Lysophosphatidic Acid Receptor 4 Is Transiently Expressed during Cardiac Differentiation and Critical for Repair of the Damaged Heart.

Efficient differentiation of pluripotent stem cells (PSCs) into cardiac cells is essential for the development of new therapeutic modalities to repair damaged heart tissue. We identified a novel cell surface marker, the G protein-coupled receptor lysophosphatidic acid receptor 4 (LPAR4), specific to cardiac progenitor cells (CPCs) and determined its functional significance and therapeutic potential. During in vitro differentiation of mouse and human PSCs toward cardiac lineage, LPAR4 expression peaked after 3-7 days of differentiation in cardiac progenitors and then declined. In vivo, LPAR4 was specifically expressed in the early stage of embryonal heart development, and as development progressed, LPAR4 expression decreased and was non-specifically distributed. We identified the effective agonist octadecenyl phosphate and a p38 MAPK blocker as the downstream signal blocker. Sequential stimulation and inhibition of LPAR4 using these agents enhanced the in vitro efficiency of cardiac differentiation from mouse and human PSCs. Importantly, in vivo, this sequential stimulation and inhibition of LPAR4 reduced the infarct size and rescued heart dysfunction in mice. In conclusion, LPAR4 is a novel CPC marker transiently expressed only in heart during embryo development. Modulation of LPAR4-positive cells may be a promising strategy for repairing myocardium after myocardial infarction.

The MicroRNA-92a/Sp1/MyoD Axis Regulates Hypoxic Stimulation of Myogenic Lineage Differentiation in Mouse Embryonic Stem Cells.

Hypoxic microenvironments exist in developing embryonic tissues and determine stem cell fate. We previously demonstrated that hypoxic priming plays roles in lineage commitment of embryonic stem cells. In the present study, we found that hypoxia-primed embryoid bodies (Hyp-EBs) efficiently differentiate into the myogenic lineage, resulting in the induction of the myogenic marker MyoD, which was not mediated by hypoxia-inducible factor 1α (HIF1α) or HIF2α, but rather by Sp1 induction and binding to the MyoD promoter. Knockdown of Sp1 in Hyp-EBs abrogated hypoxia-induced MyoD expression and myogenic differentiation. Importantly, in the cardiotoxin-muscle injury mice model, Hyp-EB transplantation facilitated muscle regeneration in vivo, whereas transplantation of Sp1-knockdown Hyp-EBs failed to do. Moreover, we compared microRNA (miRNA) expression profiles between EBs under normoxia versus hypoxia and found that hypoxia-mediated Sp1 induction was mediated by the suppression of miRNA-92a, which directly targeted the 3' untranslated region (3' UTR) of Sp1. Further, the inhibitory effect of miRNA-92a on Sp1 in luciferase assay was abolished by a point mutation in specific sequence in the Sp1 3' UTR that is required for the binding of miRNA-92a. Collectively, these results suggest that hypoxic priming enhances EB commitment to the myogenic lineage through miR-92a/Sp1/MyoD regulatory axis, suggesting a new pathway that promotes myogenic-lineage differentiation.

Identification of Adult Mesodermal Progenitor Cells and Hierarchy in Atherosclerotic Vascular Calcification.

The nature of calcifying progenitor cells remains elusive. In this study, we investigated the developmental hierarchy and dynamics of progenitor cells. In vitro and in vivo reconstitution assays demonstrated that Sca-1+/PDGFRα- cells in the bone marrow (BM) are the ancestors of Sca-1+/PDGFRα+ cells. Cells of CD29 + Sca-1+/PDGFRα- lineage in the BM showed both hematopoietic potential with osteoclastic differentiation ability as well as mesenchymal stem cell-like properties with osteoblastic differentiation potential. Clonally-isolated BM-derived artery-infiltrated Sca-1+/PDGFRα- cells maintained osteoblastic/osteoclastic bipotency but lost hematopoietic activity. In hypercholesterolemic apolipoprotein-E-deficient (Apoe-/-) mice, the mobilization from BM to peripheral circulation, followed by migration into atherosclerotic plaques of Sca-1+/PDGFRα- cells, but not Sca-1+/PDGFRα+ cells, were significantly decreased, and Interleukin-1ß (IL-1ß) and Interleukin-5 (IL-5) mediated this response. Here, we demonstrated that Sca-1+/PDGFRα- cells are mesodermal progenitor cells in adults, and the dynamics of progenitor cells were regulated by atherosclerosis-related humoral factors. These results may contribute to better understanding of vascular homeostasis and assist in the development of novel therapies for atherosclerosis. Stem Cells 2018;36:1075-1096.

