Hematopoietic interferon regulatory factor 8-deficiency accelerates atherosclerosis in mice.

OBJECTIVE: Inflammatory leukocyte accumulation drives atherosclerosis. Although monocytes/macrophages and polymorphonuclear neutrophilic leukocytes (PMN) contribute to lesion formation, sequelae of myeloproliferative disease remain to be elucidated. METHODS AND RESULTS: We used mice deficient in interferon regulatory factor 8 (IRF8(-/-)) in hematopoietic cells that develop a chronic myelogenous leukemia-like phenotype. Apolipoprotein E-deficient mice reconstituted with IRF8(-/-) or IRF8(-/-) apolipoprotein E-deficient bone marrow displayed an exacerbated atherosclerotic lesion formation compared with controls. The chronic myelogenous leukemia-like phenotype in mice with IRF8(-/-) bone marrow, reflected by an expansion of PMN in the circulation, was associated with an increased lesional accumulation and apoptosis of PMN, and enlarged necrotic cores. IRF8(-/-) compared with IRF8(+/+) PMN displayed unaffected reactive oxygen species formation and discharge of PMN granule components. In contrast, accumulating in equal numbers at sites of inflammation, IRF8(-/-) macrophages were defective in efferocytosis, lipid uptake, and interleukin-10 cytokine production. Importantly, depletion of PMN in low-density lipoprotein receptor or apolipoprotein E-deficient mice with IRF8(-/-) or IRF8(-/-) apolipoprotein E-deficient bone marrow abrogated increased lesion formation. CONCLUSIONS: These findings indicate that a chronic myelogenous leukemia-like phenotype contributes to accelerated atherosclerosis in mice. Among proatherosclerotic effects of other cell types, this, in part, is linked to an expansion of functionally intact PMN.

TGF-beta1 accelerates dendritic cell differentiation from common dendritic cell progenitors and directs subset specification toward conventional dendritic cells.

Dendritic cells (DCs) in lymphoid tissue comprise conventional DCs (cDCs) and plasmacytoid DCs (pDCs) that develop from common DC progenitors (CDPs). CDPs are Flt3(+)c-kit(int)M-CSFR(+) and reside in bone marrow. In this study, we describe a two-step culture system that recapitulates DC development from c-kit(hi)Flt3(-/lo) multipotent progenitors (MPPs) into CDPs and further into cDC and pDC subsets. MPPs and CDPs are amplified in vitro with Flt3 ligand, stem cell factor, hyper-IL-6, and insulin-like growth factor-1. The four-factor mixture readily induces self-renewal of MPPs and their progression into CDPs and has no self-renewal activity on CDPs. The amplified CDPs respond to all known DC poietins and generate all lymphoid tissue DCs in vivo and in vitro. Additionally, in vitro CDPs recapitulate the cell surface marker and gene expression profile of in vivo CDPs and possess a DC-primed transcription profile. TGF-ß1 impacts on CDPs and directs their differentiation toward cDCs. Genome-wide gene expression profiling of TGF-ß1-induced genes identified instructive transcription factors for cDC subset specification, such as IFN regulatory factor-4 and RelB. TGF-ß1 also induced the transcription factor inhibitor of differentiation/DNA binding 2 that suppresses pDC development. Thus, TGF-ß1 directs CDP differentiation into cDCs by inducing both cDC instructive factors and pDC inhibitory factors.

CD34+CD140b+ cells and circulating CXCL12 correlate with the angiographically assessed severity of cardiac allograft vasculopathy.

