Reprogramming of myeloid angiogenic cells by Bartonella henselae leads to microenvironmental regulation of pathological angiogenesis.

The contribution of myeloid cells to tumour microenvironments is a decisive factor in cancer progression. Tumour-associated macrophages (TAMs) mediate tumour invasion and angiogenesis through matrix remodelling, immune modulation and release of pro-angiogenic cytokines. Nothing is known about how pathogenic bacteria affect myeloid cells in these processes. Here we show that Bartonella henselae, a bacterial pathogen causing vasculoproliferative diseases (bacillary angiomatosis), reprogrammes human myeloid angiogenic cells (MACs), a pro-angiogenic subset of circulating progenitor cells, towards a TAM-like phenotype with increased pro-angiogenic capacity. B. henselae infection resulted in inhibition of cell death, activation of angiogenic cellular programmes and induction of M2 macrophage polarization. MACs infected with B. henselae incorporated into endothelial sprouts and increased angiogenic growth. Infected MACs developed a vascular mimicry phenotype in vitro, and expression of B. henselae adhesin A was essential in inducing these angiogenic effects. Secretome analysis revealed that increased pro-angiogenic activities were associated with the creation of a tumour-like microenvironment dominated by angiogenic inflammatory cytokines and matrix remodelling compounds. Our results demonstrate that manipulation of myeloid cells by pathogenic bacteria can contribute to microenvironmental regulation of pathological tissue growth and suggest parallels underlying both bacterial infections and cancer.

Class IIb HDAC6 regulates endothelial cell migration and angiogenesis by deacetylation of cortactin.

Histone deacetylases (HDACs) deacetylate histones and non-histone proteins, thereby affecting protein activity and gene expression. The regulation and function of the cytoplasmic class IIb HDAC6 in endothelial cells (ECs) is largely unexplored. Here, we demonstrate that HDAC6 is upregulated by hypoxia and is essential for angiogenesis. Silencing of HDAC6 in ECs decreases sprouting and migration in vitro and formation of functional vascular networks in matrigel plugs in vivo. HDAC6 regulates zebrafish vessel formation, and HDAC6-deficient mice showed a reduced formation of perfused vessels in matrigel plugs. Consistently, overexpression of wild-type HDAC6 increases sprouting from spheroids. HDAC6 function requires the catalytic activity but is independent of ubiquitin binding and deacetylation of α-tubulin. Instead, we found that HDAC6 interacts with and deacetylates the actin-remodelling protein cortactin in ECs, which is essential for zebrafish vessel formation and which mediates the angiogenic effect of HDAC6. In summary, we show that HDAC6 is necessary for angiogenesis in vivo and in vitro, involving the interaction and deacetylation of cortactin that regulates EC migration and sprouting.

MicroRNA-27a/b controls endothelial cell repulsion and angiogenesis by targeting semaphorin 6A.

MicroRNAs (miRs) are small RNAs that regulate gene expression at the posttranscriptional level. miR-27 is expressed in endothelial cells, but the specific functions of miR-27b and its family member miR-27a are largely unknown. Here we demonstrate that overexpression of miR-27a and miR-27b significantly increased endothelial cell sprouting. Inhibition of both miR-27a and miR-27b impaired endothelial cell sprout formation and induced endothelial cell repulsion in vitro. In vivo, inhibition of miR-27a/b decreased the number of perfused vessels in Matrigel plugs and impaired embryonic vessel formation in zebrafish. Mechanistically, miR-27 regulated the expression of the angiogenesis inhibitor semaphorin 6A (SEMA6A) in vitro and in vivo and targeted the 3'-untranslated region of SEMA6A. Silencing of SEMA6A partially reversed the inhibition of endothelial cell sprouting and abrogated the repulsion of endothelial cells mediated by miR-27a/b inhibition, indicating that SEMA6A is a functionally relevant miR-27 downstream target regulating endothelial cell repulsion. In summary, we show that miR-27a/b promotes angiogenesis by targeting the angiogenesis inhibitor SEMA6A, which controls repulsion of neighboring endothelial cells.

