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Judith Haendeler

Publications and source records attributed to Judith Haendeler.

At least 19 recordsLinked to original sources
PubMed2006

Sphingosine-1-phosphate stimulates the functional capacity of progenitor cells by activation of the CXCR4-dependent signaling pathway via the S1P3 receptor.

OBJECTIVE: Sphingosine-1-phosphate (S1P) is a bioactive lipid, which influences migration and proliferation of endothelial cells through activation of S1P receptors and has been shown to support SDF-1 induced migration and bone marrow homing of CD34+ progenitors. METHODS AND RESULTS: Here, we show that incubation of patient-derived endothelial progenitor cells (EPCs) with S1P or its synthetic analog FTY720 improved blood flow recovery in ischemic hind limbs. Likewise, recovery of blood flow was dramatically reduced after induction of hindlimb ischemia in mice deficient for the S1P receptor 3 (S1P3). S1P3-/- bone marrow-derived mononuclear cells (BMCs) failed to augment neovascularization after hind limb ischemia. Of note, treatment of BMCs derived from S1P3-/- mice with S1P did not rescue blood flow recovery. Mechanistically, S1P and FTY720 induced phosphorylation of CXCR4, activated the Src kinase, and stimulated phosphorylation of JAK2. The contribution of CXCR4 for S1P-mediated effects was further supported by the findings that S1P preincubation failed to stimulate invasion capacity and in vivo blood flow recovery of BMCs from CXCR4+/- mice. The activation of CXCR4 was dependent on the Src kinase family as demonstrated by preincubation with the Src inhibitor PP2. The activation of the CXCR4 signaling by S1P is mediated via the S1P3 receptor, since S1P-induced Src phosphorylation was abrogated in EPC from S1P3-/- mice. CONCLUSIONS: S1P agonists might serve as sensitizers of CXCR4-mediated signaling and may be applied in clinical progenitor cell therapy to improve EPC or BMC function in patients with coronary artery disease.

Animals↗
PubMed2006

Effects of granulocyte colony simulating factor on functional activities of endothelial progenitor cells in patients with chronic ischemic heart disease.

OBJECTIVE: Bone marrow-derived circulating endothelial progenitor cells (EPCs) may contribute to regeneration of infarcted myocardium and enhance neovascularization. Granulocyte colony-stimulating factor (G-CSF) is well-established to mobilize hematopoietic stem cells (HSCs) and might, thereby, also increase the pool of endogenously circulating EPC. Therefore, we investigated the effects of G-CSF administration on mobilization and functional activities of blood-derived EPC in patients with chronic ischemic heart disease (CIHD). METHODS AND RESULTS: Sixteen patients with CIHD received 10 microg/kg per day subcutaneous G-CSF injection for 5 days. Leukocyte counts, the number of HSCs and EPCs, and the migratory response to VEGF and SDF-1 were analyzed before and after G-CSF-therapy. At day 5 of G-CSF treatment, the number of circulating leukocytes, CD34+ CD45+ and CD34+ CD133+ cells was significantly increased. Likewise, G-CSF treatment augmented the numbers of colony forming units with endothelial cell morphology (EC-CFU). However, the functional activity of the EPC as assessed by the migratory response to VEGF and SDF-1 was significantly reduced after G-CSF treatment (P<0.01). Because G-CSF was previously shown to cleave the CXCR4 receptor, we determined the surface expression of the 6H8 epitope of the CXCR4 receptor by fluorescence-activated cell sorter (FACS) analysis. Consistent with the reduced migratory capacity, the surface expression of the functionally active CXCR4 receptor was significantly reduced. To test the functional activity of the cultivated EPCs in vivo, cells were intravenously infused in nude mice after hind limb ischemia. EPCs, which were cultivated before G-CSF administration, increased blood flow recovery and prevented limb necrosis. However, infusion of EPCs, which were isolated 5 days after G-CSF treatment from the same patient, showed a reduced capacity to augment blood flow recovery and to prevent necrosis by 27%. CONCLUSIONS: G-CSF treatment effectively mobilizes HSCs and EPCs. However, the migratory response to SDF-1 and in vivo capacity of G-CSF-mobilized EPCs was significantly reduced.