AIMS: We sought to determine whether circulating vascular progenitor cells, such as endothelial progenitor cells (EPCs) or smooth muscle progenitor cells (SPCs), were associated with the severity of cardiac allograft vasculopathy (CAV). METHODS AND RESULTS: CD34(+)CD140b(+) SPCs and CD34(+)KDR(+) EPCs were measured in the peripheral circulation of 187 adult heart transplant recipients by flow cytometry. Cardiac allograft vasculopathy was quantified by angiography using a CAV-specific scoring system. Cardiac allograft vasculopathy was present in 84 patients (44.7%) and was classified as mild in 59 and severe in 25 cases. Circulating SPCs were more frequently detectable in CAV patients than in patients without CAV. The number of CD34(+)CD140b(+) cells showed a stepwise increase in patients with moderate and severe CAV. Smooth muscle progenitor cell counts were higher in patients with coronary stent implant compared with unstented patients with CAV. In contrast, peripheral CD34(+)KDR(+) EPC counts were not changed in CAV patients. Plasma CXCL12 levels correlated with the degree of CAV and SPC counts. None of the different immunosuppressive drug regimes was related to the SPC count or the CXCL12 levels. A multivariate regression analysis revealed that the SPC count was independently associated with the presence of CAV. CONCLUSION: Circulating SPCs, but not EPCs, and plasma CXCL12 concentrations are elevated in CAV patients, indicating that they play prominent roles in transplant arteriosclerosis.

Hypoxia-induced endothelial secretion of macrophage migration inhibitory factor and role in endothelial progenitor cell recruitment.

Macrophage migration inhibitory factor (MIF) is a pleiotropic inflammatory cytokine that was recently identified as a non-cognate ligand of the CXC-family chemokine receptors 2 and 4 (CXCR2 and CXCR4). MIF is expressed and secreted from endothelial cells (ECs) following atherogenic stimulation, exhibits chemokine-like properties and promotes the recruitment of leucocytes to atherogenic endothelium. CXCR4 expressed on endothelial progenitor cells (EPCs) and EC-derived CXCL12, the cognate ligand of CXCR4, have been demonstrated to be critical when EPCs are recruited to ischemic tissues. Here we studied whether hypoxic stimulation triggers MIF secretion from ECs and whether the MIF/CXCR4 axis contributes to EPC recruitment. Exposure of human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells (HAoECs) to 1% hypoxia led to the specific release of substantial amounts of MIF. Hypoxia-induced MIF release followed a biphasic behaviour. MIF secretion in the first phase peaked at 60 min. and was inhibited by glyburide, indicating that this MIF pool was secreted by a non-classical mechanism and originated from pre-formed MIF stores. Early hypoxia-triggered MIF secretion was not inhibited by cycloheximide and echinomycin, inhibitors of general and hypoxia-inducible factor (HIF)-1α-induced protein synthesis, respectively. A second phase of MIF secretion peaked around 8 hrs and was likely due to HIF-1α-induced de novo synthesis of MIF. To functionally investigate the role of hypoxia-inducible secreted MIF on the recruitment of EPCs, we subjected human AcLDL(+) KDR(+) CD31(+) EPCs to a chemotactic MIF gradient. MIF potently promoted EPC chemotaxis in a dose-dependent bell-shaped manner (peak: 10 ng/ml MIF). Importantly, EPC migration was induced by supernatants of hypoxia-conditioned HUVECs, an effect that was completely abrogated by anti-MIF- or anti-CXCR4-antibodies. Thus, hypoxia-induced MIF secretion from ECs might play an important role in the recruitment and migration of EPCs to hypoxic tissues such as after ischemia-induced myocardial damage.

Soluble CD40 ligand impairs the function of peripheral blood angiogenic outgrowth cells and increases neointimal formation after arterial injury.

