Lothar Rössig

Reduced Number of Circulating Endothelial Progenitor Cells Predicts Future Cardiovascular Events Proof of Concept for the Clinical Importance of Endogenous Vascular Repair

Background—The maintenance of endothelial integrity plays a critical role in preventing atherosclerotic disease progression. Endothelial progenitor cells (EPCs) were experimentally shown to incorporate into sites of neovascularization and home to sites of endothelial denudation. Circulating EPCs may thus provide an endogenous repair mechanism to counteract ongoing risk factor–induced endothelial injury and to replace dysfunctional endothelium. Methods and Results—In 120 individuals (43 control subjects, 44 patients with stable coronary artery disease, and 33 patients with acute coronary syndromes), circulating EPCs were defined by the surface markers CD34+KDR+ and analyzed by flow cytometry. Cardiovascular events (cardiovascular death, unstable angina, myocardial infarction, PTCA, CABG, or ischemic stroke) served as outcome variables over a median follow-up period of 10 months. Patients suffering from cardiovascular events had significantly lower numbers of EPCs (P<0.05). Reduced numbers of EPCs were associated with a significantly higher incidence of cardiovascular events by Kaplan-Meier analysis (P=0.0009). By multivariate analysis, reduced EPC levels were a significant, independent predictor of poor prognosis, even after adjustment for traditional cardiovascular risk factors and disease activity (hazard ratio, 3.9; P<0.05). Conclusions—Reduced levels of circulating EPCs independently predict atherosclerotic disease progression, thus supporting an important role for endogenous vascular repair to modulate the clinical course of coronary artery disease.

Nitric oxide inhibits caspase-3 by S-nitrosation in vivo.

In cultured human endothelial cells, physiological levels of NO prevent apoptosis and interfere with the activation of the caspase cascade. In vitro data have demonstrated that NO inhibits the activity of caspase-3 byS-nitrosation of the enzyme. Here we present evidence for the in vivo occurrence and functional relevance of this novel antiapoptotic mechanism. To demonstrate that the cysteine residue Cys-163 of caspase-3 is S-nitrosated, cells were transfected with the Myc-tagged p17 subunit of caspase-3. After incubation of the transfected cells with different NO donors, Myc-tagged p17 was immunoprecipitated with anti-Myc antibody.S-Nitrosothiol was detected in the immunoprecipitate by electron spin resonance spectroscopy after liberation and spin trapping of NO byN-methyl-d-glucamine-dithiocarbamate-iron complex. Transfection of cells with a p17 mutant, where the essential Cys-163 was mutated into alanine, completely preventedS-nitrosation of the enzyme. As a functional correlate, in human umbilical vein endothelial cells the NO donors sodium nitroprusside or PAPA NONOate (50 μm) significantly reduced the increase in caspase-3-like activity induced by overexpressing caspase-3 by 75 and 70%, respectively. When human umbilical vein endothelial cells were cotransfected with β-galactosidase, morphological analysis of stained cells revealed that cell death induction by overexpression of caspase-3 was completely suppressed in the presence of sodium nitroprusside, PAPA NONOate, or S-nitroso-l-cysteine (50 μm). Thus, NO supplied by exogenous NO donors serves in vivo as an antiapoptotic regulator of caspase activity viaS-nitrosation of the Cys-163 residue of caspase-3.

HMG-CoA Reductase Inhibitors Reduce Senescence and Increase Proliferation of Endothelial Progenitor Cells via Regulation of Cell Cycle Regulatory Genes