Histone deacetylase 9 promotes angiogenesis by targeting the antiangiogenic microRNA-17-92 cluster in endothelial cells.

OBJECTIVE: Histone deacetylases (HDACs) modulate gene expression by deacetylation of histone and nonhistone proteins. Several HDACs control angiogenesis, but the role of HDAC9 is unclear. METHODS AND RESULTS: Here, we analyzed the function of HDAC9 in angiogenesis and its involvement in regulating microRNAs. In vitro, silencing of HDAC9 reduces endothelial cell tube formation and sprouting. Furthermore, HDAC9 silencing decreases vessel formation in a spheroid-based Matrigel plug assay in mice and disturbs vascular patterning in zebrafish embryos. Genetic deletion of HDAC9 reduces retinal vessel outgrowth and impairs blood flow recovery after hindlimb ischemia. Consistently, overexpression of HDAC9 increases endothelial cell sprouting, whereas mutant constructs lacking the catalytic domain, the nuclear localization sequence, or sumoylation site show no effect. To determine the mechanism underlying the proangiogenic effect of HDAC9, we measured the expression of the microRNA (miR)-17-92 cluster, which is known for its antiangiogenic activity. We demonstrate that silencing of HDAC9 in endothelial cells increases the expression of miR-17-92. Inhibition of miR-17-20a rescues the sprouting defects induced by HDAC9 silencing in vitro and blocking miR-17 expression partially reverses the disturbed vascular patterning of HDAC9 knockdown in zebrafish embryos. CONCLUSIONS: We found that HDAC9 promotes angiogenesis and transcriptionally represses the miR-17-92 cluster.

Epigenetic regulation of endothelial lineage committed genes in pro-angiogenic hematopoietic and endothelial progenitor cells.

RATIONALE: Proangiogenic hematopoietic and endothelial progenitor cells (EPCs) contribute to postnatal neovascularization, but the mechanisms regulating differentiation to the endothelial lineage are unclear. OBJECTIVE: To elucidate the epigenetic control of endothelial gene expression in proangiogenic cells and EPCs. METHODS AND RESULTS: Here we demonstrate that the endothelial nitric oxide synthase (eNOS) promoter is epigenetically silenced in proangiogenic cells (early EPCs), CD34(+) cells, and mesoangioblasts by DNA methylation and prominent repressive histone H3K27me3 marks. In order to reverse epigenetic silencing to facilitate endothelial commitment, we used 3-deazaneplanocin A, which inhibits the histone methyltransferase enhancer of zest homolog 2 and, thereby, reduces H3K27me3. 3-Deazaneplanocin A was not sufficient to increase eNOS expression, but the combination of 3-deazaneplanocin A and the histone deacetylase inhibitor Trichostatin A augmented eNOS expression, indicating that the concomitant inhibition of silencing histone modification and enhancement of activating histone modification facilitates eNOS expression. In ischemic tissue, hypoxia plays a role in recruiting progenitor cells. Therefore, we examined the effect of hypoxia on epigenetic modifications. Hypoxia modulated the balance of repressive to active histone marks and increased eNOS mRNA expression. The reduction of repressive H3K27me3 was associated with an increase of the histone demethylase Jmjd3. Silencing of Jmjd3 induced apoptosis and senescence in proangiogenic cells and inhibited hypoxia-mediated up-regulation of eNOS expression in mesoangioblasts. CONCLUSIONS: These findings provide evidence that histone modifications epigenetically control the eNOS promoter in proangiogenic cells.

Members of the microRNA-17-92 cluster exhibit a cell-intrinsic antiangiogenic function in endothelial cells.