BACKGROUND: Recent work has revealed an essential involvement of soluble CD40 ligand (sCD40L) in inflammation and atherosclerosis. We investigated whether sCD40L functionally affects peripheral blood-derived angiogenic early outgrowth cells (EOCs) and neointimal remodeling after arterial injury. METHODS AND RESULTS: Besides myeloid and endothelial markers, cultured human EOCs strongly expressed CD40 mRNA and protein. EOC adhesion to fibronectin, fibrinogen, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 under flow conditions, as well as their transmigration toward stromal cell-derived factor-1alpha, was dose-dependently reduced after preincubation with recombinant human sCD40L for 24 hours. Integrin expression was unaffected by sCD40L, implying that integrin adhesiveness was attenuated. Surface-immobilized CD40L supported much lower adhesion of EOCs than fibronectin. Treatment of EOCs with sCD40L increased superoxide anion production and decreased viability and proliferation. Notably, CD40(-/-) mice displayed reduced neointima and improved re-endothelialization after carotid wire injury compared with wild-type mice, and therapeutic infusion of control EOCs but not EOCs pretreated with sCD40L attenuated neointimal growth after wire injury in nude mice. Furthermore, neointimal growth was more markedly diminished by infusion of spleen-derived CD40(-/-) mouse EOCs than by that of wild-type EOCs. Preincubation of wild-type EOCs but not CD40(-/-) EOCs with sCD40L before their infusion markedly aggravated neointimal formation. Treatment with sCD40L attenuated luminal incorporation of EOCs and accelerated neointimal progression. CONCLUSIONS: Endothelial dysfunction due to persistently elevated plasma levels of sCD40L may be attributable to an impairment of EOC function. Hence, in the context of arterial injury, therapeutic blockade of sCD40L may provide a novel strategy for accelerating endothelial regeneration and attenuating neointimal remodeling.

Platelet microparticles enhance the vasoregenerative potential of angiogenic early outgrowth cells after vascular injury.

BACKGROUND: Angiogenic early outgrowth cells (EOCs) have been reported to contribute to endothelial regeneration and to limit neointima formation after vascular injury. Vascular pathologies comprise platelet activation and concomitant generation of platelet microparticles (PMPs). We hypothesized that PMPs may interact with EOCs in the context of vascular injury and modulate their regenerative potential. METHODS AND RESULTS: Using flow cytometry, confocal microscopy, and scanning electron microscopy, we demonstrated the binding of thrombin/collagen-induced PMPs to EOCs with subsequent membrane assimilation and incorporation. This interaction promoted phenotypic alterations of EOCs with increased expression of endothelial cell markers and transfer of the chemokine receptor CXCR4 to EOCs with enhanced responsiveness to its ligand CXCL12/SDF-1alpha. In addition, PMPs augmented the adhesion of EOCs to extracellular matrix components and to the injured vessel wall and accelerated cytoskeletal reorganization and migration of EOCs. PMPs induced changes in the EOC secretome toward a more proangiogenic profile and amplified the EOC-mediated induction of proliferation, migration, and capillary tube formation by mature endothelial cells. Compared with untreated EOCs, the injection of PMP-treated EOCs resulted in accelerated reendothelialization after arterial denudation injury in athymic nude mice, whereas the EOC-mediated reduction of neointima formation remained unchanged. CONCLUSIONS: Our data provide evidence that PMPs can boost the potential of EOCs to restore endothelial integrity after vascular injury. Major mechanisms involve the enhancement of EOC recruitment, migration, differentiation, and release of proangiogenic factors.

Functional alterations of myeloid cell subsets in hyperlipidaemia: relevance for atherosclerosis.

Atherosclerosis is a chronic inflammatory disease wherein the infiltration of myeloid cells of the vessel wall is a hallmark event. Lymphocytes, platelets and endothelial cells stand out as prominent suspects being involved in atherosclerosis. However, recent advances suggest a crucial role for myeloid leucocytes, specifically monocyte subsets, neutrophils, dendritic cells and endothelial progenitor cells. These cell types are not just rapidly recruited or already reside in the vascular wall, but also initiate and perpetuate core mechanisms in plaque formation and destabilization. Hyperlipidaemia is an independent risk factor for atherosclerosis. Herein, hyperlipidaemia skews myeloid cell haemostasis, phenotype and transcriptional regulation of pro-inflammatory factors ultimately promoting myeloid cell extravasation and atherosclerosis. We here review the role of myeloid cells in atherosclerosis as well as the effects of hyperlipidaemia on these cells.