Aged↗
PubMed2005

Cathepsin D and H2O2 stimulate degradation of thioredoxin-1: implication for endothelial cell apoptosis.

Cathepsin D (CatD) is a lysosomal aspartic proteinase and plays an important role in the degradation of proteins and in apoptotic processes induced by oxidative stress, cytokines, and aging. All of these stimuli are potent inducers of endothelial cell apoptosis. Therefore, we investigated the role of CatD in endothelial cell apoptosis and determined the underlying mechanisms. Incubation with 100-500 microm H2O2 for 12 h induced apoptosis in endothelial cells. To determine a role for CatD, we co-incubated endothelial cells with the CatD inhibitor pepstatin A. Pepstatin A as well as genetic knock down of CatD abolished H2O2-induced apoptosis. In contrast, overexpression of CatD wild type but not a catalytically inactive mutant of CatD (CatDD295N) induced apoptosis under basal conditions. To gain insights into the underlying mechanisms, we investigated the effect of CatD on reactive oxygen species (ROS) formation. Indeed, knocking down CatD expression reduced H2O2-induced ROS formation and apoptosis. The major redox regulator in endothelial cells is thioredoxin-1 (Trx), which plays a crucial role in apoptosis inhibition. Thus, we hypothesized that CatD may alter Trx protein levels and thereby promote formation of ROS and apoptosis. Incubation with 100 microm H2O2 for 6 h decreased Trx protein levels, whereas Trx mRNA was not altered. H2O2-induced Trx degradation was inhibited by pepstatin A and genetic knock down of CatD but not by other protease inhibitors. Incubation of unstimulated cell lysates with recombinant CatD significantly reduced Trx protein levels in vitro, which was completely blocked by pepstatin A pre-incubation. Overexpression of CatD reduced Trx protein in cells. Moreover, H2O2 incubation led to a translocation of Trx to the lysosomes prior to the induction of apoptosis. Taken together, CatD induces apoptosis via degradation of Trx protein, which is an essential anti-apoptotic and reactive oxygen species scavenging protein in endothelial cells.

Aging↗
PubMed2005

Impaired CXCR4 signaling contributes to the reduced neovascularization capacity of endothelial progenitor cells from patients with coronary artery disease.

Transplantation of bone marrow cells as well as circulating endothelial progenitor cells (EPC) enhances neovascularization after ischemia. The chemokine receptor CXCR4 is essential for migration and homing of hematopoietic stem cells. Therefore, we investigated the role of CXCR4 and its downstream signaling cascade for the angiogenic capacity of cultured human EPC. Ex vivo, differentiated EPC derived from peripheral blood abundantly expressed CXCR4. Incubation of EPC from healthy volunteers with neutralizing antibodies against CXCR4 profoundly inhibited vascular endothelial growth factor- and stromal-derived factor-1-induced migration as well as EPC-induced angiogenesis in an ex vivo assay. Preincubation of transplanted EPC with CXCR4 antibody reduced EPC incorporation and impaired blood-flow recovery in ischemic hindlimbs of nude mice (57+/-4% of normal perfusion versus untreated EPC: 80+/-11%, P<0.001). Bone marrow mononuclear cells (BM-MNC) or EPC of heterozygous CXCR4(+/-) mice displayed reduced CXCR4 expression and disclosed impaired in vivo capacity to enhance recovery of ischemic blood flow in nude mice (blood flow 27+/-11% versus 66+/-25% using wild-type cells, P<0.01). Importantly, impaired blood flow in ischemic CXCR4(+/-) mice was rescued by injection of wild-type BM-MNC. Next, we investigated the role of CXCR4 for functional capacities of EPC from patients with coronary artery disease (CAD). Surface expression of CXCR4 was similar in EPC from patients with CAD compared with healthy controls. However, basal Janus kinase (JAK)-2 phosphorylation was significantly reduced and less responsive to stromal-derived factor-1 in EPC from patients with CAD compared with healthy volunteers, indicating that CXCR4-mediated JAK-2 signaling is dysregulated in EPC from patients with CAD. The CXCR4 receptor signaling profoundly modulates the angiogenic activity and homing capacity of cultured human EPC. Disturbance of CXCR4 signaling, as demonstrated by reduced JAK-2 phosphorylation, may contribute to functional impairment of EPC from patients with CAD. Stimulating CXCR4 signaling might improve functional properties of EPC and may rescue impaired neovascularization capacity of EPC derived from patients with CAD.