Activated platelet supernatant can augment the angiogenic potential of human peripheral blood stem cells mobilized from bone marrow by G-CSF.

Platelets not only play a role in hemostasis, but they also promote angiogenesis and tissue recovery by releasing various cytokines and making an angiogenic milieu. Here, we examined autologous 'activated platelet supernatant (APS)' as a priming agent for stem cells; thereby enhance their pro-angiogenic potential and efficacy of stem cell-based therapy for ischemic diseases. The mobilized peripheral blood stem cells ((mob)PBSCs) were isolated from healthy volunteers after subcutaneous injection of granulocyte-colony stimulating factor. APS was collected separately from the platelet rich plasma after activation by thrombin. (mob)PBSCs were primed for 6h before analysis. Compared to naive platelet supernatants, APS had a higher level of various cytokines, such as IL8, IL17, PDGF and VEGF. APS-priming for 6h induced (mob)PBSCs to express key angiogenic factors, surface markers (i.e. CD34, CD31, and CXCR4) and integrins (integrins α5, ß1 and ß2). Also (mob)PBSCs were polarized toward CD14(++)/CD16(+) pro-angiogenic monocytes. The priming effect was reproduced by an in vitro reconstruction of APS. Through this phenotype, APS-priming increased cell-cell adhesion and cell-extracellular matrix adhesion. The culture supernatant of APS-primed (mob)PBSCs contained high levels of IL8, IL10, IL17 and TNFα, and augmented proliferation and capillary network formation of human umbilical vein endothelial cells. In vivo transplantation of APS-primed (mob)PBSCs into athymic mice ischemic hindlimbs and Matrigel plugs elicited vessel differentiation and tissue repair. In safety analysis, platelet activity increased after mixing with (mob)PBSCs regardless of priming, which was normalized by aspirin treatment. Collectively, our data identify that APS-priming can enhance the angiogenic potential of (mob)PBSCs, which can be used as an adjunctive strategy to improve the efficacy of cell therapy for ischemic diseases.

SOX17-mediated LPAR4 expression plays a pivotal role in cardiac development and regeneration after myocardial infarction.

Lysophosphatidic acid receptor 4 (LPAR4) exhibits transient expression at the cardiac progenitor stage during pluripotent stem cell (PSC)-derived cardiac differentiation. Using RNA sequencing, promoter analyses, and a loss-of-function study in human PSCs, we discovered that SRY-box transcription factor 17 (SOX17) is an essential upstream factor of LPAR4 during cardiac differentiation. We conducted mouse embryo analyses to further verify our human PSC in vitro findings and confirmed the transient and sequential expression of SOX17 and LPAR4 during in vivo cardiac development. In an adult bone marrow transplantation model using LPAR4 promoter-driven GFP cells, we observed two LPAR4+ cell types in the heart following myocardial infarction (MI). Cardiac differentiation potential was shown in heart-resident LPAR4+ cells, which are SOX17+, but not bone marrow-derived infiltrated LPAR4+ cells. Furthermore, we tested various strategies to enhance cardiac repair through the regulation of downstream signals of LPAR4. During the early stages following MI, the downstream inhibition of LPAR4 by a p38 mitogen-activated protein kinase (p38 MAPK) blocker improved cardiac function and reduced fibrotic scarring compared to that observed following LPAR4 stimulation. These findings improve our understanding of heart development and suggest novel therapeutic strategies that enhance repair and regeneration after injury by modulating LPAR4 signaling.