MicroRNAs are endogenously expressed small noncoding RNAs that regulate gene expression on the posttranscriptional level. The miR-17-92 cluster (encoding miR-17, -18a, -19a/b, -20a, and miR-92a) is highly expressed in tumor cells and is up-regulated by ischemia. Whereas miR-92a was recently identified as negative regulator of angiogenesis, the specific functions of the other members of the cluster are less clear. Here we demonstrate that overexpression of miR-17, -18a, -19a, and -20a significantly inhibited 3-dimensional spheroid sprouting in vitro, whereas inhibition of miR-17, -18a, and -20a augmented endothelial cell sprout formation. Inhibition of miR-17 and miR-20a in vivo using antagomirs significantly increased the number of perfused vessels in Matrigel plugs, whereas antagomirs that specifically target miR-18a and miR-19a were less effective. However, systemic inhibition of miR-17/20 did not affect tumor angiogenesis. Further mechanistic studies showed that miR-17/20 targets several proangiogenic genes. Specifically, Janus kinase 1 was shown to be a direct target of miR-17. In summary, we show that miR-17/20 exhibit a cell-intrinsic antiangiogenic activity in endothelial cells. Inhibition of miR-17/20 specifically augmented neovascularization of Matrigel plugs but did not affect tumor angiogenesis indicating a context-dependent regulation of angiogenesis by miR-17/20 in vivo.

Sox2 transduction enhances cardiovascular repair capacity of blood-derived mesoangioblasts.

RATIONALE: Complementation of pluripotency genes may improve adult stem cell functions. OBJECTIVES: Here we show that clonally expandable, telomerase expressing progenitor cells can be isolated from peripheral blood of children. The surface marker profile of the clonally expanded cells is distinct from hematopoietic or mesenchymal stromal cells, and resembles that of embryonic multipotent mesoangioblasts. Cell numbers and proliferative capacity correlated with donor age. Isolated circulating mesoangioblasts (cMABs) express the pluripotency markers Klf4, c-Myc, as well as low levels of Oct3/4, but lack Sox2. Therefore, we tested whether overexpression of Sox2 enhances pluripotency and facilitates differentiation of cMABs in cardiovascular lineages. METHODS AND RESULTS: Lentiviral transduction of Sox2 (Sox-MABs) enhanced the capacity of cMABs to differentiate into endothelial cells and cardiomyocytes in vitro. Furthermore, the number of smooth muscle actin positive cells was higher in Sox-MABs. In addition, pluripotency of Sox-MABs was shown by demonstrating the generation of endodermal and ectodermal progenies. To test whether Sox-MABs may exhibit improved therapeutic potential, we injected Sox-MABs into nude mice after acute myocardial infarction. Four weeks after cell therapy with Sox-MABs, cardiac function was significantly improved compared to mice treated with control cMABs. Furthermore, cell therapy with Sox-MABs resulted in increased number of differentiated cardiomyocytes, endothelial cells, and smooth muscle cells in vivo. CONCLUSIONS: The complementation of Sox2 in Oct3/4-, Klf4-, and c-Myc-expressing cMABs enhanced the differentiation into all 3 cardiovascular lineages and improved the functional recovery after acute myocardial infarction.

Cathepsin L is required for endothelial progenitor cell-induced neovascularization.

Infusion of endothelial progenitor cells (EPC), but not of mature endothelial cells, promotes neovascularization after ischemia. We performed gene expression profiling of EPC and endothelial cells to identify genes that might be important for the neovascularization capacity of EPC. Notably, the protease cathepsin L (CathL) was highly expressed in EPC as opposed to endothelial cells and was essential for matrix degradation and invasion by EPC in vitro. CathL-deficient mice showed impaired functional recovery following hind limb ischemia, supporting the concept of a crucial role for CathL in postnatal neovascularization. Infused CathL-deficient progenitor cells neither homed to sites of ischemia nor augmented neovascularization. Forced expression of CathL in mature endothelial cells considerably enhanced their invasive activity and sufficed to confer their capacity for neovascularization in vivo. We concluded that CathL has a critical role in the integration of circulating EPC into ischemic tissue and is required for EPC-mediated neovascularization.