C1-esterase inhibitor protects against neointima formation after arterial injury in atherosclerosis-prone mice.

BACKGROUND: Although activation of the complement system has been implicated in the progression of human atherosclerosis, its function during arterial remodeling after injury has not been investigated. Here, we examined the contribution of the complement cascade to neointima formation in apolipoprotein E-deficient mice using a C1-esterase inhibitor (C1-inhibitor). METHODS AND RESULTS: Apolipoprotein E-deficient mice fed an atherogenic diet were subjected to wire-induced endothelial denudation of the carotid artery and treated with C1-inhibitor (Berinert; 15 IU i.v.) or vehicle perioperatively and subsequently every 2 days. The effectiveness of C1-inhibitor treatment was confirmed by measurement of plasma C1-inhibitor activity. A significant reduction in serum triglyceride levels was observed in C1-inhibitor-treated mice, whereas cholesterol levels did not differ. After 3 weeks, neointimal area was significantly reduced in C1-inhibitor-treated mice versus controls, whereas medial area was unaltered. This was associated with a significant decrease in neointimal and medial macrophage and CD3+ T-cell content. Expression of C3 mRNA was significantly reduced in plaques of C1-inhibitor-treated mice 10 days after injury, as assessed by reverse-transcription polymerase chain reaction. The peak in serum C3 levels after injury was markedly downregulated by C1-inhibitor, as evidenced by ELISA. Immunohistochemistry revealed strong expression of C3 and C3c, which colocalized to plaque macrophages and was reduced in C1-inhibitor-treated mice. C1-inhibitor impaired monocyte arrest on activated endothelium and platelets under flow conditions in vitro and leukocyte recruitment to carotid arteries 1 day after injury in vivo. CONCLUSIONS: C1-inhibitor limits neointimal plaque formation and inflammation. This may involve blockade of complement activation, inhibition of leukocyte recruitment, and reduced triglyceride levels, thus providing a multimodal approach to treat arterial disease.

An optimized flow cytometry protocol for analysis of angiogenic monocytes and endothelial progenitor cells in peripheral blood.

Circulating adult CD34(+)VEGFR2(+) endothelial progenitor cells (EPCs) have been shown to differentiate into endothelial cells, thus contributing to vascular homeostasis. Furthermore, a subset of circulating CD14(+) monocytes coexpresses CD16 together with the angiopoietin receptor Tie2 and has been functionally implicated in tumor angiogenesis. However, clinically applicable protocols for flow cytometric quantification of EPCs and Tie2(+) monocytes in peripheral blood and a consensus on reference values remain elusive. The number of Tie2(+)CD14(+)CD16(mid) angiogenic monocytes and CD34(+)VEGFR2(+)CD45(low/-) EPCs was assessed in the peripheral venous blood of patients with stable coronary artery disease by three-color flow cytometry using specific monoclonal antibodies conjugated to PerCP, PE, PE-Cy7, APC, and APC-Cy7. Scatter multigating with exclusion of dead cells was performed to dissect complex mononuclear cell populations. This analysis was further refined by matching bright fluorochromes (PE-Cy7, PE, APC) with dimly expressed markers (CD34, VEGFR2, Tie2), by automatic compensation for minimizing fluorescence spillover and by using fluorescence-minus-one (FMO) controls to determine positive/negative boundaries. Presuming a Gaussian distribution, we obtained average values (mean +/- SD) of 1.45 +/- 1.29% for Tie2(+)CD14(+)CD16(mid) monocytes (n = 11, range: 0.12-3.64%) and 0.019 +/- 0.013% for CD34(+)VEGFR2(+)CD45(low/-) EPCs (n = 17, range: 0.003-0.042%). The intra- and inter-assay variability was 1.6% and 4.5%, respectively. We have optimized a fast and sensitive assay for the flow cytometric quantification of circulating angiogenic monocytes and EPCs in cardiovascular medicine. This protocol may represent a basis for standardized analysis and monitoring of these cell subsets to define their normal range and prognostic/diagnostic value in clinical use.

