James T. Willerson

Rosuvastatin to Prevent Vascular Events in Men and Women with Elevated C-Reactive Protein

BACKGROUND Increased levels of the inflammatory biomarker high-sensitivity C-reactive protein predict cardiovascular events. Since statins lower levels of high-sensitivity C-reactive protein as well as cholesterol, we hypothesized that people with elevated high-sensitivity C-reactive protein levels but without hyperlipidemia might benefit from statin treatment. METHODS We randomly assigned 17,802 apparently healthy men and women with low-density lipoprotein (LDL) cholesterol levels of less than 130 mg per deciliter (3.4 mmol per liter) and high-sensitivity C-reactive protein levels of 2.0 mg per liter or higher to rosuvastatin, 20 mg daily, or placebo and followed them for the occurrence of the combined primary end point of myocardial infarction, stroke, arterial revascularization, hospitalization for unstable angina, or death from cardiovascular causes. RESULTS The trial was stopped after a median follow-up of 1.9 years (maximum, 5.0). Rosuvastatin reduced LDL cholesterol levels by 50% and high-sensitivity C-reactive protein levels by 37%. The rates of the primary end point were 0.77 and 1.36 per 100 person-years of follow-up in the rosuvastatin and placebo groups, respectively (hazard ratio for rosuvastatin, 0.56; 95% confidence interval [CI], 0.46 to 0.69; P<0.00001), with corresponding rates of 0.17 and 0.37 for myocardial infarction (hazard ratio, 0.46; 95% CI, 0.30 to 0.70; P=0.0002), 0.18 and 0.34 for stroke (hazard ratio, 0.52; 95% CI, 0.34 to 0.79; P=0.002), 0.41 and 0.77 for revascularization or unstable angina (hazard ratio, 0.53; 95% CI, 0.40 to 0.70; P<0.00001), 0.45 and 0.85 for the combined end point of myocardial infarction, stroke, or death from cardiovascular causes (hazard ratio, 0.53; 95% CI, 0.40 to 0.69; P<0.00001), and 1.00 and 1.25 for death from any cause (hazard ratio, 0.80; 95% CI, 0.67 to 0.97; P=0.02). Consistent effects were observed in all subgroups evaluated. The rosuvastatin group did not have a significant increase in myopathy or cancer but did have a higher incidence of physician-reported diabetes. CONCLUSIONS In this trial of apparently healthy persons without hyperlipidemia but with elevated high-sensitivity C-reactive protein levels, rosuvastatin significantly reduced the incidence of major cardiovascular events. (ClinicalTrials.gov number, NCT00239681.)

Direct Proinflammatory Effect of C-Reactive Protein on Human Endothelial Cells

BackgroundThe acute-phase reactant C-reactive protein (CRP) is an important risk factor for coronary heart disease. However, the possible effects of CRP on vascular cells are not known. Methods and ResultsWe tested the effects of CRP on expression of adhesion molecules in both human umbilical vein and coronary artery endothelial cells. Expression of vascular cell adhesion molecule (VCAM-1), intercellular adhesion molecule (ICAM-1), and E-selectin was assessed by flow cytometry. Incubation with recombinant human CRP (10 &mgr;g/mL) for 24 hours induced an ≈10-fold increase in expression of ICAM-1 and a significant expression of VCAM-1, whereas a 6-hour incubation induced significant E-selectin expression. Adhesion molecule induction was similar to that observed in endothelial cells activated with interleukin-1&bgr;. In coronary artery endothelial cells, induction of ICAM-1 and VCAM-1 was already present at 5 &mgr;g/mL and reached a maximum at 50 &mgr;g/mL, at which point a substantial increase in expression of E-selectin was also evident. The CRP effect was dependent on presence of human serum in the culture medium, because no effect was seen in cells cultured with serum-free medium. In contrast, interleukin-1&bgr; was able to induce adhesion molecule expression in the absence of human serum. ConclusionsCRP induces adhesion molecule expression in human endothelial cells in the presence of serum. These findings support the hypothesis that CRP may play a direct role in promoting the inflammatory component of atherosclerosis and present a potential target for the treatment of atherosclerosis.

