Christoph D. Garlichs

Monocytes coexpress endothelial and macrophagocytic lineage markers and form cord-like structures in Matrigel® under angiogenic conditions

OBJECTIVES It has been shown that circulating human non-adherent CD34+ cells coexpressing vascular endothelial growth factor (VEGF)-R2 and AC133 have the capacity to differentiate into adherent mature endothelial cells. However, prior studies have demonstrated that a much bigger subset of primary adherent mononuclear cells can also form endothelial progenitor cells (EPC). To determine the origin of the latter cell population we tested the hypothesis: do monocytes as a firmly adherent and plastic cell type have the potential to differentiate into an endothelial phenotype. METHODS CD34-/CD14+ monocytes were isolated from human peripheral blood by adherence separation and magnetic bead selection (purity >90%) and cultured on fibronectin-coated plastic dishes (medium containing VEGF 10 ng/ml, basic fibroblast growth factor (bFGF) 2 ng/ml, insulin like growth factor (IGF-1) 1 ng/ml, 20% fetal calf serum). RESULTS After 2 weeks of culture, using fluorescence activated cell analysis we observed a new expression of the endothelial markers von Willebrand factor (vWf), VE-cadherin (VE) and ec-NOS in 45.2, 12.4 and 9.8% of the cells, respectively. The proportion of cells expressing these markers further increased after 4 weeks (94.2, 89.7 and 58.8% of these cells, respectively). The proportion of CD45 expressing cells remained unchanged during this period. However, after 14 days the specific macrophage antigen CD68 was newly expressed in 62% of the analysed cells with a further increase to 90% after 28 days of culture. In three-dimensional gel (Matrigel the formation of cord- and tubular-like structures was observed. CONCLUSION The present data indicate that under angiogenic stimulation macrophages develop an endothelial phenotype with expression of specific surface markers and even form cord- and tubular-like structures in vitro suggesting that this cell population may be recruited for vasculogenesis.

Patients with acute coronary syndromes express enhanced CD40 ligand/CD154 on platelets

OBJECTIVE To investigate whether CD40L/CD154 on platelets and soluble CD40L/CD154 may play a role in the inflammatory process of acute coronary syndromes. DESIGN AND SETTING Observational study in a university hospital. PATIENTS 15 patients with acute myocardial infarction, 25 patients with unstable angina, 15 patients with stable angina, and 12 controls. MAIN OUTCOME MEASURES CD40L/CD154 on platelets, P-selectin/CD62P on platelets, soluble CD40L/CD154 serum concentrations. RESULTS Mean (SD) CD40L/CD154 expression on platelets was 6.2 (2.8) MFI (mean fluorescence intensity) in the infarct group, 11 (3.3) MFI in the unstable angina group (p < 0.001 v infarction), 3.6 (0.9) MFI in the stable angina group (p < 0.01 vinfarction; p < 0.001 v unstable angina), and 3.2 (1.0) MFI in the controls (p < 0.01v infarction; p < 0.001v unstable angina; NSv stable angina). Soluble CD40L/CD154 concentration was 5.2 (1.1) ng/ml in the infarct group, 4.2 (0.7) ng/ml in the unstable angina group (p < 0.001v infarction), 2.9 (1.0) ng/ml in stable angina group (p < 0.001 v infarction and unstable angina), and 3.0 (0.5) ng/ml in the controls (p < 0.001v infarction and unstable angina; NSv stable angina). At a six months follow up, there was lower expression of CD40L/CD154 on platelets in patients with unstable angina (12.3 (3.6) v 3.8 (1.2) MFI, p < 0.0001) and acute myocardial infarction (6.2 (2.8)v 3.5 (0.8) MFI, p < 0.01) compared with their admission values six months earlier. Patients with unstable angina who needed redo coronary angioplasty (PTCA) or who had recurrence of angina were characterised by increased CD40L/CD154 expression on platelets compared with the remainder of the study group (recurrence of angina: 12.7 (3.2) v 9.7 (1.6) MFI, p < 0.05; re-do PTCA: 14.3 (4.2) v10.3 (2.1) MFI, p < 0.05). CONCLUSIONS Both CD40L/CD154 on platelets and soluble CD40L/CD154 are raised in patients with unstable angina and myocardial infarction. These findings suggest that CD40–CD40L/CD154 interactions may play a pathogenic role in triggering and propagation of acute coronary syndromes.