Abstract— Endothelial progenitor cells (EPCs) play an important role in postnatal neovascularization of ischemic tissue. Ex vivo expansion of EPCs might be useful for potential clinical cell therapy of myocardial ischemia. However, cultivation of primary cells leads to cellular aging (senescence), thereby severely limiting the proliferative capacity. Therefore, we investigated whether statins might be able to prevent senescence of EPCs. EPCs were isolated from peripheral blood and characterized. After ex vivo cultivation, EPCs became senescent as determined by acidic &bgr;-galactosidase staining. Atorvastatin or mevastatin dose-dependently inhibited the onset of EPC senescence in culture. Moreover, atorvastatin increased proliferation of EPCs as assessed by BrdU incorporation and colony-forming capacity. Whereas geranylgeranylpyrophosphate or farnesylpyrophosphate reduced the senescence inhibitory effect of atorvastatin, NO synthase inhibition, antioxidants, or Rho kinase inhibitors had no effect. To get further insights into the underlying downstream effects of statins, we measured telomerase activity and determined the expression of various cell cycle regulatory genes by using a microarray assay. Whereas telomerase activity did not change, atorvastatin modulated expression of cell cycle genes including upregulation of cyclins and downregulation of the cell cycle inhibitor p27Kip1. Taken together, statins inhibited senescence of EPCs independent of NO, reactive oxygen species, and Rho kinase, but dependent on geranylgeranylpyrophosphate. Atorvastatin-mediated prevention of EPC senescence appears to be mediated by the regulation of various cell cycle proteins. The inhibition of EPC senescence and induction of EPC proliferation by statins in vitro may importantly improve the functional activity of EPCs for potential cell therapy.

Akt-dependent phosphorylation of p21(Cip1) regulates PCNA binding and proliferation of endothelial cells.

ABSTRACT The protein kinase Akt is activated by growth factors and promotes cell survival and cell cycle progression. Here, we demonstrate that Akt phosphorylates the cell cycle inhibitory protein p21Cip1 at Thr 145 in vitro and in intact cells as shown by in vitro kinase assays, site-directed mutagenesis, and phospho-peptide analysis. Akt-dependent phosphorylation of p21Cip1 at Thr 145 prevents the complex formation of p21Cip1 with PCNA, which inhibits DNA replication. In addition, phosphorylation of p21Cip1 at Thr 145 decreases the binding of the cyclin-dependent kinases Cdk2 and Cdk4 to p21Cip1 and attenuates the Cdk2 inhibitory activity of p21Cip1. Immunohistochemistry and biochemical fractionation reveal that the decrease of PCNA binding and regulation of Cdk activity by p21Cip1 phosphorylation is not caused by altered intracellular localization of p21Cip1. As a functional consequence, phospho-mimetic mutagenesis of Thr 145 reverses the cell cycle-inhibitory properties of p21Cip1, whereas the nonphosphorylatable p21Cip1 T145A construct arrests cells in G0 phase. These data suggest that the modulation of p21Cip1 cell cycle functions by Akt-mediated phosphorylation regulates endothelial cell proliferation in response to stimuli that activate Akt.

Ex vivo pretreatment of bone marrow mononuclear cells with endothelial NO synthase enhancer AVE9488 enhances their functional activity for cell therapy.

Bone marrow mononuclear cells (BMC) from patients with ischemic cardiomyopathy (ICMP) show a reduced neovascularization capacity in vivo. NO plays an important role in neovascularization, and NO bioavailability is typically reduced in patients with ICMP. We investigated whether the impaired neovascularization capacity of ICMP patient-derived progenitor cells can be restored by pretreatment with the novel endothelial NO synthase (eNOS) transcription enhancer AVE9488 (AVE). Ex vivo pretreatment of BMC from patients with ICMP with AVE significantly increased eNOS mRNA expression by 2.1-fold (P < 0.05) and eNOS activity as assessed by ESR by >3-fold (P < 0.05). The increased eNOS expression was associated with an enhanced migratory capacity in vitro (P < 0.01) and improved neovascularization capacity of the infused BMC in an ischemic hind limb model in vivo (P < 0.001). The improvement in ischemic limb perfusion after infusion of AVE-pretreated BMC resulted in an increase in swimming time (P < 0.05). The enhancement of limb perfusion by AVE-treated BMC was abrogated by ex vivo pretreatment with the eNOS inhibitor NG-nitro-l-arginine methyl ester. Consistently, AVE showed no effect on the impaired migratory capacity of BMC derived from eNOS-deficient mice, documenting the specific involvement of NO. The reduced neovascularization capacity of BMC from patients with ICMP may limit their therapeutic potential in cell therapy studies. Here, we show that pharmacological enhancement of eNOS expression with AVE at least partially reverses the impaired functional activity of BMC from ICMP patients, highlighting the critical role of NO for progenitor cell function.