Kruppel-like factor 2 improves neovascularization capacity of aged proangiogenic cells.

AIMS: Coronary artery disease (CAD) patients have less circulating proangiogenic cells (PACs), formerly known as endothelial progenitor cells, which exhibit impaired neovascularization properties. Inverse correlations were also found between PAC function and risk factors like age. Krüppel-like factor 2 (KLF2) is expressed by mature endothelial cells (ECs), is induced by both shear stress and statins, and provokes endothelial functional differentiation. The aim of this study is to identify whether KLF2 can reverse negative effects of ageing on PAC function. METHODS AND RESULTS: We describe that progenitor cells in the bone marrow and PACs also express KLF2 at a comparable level to mature ECs and that senescence decreases KLF2 levels. To study the effects of ageing on KLF2 levels, we compared progenitor cells of 4 weeks and 16- to 18-month-old C57BL/6 mice. In addition to the three-fold reduction of circulating Sca1(+)/c-Kit(+)/Lin(-) progenitor cells and the 15% reduction of Sca1(+)/Flk1(+) endothelial-committed progenitor cells, the spleen-derived PACs and bone marrow-derived progenitor cells isolated from aged mice showed a lower level of KLF2 when compared with young mice. Lentiviral overexpression of KLF2 increased human PAC numbers and endothelial nitric oxide synthase expression by 60% during in vitro culture. Endothelial lineage-specific KLF2 overexpression in aged bone marrow-derived mononuclear cells strongly augments neovascularization in vivo in a murine hind-limb ischaemia model. CONCLUSION: These results imply that KLF2 is an attractive novel target to rejuvenate PACs before autologous administration to CAD patients.

Proteomic characterization of human early pro-angiogenic cells.

Early pro-angiogenic cells (EPCs) have been shown to be involved in neovascularization, angiogenesis and re-endothelialization and cathepsin L inhibition blunted their pro-angiogenic effect. In the present study, we have analysed and mapped the proteome and secretome of human EPCs, utilizing a combination of difference in-gel electrophoresis (DIGE) and shotgun proteomics. A population of 206 protein spots were analysed, with 171 being identified in the cellular proteome of EPCs. 82 proteins were identified in their conditioned medium, including the alternative macrophage markers C-C motif chemokine 18 (CCL18) and the hemoglobin scavenger receptor CD163 as well as platelet factor 4 (CXCL4) and platelet basic protein (CXCL7) with "platelet alpha granule" being returned as the top category according to the Gene Ontology Annotation. Apart from cathepsin L, the cathepsin L inhibitor also attenuated the release of a wide range of other cathepsins and lysosomal proteins such as legumain, but stimulated the secretion of members of the S100 protein family. The data presented here are the most comprehensive characterization of protein expression and secretion in human EPCs to date and highlight the potential importance of cysteine proteases in the processing of platelet factors for their pro-angiogenic potential. This article is part of a special issue entitled, "Cardiovascular Stem Cells Revisited".

Mechanism of improved cardiac function after bone marrow mononuclear cell therapy: role of cardiovascular lineage commitment.

BACKGROUND: Cell therapy is a promising option to improve functional recovery after ischemia. Several subsets of bone marrow-derived cells were shown to reduce infarct size and increase ejection fraction in experimental models of ischemia. The mechanisms underlying the functional improvement are diverse and have been shown to include paracrine effects of the injected cells, as well as a variable degree of differentiation to endothelial cells, pericytes, smooth muscle, and cardiac muscle. METHODS AND RESULTS: To elucidate the true nature of such plasticity and contribution to recovery, we engineered vectors that encoded inducible suicide genes under the control of endothelium (endothelial nitric oxide synthase)-, smooth muscle (SM22alpha)-, and cardiomyocyte (alpha-MHC)-specific promoters, thereby allowing selective depletion of the individual cell lineage acquired by the transplanted undifferentiated bone marrow-derived cells. Lentivirally delivered thymidine kinase, which converts the prodrug ganciclovir into a cytotoxic agent, was used to selectively eliminate cells 2 weeks after transplantation of bone marrow mononuclear cells in an acute myocardial infarction model. We demonstrate that elimination of transplanted endothelium-committed or SM22alpha-expressing cells, but not cardiac-committed cells, induced a significant deterioration of ejection fraction. Moreover, elimination of endothelial nitric oxide synthase-expressing cells 2 weeks after injection reduced capillary and arteriole density. CONCLUSIONS: This study demonstrates that elimination of bone marrow mononuclear cells reexpressing endothelial nitric oxide synthase particularly induced a deterioration of cardiac function, which indicates a functional contribution of the vascular cell fate decision of human bone marrow-derived mononuclear cells in vivo.

