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Background: Wnt signaling controls the balance between stem cell proliferation and differentiation and body patterning throughout development. Previous data demonstrated that non-canonical Wnts (Wnt5a, Wnt11) increased cardiac gene expression of circulating endothelial progenitor cells (EPC) and bone marrow-derived stem cells cultured in vitro. Since previous studies suggested a contribution of the protein kinase C (PKC) family to the Wnt5a-induced signalling, we investigated which PKC isoforms are activated by non-canonical Wnt5a in human EPC. Methodology/Principal Findings: Immunoblot experiments demonstrated that Wnt5a selectively activated the novel PKC isoform, PKC delta, as evidenced by phosphorylation and translocation. In contrast, the classical Ca2+-dependent PKC isoforms, PKC alpha and beta2, and one of the other novel PKC isoforms, PKC epsilon, were not activated by Wnt5a. The PKC delta inhibitor rottlerin significantly blocked co-culture-induced cardiac differentiation in vitro, whereas inhibitors directed against the classical Ca2+-dependent PKC isoforms or a PKC epsilon-inhibitory peptide did not block cardiac differentiation. In accordance, EPC derived from PKC delta heterozygous mice exhibited a significant reduction of Wnt5a-induced cardiac gene expression compared to wild type mice derived EPC. Conclusions/Significance: These data indicate that Wnt5a enhances cardiac gene expressions of EPC via an activation of PKC delta.
Background: Circulating progenitor cells (CPC) contribute to the homeostasis of the vessel wall, and a reduced CPC count predicts cardiovascular morbidity and mortality. We tested the hypothesis that CPC count improves cardiovascular risk stratification and that this is modulated by low-grade inflammation. Methodology/Principal Findings: We pooled data from 4 longitudinal studies, including a total of 1,057 patients having CPC determined and major adverse cardiovascular events (MACE) collected. We recorded cardiovascular risk factors and high-sensitive C-reactive protein (hsCRP) level. Risk estimates were derived from Cox proportional hazard analyses. CPC count and/or hsCRP level were added to a reference model including age, sex, cardiovascular risk factors, prevalent CVD, chronic renal failure (CRF) and medications. The sample was composed of high-risk individuals, as 76.3% had prevalent CVD and 31.6% had CRF. There were 331 (31.3%) incident MACE during an average 1.7±1.1 year follow-up time. CPC count was independently associated with incident MACE even after correction for hsCRP. According to C-statistics, models including CPC yielded a non-significant improvement in accuracy of MACE prediction. However, the integrated discrimination improvement index (IDI) showed better performance of models including CPC compared to the reference model and models including hsCRP in identifying MACE. CPC count also yielded significant net reclassification improvements (NRI) for CV death, non-fatal AMI and other CV events. The effect of CPC was independent of hsCRP, but there was a significant more-than-additive interaction between low CPC count and raised hsCRP level in predicting incident MACE. Conclusions/Significance: In high risk individuals, a reduced CPC count helps identifying more patients at higher risk of MACE over the short term, especially in combination with a raised hsCRP level.
Quantification of circulating endothelial progenitor cells using the modified ISHAGE protocol
(2010)
Aims: Circulating endothelial progenitor cells (EPC), involved in endothelial regeneration, neovascularisation, and determination of prognosis in cardiovascular disease can be characterised with functional assays or using immunofluorescence and flow cytometry. Combinations of markers, including CD34+KDR+ or CD133+KDR+, are used. This approach, however may not consider all characteristics of EPC. The lack of a standardised protocol with regards to reagents and gating strategies may account for the widespread inter-laboratory variations in quantification of EPC. We, therefore developed a novel protocol adapted from the standardised so-called ISHAGE protocol for enumeration of haematopoietic stem cells to enable comparison of clinical and laboratory data. Methods and Results: In 25 control subjects, 65 patients with coronary artery disease (CAD; 40 stable CAD, 25 acute coronary syndrome/acute myocardial infarction (ACS)), EPC were quantified using the following approach: Whole blood was incubated with CD45, KDR, and CD34. The ISHAGE sequential strategy was used, and finally, CD45dimCD34+ cells were quantified for KDR. A minimum of 100 CD34+ events were collected. For comparison, CD45+CD34+ and CD45-CD34+ were analysed simultaneously. The number of CD45dimCD34+KDR+ cells only were significantly higher in healthy controls compared to patients with CAD or ACS (p = 0.005 each, p<0.001 for trend). An inverse correlation of CD45dimCD34+KDR+ with disease activity (r = -0.475, p<0.001) was confirmed. Only CD45dimCD34+KDR+ correlated inversely with the number of diseased coronaries (r = -0.344; p<0.005). In a second study, a 4-week de-novo treatment of atorvastatin in stable CAD evoked an increase only of CD45dimCD34+KDR+ EPC (p<0.05). CD45+CD34+KDR+ and CD45-CD34+KDR+ were indifferent between the three groups. Conclusion: Our newly established protocol adopted from the standardised ISHAGE protocol achieved higher accuracy in EPC enumeration confirming previous findings with respect to the correlation of EPC with disease activity and the increase of EPC during statin therapy. The data of this study show the CD45dim fraction to harbour EPC.
