarly (P = .05), suggesting improved regenerative capacity.

Effects of storage-aged red blood cell transfusions on endothelial function in hospitalized patients.

BACKGROUND: Clinical and animal studies indicate that transfusions of older stored red blood cells (RBCs) impair clinical outcomes as compared to fresh RBC transfusions. It has been suggested that this effect is due to inhibition of nitric oxide (NO)-mediated vasodilation after transfusion of older RBC units. However, to date this effect has not been identified in human transfusion recipients. STUDY DESIGN AND METHODS: Forty-three hospitalized patients with transfusion orders were randomly assigned to receive either fresh (<14 days) or older stored (>21 days) RBC units. Before transfusion, and at selected time points after the start of transfusion, endothelial function was assessed using noninvasive flow-mediated dilation assays. RESULTS: After transfusion of older RBC units, there was a significant reduction in NO-mediated vasodilation at 24 hours after transfusion (p = 0.045), while fresh RBC transfusions had no effect (p = 0.231). CONCLUSIONS: This study suggests for the first time a significant inhibitory effect of transfused RBC units stored more than 21 days on NO-mediated vasodilation in anemic hospitalized patients. This finding lends further support to the hypothesis that deranged NO signaling mediates adverse clinical effects of older RBC transfusions. Future investigations will be necessary to address possible confounding factors and confirm these results.

Effect of progenitor cell mobilization with granulocyte-macrophage colony-stimulating factor in patients with peripheral artery disease: a randomized clinical trial.

IMPORTANCE: Many patients with peripheral artery disease (PAD) have walking impairment despite therapy. Experimental studies in animals demonstrate improved perfusion in ischemic hind limb after mobilization of bone marrow progenitor cells (PCs), but whether this is effective in patients with PAD is unknown. OBJECTIVE: To investigate whether therapy with granulocyte-macrophage colony-stimulating factor (GM-CSF) improves exercise capacity in patients with intermittent claudication. DESIGN, SETTING, AND PARTICIPANTS: In a phase 2 double-blind, placebo-controlled study, 159 patients (median [SD] age, 64 [8] years; 87% male, 37% with diabetes) with intermittent claudication were enrolled at medical centers affiliated with Emory University in Atlanta, Georgia, between January 2010 and July 2012. INTERVENTIONS: Participants were randomized (1:1) to received 4 weeks of subcutaneous injections of GM-CSF (leukine), 500 µg/day 3 times a week, or placebo. Both groups were encouraged to walk to claudication daily. MAIN OUTCOMES AND MEASURES: The primary outcome was peak treadmill walking time (PWT) at 3 months. Secondary outcomes were PWT at 6 months and changes in circulating PC levels, ankle brachial index (ABI), and walking impairment questionnaire (WIQ) and 36-item Short-Form Health Survey (SF-36) scores. RESULTS: Of the 159 patients randomized, 80 were assigned to the GM-CSF group. The mean (SD) PWT at 3 months increased in the GM-CSF group from 296 (151) seconds to 405 (248) seconds (mean change, 109 seconds [95% CI, 67 to 151]) and in the placebo group from 308 (161) seconds to 376 (182) seconds (change of 56 seconds [95% CI, 14 to 98]), but this difference was not significant (mean difference in change in PWT, 53 seconds [95% CI, -6 to 112], P = .08). At 3 months, compared with placebo, GM-CSF improved the physical functioning subscore of the SF-36 questionnaire by 11.4 (95% CI, 6.7 to 16.1) vs 4.8 (95% CI, -0.1 to 9.6), with a mean difference in change for GM-CSF vs placebo of 7.5 (95% CI, 1.0 to 14.0; P = .03). Similarly, the distance score of the WIQ improved by 12.5 (95% CI, 6.4 to 18.7) vs 4.8 (95% CI, -0.2 to 9.8) with GM-CSF compared with placebo (mean difference in change, 7.9 [95% CI, 0.2 to 15.7], P = .047). There were no significant differences in the ABI, WIQ distance and speed scores, claudication onset time, or mental or physical component scores of the SF-36 between the groups. CONCLUSIONS AND RELEVANCE: Therapy with GM-CSF 3 times a week did not improve treadmill walking performance at the 3-month follow-up. The improvements in some secondary outcomes with GM-CSF suggest that it may warrant further study in patients with claudication. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01041417.

Progenitor Cell Therapy to Treat Acute Myocardial Infarction: The Promise of High-Dose Autologous CD34(+) Bone Marrow Mononuclear Cells.

