A Clinical Service to Support the Return of Secondary Genomic Findings in Human Research.

Human genome and exome sequencing are powerful research tools that can generate secondary findings beyond the scope of the research. Most secondary genomic findings are of low importance, but some (for a current estimate of 1%-3% of individuals) confer high risk of a serious disease that could be mitigated by timely medical intervention. The impact and scope of secondary findings in genome and exome sequencing will only increase in the future. There is considerable agreement that high-impact findings should be returned to participants, but many researchers performing genomic research studies do not have the background, skills, or resources to identify, verify, interpret, and return such variants. Here, we introduce a proposal for the formation of a secondary-genomic-findings service (SGFS) that would support researchers by enabling the return of clinically actionable sequencing results to research participants in a standardized manner. We describe a proposed structure for such a centralized service and evaluate the advantages and challenges of the approach. We suggest that such a service would be of greater benefit to all parties involved than present practice, which is highly variable. We encourage research centers to consider the adoption of a centralized SGFS.

The ClinSeq Project: piloting large-scale genome sequencing for research in genomic medicine.

ClinSeq is a pilot project to investigate the use of whole-genome sequencing as a tool for clinical research. By piloting the acquisition of large amounts of DNA sequence data from individual human subjects, we are fostering the development of hypothesis-generating approaches for performing research in genomic medicine, including the exploration of issues related to the genetic architecture of disease, implementation of genomic technology, informed consent, disclosure of genetic information, and archiving, analyzing, and displaying sequence data. In the initial phase of ClinSeq, we are enrolling roughly 1000 participants; the evaluation of each includes obtaining a detailed family and medical history, as well as a clinical evaluation. The participants are being consented broadly for research on many traits and for whole-genome sequencing. Initially, Sanger-based sequencing of 300-400 genes thought to be relevant to atherosclerosis is being performed, with the resulting data analyzed for rare, high-penetrance variants associated with specific clinical traits. The participants are also being consented to allow the contact of family members for additional studies of sequence variants to explore their potential association with specific phenotypes. Here, we present the general considerations in designing ClinSeq, preliminary results based on the generation of an initial 826 Mb of sequence data, the findings for several genes that serve as positive controls for the project, and our views about the potential implications of ClinSeq. The early experiences with ClinSeq illustrate how large-scale medical sequencing can be a practical, productive, and critical component of research in genomic medicine.

Infrared imaging of nitric oxide-mediated blood flow in human sickle cell disease.

Vascular dysfunction is an important pathophysiologic manifestation of sickle cell disease (SCD), a condition that increases risk of pulmonary hypertension and stroke. We hypothesized that infrared (IR) imaging would detect changes in cutaneous bloodflow reflective of vascular function. We performed IR imaging and conventional strain gauge plethysmography in twenty-five adults with SCD at baseline and during intra-arterial infusions of an endothelium-dependent vasodilator acetylcholine (ACh), an endothelium-independent vasodilator sodium nitroprusside (SNP), and a NOS inhibitor L-NMMA. Skin temperature measured by IR imaging increased in a dose-dependent manner to graded infusions of ACh (+1.1°C, p<0.0001) and SNP (+0.9°C, p<0.0001), and correlated with dose-dependent increases in forearm blood flow (ACh: +19.9 mL/min/100 mL, p<0.0001; r(s)=0.57, p=0.003; SNP: +8.6 mL/min/100 mL, p<0.0001; r=0.70, p=0.0002). Although IR measurement of skin temperature accurately reflected agonist-induced increases in blood flow, it was less sensitive to decreases in blood flow caused by NOS inhibition. Baseline forearm skin temperature measured by IR imaging correlated significantly with baseline forearm blood flow (31.8±0.2°C, 6.0±0.4 mL/min/100 mL; r=0.58, p=0.003), and appeared to represent a novel biomarker of vascular function. It predicted a blunted blood flow response to SNP (r=-0.61, p=0.002), and was independently associated with a marker of pulmonary artery pressure, as well as hemoglobin level, diastolic blood pressure, homocysteine, and cholesterol (R(2)=0.84, p<0.0001 for the model). IR imaging of agonist-stimulated cutaneous blood flow represents a less cumbersome alternative to plethysmography methodology. Measurement of baseline skin temperature by IR imaging may be a useful new marker of vascular risk in adults with SCD.