Transendocardial, Autologous Bone Marrow Cell Transplantation for Severe, Chronic Ischemic Heart Failure

Background—This study evaluated the hypothesis that transendocardial injections of autologous mononuclear bone marrow cells in patients with end-stage ischemic heart disease could safely promote neovascularization and improve perfusion and myocardial contractility. Methods and Results—Twenty-one patients were enrolled in this prospective, nonrandomized, open-label study (first 14 patients, treatment; last 7 patients, control). Baseline evaluations included complete clinical and laboratory evaluations, exercise stress (ramp treadmill), 2D Doppler echocardiogram, single-photon emission computed tomography perfusion scan, and 24-hour Holter monitoring. Bone marrow mononuclear cells were harvested, isolated, washed, and resuspended in saline for injection by NOGA catheter (15 injections of 0.2 cc). Electromechanical mapping was used to identify viable myocardium (unipolar voltage ≥6.9 mV) for treatment. Treated and control patients underwent 2-month noninvasive follow-up, and treated patients alone underwent a 4-month invasive follow-up according to standard protocols and with the same procedures used as at baseline. Patient population demographics and exercise test variables did not differ significantly between the treatment and control groups; only serum creatinine and brain natriuretic peptide levels varied in laboratory evaluations at follow-up, being relatively higher in control patients. At 2 months, there was a significant reduction in total reversible defect and improvement in global left ventricular function within the treatment group and between the treatment and control groups (P =0.02) on quantitative single-photon emission computed tomography analysis. At 4 months, there was improvement in ejection fraction from a baseline of 20% to 29% (P =0.003) and a reduction in end-systolic volume (P =0.03) in the treated patients. Electromechanical mapping revealed significant mechanical improvement of the injected segments (P <0.0005) at 4 months after treatment. Conclusions—Thus, the present study demonstrates the relative safety of intramyocardial injections of bone marrow–derived stem cells in humans with severe heart failure and the potential for improving myocardial blood flow with associated enhancement of regional and global left ventricular function.

Modulation of C-Reactive Protein–Mediated Monocyte Chemoattractant Protein-1 Induction in Human Endothelial Cells by Anti-Atherosclerosis Drugs

Background—C-reactive protein (CRP) induces adhesion molecule expression by endothelial cells. However, the effects of CRP on chemokine expression by endothelial cells are not known. Methods and Results—We tested the effects of CRP on the production of the chemokines monocyte chemoattractant protein-1 (MCP-1) and RANTES in cultured human umbilical vein endothelial cells. The secretion of chemokines was assessed by ELISA. Incubation with 100 &mgr;g/mL recombinant human CRP induced a 7-fold increase in MCP-1 but no change in RANTES secretion. We showed that the effect of CRP on MCP-1 was present even at 5 &mgr;g/mL CRP, with stepwise increases as the CRP concentration was increased to 10, 50, and 100 &mgr;g/mL. The effect of CRP on MCP-1 induction was not influenced by aspirin (at concentrations up to 1 mmol/L), but it was significantly inhibited by 5 &mgr;mol/L simvastatin. The peroxisome proliferator-activated receptor-&agr; activators fenofibrate (100 &mgr;mol/L) and Wy-14649 (100 &mgr;mol/L) almost completely abolished the induction of MCP-1, but the peroxisome proliferator-activated receptor-&ggr; activator ciglitazone had only a moderate effect. Conclusions—These results further strengthen the role of CRP in the pathogenesis of vascular inflammation and, likely, atherosclerosis and provide a crucial insight into a novel mechanism of action of anti-atherosclerosis drugs such as simvastatin and fenofibrate.

Reduction in C-reactive protein and LDL cholesterol and cardiovascular event rates after initiation of rosuvastatin: a prospective study of the JUPITER trial

BACKGROUND Statins lower high-sensitivity C-reactive protein (hsCRP) and cholesterol concentrations, and hypothesis generating analyses suggest that clinical outcomes improve in patients given statins who achieve hsCRP concentrations less than 2 mg/L in addition to LDL cholesterol less than 1.8 mmol/L (<70 mg/dL). However, the benefit of lowering both LDL cholesterol and hsCRP after the start of statin therapy is controversial. We prospectively tested this hypothesis. METHODS In an analysis of 15 548 initially healthy men and women participating in the JUPITER trial (87% of full cohort), we prospectively assessed the effects of rosuvastatin 20 mg versus placebo on rates of non-fatal myocardial infarction, non-fatal stroke, admission for unstable angina, arterial revascularisation, or cardiovascular death (prespecified endpoints) during a maximum follow-up of 5 years (median 1.9 years), according to on-treatment concentrations of LDL cholesterol (>/=1.8 mmol/L or <1.8 mmol/L) and hsCRP (>/=2 mg/L or <2 mg/L). We included all events occurring after randomisation. This trial is registered with ClinicalTrials.gov, number NCT00239681. FINDINGS Compared with placebo, participants allocated to rosuvastatin who achieved LDL cholesterol less than 1.8 mmol/L had a 55% reduction in vascular events (event rate 1.11 vs 0.51 per 100 person-years; hazard ratio [HR] 0.45, 95% CI 0.34-0.60, p<0.0001), and those achieving hsCRP less than 2 mg/L a 62% reduction (event rate 0.42 per 100 person-years; HR 0.38, 95% CI 0.26-0.56, p<0.0001). Although LDL cholesterol and hsCRP reductions were only weakly correlated in individual patients (r values <0.15), we recorded a 65% reduction in vascular events in participants allocated to rosuvastatin who achieved both LDL cholesterol less than 1.8 mmol/L and hsCRP less than 2 mg/L (event rate 0.38 per 100 person-years; adjusted HR 0.35, 95% CI 0.23-0.54), versus a 33% reduction in those who achieved one or neither target (event rate 0.74 per 100 person-years; HR 0.67, 95% CI 0.52-0.87) (p across treatment groups <0.0001). In participants who achieved LDL cholesterol less than 1.8 mmol/L and hsCRP less than 1 mg/L, we noted a 79% reduction (event rate 0.24 per 100 person-years; HR 0.21, 95% CI 0.09-0.52). Achieved hsCRP concentrations were predictive of event rates irrespective of the lipid endpoint used, including the apolipoprotein B to apolipoprotein AI ratio. INTERPRETATION For people choosing to start pharmacological prophylaxis, reduction in both LDL cholesterol and hsCRP are indicators of successful treatment with rosuvastatin.