Importance of CXC chemokine receptor 2 in the homing of human peripheral blood endothelial progenitor cells to sites of arterial injury.

Circulating endothelial progenitor cells (EPCs) may contribute to endothelial regeneration; however, the exact mechanisms of their arterial homing remain elusive. We examined the role of the angiogenic chemokine receptor CXCR2 in the homing of human EPCs. Isolated EPCs expressed CXCR2 together with kinase insert domain-containing receptor, CD31, vascular endothelial cadherin, and CXCR4. Adhesion assays under flow conditions showed that EPCs preferentially adhered to beta(2)-integrin ligands, that firm arrest on fibronectin or fibrinogen was enhanced by the CXCR2 ligands CXCL1 or CXCL7, and that blockade of CXCR2 significantly reduced EPC adhesion on platelet-coated endothelial matrix. This was corroborated by the involvement of CXCR2 in EPC recruitment to denuded areas of murine carotid arteries ex vivo and in vivo. Notably, blocking CXCR2 inhibited the incorporation of human EPCs expressing CXCR2 at sites of arterial injury in athymic nude mice. Immunoreactivity for the beta-thromboglobulin isoform CXCL7 was observed in murine platelets and denuded smooth muscle cells (SMCs) early after wire injury, and transcripts for CXCL7 and CXCL1 were detected in isolated human arterial SMCs. Human KDR(+)CXCR2(+) cells showed better in situ adhesion to injured murine carotid arteries than KDR(+)CXCR2(-) cells, were predominantly CD14(+), and improved CXCR2-dependent endothelial recovery after injury in nude mice. In conclusion, our data clearly demonstrate the importance of CXCR2 for the homing of circulating EPCs to sites of arterial injury and for endothelial recovery in vivo.

No evidence for a role of cosmc-chaperone mutations in European IgA nephropathy patients.

BACKGROUND: Altered IgA1 galactosylation is involved in the pathogenesis of IgA nephropathy (IgAN). The galactosyltransferase core-1 beta3-galactosyltransferase-1 (C1GALT1) and its chaperone cosmc are specifically required for O-galactosylation of the IgA1 hinge region. Mutations in the cosmc gene result in a secondary loss of function of C1GALT1 with subsequent undergalactosylation of glycoproteins. Mosaic mutations of cosmc have been shown to result in autoimmune disease. We hypothesized that cosmc mutations might contribute to the altered IgA1 galactosylation in IgAN patients. METHODS: We studied cosmc gene sequences in genomic DNA obtained from male patients with biopsy-proven sporadic (n = 33) and familial IgAN (n = 6 patients from different families). To account for a potential mosaicism we sequenced cosmc in 10 different peripheral blood mononuclear cell DNA clones of every patient. To specifically assess potential mosaic mutations in IgA-producing cells, cosmc mutations were also analysed in DNA isolated from CD20+ B-lymphocytes from three male IgAN patients. RESULTS: Despite our extensive genomic analysis, the data revealed no functionally relevant cosmc gene variants in sporadic or familial IgAN cases. A cosmc gene polymorphism, rs17261572, was identified in these IgAN patients in a similar frequency as previously reported in healthy adults. A functional consequence of this polymorphism has not yet been determined. CONCLUSION: Although decreased C1GALT1 activity has been implicated in the IgAN pathogenesis and cosmc chaperone mutations can cause autoimmune disease, our data provide no evidence for a relevant role of cosmc gene mutations in European patients with sporadic or familial IgAN.

High-reproducible flow cytometric endothelial progenitor cell determination in human peripheral blood as CD34+/CD144+/CD3- lymphocyte sub-population.