Phenotype and course of Hutchinson-Gilford progeria syndrome.

BACKGROUND: Hutchinson-Gilford progeria syndrome is a rare, sporadic, autosomal dominant syndrome that involves premature aging, generally leading to death at approximately 13 years of age due to myocardial infarction or stroke. The genetic basis of most cases of this syndrome is a change from glycine GGC to glycine GGT in codon 608 of the lamin A (LMNA) gene, which activates a cryptic splice donor site to produce abnormal lamin A; this disrupts the nuclear membrane and alters transcription. METHODS: We enrolled 15 children between 1 and 17 years of age, representing nearly half of the world's known patients with Hutchinson-Gilford progeria syndrome, in a comprehensive clinical protocol between February 2005 and May 2006. RESULTS: Clinical investigations confirmed sclerotic skin, joint contractures, bone abnormalities, alopecia, and growth impairment in all 15 patients; cardiovascular and central nervous system sequelae were also documented. Previously unrecognized findings included prolonged prothrombin times, elevated platelet counts and serum phosphorus levels, measured reductions in joint range of motion, low-frequency conductive hearing loss, and functional oral deficits. Growth impairment was not related to inadequate nutrition, insulin unresponsiveness, or growth hormone deficiency. Growth hormone treatment in a few patients increased height growth by 10% and weight growth by 50%. Cardiovascular studies revealed diminishing vascular function with age, including elevated blood pressure, reduced vascular compliance, decreased ankle-brachial indexes, and adventitial thickening. CONCLUSIONS: Establishing the detailed phenotype of Hutchinson-Gilford progeria syndrome is important because advances in understanding this syndrome may offer insight into normal aging. Abnormal lamin A (progerin) appears to accumulate with aging in normal cells. (ClinicalTrials.gov number, NCT00094393.)

Transcriptional profiling of CD133(+) cells in coronary artery disease and effects of exercise on gene expression.

BACKGROUND AIMS: Bone marrow (BM)-derived progenitor cells are under investigation for cardiovascular repair but may be altered by disease. Our aim was to identify differences in gene expression in CD133(+) cells of patients with coronary artery disease (CAD) and healthy controls, and determine whether exercise modifies gene expression. METHODS: CD133(+) cells were flow-sorted from 10 CAD patients and four controls, and total RNA was isolated for microarray-based gene expression profiling. Genes that were found to be differentially regulated in patients were analyzed further to investigate whether exercise had any normalizing effect on CD133(+) cells in CAD patients following 3 months of an exercise program. RESULTS: Improvement in effort tolerance and increases in the number of CD133(+) cells were observed in CAD patients after 3 months of exercise. Gene expression analysis of the CD133(+) cells identified 82 differentially expressed genes (2-fold cut-off, 25% false-discovery rate and % present calls) in patients compared with controls, of which 59 were found to be up-regulated and 23 down-regulated. These genes were found to be involved in carbohydrate metabolism, cell cycle, cellular development and signaling, and molecular transport. Following completion of the exercise program, gene expression patterns resembled those of controls in seven of 10 patients. CONCLUSIONS: Alterations in gene expression of BM-derived CD133(+) progenitor cells were found in CAD patients, which in part may be normalized by exercise.

Cell-free hemoglobin limits nitric oxide bioavailability in sickle-cell disease.

Although the deleterious vasoconstrictive effects of cell-free, hemoglobin-based blood substitutes have been appreciated, the systemic effects of chronic hemolysis on nitric oxide bioavailability have not been considered or quantified. Central to this investigation is the understanding that nitric oxide reacts at least 1,000 times more rapidly with free hemoglobin solutions than with erythrocytes. We hypothesized that decompartmentalization of hemoglobin into plasma would divert nitric oxide from homeostatic vascular function. We demonstrate here that plasma from patients with sickle-cell disease contains cell-free ferrous hemoglobin, which stoichiometrically consumes micromolar quantities of nitric oxide and abrogates forearm blood flow responses to nitric oxide donor infusions. Therapies that inactivate plasma hemoglobin by oxidation or nitric oxide ligation restore nitric oxide bioavailability. Decompartmentalization of hemoglobin and subsequent dioxygenation of nitric oxide may explain the vascular complications shared by acute and chronic hemolytic disorders.