Mesenchymal Stem Cells Differentiate into an Endothelial Phenotype, Enhance Vascular Density, and Improve Heart Function in a Canine Chronic Ischemia Model

Background—Bone marrow–derived stem cells are under investigation as a treatment for ischemic heart disease. Mesenchymal stem cells (MSCs) have been used preferentially in the acute ischemia model; data in the chronic ischemia model are lacking. Methods and Results—Twelve dogs underwent ameroid constrictor placement. Thirty days later, they received intramyocardial injections of either MSCs (100×106 MSCs/10 mL saline) (n=6) or saline only (10 mL) (controls) (n=6). All were euthanized at 60 days. Resting and stress 2D echocardiography was performed at 30 and 60 days after ameroid placement. White blood cell count (WBC), C-reactive protein (CRP), creatine kinase MB (CK-MB), and troponin I levels were measured. Histopathological and immunohistochemical analyses were performed. Mean left ventricular ejection fraction was similar in both groups at baseline but significantly higher in treated dogs at 60 days. WBC and CRP levels were similar over time in both groups. CK-MB and troponin I increased from baseline to 48 hours, eventually returning to baseline. There was a trend toward reduced fibrosis and greater vascular density in the treated group. MSCs colocalized with endothelial and smooth muscle cells but not with myocytes. Conclusions—In a canine chronic ischemia model, MSCs differentiated into smooth muscle cells and endothelial cells, resulting in increased vascularity and improved cardiac function.

Modulation of Vascular Inflammation In Vitro and In Vivo by Peroxisome Proliferator–Activated Receptor-γ Activators

BACKGROUND Peroxisome proliferator-activated receptor-gamma (PPARgamma) is expressed in atherosclerotic plaques and in endothelial cells. The possible effects of PPARgamma activators on endothelial activation and inflammatory response within the plaque are currently unknown. METHODS AND RESULTS We tested the hypothesis that PPARgamma activators inhibit vascular cell adhesion molecule (VCAM-1) and intercellular adhesion molecule (ICAM-1) expression in cultured endothelial cells (evaluated by flow cytometry) and homing of monocyte/macrophages to atherosclerotic plaques in vivo. In endothelial cells, the PPARgamma agonists troglitazone at 100 micromol/L and 15-deoxy-(Delta12,14)-prostaglandin J(2) (15d-PGJ2) at 20 micromol/L markedly attenuated the tumor necrosis factor-induced expression of VCAM-1 and ICAM-1. A significant inhibition of VCAM-1 expression was also evident at 5 and 10 micromol/L 15d-PGJ2 and 20 micromol/L troglitazone. Expression of E-selectin and PECAM-1 was not altered. To confirm the biological relevance of these results, we assessed the effects of troglitazone on monocyte/macrophage homing to atherosclerotic plaques in apoE-deficient mice. A 7-day treatment with troglitazone (400 mg/kg) significantly reduced monocyte/macrophage homing to atherosclerotic plaques (236+/-77 versus 177+/-43 macrophages, P=0.03); an even more striking inhibition was found at 3200 mg/kg troglitazone (344+/-76 versus 172+/-83 macrophages, P=0.005). CONCLUSIONS PPARgamma activators inhibit expression of VCAM-1 and ICAM-1 in activated endothelial cells and significantly reduce monocyte/macrophage homing to atherosclerotic plaques. These findings suggest that PPARgamma activators, currently used in treatment of type II diabetes, may have beneficial effects in modulating inflammatory response in atherosclerosis.

Transdifferentiation of human peripheral blood CD34+-enriched cell population into cardiomyocytes, endothelial cells, and smooth muscle cells in vivo.

