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OBJECTIVE: We examined the effect of the vasoactive agents carbon monoxide (CO) and nitric oxide (NO) : n the phosphorylation and intracellular redistribution of vasodilator-stimulated phosphoprotein (VASP),a critical actin motor protein required for cell migration that also controls vasodilation and platelet aggregation. RESEARCH DESIGN AND METHODS: We examined the effect of donor-released CO and NO in endothelial progenitor cells (EPCs) and platelets from nondiabetic and diabetic subjects and in human microvascular endothelial cells (HMECs) cultured under low (5.5 mmol/l) or high (25 mmol/l) glucose conditions. VASP phosphorylation was evaluated using phosphorylation site-specific antibodies. RESULTS: In control platelets,CO selectively promotes phosphorylation at VASP Ser-157,whereas NO promotes phosphorylation primarily at Ser-157 and also at Ser-239,with maximal responses at 1 min with both agents on Ser-157 and at 15 min on Ser-239 with NO treatment. In diabetic platelets,neither agent resulted in VASP phosphorylation. In nondiabetic EPCs,NO and CO increased phosphorylation at Ser-239 and Ser-157,respectively,but this response was markedly reduced in diabetic EPCs. In endothelial cells cultured under low glucose conditions,both CO and NO induced phosphorylation at Ser-157 and Ser-239; however,this response was completely lost when cells were cultured under high glucose conditions. In control EPCs and in HMECs exposed to low glucose,VASP was redistributed to filopodia-like structures following CO or NO exposure; however,redistribution was dramatically attenuated under high glucose conditions. CONCLUSIONS: Vasoactive gases CO and NO promote cytoskeletal changes through site- and cell type-specific VASP phosphorylation,and in diabetes,blunted responses to these agents may lead to reduced vascular repair and tissue perfusion. View Publication

This study investigated the factors responsible for migration and homing of magnetically labeled AC133(+) cells at the sites of active angiogenesis in tumor. AC133(+) cells labeled with ferumoxide-protamine sulfate were mixed with either rat glioma or human melanoma cells and implanted in flank of nude mice. An MRI of the tumors including surrounding tissues was performed. Tumor sections were stained for Prussian blue (PB),platelet-derived growth factor (PDGF),hypoxia-inducible factor-1alpha (HIF-1alpha),stromal cell derived factor-1 (SDF-1),matrix metalloproteinase-2 (MMP-2),vascular endothelial growth factor (VEGF),and endothelial markers. Fresh snap-frozen strips from the central and peripheral parts of the tumor were collected for Western blotting. MRIs demonstrated hypointense regions at the periphery of the tumors where the PB(+)/AC133(+) cells were positive for endothelial cells markers. At the sites of PB(+)/AC133(+) cells,both HIF-1alpha and SDF-1 were strongly positive and PDGF and MMP-2 showed generalized expression in the tumor and surrounding tissues. There was no significant association of PB(+)/AC133(+) cell localization and VEGF expression in tumor cells. Western blot demonstrated strong expression of the SDF-1,MMP-2,and PDGF at the peripheral parts of the tumors. HIF-1alpha was expressed at both the periphery and central parts of the tumor. This work demonstrates that magnetically labeled cells can be used as probes for MRI and histological identification of administered cells. View Publication

OBJECTIVE: In this study,the alternative splicing product of vasohibin 1 (VASH1B) was analyzed in direct comparison to the major isoform (VASH1A) for antiangiogenic effects on endothelial colony forming cells (ECFCs) from peripheral blood and on human umbilical vein endothelial cells (HUVECs). METHODS AND RESULTS: Expression studies in primary human endothelial cells revealed that both vasohibin proteins,hVASH1A and hVASH1B,localized in the nucleus and cytoplasm. Adenoviruses carrying the cDNA for VASH1A/B and purified recombinant proteins were used to study the function of both molecules in ECFCs and HUVECs. Recombinant VASH1A protein did not inhibit cell proliferation,tube formation,or vessel growth in vivo in the chick chorioallantoic membrane (CAM) assay,but promoted endothelial cell migration in vitro. The VASH1B protein had an inhibitory effect on cell proliferation,migration,tube formation,and inhibited blood vessel formation in the CAM assay. Adenoviral overexpression of VASH1B,but not of VASH1A,resulted in inhibition of endothelial cell growth,migration,and capillary formation. Interestingly,overexpression of VASH1A and B induced apoptosis in proliferating human fibroblasts,but did not affect cell growth of keratinocytes. CONCLUSIONS: Our data point out that alternative splicing of the VASH1 pre-mRNA transcript generates a potent antiangiogenic protein. View Publication