ST elevation myocardial infarction (STEMI) is associated with an increased risk for congestive heart failure and long-term mortality despite the widespread use of thrombolysis and catheter-based revascularization. The need for improved post-STEMI therapies has led to a surge of novel therapeutics, especially regenerative approaches using autologous mononuclear cells. Indeed, the past decade has been marked by a number of human trials studying the safety and efficacy of progenitor cell delivery in the post-STEMI setting. While a variety of cell types and delivery techniques have been utilized, directed therapy to the infarct-related artery has been the most widely used approach. From over 1300 subjects randomized in these studies, there is sufficient evidence to conclude that cell therapy after STEMI is uniformly safe, while the efficacy of this intervention for improving outcomes is less clear. Recent meta-analyses have highlighted the importance of both timing of cell delivery, as well as the type, quantity, and mobility of delivered cells as determinants of response. Here, we show the case in which higher doses of CD34(+) cells, which are more potent in terms of their migratory capacity, offer the best hope for preserving cardiac function following STEMI.

Racial differences in arterial stiffness and microcirculatory function between Black and White Americans.

BACKGROUND: Compared with whites, black Americans suffer from a disproportionate burden of cardiovascular disease (CVD). We hypothesized that racial differences in the prevalence of CVD could be attributed, in part, to impaired vascular function in blacks after adjustment for differences in risk factor burden. METHODS AND RESULTS: We assessed vascular function in 385 black and 470 white subjects (mean age, 48±11 years; 45% male). Using digital pulse amplitude tonometry (EndoPAT) we estimated the reactive hyperemia index (RHI), a measure of microvascular endothelial function, and peripheral augmentation index (PAT-AIx). Central augmentation index (C-AIx) and pulse-wave velocity (PWV) were measured as indices of wave reflections and arterial stiffness, respectively, using applanation tonometry (Sphygmocor). Compared with whites, blacks had lower RHI (2.1±0.6 versus 2.3±0.6, P<0.001), greater arterial wave reflections assessed as both PAT-AIx (20.4±21.5 versus 17.0±22.4, P=0.01) and CAIx (20.8±12.3 versus 17.5±13.3, P=0.001), and greater arterial stiffness, measured as PWV (7.4±1.6 versus 7.1±1.6 m/s, P=0.001). After adjustment for traditional CVD risk factors, black race remained a significant predictor of lower RHI and higher PAT-AIx and CAIx (all P<0.001) in all subjects and of higher PWV in men (P=0.01). Furthermore, these associations persisted in a subgroup analysis of "healthy" individuals free of CVD risk factors. CONCLUSION: Black race is associated with impaired microvascular vasodilatory function, and greater large arterial wave reflections and stiffness. Because impairment in these vascular indices may be associated with worse long-term outcomes, they may represent underlying mechanisms for the increased CVD risk in blacks.

Ethnic and gender differences in ambulatory blood pressure trajectories: results from a 15-year longitudinal study in youth and young adults.

BACKGROUND: Cross-sectional studies demonstrated ethnic and gender differences in ambulatory blood pressure patterns, but little is known about the longitudinal development of these differences. METHODS AND RESULTS: Twenty-four-hour ambulatory blood pressure was measured up to 12 times (5 times on average) over a 15-year period in 312 African Americans (AAs) and 351 European Americans aged 7 to 30 years. Multivariate individual growth curves across age were created for daytime and nighttime blood pressure jointly. For both daytime and nighttime systolic blood pressure (SBP), AAs and males had higher levels (P<0.001) than European Americans and females. Males also showed a greater increase with age (P<0.001) than females. For nighttime SBP, a faster increase of SBP with age (P<0.01) in AAs was additionally observed. The ethnic difference in nighttime SBP levels and its increase with age were significantly larger than in daytime SBP. For daytime and nighttime diastolic blood pressure, AAs had higher levels than European Americans (P<0.001), and this difference was significantly larger at night. From late adolescence onward, males showed a greater increase in diastolic blood pressure with age than females. Ethnic and gender differences persisted after adjustment for height, body mass index, socioeconomic status, and stress-related coping styles. Family history of essential hypertension explained ethnic differences in daytime SBP. CONCLUSIONS: We observed significant ethnic and gender differences in longitudinal trajectories of ambulatory blood pressure in youth and young adults. The blunted nocturnal decline and its exacerbation with age in AAs corroborate and extend findings of cross-sectional studies.

Anger suppression and adiposity modulate association between ADRB2 haplotype and cardiovascular stress reactivity.