Background—Adult human peripheral blood cells have been shown to differentiate into mature cells of nonhematopoietic tissues, such as hepatocytes and epithelial cells of the skin and gastrointestinal track. We investigated whether these cells could also transdifferentiate into human cardiomyocytes, mature endothelial cells, and smooth muscle cells in vivo. Methods and Results—Myocardial infarction was created in SCID mice by occluding the left anterior descending coronary artery, after which adult peripheral blood CD34+ cells were injected into the tail vein. Hearts were harvested 2 months after injection and stained for human leukocyte antigen (HLA) and markers for cardiomyocytes, endothelial cells, and smooth muscle cells. Cardiomyocytes, endothelial cells, and smooth muscle cells that bear HLA were identified in the infarct and peri-infarct regions of the mouse hearts. In a separate experiment, CD34+ cells were injected intraventricularly into mice without experimental myocardial infarction. HLA-positive myocytes and smooth muscle cells could only be identified in 1 of these mice killed at different time points. Conclusions—Adult peripheral blood CD34+ cells can transdifferentiate into cardiomyocytes, mature endothelial cells, and smooth muscle cells in vivo. However, transdifferentiation is augmented significantly by local tissue injury. The use of peripheral blood CD34+ cells for cell-based therapy should greatly simplify the procurement of cells for the regeneration of damaged myocardium.

Inflammatory Cytokines Stimulated C-Reactive Protein Production by Human Coronary Artery Smooth Muscle Cells

Background—Serum C-reactive protein (CRP) levels are good predictors of the development of cardiovascular events in apparently healthy men and women. CRP has been believed to be produced exclusively by hepatocytes during the acute-phase response. Several lines of evidence have suggested that atherosclerotic arteries can also produce CRP. However, the cell types that produce CRP locally in the atherosclerotic arterial wall have not been clearly identified. Methods and Results—Human coronary artery smooth muscle cells (HCASMCs) and human umbilical vein endothelial cells (HUVECs) were incubated with interleukin-1&bgr; (IL-1&bgr;), IL-6, their combination, tumor necrosis factor-&agr; (TNF-&agr;), or lipopolysaccharide (LPS) at different concentrations. The supernatants were concentrated and analyzed by a high-sensitivity enzyme-linked immunosorbent assay specific for human CRP. RNA was extracted from the HCASMCs for reverse transcriptase-polymerase chain reaction (RT-PCR) using specific primers for the CRP. Maximal CRP production was observed in HCASMCs after 48 hours of incubation with the combination of 25 ng/mL of IL-1&bgr; and 10 ng/mL of IL-6, whereas incubation with IL-1&bgr; or IL-6 alone only modestly induced CRP. Incubation with TNF-&agr; (50 ng/mL) or LPS (1000 EU/mL) resulted in an increase in CRP production comparable to the IL-1&bgr; and IL-6 combination. The induction of CRP in HCASMCs was independently confirmed by RT-PCR comparing the relative CRP mRNA levels. The induction of CRP production by HCASMCs was not reproduced in HUVECs, however. Conclusions—These results demonstrated that HCASMCs, but not HUVECs, could produce CRP in response to inflammatory cytokines. The locally produced CRP could directly participate in atherogenesis and the development of cardiovascular complications.

Suppression of canonical Wnt/β-catenin signaling by nuclear plakoglobin recapitulates phenotype of arrhythmogenic right ventricular cardiomyopathy

Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVC) is a genetic disease caused by mutations in desmosomal proteins. The phenotypic hallmark of ARVC is fibroadipocytic replacement of cardiac myocytes, which is a unique phenotype with a yet-to-be-defined molecular mechanism. We established atrial myocyte cell lines expressing siRNA against desmoplakin (DP), responsible for human ARVC. We show suppression of DP expression leads to nuclear localization of the desmosomal protein plakoglobin and a 2-fold reduction in canonical Wnt/beta-catenin signaling through Tcf/Lef1 transcription factors. The ensuing phenotype is increased expression of adipogenic and fibrogenic genes and accumulation of fat droplets. We further show that cardiac-restricted deletion of Dsp, encoding DP, impairs cardiac morphogenesis and leads to high embryonic lethality in the homozygous state. Heterozygous DP-deficient mice exhibited excess adipocytes and fibrosis in the myocardium, increased myocyte apoptosis, cardiac dysfunction, and ventricular arrhythmias, thus recapitulating the phenotype of human ARVC. We believe our results provide for a novel molecular mechanism for the pathogenesis of ARVC and establish cardiac-restricted DP-deficient mice as a model for human ARVC. These findings could provide for the opportunity to identify new diagnostic markers and therapeutic targets in patients with ARVC.