Induction of pluripotent stem cells from adult somatic cells by protein-based reprogramming without genetic manipulation.

The concept of reprogramming of somatic cells has opened a new era in regenerative medicine. Transduction of defined factors has successfully achieved pluripotency. However, during the generation process of induced pluripotent stem (iPS) cells, genetic manipulation of certain factors may cause tumorigenicity, which limits further application. We report that that a single transfer of embryonic stem (ES) cell-derived proteins into primarily cultured adult mouse fibroblasts, rather than repeated transfer or prolonged exposure to materials, can achieve full reprogramming up to the pluripotent state without the forced expression of ectopic transgenes. During the process, gene expression and epigenetic status were converted from somatic to ES-equivalent status. We verified that protein-based reprogramming was neither by the contamination of protein donor ES cell nor by DNA/RNA from donor ES cell. Protein-iPS cells were biologically and functionally very similar to ES cells and differentiated into 3 germ layers in vitro. Furthermore, protein-iPS cells possessed in vivo differentiation (well-differentiated teratoma formation) and development (chimeric mice generation and a tetraploid blastocyst complementation) potentials. Our results provide an alternative and safe strategy for the reprogramming of somatic cells that can be used to facilitate pluripotent stem cell-based cell therapy.

Cardiovascular Regeneration via Stem Cells and Direct Reprogramming: A Review.

Cardiovascular disease (CVD) is the leading causes of morbidity and death globally. In particular, a heart failure remains a major problem that contributes to global mortality. Considerable advancements have been made in conventional pharmacological therapies and coronary intervention surgery for cardiac disorder treatment. However, more than 15% of patients continuously progress to end-stage heart failure and eventually require heart transplantation. Over the past year, numerous numbers of protocols to generate cardiomyocytes (CMCs) from human pluripotent stem cells (hPSCs) have been developed and applied in clinical settings. Number of studies have described the therapeutic effects of hPSCs in animal models and revealed the underlying repair mechanisms of cardiac regeneration. In addition, biomedical engineering technologies have improved the therapeutic potential of hPSC-derived CMCs in vivo. Recently substantial progress has been made in driving the direct differentiation of somatic cells into mature CMCs, wherein an intermediate cellular reprogramming stage can be bypassed. This review provides information on the role of hPSCs in cardiac regeneration and discusses the practical applications of hPSC-derived CMCs; furthermore, it outlines the relevance of directly reprogrammed CMCs in regenerative medicine.

A subset of macrophages and monocytes in the mouse bone marrow express atypical chemokine receptor 1.

Duffy antigen receptor for chemokines (DARC)/CD234, also known as atypical chemokine receptor 1 (ACKR1), is a seven-transmembrane domain protein expressed on erythrocytes, vascular endothelium, and a subset of epithelial cells (Peiper et al., 1995). Previously, we reported that ACKR1 was expressed in bone marrow macrophages. ACKR1 interacts with CD82 on long-term repopulating hematopoietic stem cells (LT-HSCs) to maintain the dormancy of LT-HSCs during homeostasis (Hur et al., 2016). We also demonstrated that ACKR1 interacts with CD82 in HSCs from human umbilical cord blood (hUCB). These findings demonstrated that CD82 is a functional surface marker of LT-HSCs and this molecule maintains LT-HSC quiescence by interactions with ACKR1-expressing macrophages in mice and humans.

The G Protein-Coupled Receptor Latrophilin-2, A Marker for Heart Development, Induces Myocardial Repair After Infarction.