Animals↗
PubMed2005

Differentiation of circulating endothelial progenitor cells to a cardiomyogenic phenotype depends on E-cadherin.

Progenitor cells may contribute to cardiac regeneration. Here, we investigated the role of cadherins and integrins for differentiation of human adult circulating endothelial progenitor cells (EPCs) into cardiomyocytes (CM) in a co-culture system. N- and E-cadherin were expressed in EPCs and were localized at the interface between EPCs and CM. Incubation of a blocking antibody against E-cadherin reduced the expression of CM marker protein in EPCs. Blocking antibodies against N- or P-cadherin or the beta1- and beta2-integrins were not effective. These data suggested that cell-to-cell communication mediated by E-cadherin contributes to the acquirement of a cardiomyogenic phenotype of human endothelial progenitor cells.

Antibodies, Blocking↗
PubMed2005

The role of junctional adhesion molecule-C (JAM-C) in oxidized LDL-mediated leukocyte recruitment.

The junctional adhesion molecule-C (JAM-C) was recently shown to be a counter receptor for the leukocyte beta2-integrin Mac-1 (CD11b/CD18), thereby mediating interactions between vascular cells, particularly in inflammatory cell recruitment. Here, we investigated the role of JAM-C in oxidized low-density lipoprotein (LDL)-mediated leukocyte recruitment. As compared with normal arteries, immunostaining of atherosclerotic vessels revealed a high expression of JAM-C in association with neointimal smooth muscle cells and the endothelium. Moreover, JAM-C was strongly up-regulated in the spontaneous early lesions in ApoE -/- mice. In vitro, cultured human arterial smooth muscle cells (HASMC) were found to express JAM-C, and oxLDL, as well as enzymatically modified LDL (eLDL) significantly up-regulated JAM-C on both HASMC and endothelial cells in a time- and dose-dependent manner. Although under quiescent conditions, JAM-C predominantly localized to interendothelial cell-cell contacts in close proximity to zonula occludens-1 (ZO-1), oxLDL treatment induced a disorganization of JAM-C localization that was no more restricted to the interendothelial junctions. JAM-C thereby mediated both leukocyte adhesion and leukocyte transendothelial migration upon oxLDL treatment of endothelial cells, whereas JAM-C on quiescent endothelial cells only mediates leukocyte transmigration. Thus, upon oxLDL stimulation endothelial JAM-C functions as both an adhesion, as well as a transmigration receptor for leukocytes. Taken together, JAM-C is up-regulated by oxLDL and may thereby contribute to increased inflammatory cell recruitment during atherosclerosis. JAM-C may therefore provide a novel molecular target for antagonizing interactions between vascular cells in atherosclerosis.

Aged↗
PubMed2005

Cell-to-cell connection of endothelial progenitor cells with cardiac myocytes by nanotubes: a novel mechanism for cell fate changes?