Upregulation of CD40-CD40 Ligand (CD154) in Patients With Acute Cerebral Ischemia

Background and Purpose— Inflammation and hypercoagulability contribute to the development of acute cerebral ischemia. Both can be mediated by the CD40 system. This study investigated whether the CD40 system and related mediators are upregulated in patients with transient ischemic attack (TIA) or stroke. Methods— Seventeen patients with TIA, 60 patients with complete stroke, and 15 control subjects were investigated. CD154 and P-selectin were analyzed on platelets and CD40 on monocytes during and 3 months after acute cerebral ischemia by double-label flow cytometry. Blood concentrations of soluble CD154 and monocyte chemoattractant protein-1 (MCP-1) were evaluated. Results— Our main findings are as follows: (1) patients with acute cerebral ischemia showed a significant increase of CD154 on platelets and CD40 on monocytes compared with controls; (2) plasma levels of soluble CD154 were significantly higher in these patients; (3) these patients had significantly higher numbers of prothrombotic platelet-monocyte aggregates; (4) the chemoattractant MCP-1 was significantly elevated in cerebral ischemia; and (5) at 3 months’ follow-up, upregulation of CD154 still persisted in patients with previous acute cerebral ischemia. Conclusions— Patients with acute cerebral ischemia show upregulation of the CD40 system, which might contribute to the known proinflammatory, proatherogenic, and prothrombotic milieu found in these patients.

Distinct functions of chemokine receptor axes in the atherogenic mobilization and recruitment of classical monocytes

We used a novel approach of cytostatically induced leucocyte depletion and subsequent reconstitution with leucocytes deprived of classical (inflammatory/Gr1hi) or non‐classical (resident/Gr1lo) monocytes to dissect their differential role in atheroprogression under high‐fat diet (HFD). Apolipoprotein E‐deficient (Apoe−/−) mice lacking classical but not non‐classical monocytes displayed reduced lesion size and macrophage and apoptotic cell content. Conversely, HFD induced a selective expansion of classical monocytes in blood and bone marrow. Increased CXCL1 levels accompanied by higher expression of its receptor CXCR2 on classical monocytes and inhibition of monocytosis by CXCL1‐neutralization indicated a preferential role for the CXCL1/CXCR2 axis in mobilizing classical monocytes during hypercholesterolemia. Studies correlating circulating and lesional classical monocytes in gene‐deficient Apoe−/− mice, adoptive transfer of gene‐deficient cells and pharmacological modulation during intravital microscopy of the carotid artery revealed a crucial function of CCR1 and CCR5 but not CCR2 or CX3CR1 in classical monocyte recruitment to atherosclerotic vessels. Collectively, these data establish the impact of classical monocytes on atheroprogression, identify a sequential role of CXCL1 in their mobilization and CCR1/CCR5 in their recruitment.

Genetic Regulation of Serum Phytosterol Levels and Risk of Coronary Artery Disease

Background—Phytosterols are plant-derived sterols that are taken up from food and can serve as biomarkers of cholesterol uptake. Serum levels are under tight genetic control. We used a genomic approach to study the molecular regulation of serum phytosterol levels and potential links to coronary artery disease (CAD). Methods and Results—A genome-wide association study for serum phytosterols (campesterol, sitosterol, brassicasterol) was conducted in a population-based sample from KORA (Cooperative Research in the Region of Augsburg) (n=1495) with subsequent replication in 2 additional samples (n=1157 and n=1760). Replicated single-nucleotide polymorphisms (SNPs) were tested for association with premature CAD in a metaanalysis of 11 different samples comprising 13 764 CAD cases and 13 630 healthy controls. Genetic variants in the ATP-binding hemitransporter ABCG8 and at the blood group ABO locus were significantly associated with serum phytosterols. Effects in ABCG8 were independently related to SNPs rs4245791 and rs41360247 (combined P=1.6×10−50 and 6.2×10−25, respectively; n=4412). Serum campesterol was elevated 12% for each rs4245791 T-allele. The same allele was associated with 40% decreased hepatic ABCG8 mRNA expression (P=0.009). Effects at the ABO locus were related to SNP rs657152 (combined P=9.4×10−13). Alleles of ABCG8 and ABO associated with elevated phytosterol levels displayed significant associations with increased CAD risk (rs4245791 odds ratio, 1.10; 95% CI, 1.06 to 1.14; P=2.2×10−6; rs657152 odds ratio, 1.13; 95% CI, 1.07 to 1.19; P=9.4×10−6), whereas alleles at ABCG8 associated with reduced phytosterol levels were associated with reduced CAD risk (rs41360247 odds ratio, 0.84; 95% CI, 0.78 to 0.91; P=1.3×10−5). Conclusion—Common variants in ABCG8 and ABO are strongly associated with serum phytosterol levels and show concordant and previously unknown associations with CAD.