Intracarotid administration of human bone marrow mononuclear cells in rat photothrombotic ischemia
(2010)
Background: Increasing evidence suggests that cell therapy improves functional recovery in experimental models of stroke and myocardial infarction. So far only small pilot trials tested the effects of cell therapy in stroke patients, whereas large clinical trials were conducted in patients with ischemic heart disease. To investigate the therapeutic benefit of cell therapy to improve the recovery after stroke, we determined the efficacy of bone marrow derived mononuclear cells, which were shown to improve the recovery in experimental and clinical acute myocardial infarction studies, in a rat stroke model. Methods: Adult male Wistar rats were randomly assigned to receive either five million human bone marrow mononuclear cells (hBMC) or placebo intraarterially 3 days after photothrombotic ischemia. For immunosuppression the animals received daily injections of cyclosporine throughout the experiment, commencing 24 hours before the cell transplantation. A battery of behavioural tests was performed before and up to 4 weeks after ischemia. Results: Body temperature and body weight revealed no difference between groups. Neurological deficits measured by the Rotarod test, the adhesive-removal test and the cylinder test were not improved by hBMC transplantation compared to placebo. Conclusions: This study demonstrates that hBMC do not improve functional recovery when transplanted intraaterially 3 days after the onset of focal cerebral ischemia. A possible reason for the failed neurological improvement after cell therapy might be the delayed treatment initiation compared to other experimental stroke studies that showed efficacy of bone marrow mononuclear cells.
Background: Adaptation to low oxygen by changing gene expression is vitally important for cell survival and tissue development. The sprouting of new blood vessels, initiated from endothelial cells, restores the oxygen supply of ischemic tissues. In contrast to the transcriptional response induced by hypoxia, which is mainly mediated by members of the HIF family, there are only few studies investigating alternative splicing events. Therefore, we performed an exon array for the genome-wide analysis of hypoxia-related changes of alternative splicing in endothelial cells.
Methodology/Principal findings: Human umbilical vein endothelial cells (HUVECs) were incubated under hypoxic conditions (1% O(2)) for 48 h. Genome-wide transcript and exon expression levels were assessed using the Affymetrix GeneChip Human Exon 1.0 ST Array. We found altered expression of 294 genes after hypoxia treatment. Upregulated genes are highly enriched in glucose metabolism and angiogenesis related processes, whereas downregulated genes are mainly connected to cell cycle and DNA repair. Thus, gene expression patterns recapitulate known adaptations to low oxygen supply. Alternative splicing events, until now not related to hypoxia, are shown for nine genes: six which are implicated in angiogenesis-mediated cytoskeleton remodeling (cask, itsn1, larp6, sptan1, tpm1 and robo1); one, which is involved in the synthesis of membrane-anchors (pign) and two universal regulators of gene expression (cugbp1 and max).
Conclusions/Significance: For the first time, this study investigates changes in splicing in the physiological response to hypoxia on a genome-wide scale. Nine alternative splicing events, until now not related to hypoxia, are reported, considerably expanding the information on splicing changes due to low oxygen supply. Therefore, this study provides further knowledge on hypoxia induced gene expression changes and presents new starting points to study the hypoxia adaptation of endothelial cells.