The regeneration of new myocardium by stem or progenitor cells is an important therapeutic option. Cellular or nuclear fusion is considered as an alternative to cell reprogramming by transdifferentiation. However, the generation of hybrid cells may also be a consequence of a transient transmembrane exchange of proteins and organelles between cells. Therefore, we investigated the formation of intercellular connections, which may allow the transport of macromolecular structures between labeled adult human endothelial progenitor cells (EPC) and GFP-expressing neonatal rat cardiomyocytes (CM) in a coculture system. FACS analysis revealed that, 6 days after initiation of coculture, 2.1+/-0.4% of the cells stained positive for GFP and Dil-ac-LDL. 6 hours after initiation of the coculture, ultrafine intercellular structures between Dil-ac-LDL-labeled EPC and GFP-expressing CM were observed. The number of EPC, which established nanotubular connections with CM increased from 0.5+/-0.2% after 6 hours to 2.6+/-0.3% after 24 hours of coculture. The intercellular connections had a diameter from 50 to 800 nm, a length of 5 to 120 mum, and were only transiently established. To determine whether the nanotubular structures allowed the transport of organelles, we labeled CM with a mitochondrial live tracker (MitoTracker). Using time-lapse video microscopy, we observed the transport of stained complexes between CM and EPC resulting in up-take of MitoTracker-positive structures in EPC. Thus, the present study shows a novel type of cell-to-cell communication between progenitor cells and CM in vitro, which may contribute to the acquisition of a cardiomyogenic phenotype independent of permanent cellular or nuclear fusion.

Animals↗
PubMed2005

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-alpha, 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-alpha 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-alpha 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-alpha and glucose on the number of EPCs and may be useful to improve the number of EPCs for potential cell therapy.

Activating Transcription Factor 2↗
PubMed2005

Non-canonical Wnt signaling enhances differentiation of human circulating progenitor cells to cardiomyogenic cells.

Human endothelial circulating progenitor cells (CPCs) can differentiate to cardiomyogenic cells during co-culture with neonatal rat cardiomyocytes. Wnt proteins induce myogenic specification and cardiac myogenesis. Here, we elucidated the effect of Wnts on differentiation of CPCs to cardiomyogenic cells. CPCs from peripheral blood mononuclear cells were isolated from healthy volunteers and co-cultured with neonatal rat cardiomyocytes. 6-10 days after co-culture, cardiac differentiation was determined by alpha-sarcomeric actinin staining of human lymphocyte antigen-positive cells (fluorescence-activated cell-sorting analysis) and mRNA expression of human myosin heavy chain and atrial natriuretic peptide. Supplementation of co-cultures with Wnt11-conditioned medium significantly enhanced the differentiation of CPCs to cardiomyocytes (1.7+/-0.3-fold), whereas Wnt3A-conditioned medium showed no effect. Cell fusion was not affected by Wnt11-conditioned medium. Because Wnts inhibit glycogen synthase kinase-3beta, we further determined whether the glycogen synthase kinase-3beta inhibitor LiCl also enhanced cardiac differentiation of CPCs. However, LiCl (10 mM) did not affect CPC differentiation. In contrast, Wnt11-conditioned medium time-dependently activated protein kinase C (PKC). Moreover, the PKC inhibitors bisindolylmaleimide I and III significantly blocked differentiation of CPCs to cardiomyocytes. PKC activation by phorbol 12-myristate 13-acetate significantly increased CPC differentiation to a similar extent as compared with Wnt11-conditioned medium. Our data demonstrate that Wnt11, but not Wnt3A, augments cardiomyogenic differentiation of human CPCs. Wnt11 promotes cardiac differentiation via the non-canonical PKC-dependent signaling pathway.

Animals↗
PubMed2004

Low doses of reactive oxygen species protect endothelial cells from apoptosis by increasing thioredoxin-1 expression.

The redox regulator thioredoxin-1 (Trx-1) is required for the redox potential of the cell and exerts important functions in cell growth and apoptosis. Severe oxidative stress has been implicated in the oxidation of proteins and cell death. However, the role of low doses of reactive oxygen species (ROS) is poorly understood. Here, we show that 10 and 50 microM H2O2 and short-term exposure to shear stress significantly increased Trx-1 mRNA and protein levels in endothelial cells. Since it is known that Trx-1 exerts anti-apoptotic functions, we next investigated whether low doses of ROS can inhibit basal and serum-depletion induced endothelial cell apoptosis. Indeed, treatment of endothelial cells with 10 and 50 microM H2O2 significantly reduced apoptosis induction. Reduction of Trx-1 expression using an antisense oligonucleotide approach resulted in the induction of apoptosis and abolished the inhibitory effect of low doses of H2O2. Taken together, our results demonstrate that low doses of ROS act as signaling molecules and exert anti-apoptotic functions in endothelial cells via upregulation of the redox-regulator Trx-1.