Nitrite reduction to nitric oxide by deoxyhemoglobin vasodilates the human circulation.

Nitrite anions comprise the largest vascular storage pool of nitric oxide (NO), provided that physiological mechanisms exist to reduce nitrite to NO. We evaluated the vasodilator properties and mechanisms for bioactivation of nitrite in the human forearm. Nitrite infusions of 36 and 0.36 micromol/min into the forearm brachial artery resulted in supra- and near-physiologic intravascular nitrite concentrations, respectively, and increased forearm blood flow before and during exercise, with or without NO synthase inhibition. Nitrite infusions were associated with rapid formation of erythrocyte iron-nitrosylated hemoglobin and, to a lesser extent, S-nitroso-hemoglobin. NO-modified hemoglobin formation was inversely proportional to oxyhemoglobin saturation. Vasodilation of rat aortic rings and formation of both NO gas and NO-modified hemoglobin resulted from the nitrite reductase activity of deoxyhemoglobin and deoxygenated erythrocytes. This finding links tissue hypoxia, hemoglobin allostery and nitrite bioactivation. These results suggest that nitrite represents a major bioavailable pool of NO, and describe a new physiological function for hemoglobin as a nitrite reductase, potentially contributing to hypoxic vasodilation.

Microarray-based characterization of a colony assay used to investigate endothelial progenitor cells and relevance to endothelial function in humans.

OBJECTIVE: An assay proposed to quantify endothelial progenitor cell (EPC) colonies in humans was investigated to determine the phenotype of recovered cells and their relevance to in vivo endothelial function. METHODS AND RESULTS: Twelve sedentary subjects participating in a worksite wellness program underwent endothelial flow-mediated dilation (FMD) testing of the brachial artery and blood sampling for EPC colony assay. Microarray-based genotypic characterization of colonies showed surface markers consistent with T lymphocyte phenotype, but not with an EPC (CD34, CD133, VEGFR-2) or endothelial (CD146) phenotype. Gene expression patterns more closely matched T lymphocytes (r=0.87) than endothelial cells (r=0.66) in our microarray database. Flow cytometry of colonies confirmed large populations of CD3+CD45+ T cells (>75%) and few CD146+CD45- endothelial cells (<1%). Further, there was no correlation between colony number and the magnitude of FMD (r=-0.1512, P=0.6389). After exercise training, subjects improved FMD, from 6.7+/-2.0 to 8.7+/-1.9% (P=0.0043). Colonies also increased (P=0.0210), but without relation to FMD (r=0.1074, P=0.7396). T lymphocyte phenotype persisted after exercise (r=0.87). CONCLUSIONS: Cells in a commonly used EPC colony assay have a gene expression and cell surface marker profile consistent with a predominance of T lymphocytes and have an unclear relevance to endothelial function, either before or after exercise training.

Nitrite anion provides potent cytoprotective and antiapoptotic effects as adjunctive therapy to reperfusion for acute myocardial infarction.

BACKGROUND: Accumulating evidence suggests that the ubiquitous anion nitrite (NO2-) is a physiological signaling molecule, with roles in intravascular endocrine nitric oxide transport, hypoxic vasodilation, signaling, and cytoprotection. Thus, nitrite could enhance the efficacy of reperfusion therapy for acute myocardial infarction. The specific aims of this study were (1) to assess the efficacy of nitrite in reducing necrosis and apoptosis in canine myocardial infarction and (2) to determine the relative role of nitrite versus chemical intermediates, such as S-nitrosothiols. METHODS AND RESULTS: We evaluated infarct size, microvascular perfusion, and left ventricular function by histopathology, microspheres, and magnetic resonance imaging in 27 canines subjected to 120 minutes of coronary artery occlusion. This was a blinded, prospective study comparing a saline control group (n=9) with intravenous nitrite during the last 60 minutes of ischemia (n=9) and during the last 5 minutes of ischemia (n=9). In saline-treated control animals, 70+/-10% of the area at risk was infarcted compared with 23+/-5% in animals treated with a 60-minute nitrite infusion. Remarkably, a nitrite infusion in the last 5 minutes of ischemia also limited the extent of infarction (36+/-8% of area at risk). Nitrite improved microvascular perfusion, reduced apoptosis, and improved contractile function. S-Nitrosothiol and iron-nitrosyl-protein adducts did not accumulate in the 5-minute nitrite infusion, suggesting that nitrite is the bioactive intravascular nitric oxide species accounting for cardioprotection. CONCLUSIONS: Nitrite has significant potential as adjunctive therapy to enhance the efficacy of reperfusion therapy for acute myocardial infarction.