p38 Mitogen-Activated Protein Kinase Downregulates Endothelial Progenitor Cells

Background—Transplantation of endothelial progenitor cells (EPCs) improves neovascularization after ischemia, but patients with coronary artery disease (CAD) or diabetes mellitus show a reduced number of EPCs and impaired functional activity. Therefore, we investigated the effects of risk factors, such as glucose and TNF-&agr;, on the number of EPCs in vitro to elucidate the underlying mechanisms. Methods and Results—EPCs of patients or healthy subjects were isolated from peripheral blood. Incubation with glucose or TNF-&agr; dose-dependently reduced the number of EPCs (79.9±1.3% and 74.3±8.1% of control; P<0.05, respectively). This reduction was not caused by apoptosis. TNF-&agr; and glucose induced a dose- and time-dependent activation of the p38 MAP kinase, the downstream kinase mitogen- and stress-activated kinase 1, and the transcription factor cAMP-responsive element–binding protein (CREB), in EPCs. Moreover, EPCs from CAD patients had significantly higher basal p38-phosphorylation levels (1.83±0.2-fold increase; P<0.05) compared with healthy subjects. The inhibition of the p38-kinase by SB203580 or infection with a dominant negative p38 kinase adenovirus significantly increased basal number of EPCs (136.7±6.3% and 142.9±18% versus control, respectively). Likewise, ex vivo cultivation of EPCs from patients with CAD with SB203580 significantly increased the number of EPCs and partially reversed the impaired capacity for neovascularization of EPCs in vivo (relative blood flow: 0.40±0.03 versus 0.64±0.08, P<0.05). The increased numbers of EPCs by SB203580 were associated with an augmentation of EPC proliferation and a reduction of cells expressing the monocytic marker proteins CD14 and CD64, suggesting that p38 regulates proliferation and differentiation events. Conclusions—These results demonstrate that p38 MAP kinase plays a pivotal role in the signal transduction pathways regulating the number of EPCs ex vivo. SB203580 can prevent the negative effects of TNF-&agr; and glucose on the number of EPCs and may be useful to improve the number of EPCs for potential cell therapy.

Glycogen synthase kinase-3 couples AKT-dependent signaling to the regulation of p21Cip1 degradation.

Signaling via the phosphoinositide 3-kinase (PI3K)/AKT pathway is crucial for the regulation of endothelial cell (EC) proliferation and survival, which involves the AKT-dependent phosphorylation of the DNA repair protein p21Cip1 at Thr-145. Because p21Cip1 is a short-lived protein with a high proteasomal degradation rate, we investigated the regulation of p21Cip1protein levels by PI3K/AKT-dependent signaling. The PI3K inhibitors Ly294002 and wortmannin reduced p21Cip1 protein abundance in human umbilical vein EC. However, mutation of the AKT site Thr-145 into aspartate (T145D) did not increase its protein half-life. We therefore investigated whether a kinase downstream of AKT regulates p21Cip1 protein levels. In various cell types, AKT phosphorylates and inhibits glycogen synthase kinase-3 (GSK-3). Upon serum stimulation of EC, GSK-3β was phosphorylated at Ser-9. Site-directed mutagenesis revealed that GSK-3 in vitrophosphorylated p21Cip1 specifically at Thr-57 within the Cdk binding domain. Overexpression of GSK-3β decreased p21Cip1 protein levels in EC, whereas the specific inhibition of GSK-3 with lithium chloride interfered with p21Cip1 degradation and increased p21Cip1protein about 10-fold in EC and cardiac myocytes (30 mm,p < 0.001). These data indicate that GSK-3 triggers p21Cip1 degradation. In contrast, stimulation of AKT increases p21Cip1 via inhibitory phosphorylation of GSK-3.