Although determination of circulating endothelial progenitor cell (EPC) in peripheral blood by flow cytometry is an emerging marker for cardiovascular medicine, a common standardized protocol is still not available, due to the low numbers achieved in peripheral blood. In the present paper we describe a novel technique for EPC quantification as CD34+/CD144+/CD3- cells within the lymphocyte gate, which increases the percentages of EPC positivity described before and also offers high intra-assay reproducibility. These improvements are based on a gating strategy for big-sized lymphocytes, smooth fixation and cytometric clearance of CD3+ lymphocytes (T-cells). This last procedure is able to increase intra-assay Pearson's correlation from 0.8517 to 0.8908. Therefore, the technical setting described here offers a high-performance and clinically oriented EPC determination strategy in human peripheral blood.

Predictors of low circulating endothelial progenitor cell numbers in haemodialysis patients.

BACKGROUND: End-stage renal disease (ESRD) patients exhibit increased cardiovascular mortality associated with cardiovascular calcifications and endothelial dysfunction. As circulating endothelial progenitor cells (EPCs) harbour vascular regenerative potential and are altered in uraemia, we examined clinical and biochemical factors influencing EPC levels as well as the relation between EPC numbers and function and uraemic cardiovascular calcifications. METHODS: Sixty-five haemodialysis patients were investigated. Cardiovascular calcifications were assessed by multi-slice spiral CT (MSCT, n = 44) with the calculation of coronary Agatston scores and indirectly by carotid-femoral pulse wave velocity (PWV, n = 61). EPCs were quantified in peripheral blood (CD34(+)/KDR(+)) and at day 7 after ex vivo cultivation (ac-LDL(+)/lectin(+)) by flow cytometry. In addition, colony-forming units (CFUs), migratory activity, adhesion and viability of isolated EPCs were analysed. RESULTS: EPC numbers were reduced (P < 0.001) compared to 27 healthy controls (-64%) or 81 patients with documented coronary artery disease and normal renal function (-58%). Coronary calcifications did not exhibit a significant association with the numbers of circulating CD34(+)/KDR(+) or isolated ac-LDL(+)/lectin(+) EPCs. No difference in EPC functions was observed between the 10 patients with the lowest Agatston scores (range 0-41) versus those with the highest scores (range 1181-3736). Multivariate analysis revealed low fetuin-A serum levels to be a positive predictor, while haematocrit and reticulocytes were negative predictors of reduced ac-LDL(+)/lectin(+) EPC numbers. CONCLUSIONS: EPC numbers and function did not correlate with the degree of coronary calcifications in haemodialysis patients. Rather they appear to be related to serum fetuin-A levels, haematocrit and reticulocytes.

Expression of HIF-1alpha in injured arteries controls SDF-1alpha mediated neointima formation in apolipoprotein E deficient mice.

OBJECTIVE: Hypoxia-inducible factor (HIF)-1alpha is the regulatory subunit of a transcriptional complex, which controls the recruitment of multipotent progenitor cells and tissue repair in ischemic tissue by inducing stromal cell-derived factor (SDF)-1alpha expression. Because HIF-1alpha can be activated under normoxic conditions in smooth muscle cells (SMCs) by platelet products, we investigated the role of HIF-1alpha in SDF-1alpha-mediated neointima formation after vascular injury. METHODS AND RESULTS: Wire-induced injury of the left carotid artery was performed in apolipoprotein E-deficient mice. HIF-1alpha expression was increased in the media as early as 1 day after injury, predominantly in SMCs. Nuclear translocation of HIF-1alpha and colocalization with SDF-1alpha was detected in neointimal cells after 2 weeks. HIF-1alpha mRNA expression was induced at 6 hours after injury as determined by real-time RT-PCR. Inhibition of HIF-1alpha expression by local application of HIF-1alpha-siRNA reduced the neointimal area by 49% and significantly decreased the neointimal SMCs content compared with control-siRNA. HIF-1alpha and SDF-1alpha expression were clearly diminished in neointimal cells of HIF-1alpha-siRNA treated arteries. CONCLUSIONS: HIF-1alpha expression is directly involved in neointimal formation after vascular injury and mediates the upregulation of SDF-1alpha, which may affect the stem cell-based repair of injured arteries.