Apoptosis↗
PubMed2004

Statins enhance migratory capacity by upregulation of the telomere repeat-binding factor TRF2 in endothelial progenitor cells.

BACKGROUND: Cultivation of endothelial progenitor cells (EPCs) leads to premature replicative senescence, limiting ex vivo expansion for potential clinical cell therapy. Recent studies have linked senescence to the dysfunction of telomeres, the "ends" of chromosomes, via the so-called mitotic clock or culture-induced stress. The purpose of this study was to elucidate a possible role of telomere biology in the functional augmentation of EPCs by statins. METHODS AND RESULTS: Human EPCs were isolated from peripheral blood. Using flow cytometry after fluorescence in situ hybridization with a telomere-specific (C3TA2)3 peptide nucleic acid probe (Flow-FISH), we found mean telomere length in untreated EPCs from healthy subjects to range between 8.5+/-0.2 and 11.1+/-0.5 kb with no change over 6 days of culture, excluding telomere erosion as one cause for premature senescence. Although mean telomere length did not differ between statin-treated and untreated EPCs, atorvastatin (0.1 micromol/L) and mevastatin (1.0 micromol/L) both led to a more than 3-fold increase in the expression of the telomere capping protein TRF2 (telomere repeat-binding factor), as shown by immunoblotting, whereas quantitative reverse transcription-polymerase chain reaction demonstrated no increase in TRF2 mRNA. Telomere dysfunction of EPCs was also paralleled by a 4-fold increase in the DNA damage checkpoint-kinase 2 (Chk2). Conversely, statin cotreatment or overexpression of TRF2 completely suppressed Chk2 induction. Finally, overexpression of a dominant negative mutant of the TRF2 protein abrogated statin-induced enhancement of migratory activity down to baseline values. CONCLUSIONS: Ex vivo culturing of EPCs leads to "uncapping" of telomeres, indicated by the loss of TRF2. Statin cotreatment of EPCs prevents impairment of their functional capacity by a TRF2-dependent, posttranscriptional mechanism. This is the first time a beneficial effect of statins on telomere biology has been described.

Adenoviridae↗
PubMed2004

Antioxidant effects of statins via S-nitrosylation and activation of thioredoxin in endothelial cells: a novel vasculoprotective function of statins.

BACKGROUND: HMG-CoA reductase inhibitors (statins) are lipid-lowering drugs that also exert pleiotropic vasculoprotective effects via activation of the endothelial NO synthesis. NO induces S-nitrosylation of target proteins. S-Nitrosylation of the antioxidant enzyme thioredoxin was recently shown to enhance its activity, thereby reducing intracellular reactive oxygen species. Therefore, we investigated whether statins may exert an antioxidant activity in endothelial cells via S-nitrosylation of thioredoxin. METHODS AND RESULTS: Statins dose- and time-dependently increased the overall level of S-nitrosylated proteins in endothelial cells (atorvastatin 0.1 micromol/L, 206+/-30% increase; simvastatin 1 micromol/L, 214+/-19% increase; mevastatin 1 micromol/L, 191+/-10% increase). The increased S-nitrosylation was blocked by an NO-synthase inhibitor and mevalonate. Moreover, S-nitrosylation of thioredoxin was also significantly augmented after atorvastatin treatment. The atorvastatin-mediated increase in S-nitrosylation was associated with an enhanced enzymatic activity of thioredoxin (atorvastatin, 157+/-9% increase). This resulted in a significant reduction of intracellular reactive oxygen species within the endothelial cells. In contrast, in endothelial cells overexpressing a thioredoxin construct in which the S-nitrosylation acceptor amino acid cysteine 69 was replaced by serine [TRX(C69S)], atorvastatin did not activate the redox-regulatory activity of thioredoxin. Moreover, overexpression of the non-nitrosylatable thioredoxin TRX(C69S) abolished atorvastatin-mediated reduction of reactive oxygen species. CONCLUSIONS: Here, we demonstrate a novel antioxidant mechanism by which statins reduce reactive oxygen species in endothelial cells. Statin-mediated S-nitrosylation of thioredoxin enhanced the enzymatic activity of thioredoxin, resulting in a significant reduction in intracellular reactive oxygen species.