Transradial versus transfemoral approach for coronary angiography and intervention in patients above 75 years of age

Aims: The transradial (TR) approach has potentially lower complication rates than transfemoral (TF) approach coronary angiography. However, it may be technically more challenging, especially in elderly patients with alterations in vascular anatomy. We thus determined success rates, procedural data, and complication rates of TR angiography in comparison to the TF approach in elderly patients in a randomized, prospective trial. Methods and results: Four hundred consecutive patients ≥75 years with known or suspected coronary artery disease were included in the study. After exclusion of 93 patients with contraindications to the radial approach, 152 patients were randomized to the TR and 155 to TF coronary angiography and intervention. In 13 patients randomized to TR, cross‐over to TF was necessary (9%). Total examination time was significantly longer for the TR approach (18.1 vs. 15.0 min, P = 0.009), but no difference was found for fluoroscopy time, number of catheters used, or amount of contrast agent. The rate of major complications (bleeding requiring surgery or transfusion, stroke) was 0% for TR and 3.2% for TF approach (P < 0.001). Minor complications occurred in 1.3% versus 5.8% of patients (P < 0.001). Conclusion: In elderly patients, TR coronary angiography and intervention has a high technical success rate and lower complication rates than the TF approach.

Differential effects of statins on relevant functions of human monocyte-derived dendritic cells

Statins were shown to possess immunomodulating properties, but the mechanisms of statin effects on the immune system are poorly understood. We analyzed the influence of statins on professional antigen‐presenting dendritic cells (DC). Immature DC were cultivated from monocytes of healthy donors. DC maturation was induced by lipopolysaccharide (LPS; 1 μg/mL). Unstimulated and LPS‐stimulated DC were treated with simvastatin or atorvastatin (0.1–1 μM). The expression of CD40, CD83, CD86, and human leukocyte antigen‐DR on unstimulated and LPS‐stimulated DC was reduced significantly by statins, and the expression of Toll‐like receptor 2 (TLR2) and TLR4 on LPS‐stimulated DC was enhanced temporarily. Statins caused a significant reduction of endocytosis of fluorescein isothiocyanate‐dextran by DC. Statins significantly inhibited the basal secretion of interleukin (IL)‐6, IL‐8, IL‐12, and tumor necrosis factor α from unstimulated DC, and their release from LPS‐stimulated DC was enhanced. In mixed leukocyte reaction, preincubation of LPS‐stimulated DC with statins significantly suppressed their clustering with T cells and their ability to induce T cell proliferation, CD71, and CD25 up‐regulation on T cells and the secretion of interferon‐γ and IL‐2 from T cells. In conclusion, this study showed that statins suppressed endocytosis, basal secretion of proinflammatory cytokines, and the ability of DC to induce T cell proliferation, activation, and T helper cell type 1 differentiation. However, statin preincubation of LPS‐stimulated DC caused a further increase in their secretion of proinflammatory cytokines.

Connective Tissue Growth Factor Is Overexpressed in Complicated Atherosclerotic Plaques and Induces Mononuclear Cell Chemotaxis In Vitro

Objective—Atherosclerotic blood vessels overexpress connective tissue growth factor (CTGF) mRNA, but the role of CTGF in atherosclerosis remains controversial. To assess the hypothesis that CTGF is involved in atherosclerotic plaque progression, we investigated CTGF protein expression and distribution in the different types of plaque morphology. Methods and Results—Serial cross-sections of 45 human carotid plaques were immunohistochemically analyzed for the presence of CTGF protein, neovascularization (von Willebrand factor), macrophages (CD68), and T cells (CD3). The lesions were categorized according to American Heart Association (AHA) classification as fibrous (type IV and V) or complicated plaques (type VI). The levels of CTGF were significantly higher in complicated compared with fibrous plaques (P=0.002). CTGF accumulated particularly in the rupture-prone plaque shoulder and in the areas of neovascularization or infiltration with inflammatory cells. Macrophage-like cells stained positive for CTGF protein in plaques. Subsequent in vitro studies showed that although monocyte-derived macrophages do not produce CTGF on stimulation with transforming growth factor-β, lipopolysaccharide, or thrombin, they take it up from culture medium. Furthermore, CTGF induces mononuclear cell chemotaxis in a dose-dependent manner. ConclusionCTGF protein is significantly increased in complicated compared with fibrous plaques and may enhance monocyte migration into atherosclerotic lesions, thus contributing to atherogenesis.