Targeted antagonism of CXCR4 mobilizes progenitor cells under investigation for cardiovascular disease.

BACKGROUND AIMS: Bone marrow (BM)-derived cells may repair cardiovascular injury but populations of interest circulate in small numbers. Cytokines such as granulocyte-colony-stimulating factor mobilize cells under investigation for this purpose, including CD133+ but require injections over multiple days and may promote inflammation. The purpose of this study was to evaluate the effects of a novel CXCR4 inhibitor (plerixafor), previously shown to mobilize CD34+ stem cells, on CD133+ mobilization and markers of inflammation. METHODS: Healthy subjects received a single subcutaneous injection of plerixafor in escalating doses: 240 mcg/kg (n = 3), 320 mcg/kg (n = 5) and 400 mcg/kg (n = 7). CD133+ and CD133+/VEGFR-2+ cells were measured by flow cytometry at baseline, then 4-6 h following plerixafor injection. Markers of inflammation in serum were measured at baseline, then again 10 h following injection of the 400 mcg/kg dose. RESULTS: Across all doses, white blood cells increased on average three-fold from baseline values. CD133+ cells increased on average 24-fold (from 616 +/- 141 cells/mL to 14 713 +/- 4423 cells/mL, P = 0.0064) without clear evidence of a dose effect. CD133+/VEGFR-2+ cells ranged from 0 to 20 cells/mL at baseline and from 0 to 124 cells/mL following plerixafor administration, although the rarity of these cells precluded a statistical analysis of this population. C-reactive protein and serum amyloid type A were not increased after the 400 mcg/kg dose. Pro-inflammatory cytokine levels were undetectable before and after plerixafor, except for macrophage inflammatory protein-1 beta, which increased slightly but significantly after the 400 mcg/kg dose of plerixafor (P = 0.0156). CONCLUSIONS: CD133+ cells are mobilized into the circulation following a single injection of the CXCR4 antagonist plerixafor, without clear evidence for systemic activation of inflammation. This effect may be of importance in cell-based approaches for treating cardiovascular diseases.

Variability of C-reactive protein levels among patients with stable coronary artery disease and on statin therapy.

BACKGROUND: High sensitivity C-reactive protein, a marker of inflammation, has been proposed to stratify coronary artery disease risk and is lowered by HMG-CoA reductase (statin) therapy. However, the reproducibility of persistently elevated hs-CRP levels and association with other markers of inflammation in patients with stable CAD on aggressive statin therapy is unknown. OBJECTIVES: To determine the reproducibility of hs-CRP levels measured within 2 weeks in patients with documented CAD with stable symptoms and to identify associations with other markers of inflammation. METHODS: Levels of hs-CRP were measured twice within 14 days (7 +/- 4) in 23 patients (22 males and 1 female, average age 66 +/- 10 years) with stable CAD and hs-CRP > or = 2.0 mg/L but < or = 10 mg/L at visit 1. All patients had received statins for cholesterol management (low density lipoprotein-cholesterol 84 +/- 25 mg/dl) with no dose change for > 3 months. None had a history or evidence of malignancy, chronic infection or inflammation, or recent trauma. There was no change in medications between visits 1 and 2, and no patient reported a change in symptoms or general health during this interval. White blood cell count and pro- and anti-inflammatory cytokines were measured at both visits. RESULTS: hs-CRP levels tended to be lower at visit 2 (median 2.4 mg/L, range 0.8-11 mg/L) than at visit 1 (median 3.3 mg/L, range 2.0-9.7; P = 0.1793). However, between the two visits hs-CRP levels decreased by more than 1.0 mg/L in 10 patients and increased by more than 1.0 mg/L in 4 patients. Changes in hs-CRP levels were unrelated to changes in levels of white blood cells (P = 0.4353). Of the cytokines tested, only the antiinflammatory cytokine interleukin-1 receptor antagonist and the pro-inflammatory cytokine interleukin-8 were above lower limits of detection, but there were no correlations between changes in these values and changes in hs-CRP (both P > 0.5). CONCLUSIONS: In stable CAD patients on aggressive statin therapy, hs-CRP levels may fluctuate over brief periods in the absence of changes in health, cardiac symptom status and medications, and without corroboration with other measures of inflammation. Accordingly, elevated hs-CRP levels should be interpreted with caution in this setting.