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.

Role of beta2-integrins for homing and neovascularization capacity of endothelial progenitor cells.

The mechanisms of homing of endothelial progenitor cells (EPCs) to sites of ischemia are unclear. Here, we demonstrate that ex vivo-expanded EPCs as well as murine hematopoietic Sca-1+/Lin- progenitor cells express beta2-integrins, which mediate the adhesion of EPCs to endothelial cell monolayers and their chemokine-induced transendothelial migration in vitro. In a murine model of hind limb ischemia, Sca-1+/Lin- hematopoietic progenitor cells from beta2-integrin-deficient mice are less capable of homing to sites of ischemia and of improving neovascularization. Preactivation of the beta2-integrins expressed on EPCs by activating antibodies augments the EPC-induced neovascularization in vivo. These results provide evidence for a novel function of beta2-integrins in postnatal vasculogenesis.

Proteomic analysis reveals presence of platelet microparticles in endothelial progenitor cell cultures.

The concept of endothelial progenitor cells (EPCs) has attracted considerable interest in cardiovascular research, but despite a decade of research there are still no specific markers for EPCs and results from clinical trials remain controversial. Using liquid chromatography-tandem mass spectrometry, we analyzed the protein composition of microparticles (MPs) originating from the cell surface of EPC cultures. Our data revealed that the conventional methods for isolating mononuclear cells lead to a contamination with platelet proteins. Notably, platelets readily disintegrate into platelet MPs. These platelet MPs are taken up by the mononuclear cell population, which acquires "endothelial" characteristics (CD31, von Willebrand factor [VWF], lectin-binding), and angiogenic properties. In a large population-based study (n = 526), platelets emerged as a positive predictor for the number of colony-forming units and early outgrowth EPCs. Our study provides the first evidence that the cell type consistent with current definitions of an EPC phenotype may arise from an uptake of platelet MPs by mononuclear cells resulting in a gross misinterpretation of their cellular progeny. These findings demonstrate the advantage of using an unbiased proteomic approach to assess cellular phenotypes and advise caution in attributing the benefits in clinical trials using unselected bone marrow mononuclear cells (BMCs) to stem cell-mediated repair.

HDAC5 is a repressor of angiogenesis and determines the angiogenic gene expression pattern of endothelial cells.

Class IIa histone deacetylases (HDACs) are signal-responsive regulators of gene expression involved in vascular homeostasis. To investigate the differential role of class IIa HDACs for the regulation of angiogenesis, we used siRNA to specifically suppress the individual HDAC isoenzymes. Silencing of HDAC5 exhibited a unique pro-angiogenic effect evidenced by increased endothelial cell migration, sprouting, and tube formation. Consistently, overexpression of HDAC5 decreased sprout formation, indicating that HDAC5 is a negative regulator of angiogenesis. The antiangiogenic activity of HDAC5 was independent of myocyte enhancer factor-2 binding and its deacetylase activity but required a nuclear localization indicating that HDAC5 might affect the transcriptional regulation of gene expression. To identify putative HDAC5 targets, we performed microarray expression analysis. Silencing of HDAC5 increased the expression of fibroblast growth factor 2 (FGF2) and angiogenic guidance factors, including Slit2. Antagonization of FGF2 or Slit2 reduced sprout induction in response to HDAC5 siRNA. Chromatin immunoprecipitation assays demonstrate that HDAC5 binds to the promoter of FGF2 and Slit2. In summary, HDAC5 represses angiogenic genes, such as FGF2 and Slit2, which causally contribute to capillary-like sprouting of endothelial cells. The derepression of angiogenic genes by HDAC5 inactivation may provide a useful therapeutic target for induction of angiogenesis.