Reduced numbers of circulating endothelial progenitor cells in patients with coronary artery disease associated with long-term statin treatment.

While statin treatment may transiently mobilize endothelial progenitor cells (EPCs), the dose-dependent effects of a continuous statin therapy on EPCs in patients with chronic coronary artery disease (CAD) have not been analyzed. In 209 patients with angiographically documented CAD, 144 of which received 10-40 mg/day of statins for >8 weeks, the EPC number was determined by flow cytometry directly (CD34(+)/KDR(+), n=58) and after in vitro-culture (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-labeled Ac-LDL (DiI-Ac-LDL(+))/lectin(+), n=209). EPC function was assessed by the formation of colony forming units (CFUs). Univariate analysis revealed that the dose of continuous statin therapy inversely correlated with the EPC number. Treatment with 40 mg/day significantly reduced EPC counts. Multivariate analysis unveiled the statin dose and extent of CAD as independent predictors of reduced EPC numbers. Conversely, obesity predicted increased counts, while CFU development was not detectable in all patients and augmented in females and smokers but not in statin-treated patients. Compared with matched controls, statin-treated patients showed significantly reduced absolute and relative EPC counts. In a prospective analysis, initiation of statin therapy significantly diminished the number of circulating and isolated EPCs after 3 but not after 1 month(s). Thus, the statin dose during chronic and continuous treatment independently predicts reduced numbers of circulating as well as isolated EPCs in patients with CAD.

Regulation of endothelial progenitor cell homing after arterial injury.

Adult bone marrow and peripheral blood contain sub-populations of vascular precursor cells, which can differentiate into mature endothelial cells and have therefore been commonly termed endothelial progenitor cells (EPCs). Although EPCs encompass rather heterogeneous cell sub-populations of multiple origins and localization, these cells were basically characterized by expression of progenitor markers and by the development of colony-forming units and late endothelial outgrowth with terminal differentiation into mature endothelial cells. Notably, functional studies in vivo have implied the contribution of EPCs to therapeutic reendothelialization and inhibition of neointimal growth following endothelial injury. In the context of this regenerative arterial remodeling, an adequate homing of EPCs plays a central role. This multi-step process of EPC mobilization, recruitment and firm adhesion is regulated by key angiogenic chemokines (CCL2, CXCL1, CXCL7, CXCL12) and their respective receptors (CCR2, CXCR2, CXCR4). Furthermore, the recruitment of circulating EPCs to sites of arterial injury is synchronized by activated platelets and adhesion molecules of the selectin and integrin family. Thus, translating this molecular knowledge to interventional cardiovascular medicine, such a detailed understanding in the complex regulation of EPC homing may be helpful for more effectively preventing "in-stent" stenosis by facilitating stent endothelialization.

Biphasic effect of pioglitazone on isolated human endothelial progenitor cells: involvement of peroxisome proliferator-activated receptor-gamma and transforming growth factor-beta1.

Endothelial progenitor cells (EPCs) have been implicated in vascular repair and found to be functionally impaired in patients with diabetes. We evaluated the effects of the anti-diabetic drug pioglitazone on human EPC function and the involvement of PPAR-gamma and TGF-beta1. EPCs in culture were characterized at day 7 by the development of colony-forming units (CFUs) and flow cytometry assessment of differentiation marker (DiI-ac-LDL/lectin, KDR and CD31). Adhesion on fibronectin and fibrinogen in flow was analyzed as functional parameter. Treatment with pioglitazone for 72 hours increased the number of EPC-CFUs, DiI-ac-LDL(+)/lectin(+), CD31(+) and KDR(+) EPCs at 1 microM but not at 10 microM. Since pioglitazone did not significantly alter proliferation and apoptosis in cultured EPCs, the increase in EPC number was most likely attributable to augmented adhesion and differentiation. Indeed, pioglitazone increased EPC adhesion in flow at 1 microM, an effect prevented by PPAR-gamma and beta2-integrin blockade. In contrast, pioglitazone did not promote EPC adhesion at 10 microM; however, increased adhesion became evident by co-incubation with a blocking TGF-beta1 antibody. As determined by ELISA, pioglitazone induced a persistent increase in TGF-beta1 secretion only at 10 microM when a significantly elevated expression of endoglin, the accessory receptor for TGF-beta1, was also observed. Taken together, pioglitazone exerts biphasic effects on the function of isolated EPCs, causing a PPAR-gamma-dependent stimulation at 1 microM and a TGF-beta1-mediated suppression at 10 microM. These results may help to define optimal therapeutic doses of pioglitazone for improving endothelial dysfunction.