Nitrite infusion in humans and nonhuman primates: endocrine effects, pharmacokinetics, and tolerance formation.

BACKGROUND: The recent discovery that nitrite is an intrinsic vasodilator and signaling molecule at near-physiological concentrations has raised the possibility that nitrite contributes to hypoxic vasodilation and to the bioactivity of nitroglycerin and mediates the cardiovascular protective effects of nitrate in the Mediterranean diet. However, important questions of potency, kinetics, mechanism of action, and possible induction of tolerance remain unanswered. METHODS AND RESULTS: In the present study, we performed biochemical, physiological, and pharmacological studies using nitrite infusion protocols in 20 normal human volunteers and in nonhuman primates to answer these questions, and we specifically tested 3 proposed mechanisms of bioactivation: reduction to nitric oxide by xanthine oxidoreductase, nonenzymatic disproportionation, and reduction by deoxyhemoglobin. We found that (1) nitrite is a relatively potent and fast vasodilator at near-physiological concentrations; (2) nitrite functions as an endocrine reservoir of nitric oxide, producing remote vasodilation during first-pass perfusion of the opposite limb; (3) nitrite is reduced to nitric oxide by intravascular reactions with hemoglobin and with intravascular reductants (ie, ascorbate); (4) inhibition of xanthine oxidoreductase with oxypurinol does not inhibit nitrite-dependent vasodilation but potentiates it; and (5) nitrite does not induce tolerance as observed with the organic nitrates. CONCLUSIONS: We propose that nitrite functions as a physiological regulator of vascular function and endocrine nitric oxide homeostasis and suggest that it is an active metabolite of the organic nitrates that can be used therapeutically to bypass enzymatic tolerance.

Relation of endothelial function to cardiovascular risk in women with sedentary occupations and without known cardiovascular disease.

Our purpose was to determine predictors of endothelial function and potential association with cardiovascular risk in women with sedentary occupations, in whom obesity-associated risk factors may contribute to excess morbidity and mortality. Ninety consecutive women (age range 22 to 63 years, 22 overweight (body mass index [BMI] > or =25 to 29.9 kg/m(2)) and 42 obese (BMI > or = 30 kg/m(2)), had vital signs, lipids, insulin, glucose, high-sensitivity C-reactive protein, and sex hormones measured. Endothelial function was determined using brachial artery flow-mediated dilation after 5 minutes of forearm ischemia. Treadmill stress testing was performed with gas exchange analysis at peak exercise (peak oxygen consumption [Vo(2)]) to assess cardiorespiratory fitness. Brachial artery reactivity was negatively associated with Framingham risk score (r = -0.3542, p = 0.0007). Univariate predictors of endothelial function included peak Vo(2) (r = 0.4483, p <0.0001), age (r = -0.3420, p = 0.0010), BMI (r = -0.3065, p = 0.0035), and high-sensitivity C-reactive protein (r = -0.2220, p = 0.0400). Using multiple linear regression analysis with stepwise modeling, peak Vo(2) (p = 0.0003) was the best independent predictor of brachial artery reactivity, with age as the only other variable reaching statistical significance (p = 0.0436) in this model. In conclusion, endothelial function was significantly associated with cardiovascular risk in women with sedentary occupations, who were commonly overweight or obese. Even in the absence of routine exercise, cardiorespiratory fitness, rather than conventional risk factors or body mass, is the dominant predictor of endothelial function and suggests a modifiable approach to risk.