OBJECTIVE: Studies in pulmonary arteries (PA) of patients with chronic obstructive pulmonary disease (COPD) suggest that bone marrow-derived endothelial progenitor cells (CD133(+)) may infiltrate the intima and differentiate into smooth muscle cells (SMC). This study aimed to evaluate the plasticity of CD133(+) cells to differentiate into SMC and endothelial cells (EC) in both cell culture and human isolated PA. METHODS: Plasticity of granulocyte-colony stimulator factor (G-CSF)-mobilized peripheral blood CD133(+) cells was assessed in co-cultures with primary lines of human PA endothelial cells (PAEC) or SMC (PASMC) and in isolated human PA. We also evaluated if the phenotype of differentiated progenitor cells was acquired by fusion or differentiation. RESULTS: The in vitro studies demonstrated CD133(+) cells may acquire the morphology and phenotype of the cells they were co-cultured with. CD133(+) cells co-incubated with human isolated PA were able to migrate into the intima and differentiate into SMC. Progenitor cell differentiation was produced without fusion with mature cells. CONCLUSIONS: We provide evidence of plasticity of CD133(+) cells to differentiate into both endothelial cells and SMC,reinforcing the idea of their potential role in the remodeling process of PA in COPD. This process was conducted by transdifferentiation and not by cell fusion. View Publication

BACKGROUND AND OBJECTIVES: Chemokines are soluble mediators involved in angiogenesis,cellular growth control and immunomodulation. In the present study we investigated the effects of various chemokines on proliferation of acute myelogenous leukemia (AML) cells and constitutive chemokine release by primary AML cells. DESIGN AND METHODS: Native human AML cells derived from 68 consecutive patients were cultured in vitro. We investigated AML cell proliferation (3H-thymidine incorporation,colony formation),chemokine receptor expression,constitutive chemokine release and chemotaxis of normal peripheral blood mononuclear cells. RESULTS: Exogenous chemokines usually did not have any effect on AML blast proliferation in the absence of hematopoietic growth factors,but when investigating growth factor-dependent (interleukin 3 + granulocyte-macrophage colony-stimulating factor + stem cell factor) proliferation in suspension cultures the following patient subsets were identified: (i) patients whose cells showed chemokine-induced growth enhancement (8 patients); (ii) divergent effects on proliferation (15 patients); and (iii) no effect (most patients). These patient subsets did not differ in chemokine receptor expression,but,compared to CD34- AML cells,CD34+ cells showed higher expression of several receptors. Chemokines also increased the proliferation of clonogenic AML cells from the first subset of patients. Furthermore,a broad constitutive chemokine release profile was detected for most patients,and the following chemokine clusters could be identified: CCL2-4/CXCL1/8,CCL5/CXCL9-11 (possibly also CCL23) and CCL13/17/22/24/CXCL5 (possibly also CXCL6). Only the CCL2-4/CXCL1/8 cluster showed significant correlations between corresponding mRNA levels and NFkB levels/activation. The chemotaxis of normal immunocompetent cells for patients without constitutive chemokine release was observed to be decreased. INTERPRETATION AND CONCLUSIONS: Differences in chemokine responsiveness as well as chemokine release contribute to patient heterogeneity in AML. Patients with AML can be classified into distinct subsets according to their chemokine responsiveness and chemokine release profile. View Publication