1993 stellte die Entdeckung winziger Stückchen von Ribonukleinsäuren, heute als microRNAs bekannt, die Wissenschaftler vor ein Rätsel. Erstmals beobachtet wurden sie in dem Fadenwurm C. elegans, einem einfachen, vergleichsweise leicht durchschaubaren Organismus. Was die Wissenschaftler verwirrte, war die Tatsache, dass diese microRNAs ganz offensichtlich nicht für Proteine kodierten. Welche Funktion haben sie dann? Inzwischen weiß man, dass sie eine wichtige Rolle bei der Genregulation spielen. Und das nicht nur im Fadenwurm: MicroRNAs sind evolutionär hoch konserviert, sie kommen auch in höheren Organismen vor. Im Menschen sind mehr als 1500 microRNAs beschrieben, und man geht davon aus, dass mindestens 30 Prozent der Gene direkt durch microRNAs reguliert werden. Das lässt sich auch für therapeutische Zwecke nutzen. In unserer Arbeitsgruppe erforschen wir insbesondere die Rolle der microRNAs bei Herz- und Gefäß-Erkrankungen.
Cytokines play an important role in ischemic injury and repair. However, little is known about their prognostic value in cardiovascular disease. The aim of this study was to investigate the prognostic importance of chemokines CCL3/MIP-1α, CCL5/RANTES and CCL18/PARC for the risk of future cardiovascular events in patients with acute coronary syndromes (ACS). Baseline levels of CCL3/MIP-1α, CCL5/RANTES and CCL18/PARC were determined in ACS patients from the Bad Nauheim ACS II registry (n = 609). During the following 200 days, patients were monitored for the occurrence of fatal and non-fatal cardiovascular events. Patients with CCL3/MIP1α, CCL5/RANTES and CCL18/PARC concentrations in the highest tertile were associated with an increased risk of a fatal event during follow-up (HR: 2.19, 95%CI: 1.04–4.61 for CCL3/MIP1α, HR: 3.45, 95%CI: 1.54–7.72 for CCL5/RANTES and HR: 3.14, 95%CI: 1.33–7.46 for CCL18/PARC). This risk was highest for patients with all three biomarkers concentrations in the upper tertile (HR: 2.52, 95%CI: 1.11–5.65). Together with known risk predictors of cardiovascular events, CCL3/MIP-1α, CCL5/RANTES and CCL18/PARC combined improved the c-statistics from 0.74 to 0.81 (p = 0.007). In conclusion, CCL3/MIP-1α, CCL5/RANTES and CCL18/PARC are independently associated with the risk of short-term mortality in ACS patients. Combining all three biomarkers further increased their prognostic value.
Endothelial Wnt/β-catenin signaling is necessary for angiogenesis of the central nervous system and blood–brain barrier (BBB) differentiation, but its relevance for glioma vascularization is unknown. In this study, we show that doxycycline-dependent Wnt1 expression in subcutaneous and intracranial mouse glioma models induced endothelial Wnt/β-catenin signaling and led to diminished tumor growth, reduced vascular density, and normalized vessels with increased mural cell attachment. These findings were corroborated in GL261 glioma cells intracranially transplanted in mice expressing dominant-active β-catenin specifically in the endothelium. Enforced endothelial β-catenin signaling restored BBB characteristics, whereas inhibition by Dkk1 (Dickkopf-1) had opposing effects. By overactivating the Wnt pathway, we induced the Wnt/β-catenin–Dll4/Notch signaling cascade in tumor endothelia, blocking an angiogenic and favoring a quiescent vascular phenotype, indicated by induction of stalk cell genes. We show that β-catenin transcriptional activity directly regulated endothelial expression of platelet-derived growth factor B (PDGF-B), leading to mural cell recruitment thereby contributing to vascular quiescence and barrier function. We propose that reinforced Wnt/β-catenin signaling leads to inhibition of angiogenesis with normalized and less permeable vessels, which might prove to be a valuable therapeutic target for antiangiogenic and edema glioma therapy.