Predictors of endothelial function in employees with sedentary occupations in a worksite exercise program.

A sedentary workforce may be at increased risk for future cardiovascular disease. Exercise at the work site has been advocated, but effects on endothelium as a biomarker of risk and relation to weight loss, lipid changes, or circulating endothelial progenitor cells (EPCs) have not been reported. Seventy-two office and laboratory employees (58 women; average age 45 years, range 22 to 62; 26 with body mass index values >30 kg/m(2)) completed 3 months of participation in the National Heart, Lung, and Blood Institute's Keep the Beat program, with the determination of vital signs, laboratory data, and peak oxygen consumption (VO(2)) during treadmill exercise. Brachial artery endothelium was tested by flow-mediated dilation (FMD), which at baseline was inversely associated with Framingham risk score (r = -0.3689, p <0.0001). EPCs were quantified by colony assay. With exercise averaging 98 +/- 47 minutes each workweek, there was improvement in FMD (from 7.8 +/- 3.4% to 8.5 +/- 3.0%, p = 0.0096) and peak VO(2) (+1.2 +/- 3.1 ml O(2)/kg/min, p = 0.0028), with reductions in diastolic blood pressure (-2 +/- 8 mm Hg, p = 0.0478), total cholesterol (-8 +/- 25 mg/dl, p = 0.0131), and low-density lipoprotein cholesterol (-7 +/- 19 mg/dl, p = 0.0044) but with a marginal reduction in weight (-0.5 +/- 2.1 kg, p = 0.0565). By multiple regression modeling, lower baseline FMD, greater age, reductions in total and low-density lipoprotein cholesterol and diastolic blood pressure, and increases in EPC colonies and peak VO(2) were jointly statistically significant predictors of change in FMD and accounted for 47% of the variability in FMD improvement with program participation. Results were similar when modeling was performed for women only. In contrast, neither adiposity at baseline nor change in weight was a predictor of improved endothelial function. In conclusion, daily exercise achievable at their work sites by employees with sedentary occupations improves endothelial function, even with the absence of weight loss, which may decrease cardiovascular risk, if sustained.

First-in-human experience of an antidote-controlled anticoagulant using RNA aptamer technology: a phase 1a pharmacodynamic evaluation of a drug-antidote pair for the controlled regulation of factor IXa activity.

BACKGROUND: Selectivity, titratability, rapidity of onset, and active reversibility are desirable pharmacological properties of anticoagulant therapy administered for acute indications and collectively represent an attractive platform to maximize patient safety. A novel anticoagulation system (REG1, Regado Biosciences), developed using a protein-binding oligonucleotide to factor IXa (drug, RB006) and its complementary oligonucleotide antidote (RB007), was evaluated in healthy volunteers. The primary objective was to determine the safety profile and to characterize the pharmacodynamic responses in this first-in-human study. METHODS AND RESULTS: Regado 1a was a subject-blinded, dose-escalation, placebo-controlled study that randomized 85 healthy volunteers to receive a bolus of drug or placebo followed 3 hours later by a bolus of antidote or placebo. Pharmacodynamic samples were collected serially. Subject characteristics were the following: median age, 32 years (interquartile range, 23 to 39 years); female gender, 35%; and median weight, 79 kg (interquartile range, 70 to 87 kg). No significant differences were found in median hemoglobin, platelet, creatinine, or liver function studies. There were no significant bleeding signals associated with RB006, and overall, both drug and antidote were well tolerated. One serious adverse event, an episode of transient encephalopathy, occurred in a subject receiving the low intermediate dose of RB006. The subject's symptoms resolved rapidly, and no further sequelae occurred. A predictable dose-pharmacodynamic response, reflected in activated partial thromboplastin time measurements, was seen after administration of the bolus of drug, with a clear correlation between the peak posttreatment activated partial thromboplastin time and post hoc weight-adjusted dose of drug (correlation coefficient, 0.725; P<0.001). In subjects treated with drug, antidote administration reversed the pharmacological activity of the drug, with a rapid (mean time, 1 to 5 minutes across all dose levels) and sustained return of activated partial thromboplastin time to within the normal range. The activated clotting time followed a similar anticoagulant response and reversal pattern. As anticipated, prothrombin time remained unchanged compared with baseline. CONCLUSIONS: These observations represent a first-in-human experience of an RNA aptamer and its complementary oligonucleotide antidote used as an anticoagulant system. The findings contribute to an emerging platform of selective, actively reversible anticoagulant drugs for use among patients with thrombotic disorders of the venous and arterial circulations.