OBJECTIVE: To investigate the kinetics of myocardial engraftment of bone marrow-derived mononuclear cells (BMNCs) after intracoronary injection using 99mTc-d,l-hexamethylpropylene amine oxime (99mTc-HMPAO) nuclear imaging in patients with acute and chronic anterior myocardial infarction. DESIGN: Nuclear imaging-derived tracking of BMNCs at 2 and 20 h after injection in the left anterior descending (LAD) coronary artery. SETTING: Academical cardiocentre. PATIENTS: Five patients with acute (mean (SD) age 58 (11) years; ejection fraction range 33-45%) and five patients with chronic (mean (SD) age 50 (6) years; ejection fraction range 28-34%) anterior myocardial infarction. INTERVENTIONS: A total of 24.2 x 10(8)-57.0 x 10(8) BMNCs (20% labelled with 700-1000 MBq 99mTc-HMPAO) were injected in the LAD coronary artery. RESULTS: At 2 h after BMNC injection,myocardial activity was observed in all patients with acute (range 1.31-5.10%) and in all but one patient with chronic infarction (range 1.10-3.0%). At 20 h,myocardial engraftment was noted only in three patients with acute myocardial infarction,whereas no myocardial activity was noted in any patient with chronic infarction. CONCLUSIONS: Engraftment of BMNCs shows dynamic changes within the first 20 h after intracoronary injection. Persistent myocardial engraftment was noted only in a subset of patients with acute myocardial infarction. View Publication

In vivo endothelial commitment of adipose stem cells (ASCs) has scarcely been reported,and controversy remains on the contribution of ASCs to vascularization. We address the epigenetic commitment of ASCs to the endothelial lineage. We report a bisulfite sequencing analysis of CpG methylation in the promoters of two endothelial-cell-specific genes,CD31 and CD144,in freshly isolated and in cultures of ASCs before and after induction of endothelial differentiation. In contrast to adipose tissue-derived endothelial (CD31(+)) cells,freshly isolated ASCs display a heavily methylated CD31 promoter and a mosaically methylated CD144 promoter despite basal transcription of both genes. Methylation state of both promoters remains globally stable upon culture. Endothelial stimulation of ASCs in methylcellulose elicits phenotypic changes,marginal upregulation of CD31,and CD144 expression and restrictive induction of a CD31(+)CD144(+) immunophenotype. These events are accompanied by discrete changes in CpG methylation in CD31 and CD144 promoters; however,no global demethylation that marks CD31(+) cells and human umbilical vein endothelial cells occurs. Immunoselection of CD31(+) cells after endothelial stimulation reveals consistent demethylation of one CpG immediately 3' of the transcription start site of the CD31 promoter. Adipogenic or osteogenic differentiation maintains CD31 and CD144 methylation patterns of undifferentiated cells. Methylation profiles of CD31 and CD144 promoters suggest a limited commitment of ASCs to the endothelial lineage. This contrasts with the reported hypomethylation of adipogenic promoters,which reflects a propensity of ASCs toward adipogenic differentiation. Analysis of CpG methylation at lineage-specific promoters provides a robust assessment of epigenetic commitment of stem cells to a specific lineage. View Publication

Reduced microcirculation and diminished expression of growth factors contribute to wound healing impairment in diabetes. Placenta growth factor (PlGF),an angiogenic mediator promoting pathophysiological neovascularization,is expressed during cutaneous wound healing and improves wound closure by enhancing angiogenesis. By using streptozotocin-induced diabetic mice,we here demonstrate that PlGF induction is strongly reduced in diabetic wounds. Diabetic transgenic mice overexpressing PlGF in the skin displayed accelerated wound closure compared with diabetic wild-type littermates. Moreover,diabetic wound treatment with an adenovirus vector expressing the human PlGF gene (AdCMV.PlGF) significantly accelerated the healing process compared with wounds treated with a control vector. The analysis of treated wounds showed that PlGF gene transfer improved granulation tissue formation,maturation,and vascularization,as well as monocytes/macrophages local recruitment. Platelet-derived growth factor,fibroblast growth factor-2,and vascular endothelial growth factor mRNA levels were increased in AdCMV.PlGF-treated wounds,possibly enhancing PlGF-mediated effects. Finally,PlGF treatment stimulated cultured dermal fibroblast migration,pointing to a direct role of PlGF in accelerating granulation tissue maturation. In conclusion,our data indicate that reduced PlGF expression contributes to impaired wound healing in diabetes and that PlGF gene transfer to diabetic wounds exerts therapeutic activity by promoting different aspects of the repair process. View Publication