OBJECTIVES: The purpose of this study was to examine how variation in the beta-2 adrenergic receptor gene (ADRB2), in combination with the moderating influences of race, body mass index (BMI), and anger expression style (anger-in, anger-out), affects blood pressure (BP) at rest and in response to acute laboratory stress. METHODS: Four hundred fifty adolescents (mean age = 18.5 +/- 2.7 years; 228 [124 males] whites and 222 [110 males] blacks completed two stressors (video game challenge, forehead cold pressor). Hemodynamic measures were taken before, during, and after each stressor. Stressors were separated by a 20-minute rest period. RESULTS: Frequency of detrimental haplotype (Gly16/Glu27) carrier status was greater among whites than blacks (p < .05). A significant three-way interaction among haplotype, BMI, and race for resting systolic blood pressure (SBP) found the highest BP level to be among high BMI carriers, but only for whites. A separate three-way interaction was found to be significant for haplotype, anger-in and race such that high anger-in carriers showed the highest level of resting SBP (p < .05) and total peripheral resistance (TPR) (p < .05) and the greatest TPR reactivity to the cold pressor task (p < .01). Post hoc analyses revealed these interactions with anger-in were only present among blacks. No significant interactions with anger-out for either ethnic group were observed. CONCLUSIONS: This study demonstrates modulating influences of BMI and anger expression styles on ADRB2 gene associations with hemodynamic function at rest and in response to laboratory stress. These findings support the hypothesis that consideration of gene-environment interactions may better characterize the role of ADRB2 variation in the development of stress-induced essential hypertension.

Sympathetic nervous system, genes and human essential hypertension.

The sympathetic nervous system (SNS) is the first line of defense in the response to environmental stress through its regulation of second-to-second changes in blood pressure (BP). Both the activity of the SNS and the therapeutic responses to SNS agonists and antagonists are known to be highly variable in the population. "Small" changes caused by single nucleotide polymorphisms (SNPs) of SNS genes may have considerable impact on SNS function and individualized hypertension treatment. In this review, we first describe the physiology of the SNS and its influence on cardiovascular and renal mechanisms of BP regulation. A thorough review of the role of genetic variability of various SNS genes in relation to the development of BP and essential hypertension (EH) follows. Given the vast number of SNS components, evaluations of multiple SNPs from multiple SNS genes are necessary for future association studies of BP and EH. One way to surpass the limitations and inconsistencies of previous association studies is to use a gene-based approach also referred to as indirect association, which takes all common variation within a candidate gene into account. In order to determine how SNS genes are differentially expressed or silenced, activated or inactivated against various environmental backgrounds, it is important to assess not only environmental and lifestyle risk factors such as diet, climate, chronic stress, but also personality characteristics such as hostility and coping styles. Uncovering relevant gene-gene and gene-environment interactions within the SNS cascade will not only enable early detection of EH risk but will also aid in the treatment of hypertensives through both non-pharmacological and pharmacological means.

Interactive effects of anger expression and ET-1 Lys198Asn polymorphism on vasoconstriction reactivity to behavioral stress.

BACKGROUND: Ineffective anger expression has been associated with essential hypertension (EH) and with blood pressure (BP) reactivity to stress. The ET-1/Lys198Asn polymorphism has been associated with increased resting BP and exaggerated vasoconstrictive mediated BP reactivity. African Americans (AAs) are at particular risk for development of EH, report greater anger difficulties, and exhibit greater vasoconstrictive reactivity than their European American (EA) counterparts. PURPOSE: The objective is to investigate a gene-environment model of stress reactivity in which anger expression, particularly in combination with ET-1 T allele carrier status and AA ethnicity, would be associated with the greatest vasoconstrictive reactivity in response to a behavioral stressor. METHODS: One hundred ninety-one AA and 197 EA normotensive young adults (M age=18.8+/-2.5 years) participated in the study. Total peripheral resistance index (TPRI) reactivity was assessed during a 10-min video game challenge. Anger expression was measured using Spielberger's Anger Expression Scale. RESULTS: A multiple regression model with TPRI reactivity as the dependent variable revealed a three-way interaction effect for anger management (i.e., AM=anger control minus anger out scores), ethnicity, and ET-1 polymorphism. Specifically, AA carriers of the ET-1 polymorphism with poor AM skills exhibited the greatest TPRI reactivity. CONCLUSIONS: Individuals with a genetic predisposition for exaggerated vasoconstriction who also display low AM skills may be at particular risk for development of stress-induced EH. Such individuals may particularly benefit from anger management training.

A gene-environment interaction model of stress-induced hypertension.

The case for a gene-environment interaction model of stress-induced hypertension is detailed in this paper. We hypothesize that repeated exposure to stress in combination with an environmentally and/or genetically mediated susceptibility may lead to the development of essential hypertension. Previously, we reviewed the evidence for a genetic influence on the two major intermediate phenotypes of our model: cardiovascular reactivity to psychological stress and stress-induced sodium retention, representing the cardiovascular and renal stress response, respectively. Here we first describe how genes underlying the physiological systems mediating the stress response of heart, vasculature, and kidney (i.e., the sympathetic nervous system, renin-angiotensin- aldosterone system and sodium reabsorption, and the endothelial system) may increase vulnerability to stress and confer susceptibility to development of essential hypertension. Next, we extend our model and review genes underlying three additional systems that may mediate the influence of stress on the development of essential hypertension: the parasympathetic nervous system, the serotonergic system, and the hypothamamus-pituitary-adrenal axis. The elucidation of our gene-environment interaction model of stress-induced essential hypertension will improve the understanding of the contribution of stress to the development of essential hypertension. This knowledge may lead to more effective primary and secondary prevention programs involving lifestyle interventions in which the role of stress, both acute and chronic, will be taken into account, particularly for individuals at increased genetic risk of essential hypertension.