Granulocyte colony-stimulating factor mobilizes functional endothelial progenitor cells in patients with coronary artery disease.

OBJECTIVE: Endothelial progenitor cells (EPCs) that may repair vascular injury are reduced in patients with coronary artery disease (CAD). We reasoned that EPC number and function may be increased by granulocyte colony-stimulating factor (G-CSF) used to mobilize hematopoietic progenitor cells in healthy donors. METHODS AND RESULTS: Sixteen CAD patients had reduced CD34(+)/CD133(+) (0.0224+/-0.0063% versus 0.121+/-0.038% mononuclear cells [MNCs], P<0.01) and CD133(+)/VEGFR-2(+) cells, consistent with EPC phenotype (0.00033+/-0.00015% versus 0.0017+/-0.0006% MNCs, P<0.01), compared with 7 healthy controls. Patients also had fewer clusters of cells in culture, with out-growth consistent with mature endothelial phenotype (2+/-1/well) compared with 16 healthy subjects at high risk (13+/-4/well, P<0.05) or 14 at low risk (22+/-3/well, P<0.001) for CAD. G-CSF 10 microg/kg per day for 5 days increased CD34(+)/CD133(+) cells from 0.5+/-0.2/microL to 59.5+/-10.6/microL and CD133(+)/ VEGFR-2(+) cells from 0.007+/-0.004/microL to 1.9+/-0.6/microL (both P<0.001). Also increased were CD133(+) cells that coexpressed the homing receptor CXCR4 (30.4+/-8.3/microL, P<0.05). Endothelial cell-forming clusters in 10 patients increased to 27+/-9/well after treatment (P<0.05), with a decline to 9+/-4/well at 2 weeks (P=0.06). CONCLUSIONS: Despite reduced EPCs compared with healthy controls, patients with CAD respond to G-CSF with increases in EPC number and homing receptor expression in the circulation and endothelial out-growth in culture. Endothelial progenitor cells (EPCs) are reduced in coronary artery disease. Granulocyte colony-stimulating factor (CSF) administered to patients increased: (1) CD133+/VEGFR-2+ cells consistent with EPC phenotype; (2) CD133+ cells coexpressing the chemokine receptor CXCR4, important for homing of EPCs to ischemic tissue; and (3) endothelial cell-forming clusters in culture. Whether EPCs mobilized into the circulation will be useful for the purpose of initiating vascular growth and myocyte repair in coronary artery disease patients must be tested in clinical trials.

Statin attenuates increase in C-reactive protein during estrogen replacement therapy in postmenopausal women.

BACKGROUND: HMG-CoA reductase inhibitor (statin) therapy reduces cardiovascular risk, mechanisms of which may include diminished arterial inflammation, as suggested by reduction in levels of C-reactive protein (CRP). Because oral estrogens increase CRP in postmenopausal women, with potential inflammatory and thrombotic consequences that could compromise any benefit to cardiovascular risk, we determined whether the coadministration of a statin might modify the estrogenic effect on CRP. METHODS AND RESULTS: In a double-blind, 3-period crossover study, 28 postmenopausal women (average LDL cholesterol 163+/-36 mg/dL) were randomly assigned to daily conjugated equine estrogens (CEEs) 0.625 mg, simvastatin 10 mg, or their combination for 6 weeks, with each treatment period separated by 6 weeks. CEEs increased median CRP levels from 0.27 to 0.46 mg/dL, simvastatin decreased CRP from 0.29 to 0.28 mg/dL, and the therapies combined increased CRP from 0.28 to 0.36 mg/dL (all P< or =0.02 versus respective baseline values). Post hoc testing showed that the 29% increase in CRP on the combination of CEEs with simvastatin was significantly less than the 70% increase in CRP on CEEs alone (P<0.05). The effect of combination therapy on CRP levels did not correlate with baseline CRP or with baseline or treatment-induced changes in levels of interleukin-6, lipoproteins, or flow-mediated dilation of the brachial artery as a measure of nitric oxide bioactivity. CONCLUSIONS: The combination of statin with estrogen may attenuate the potential harmful effects of estrogen therapy in postmenopausal women and maximize any benefit to cardiovascular risk.