Comparative proteomics profiling reveals role of smooth muscle progenitors in extracellular matrix production.

OBJECTIVE: Recent studies on cardiovascular progenitors have led to a new appreciation that paracrine factors may support the regeneration of damaged tissues. METHODS AND RESULTS: We used a shotgun proteomics strategy to compare the secretome of peripheral blood-derived smooth muscle progenitors (SPCs) with human aortic smooth muscle cells. The late-outgrowth SPCs produced fewer proteolytic enzymes and inflammatory cytokines and showed reduced invasive capacity. Similar to smooth muscle cells, SPCs secreted extracellular matrix. However, SPCs produced different matrix proteins, as evidenced by the truncation of proangiogenic domains in collagen alpha-1 (I) and increased production of periostin. Moreover, SPCs retained serum proteins, including proteoglycans, regulating collagen assembly; and pigment epithelium-derived factor, a potent inhibitor of angiogenesis. As a functional consequence, their conditioned medium was less angiogenic, as demonstrated by endothelial tube formation assays in vitro and implantation of Matrigel plugs into nude, severe combined immunodeficient mice (NOD/SCID). CONCLUSIONS: The present study represents an important conceptual development, suggesting that SPCs may contribute to extracellular matrix production.

Essential role of endothelial nitric oxide synthase for mobilization of stem and progenitor cells.

Endothelial nitric oxide synthase (eNOS) is essential for neovascularization. Here we show that the impaired neovascularization in mice lacking eNOS is related to a defect in progenitor cell mobilization. Mice deficient in eNOS (Nos3(-/-)) show reduced vascular endothelial growth factor (VEGF)-induced mobilization of endothelial progenitor cells (EPCs) and increased mortality after myelosuppression. Intravenous infusion of wild-type progenitor cells, but not bone marrow transplantation, rescued the defective neovascularization of Nos3(-/-) mice in a model of hind-limb ischemia, suggesting that progenitor mobilization from the bone marrow is impaired in Nos3(-/-) mice. Mechanistically, matrix metalloproteinase-9 (MMP-9), which is required for stem cell mobilization, was reduced in the bone marrow of Nos3(-/-) mice. These findings indicate that eNOS expressed by bone marrow stromal cells influences recruitment of stem and progenitor cells. This may contribute to impaired regeneration processes in ischemic heart disease patients, who are characterized by a reduced systemic NO bioactivity.

Phosphatidylinositol-3-kinase-gamma is integral to homing functions of progenitor cells.

Endothelial progenitor cells (EPCs) and hematopoietic progenitor cells are recruited to ischemic regions, improving neovascularization. beta1 and beta2 integrins play a crucial role for progenitor cell homing to ischemic tissues. Integrin activity is regulated by chemokines and their respective G protein-coupled receptors. The phosphatidylinositol-3-kinase catalytic subunit gamma (PI3Kgamma) is the PI3K isoform that selectively transduces signals from G protein-coupled receptors. Here, we investigated the role of PI3Kgamma as a signaling intermediate in the chemokine-induced integrin-dependent homing functions of progenitor cells. A pharmacological PI3Kgamma inhibitor significantly reduced chemokine-induced chemotaxis and stromal cell-derived factor (SDF)1alpha-induced transmigration of human EPCs. Moreover, the PI3Kgamma inhibitor significantly reduced SDF1alpha-induced adhesion of EPCs to intercellular adhesion molecule-1 and human umbilical vein endothelial cell monolayers. These findings were corroborated with Lin(-) bone marrow-derived progenitor cells from PI3Kgamma-deficient mice that displayed reduced SDF1alpha-induced migration and intercellular adhesion molecule-1 adhesion as compared with wild-type cells. Pharmacological inhibition or genetic ablation of PI3Kgamma reduced SDF1alpha-induced integrin activation in human EPCs and in murine Lin(-) BM-derived progenitor cells, respectively. In vivo, the homing of PI3Kgamma-deficient Lin(-) progenitor cells to ischemic muscles after intravenous infusion in the model of hindlimb ischemia and their neovascularization-promoting capacity was reduced as compared with wild-type cells. In conclusion, PI3Kgamma is integral to the integrin-dependent homing of progenitor cells.