Effects of NOS3 Glu298Asp polymorphism on hemodynamic reactivity to stress: influences of ethnicity and obesity.

Studies on the associations between the nitric oxide synthase gene (NOS3) Glu298Asp polymorphism and hypertension status or blood pressure (BP) levels have had inconsistent results. Potential moderating influences of ethnicity, sex, and obesity on the effects of the NOS3 polymorphism have not been examined. We evaluated the influence of these factors on associations between the NOS3 polymorphism, nitric oxide metabolites (NOx), and hemodynamics at rest and during stress. Subjects were 235 African American (AA) and 262 European American (EA) young adults (18.5+/-2.6 years). Hemodynamic measurements and blood samples for NOx assays were taken before and after a competitive video game challenge. Glu298Asp polymorphism was detected by polymerase chain reaction-restriction enzyme digestion assay. A regression model was built using genotypes, ethnicity, sex, and obesity (body mass index >85th percentile) and their interactions controlling for age; 20.1% of AAs and 49.8% of EAs were carriers of the Asp allele. AAs, regardless of obesity status, exhibited high diastolic blood pressure (DBP) reactivity unless they were nonobese and noncarriers of the Asp allele. EAs exhibited lower DBP reactivity unless they were obese Asp allele carriers. AA nonobese carriers exhibited the greatest total peripheral resistance reactivity. Obese Asp allele carriers exhibited the greatest increases in cardiac output and the greatest decrease in NOx to the stressor. Results indicate the importance of examining impact of BP control-related genetic polymorphisms within the context of moderating factors such as adiposity and ethnicity.

Control mechanisms in the regulation of telomerase reverse transcriptase expression in differentiating human teratocarcinoma cells.

Telomerase is active in about 90% of cancers and contributes to the immortality of cancer cells by maintaining the lengths of the ends of chromosomes. Undifferentiated embryonic human teratocarcinoma (HT) cells were found to express high levels of hTERT, the catalytic subunit of telomerase, and the hTERT promoter was unmethylated in these cells. Retinoic acid (RA)-induced differentiation led to hTERT gene silencing and increased methylation of the hTERT promoter. Treatment with trichostatin A, a histone deacetylase inhibitor, resulted in hTERT reactivation only in very early differentiating HT cells. After methylation patterns had been established within the hTERT promoter region in late differentiating cells, 5-azacytidine, a common demethylating agent, activated the hTERT gene but trichostatin A had no effect on hTERT transcription. These studies suggest that histone deacetylation is involved in early hTERT gene down-regulation and that DNA methylation may maintain silencing of the hTERT gene in these cells.

Differential maintenance and de novo methylating activity by three DNA methyltransferases in aging and immortalized fibroblasts.

Genomic methylation, which influences many cellular processes such as gene expression and chromatin organization, generally declines with cellular senescence although some genes undergo paradoxical hypermethylation during cellular aging and immortalization. To explore potential mechanisms for this process, we analyzed the methylating activity of three DNA methyltransferases (Dnmts) in aging and immortalized WI-38 fibroblasts. Overall maintenance methylating activity by the Dnmts greatly decreased during cellular senescence. In immortalized WI-38 cells, maintenance methylating activity was similar to that of normal young cells. Combined de novo methylation activity of the Dnmts initially decreased but later increased as WI-38 cells aged and was strikingly elevated in immortalized cells. To further elucidate the mechanisms for changes in DNA methylation in aging and immortalized cells, the individual Dnmts were separated and individually assessed for maintenance and de novo methylating activity. We resolved three Dnmt fractions, one of which was the major maintenance methyltransferase, Dnmt1, which declined steadily in activity with cellular senescence and immortalization. However, a more basic Dnmt, which has significant de novo methylating activity, increased markedly in activity in aging and immortalized cells. We have identified this methyltransferase as Dnmt3b which has an important role in neoplastic transformation but its role in cellular senescence and immortalization has not previously been reported. An acidic Dnmt we isolated also had increased de novo methylating activity in senescent and immortalized WI-38 cells. These studies indicate that reduced genome-wide methylation in aging cells may be attributed to attenuated Dnmt1 activity but that regional or gene-localized hypermethylation in aging and immortalized cells may be linked to increased de novo methylation by Dnmts other than the maintenance methyltransferase.