Cardiac sympathetic dysautonomia in chronic orthostatic intolerance syndromes.

BACKGROUND: In postural tachycardia syndrome (POTS) and repeated neurocardiogenic presyncope (NCS), orthostatic intolerance occurs without persistent sympathetic neurocirculatory failure. Whether these conditions involve abnormal cardiac sympathetic innervation or function has been unclear. METHODS AND RESULTS: Patients with POTS or NCS underwent measurements of neurochemical indices of cardiac release, reuptake, and synthesis of the sympathetic neurotransmitter norepinephrine based on entry of norepinephrine into the cardiac venous drainage (cardiac norepinephrine spillover), cardiac extraction of circulating (3)H-norepinephrine, and cardiac production of dihydroxyphenylalanine and measurement of left ventricular myocardial innervation density using 6-[(18)F]fluorodopamine positron emission tomographic scanning. Mean cardiac norepinephrine spillover in POTS (171+/-30 pmol/min, N=16) was higher and in NCS (62+/-9 pmol/min, N=20) was lower than in a large group of healthy volunteers (102+/-9 pmol/min, N=52) and in a subgroup of age-matched healthy women (106+/-18 pmol/min, N=11). Both patient groups had normal cardiac extraction of (3)H-norepinephrine, normal cardiac production of dihydroxyphenylalanine, and normal myocardial 6-[(18)F]fluorodopamine-derived radioactivity. CONCLUSIONS: POTS and NCS differ in tonic cardiac sympathetic function, with increased cardiac norepinephrine release in the former and decreased release in the latter. Both groups had normal values for indices of function of the cell membrane norepinephrine transporter, norepinephrine synthesis, and density of myocardial sympathetic innervation. Because POTS and NCS both include specific abnormalities of cardiac sympathetic function, both can be considered forms of dysautonomia.

Divergent nitric oxide bioavailability in men and women with sickle cell disease.

BACKGROUND: Although reduced endothelial nitric oxide (NO) bioavailability has been demonstrated in arteriosclerotic vascular disease, the integrity of this system in sickle cell disease remains uncertain. METHODS AND RESULTS: We measured forearm blood flow in 21 patients with sickle cell disease (hemoglobin SS genotype) and 18 black control subjects before and after intra-arterial infusions of acetylcholine, nitroprusside, and the NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA). Endothelium-dependent vasodilation, measured by the percent increase in flow induced by acetylcholine infusion, was significantly greater than in controls (252+/-37% for patients versus 134+/-24% for controls; P<0.0001). However, there was a large sex difference in blood flow responses between female and male patients (340+/-46% versus 173+/-41%; P=0.035). Similarly, basal NO bioactivity, as measured by the percent decrease in flow induced by L-NMMA, was depressed in male compared with female patients (-17+/-5% versus -34+/-4%; P=0.01), as was the response to nitroprusside (86+/-21% versus 171+/-22%; P=0.008). L-NMMA reduced the blood flow response to acetylcholine in women, but not in men. Sex differences in vascular cell adhesion molecule-1 were appreciated, with significant correlations between levels of soluble vascular cell adhesion molecule-1 and blood flow responses to L-NMMA and nitroprusside (r=0.53, P=0.004 and r=-0.66, P<0.001, respectively). CONCLUSIONS: NO bioavailability and NO responsiveness are greater in women than in men with sickle cell disease and determines adhesion molecule expression. Endothelium-dependent blood flows are largely non-NO mediated in male patients. These results provide a possible mechanism for reported sex differences in sickle cell disease morbidity and mortality and provide a basis for novel pharmacological interventions.