Amino Acid Substitution↗
PubMed2004

GIT1 mediates thrombin signaling in endothelial cells: role in turnover of RhoA-type focal adhesions.

Thrombin mediates changes in endothelial barrier function and increases endothelial permeability. A feature of thrombin-enhanced endothelial hyperpermeability is contraction of endothelial cells (ECs), accompanied by formation of focal adhesions (FAs). Recently, a G protein-coupled receptor kinase-interacting protein, GIT1, was shown to regulate FA disassembly. We hypothesized that GIT1 modulates thrombin-induced changes in FAs. In human umbilical vein ECs (HUVECs), thrombin recruited GIT1 to FAs, where GIT1 colocalized with FAK and vinculin. Recruitment of GIT1 to FAs was dependent on activation of the small GTPase RhoA, and Rho kinase, as demonstrated by adenoviral transfection of dominant-negative RhoA and treatment with Y-27632. Thrombin stimulated GIT1 tyrosine phosphorylation with a time course similar to FAK phosphorylation in a Rho kinase- and Src-dependent manner. Depletion of GIT1 with antisense GIT1 oligonucleotides had no effect on basal cell morphology, but increased cell rounding and contraction of HUVECs, increased FA formation, and increased FAK tyrosine phosphorylation in response to thrombin, concomitant with increased endothelial hyperpermeability. These data identify GIT1 as a novel mediator in agonist-dependent signaling in ECs, demonstrate that GIT1 is involved in cell shape changes, and suggest a role for GIT1 as a negative feedback regulator that augments recovery of cell contraction.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Antioxidants inhibit nuclear export of telomerase reverse transcriptase and delay replicative senescence of endothelial cells.

Aging is associated with a rise in intracellular reactive oxygen species (ROS) and a loss of telomerase reverse transcriptase activity. Incubation with H2O2 induced the nuclear export of telomerase reverse transcriptase (TERT) into the cytosol in a Src-family kinase-dependent manner. Therefore, we investigated the hypothesis that age-related increase in reactive oxygen species (ROS) may induce the nuclear export of TERT and contribute to endothelial cell senescence. Continuous cultivation of endothelial cells resulted in an increased endogenous formation of ROS starting after 29 population doublings (PDL). This increase was accompanied by mitochondrial DNA damage and preceded the onset of replicative senescence at PDL 37. Along with the enhanced formation of ROS, we detected an export of nuclear TERT protein from the nucleus into the cytoplasm and an activation of the Src-kinase. Moreover, the induction of premature senescence by low concentrations of H2O2 was completely blocked with the Src-family kinase inhibitor PP2, suggesting a crucial role for Src-family kinases in the induction of endothelial cell aging. Incubation with the antioxidant N-acetylcysteine, from PDL 26, reduced the intracellular ROS formation and prevented mitochondrial DNA damage. Likewise, nuclear export of TERT protein, loss in the overall TERT activity, and the onset of replicative senescence were delayed by incubation with N-acetylcysteine. Low doses of the statin, atorvastatin (0.1 micromol/L), had also effects similar to those of N-acetylcysteine. We conclude that both antioxidants and statins can delay the onset of replicative senescence by counteracting the increased ROS production linked to aging of endothelial cells.

Acetylcysteine↗
PubMed2004

p21Cip1 levels differentially regulate turnover of mature endothelial cells, endothelial progenitor cells, and in vivo neovascularization.