The Wnt antagonist Dickkopf-1 mobilizes vasculogenic progenitor cells via activation of the bone marrow endosteal stem cell niche.

Therapeutic mobilization of vasculogenic progenitor cells is a novel strategy to enhance neovascularization for tissue repair. Prototypical mobilizing agents such as granulocyte colony-stimulating factor mobilize vasculogenic progenitor cells from the bone marrow concomitantly with inflammatory cells. In the bone marrow, mobilization is regulated in the stem cell niche, in which endosteal cells such as osteoblasts and osteoclasts play a key role. Because Wnt signaling regulates endosteal cells, we examined whether the Wnt signaling antagonist Dickkopf (Dkk)-1 is involved in the mobilization of vasculogenic progenitor cells. Using TOP-GAL transgenic mice to determine activation of beta-catenin, we demonstrate that Dkk-1 regulates endosteal cells in the bone marrow stem cell niche and subsequently mobilizes vasculogenic and hematopoietic progenitors cells without concomitant mobilization of inflammatory neutrophils. The mobilization of vasculogenic progenitors required the presence of functionally active osteoclasts, as demonstrated in PTPepsilon-deficient mice with defective osteoclast function. Mechanistically, Dkk-1 induced the osteoclast differentiation factor RANKL, which subsequently stimulated the release of the major bone-resorbing protease cathepsin K. Eventually, the Dkk-1-induced mobilization of bone marrow-derived vasculogenic progenitors enhanced neovascularization in Matrigel plugs. Thus, these data show that Dkk-1 is a mobilizer of vasculogenic progenitors but not of inflammatory cells, which could be of great clinical importance to enhance regenerative cell therapy.

Interleukin-10 from transplanted bone marrow mononuclear cells contributes to cardiac protection after myocardial infarction.

Bone marrow mononuclear cells (BM-MNCs) have successfully been used as a therapy for the improvement of left ventricular (LV) function after myocardial infarction (MI). It has been suggested that paracrine factors from BM-MNCs may be a key mechanism mediating cardiac protection. We previously performed microarray analysis and found that the pleiotropic cytokine interleukin (IL)-10 was highly upregulated in human progenitor cells in comparison with adult endothelial cells and CD14+ cells. Moreover, BM-MNCs secrete significant amounts of IL-10, and IL-10 could be detected from progenitor cells transplanted in infarcted mouse hearts. Specifically, intramyocardial injection of wild-type BM-MNCs led to a significant decrease in LV end-diastolic pressure (LVEDP) and LV end-systolic volume (LVESV) compared to hearts injected with either diluent or IL-10 knock-out BM-MNCs. Furthermore, intramyocardial injection of wild-type BM-MNCs led to a significant increase in stroke volume (SV) and rate of the development of pressure over time (+dP/dt) compared to hearts injected with either diluent or IL-10 knock-out BM-MNCs. The IL-10-dependent improvement provided by transplanted cells was not caused by reduced infarct size, neutrophil infiltration, or capillary density, but rather was associated with decreased T lymphocyte accumulation, reactive hypertrophy, and myocardial collagen deposition. These results suggest that BM-MNCs mediate cardiac protection after myocardial infarction and this is, at least in part, dependent on IL-10.