Establishment of angiogenic circuits that orchestrate blood vessel development and remodeling requires an exquisite balance between the activities of pro- and antiangiogenic factors. However,the logic that permits complex signal integration by vascular endothelium is poorly understood. We demonstrate that a neuropeptide� View Publication

Definitive mesoderm arises from a bipotent mesendodermal population,and to study processes controlling its development at this stage,embryonic stem (ES) cells can be employed. SHB (Src homology 2 protein in beta-cells) is an adapter protein previously found to be involved in ES cell differentiation to mesoderm. To further study the role of SHB in this context,we have established ES cell lines deficient for one (SHB+/-) or both SHB alleles (SHB-/-). Differentiating embryoid bodies (EBs) derived from these ES cell lines were used for gene expression analysis. Alternatively,EBs were stained for the blood vessel marker CD31. For hematopoietic differentiation,EBs were differentiated in methylcellulose. SHB-/- EBs exhibited delayed down-regulation of the early mesodermal marker Brachyury. Later mesodermal markers relatively specific for the hematopoietic,vascular,and cardiac lineages were expressed at lower levels on day 6 or 8 of differentiation in EBs lacking SHB. The expression of vascular endothelial growth factor receptor-2 and fibroblast growth factor receptor-1 was also reduced in SHB-/- EBs. SHB-/- EBs demonstrated impaired blood vessel formation after vascular endothelial growth factor stimulation. In addition,the SHB-/- ES cells formed fewer blood cell colonies than SHB+/+ ES cells. It is concluded that SHB is required for appropriate hematopoietic and vascular differentiation and that delayed down-regulation of Brachyury expression may play a role in this context. View Publication

Hematopoietic progenitor cell trafficking is an important phenomenon throughout life. It is thought to occur in sequential steps,similar to what has been described for mature leukocytes. Molecular actors have been identified for each step of leukocyte migration; recently,CD99 was shown to play a part during transendothelial migration. We explored the expression and role of CD99 on human hematopoietic progenitors. We demonstrate that (1) CD34+ cells express CD99,albeit with various intensities; (2) subsets of CD34+ cells with high or low levels of CD99 expression produce different numbers of erythroid,natural killer (NK),or dendritic cells in the in vitro differentiation assays; (3) the level of CD99 expression is related to the ability to differentiate toward B cells; (4) CD34+ cells that migrate through an endothelial monolayer in response to SDF-1alpha and SCF display the highest level of CD99 expression; (5) binding of a neutralizing antibody to CD99 partially inhibits transendothelial migration of CD34+ progenitors in an in vitro assay; and (6) binding of a neutralizing antibody to CD99 reduces homing of CD34+ progenitors xenotransplanted in NOD-SCID mice. We conclude that expression of CD99 on human CD34+ progenitors has functional significance and that CD99 may be involved in transendothelial migration of progenitors. View Publication

Current therapies for delayed- or nonunion bone fractures are still largely ineffective. Previous studies indicated that the VEGF homolog placental growth factor (PlGF) has a more significant role in disease than in health. Therefore we investigated the role of PlGF in a model of semi-stabilized bone fracture healing. Fracture repair in mice lacking PlGF was impaired and characterized by a massive accumulation of cartilage in the callus,reminiscent of delayed- or nonunion fractures. PlGF was required for the early recruitment of inflammatory cells and the vascularization of the fracture wound. Interestingly,however,PlGF also played a role in the subsequent stages of the repair process. Indeed in vivo and in vitro findings indicated that PlGF induced the proliferation and osteogenic differentiation of mesenchymal progenitors and stimulated cartilage turnover by particular MMPs. Later in the process,PlGF was required for the remodeling of the newly formed bone by stimulating osteoclast differentiation. As PlGF expression was increased throughout the process of bone repair and all the important cell types involved expressed its receptor VEGFR-1,the present data suggest that PlGF is required for mediating and coordinating the key aspects of fracture repair. Therefore PlGF may potentially offer therapeutic advantages for fracture repair. View Publication