[The therapeutic effect of autologous bone marrow cells in ischemic heart disease]. / Die therapeutische Wirkkraft autologer Knochenmarkzellen bei ischämischen Herzerkrankungen.

Adult human bone marrow and peripheral blood contain diverse stem and progenitor cells with some properties resembling those of embryonic stem cells, as this has been revealed by an increasing body of evidence within the near past. Numerous in vitro experiments and subsequent animal studies have already demonstrated that these adult progenitor cells considerably contribute to the regeneration of ischemic or injured tissue. Over the last 4 years, several clinical studies employing such a hypothesis in the context of myocardial repair after acute infarction or during chronic ischemic heart disease have been published. These studies have used autologous bone marrow- as well as peripheral blood-derived progenitor cells, which were delivered via intracoronary or intramyocardial routes near the ischemic area. The initial results demonstrated the safety and possible benefit of this strategy, which appears to be relatively inexpensive and free of side effects. However, the present clinical studies were small in size so that the overall therapeutic efficacy remains open to debate and evaluation. Furthermore, a major part of the underlying repair mechanisms has been proposed but not yet elaborated. Hence, larger case-controlled, randomized and double-blinded trials in addition to experimental investigations on the primary molecular mechanisms of myocardial repair are crucial for the future.

Endothelial progenitor cells: mobilization, differentiation, and homing.

Postnatal bone marrow contains a subtype of progenitor cells that have the capacity to migrate to the peripheral circulation and to differentiate into mature endothelial cells. Therefore, these cells have been termed endothelial progenitor cells (EPCs). The isolation of EPCs by adherence culture or magnetic microbeads has been described. In general, EPCs are characterized by the expression of 3 markers, CD133, CD34, and the vascular endothelial growth factor receptor-2. During differentiation, EPCs obviously lose CD133 and start to express CD31, vascular endothelial cadherin, and von Willebrand factor. EPCs seem to participate in endothelial repair and neovascularization of ischemic organs. Clinical studies using EPCs for neovascularization have just been started; however, the mechanisms stimulating or inhibiting the differentiation of EPC in vivo and the signals causing their migration and homing to sites of injured endothelium or extravascular tissue are largely unknown at present. Thus, future studies will help to explore areas of potential basic research and clinical application of EPCs.

Endothelial progenitor cells: isolation and characterization.

Bone marrow of adults contains a subtype of progenitor cells that have the capacity to differentiate into mature endothelial cells and have therefore been termed endothelial progenitor cells (EPCs). Of the three cell markers (CD133, CD34, and the vascular endothelial growth factor receptor 2) that characterize the early functional EPCs, located predominantly in the bone marrow, EPCs obviously lose CD133/CD34 and start to express CD31, vascular endothelial cadherin, and von Willebrand factor when migrating to the circulation. Various isolation procedures of EPCs from different sources by using adherence culture or magnetic microbeads have been described, but published findings with regard to the number of EPCs in the peripheral circulation of healthy adults are scanty and no data regarding the lifetime of EPCs in vivo exist. Clinical studies employing EPCs for neovascularization of ischemic organs have just been started; however, the mechanisms stimulating or inhibiting the differentiation of bone marrow-derived EPCs in vivo and the signals causing their adhesion, migration, and homing to sites of injured tissue are largely unknown at present.