Inhibitors of Histone Deacetylation Downregulate the Expression of Endothelial Nitric Oxide Synthase and Compromise Endothelial Cell Function in Vasorelaxation and Angiogenesis

Abstract— The histone deacetylase (HDAC) inhibitor trichostatin A (TSA) inhibits hypoxia-stimulated angiogenesis. Endothelial nitric oxide synthase (eNOS)–derived NO is central to angiogenesis signaling in endothelial cells (ECs). We hypothesized that the HDAC-dependent regulation of angiogenesis may involve a modulatory effect on eNOS expression. The HDAC inhibitors TSA, butyric acid (BuA), and MS-275 time- and concentration-dependently suppressed eNOS protein levels to 41±2%, 46±12%, and 40±12% of control, respectively. In parallel, TSA and BuA also downregulated eNOS mRNA expression to 21±4% and 37±4% of control. TSA also attenuated the NO-dependent relaxation of porcine coronary arteries (P <0.0001, TSA 1 &mgr;mol/L) and prevented tube formation in a human angiogenesis assay. Although vascular endothelial growth factor substitution did not compensate for the inhibitory effect of TSA, exogenous NO reversed the inhibition of angiogenesis by TSA. To address the underlying signaling mechanism, we characterized the effect of TSA on eNOS gene transcription and mRNA half-life. Although TSA decreased both eNOS protein and mRNA levels, TSA paradoxically enhanced the activity of the eNOS promoter, and did not alter the eNOS transcription rate in nuclear run-on experiments, suggesting that TSA posttranscriptionally targets eNOS mRNA. These data indicate that HDAC-dependent mechanisms contribute to the regulation of eNOS expression in ECs.

Histone deacetylase activity is essential for the expression of HoxA9 and for endothelial commitment of progenitor cells

The regulation of acetylation is central for the epigenetic control of lineage-specific gene expression and determines cell fate decisions. We provide evidence that the inhibition of histone deacetylases (HDACs) blocks the endothelial differentiation of adult progenitor cells. To define the mechanisms by which HDAC inhibition prevents endothelial differentiation, we determined the expression of homeobox transcription factors and demonstrated that HoxA9 expression is down-regulated by HDAC inhibitors. The causal involvement of HoxA9 in the endothelial differentiation of adult progenitor cells is supported by the finding that HoxA9 overexpression partially rescued the endothelial differentiation blockade induced by HDAC inhibitors. Knockdown and overexpression studies revealed that HoxA9 acts as a master switch to regulate the expression of prototypical endothelial-committed genes such as endothelial nitric oxide synthase, VEGF-R2, and VE-cadherin, and mediates the shear stress–induced maturation of endothelial cells. Consistently, HoxA9-deficient mice exhibited lower numbers of endothelial progenitor cells and showed an impaired postnatal neovascularization capacity after the induction of ischemia. Thus, HoxA9 is regulated by HDACs and is critical for postnatal neovascularization.

Apoptosis in the vascular wall and atherosclerosis.

Abstract Apoptosis, programmed cell death, has emerged as a key element in the complex pathophysiology underlying the development as well as the progression of atherosclerosis. A number of recent reports provided evidence for both in vivo and in vitro occurrence of apoptotic cell death of vascular cells, namely endothelial cells, macrophages, and vascular smooth muscle cells. In addition, functional studies in disease models underscore the relevance of these findings for the understanding of processes which lead to lesion development, plaque rupture, and thrombus formation. Pathomechanistic in vitro investigations provided an increasingly detailed picture of the involved intracellular signaling pathways that regulate onset and execution of apoptosis. These insights offer the potential of therapeutic interventions targeted to interfere with the molecular processes involving apoptotic cell death in the vascular wall.