Decrease of serum levels of the anti-inflammatory cytokine interleukin-10 in patients with advanced chronic heart failure

Inflammation plays a significant contributory role in the pathogenesis of chronic heart failure (CHF). Many studies have shown enhanced plasma levels of proinflammatory cytokines [i.e. tumour necrosis factor-alpha (TNF-alpha) and interleukin (IL)-6] in patients with CHF. However, there are only few reports on the regulation of anti-inflammatory cytokines such as IL-10. IL-10 has potent deactivating properties in macrophages and T-cells and thus acts as a down-regulator of cell-mediated immune responses. The aim of the present study was to assess whether serum concentrations of IL-10 significantly differ between patients with CHF and healthy control subjects. Patients with CHF [ n =50; 66.9+/-12.6 years; mean ejection fraction, 22.1+/-9.2%; New York Heart Association (NYHA) class II-IV] and 25 healthy controls (63.6+/-10.2 years) were examined. Of the 50 patients with CHF, 32 patients were taking aspirin (100 mg/day) and 33 patients had lipid-lowering therapy with a statin. Serum IL-10 as well as TNF-alpha concentrations were measured using commercially available immunoassays. Patients with CHF showed significantly lower IL-10 concentrations (2.3+/-1.9 compared with 5.2+/-2.3 pg/ml; P <0.001). Patients with advanced CHF (NYHA class III and IV) had the lowest IL-10 plasma levels. Aspirin and statin therapy did not significantly influence serum levels of IL-10. The ratio of TNF-alpha to IL-10 was significantly higher in patients with advanced CHF (NYHA class III and IV, ratio 3.2+/-1.2 and 3.1+/-1.1 respectively, compared with control 0.4+/-0.2; P <0.01). Our present study demonstrates significantly decreased serum levels of IL-10 in patients with advanced CHF. Since IL-10 is known as a potent anti-inflammatory cytokine, its decrease in advanced CHF may favour the inflammatory milieu in CHF.

Genome-wide association study identifies a new locus for coronary artery disease on chromosome 10p11.23

AIMS Recent genome-wide association (GWA) studies identified 10 chromosomal loci for coronary artery disease (CAD) or myocardial infarction (MI). However, these loci explain only a small proportion of the genetic variability of these pertinent diseases. We sought to identify additional CAD/MI loci by applying a three-stage approach. METHODS AND RESULTS We genotyped n = 1157 MI cases and n = 1748 controls from a population-based study population [German MI Family Study (GerMIFS) III (KORA)] with genome-wide SNP arrays. At this first stage, n = 462 SNPs showed association with MI at P<1 × 10(-3) in two-sided logistic regression. In a second stage, 415 of these SNPs were evaluated in silico in two independent GWA samples, the GerMIFS I (875 cases/1644 controls) and GerMIFS II (1222 cases/1298 controls). Nine SNPs, representing three regions, displayed consistent replication in this in silico analysis (P<0.05 for each GWA sample): five SNPs at 9p21.3, a well-known CAD/MI locus, two SNPs at 10p11.21, and two SNPs at 2p24.3. Wet-lab replication, i.e. the third stage, of SNP rs3739998 (representing the novel locus at 10p11.21, p.S1002T in the KIAA1462 gene) in additional 5790 cases and 5302 controls confirmed the association (P=9.54 × 10(-4)), but not for the 2p24.3 locus. The combined P-value across all stages for SNP rs3739998 is P=1.27 × 10(-11) [odds ratio (OR) = 1.15 (1.11-1.20)]. CONCLUSION Analysis of a GWA study followed by in silico and wet-lab replication steps identified the KIAA1462 gene, encoding a yet uncharacterized protein, on chromosome 10p11.23 with genome-wide significant association for CAD/MI. Further studies are needed to characterize the functional role of this locus in the aetiology of these diseases.