p21(Cip1) (p21) controls cell cycle progression and apoptosis in mature endothelial cells (ECs) and regulates size and cycling of the hematopoietic progenitor cell pool. Because circulating endothelial progenitor cells (EPCs) contribute to postnatal neovascularization in addition to mature ECs, we investigated the regulation of ECs and EPCs in p21-deficient mice. Mature aortic EC proliferation was increased in homozygous p21(-/-) and heterozygous p21(+/-) mice, in which p21 protein levels are reduced to one third of wild-type (WT). In contrast, apoptosis sensitivity was increased by 3.5-fold only in p21(-/-), but not in p21(+/-) mice. Consistently, in vivo apoptosis of ECs within areas of neovascularization was elevated in p21(-/-) but not in p21(+/-) mice. EPC numbers were elevated 2-fold in p21(-/-) mice compared with WT (P<0.001), and clonal expansion capacity of EPCs was increased from 25+/-4 (WT) to 57+/-8 colony-forming units in p21(-/-) mice (P<0.005). EPC numbers and expansion were likewise increased in p21(+/-) mice. As the integrative endpoint, in vivo neovascularization reflecting all p21-affected parameters was increased over WT only in p21(+/-) (P<0.001), but not in p21(-/-) mice. In conclusion, reduced p21 protein levels of mice lacking one p21 allele are associated with increased proliferation of ECs and EPCs, whereas survival of ECs to apoptotic stimuli in vitro and in vivo is not impaired. Under these conditions, neovascularization was increased. In contrast, complete p21 deficiency did not result in an increased neovascularization despite increased mature EC and EPC proliferation. This may be due to the sensitization of ECs against apoptosis.

Alleles↗
PubMed2004

Gas6 inhibits apoptosis in vascular smooth muscle: role of Axl kinase and Akt.

Axl is a receptor tyrosine kinase originally identified as a transforming gene product in human myeloid leukemia cells. Previously, we showed that Axl expression correlated with neointima formation in balloon-injured rat carotid, and that Axl expression was highly regulated by angiotensin II. In the present study we tested the mechanisms by which Axl regulates vascular smooth muscle cell (VSMC) growth focusing on its ability to inhibit apoptosis. Treatment of cultured rat aortic VSMC for 24 h with 0% serum resulted in 19.8 +/- 1.4% apoptotic cells. Treatment of VSMC with 100 ng/ml Gas6 (the putative ligand for Axl) decreased apoptosis to 8.9 +/- 0.7% (P = 0.002, N = 17) as compared to a decrease with 10% serum to 3.0 +/- 0.2% (P = 0.001, N = 17). The ability of Gas6 to prevent apoptosis required both Gas6 binding to Axl and Axl kinase activity since treatment with a soluble, competitive Axl extracellular domain protein or transfection of a kinase inactive mutant (Axl-K567R) completely prevented the anti-apoptotic effect. Prevention of apoptosis by Gas6-Axl required activation of phosphatidyl inositol 3-kinase (PI3K) as shown by treatment with LY294002 or transfection of an Axl deletion mutant that does not bind PI3K (Axl- triangle up PI3K). There was no significant role for ERK1/2 in the anti-apoptotic effects of Gas6-Axl since ERK1/2 activity was maintained in cells transfected with Axl- triangle up PI3K and Axl-K567R. These findings establish the Gas6-Axl-PI3K-Akt pathway as an anti-apoptotic mechanism for VSMC that may be important in the response to vascular injury.

Animals↗
PubMed2004

GIT1 functions as a scaffold for MEK1-extracellular signal-regulated kinase 1 and 2 activation by angiotensin II and epidermal growth factor.

Activation of the mitogen-activated protein kinase pathway represented by extracellular signal-regulated kinases (ERK1/2) and activation of the upstream kinase (MEK1) are critical events for growth factor signal transduction. c-Src has been proposed as a common mediator for these signals in response to both G protein-coupled receptors (GPCRs) and tyrosine kinase-coupled receptors (TKRs). Here we show that the GPCR kinase-interacting protein 1 (GIT1) is a substrate for c-Src that associates with MEK1 in vascular smooth-muscle cells and human embryonic kidney 293 cells. GIT1 binding via coiled-coil domains and a Spa2 homology domain is required for sustained activation of MEK1-ERK1/2 after stimulation with angiotensin II and epidermal growth factor. We propose that GIT1 serves as a scaffold protein to facilitate c-Src-dependent activation of MEK1-ERK1/2 in response to both GPCRs and TKRs.

Adaptor Proteins, Signal Transducing↗