Discovering cell-surface markers based on a comprehensive understanding of development is utilized to isolate a particular cell type with high purity for therapeutic purposes. Given that latrophilin-2 (Lphn2) substantially contributes to cardiac differentiation, we examined whether Lphn2 regulates functional significance in heart development and repair. We performed whole-mount immunostaining followed by clearing technique of embryo, RNA sequencing related to Lphn2-knockout (KO) embryo, and in vivo functional analyses of Lphn2+ cells using echocardiography. After immunostaining the cleared embryo sample, Lphn2 was exclusively observed in cardiac cells expressing α-sarcomeric actinin at embryonic days E9.5 and E10.5. Homozygous Lphn2-KO mice were embryonically lethal and showed underdevelopment of the ventricular myocardium. However, Lphn2 was not required to develop vessels, including endothelial cells and smooth muscle cells. For the purpose of cardiac regeneration, we transplanted pluripotent stem cell (PSC)-derived Lphn2+ cells into the infarcted heart. PSC-derived Lphn2+ cells differentiated into cardiomyocytes and regenerated the myocardium when transplanted into the infarcted heart, unlike Lphn2- cells. Transplanted Lphn2+ cells improved left-ventricle systolic function and reduced infarct size. We demonstrated that Lphn2 exhibits potential as a cardiomyogenic marker to facilitate targeted stem cell therapy for heart repair in clinical practice.

Adhesion GPCR Latrophilin-2 Specifies Cardiac Lineage Commitment through CDK5, Src, and P38MAPK.

Identifying lineage-specific markers is pivotal for understanding developmental processes and developing cell therapies. Here, we investigated the functioning of a cardiomyogenic cell-surface marker, latrophilin-2 (LPHN2), an adhesion G-protein-coupled receptor, in cardiac differentiation. LPHN2 was selectively expressed in cardiac progenitor cells (CPCs) and cardiomyocytes (CMCs) during mouse and human pluripotent stem cell (PSC) differentiation; cell sorting with an anti-LPHN2 antibody promoted the isolation of populations highly enriched in CPCs and CMCs. Lphn2 knockdown or knockout PSCs did not express cardiac genes. We used the Phospho Explorer Antibody Array, which encompasses nearly all known signaling pathways, to assess molecular mechanisms underlying LPHN2-induced cardiac differentiation. LPHN2-dependent phosphorylation was the strongest for cyclin-dependent kinase 5 (CDK5) at Tyr15. We identified CDK5, Src, and P38MAPK as key downstream molecules of LPHN2 signaling. These findings provide a valuable strategy for isolating CPCs and CMCs from PSCs and insights into the still-unknown cardiac differentiation mechanisms.

Priming with angiopoietin-1 augments the vasculogenic potential of the peripheral blood stem cells mobilized with granulocyte colony-stimulating factor through a novel Tie2/Ets-1 pathway.

BACKGROUND: The low engraftment rate of stem/progenitor cells infused via the intracoronary route to the ischemic myocardium is one of the most important factors limiting the efficacy of cell therapy. We investigated the concept of priming peripheral blood stem cells enriched by granulocyte colony-stimulating factor mobilization and apheresis ((mob)PBSCs) with angiopoietin-1 (Ang1), to enhance the engraftment into the ischemic tissue and neovasculogenic potential. METHODS AND RESULTS: The expression of Tie2, the Ang1 receptor, was significantly higher in (mob)PBSCs than naïve peripheral blood mononuclear cells (19.2+/-3.0% versus 1.2+/-0.8% versus 1.2+/-0.2%; P<0.001 for (mob)PBSCs from acute myocardial infarction (AMI) patients with granulocyte colony-stimulating factor treatment for 3 days versus peripheral blood mononuclear cells from AMI patients versus peripheral blood mononuclear cells from stable angina patients). After 4 hours of cartilage oligomeric matrix protein (COMP)-Ang1 stimulation, (mob)PBSCs committed to the endothelial lineage with the induction of CD31 and VE-cadherin expression, mediated by Tie2/Ets-1 pathway. Priming of (mob)PBSCs with COMP-Ang1 induced the expression of alpha4beta1 and alpha5beta1 integrins, which are also Ets-1 downstream molecules, leading to enhanced adhesion to endothelial cells or fibronectin. In a rabbit ear ischemia/reperfusion model, priming of (mob)PBSCs with COMP-Ang1 improved first-pass engraftment to the distal vascular bed after intraarterial delivery. In a murine ischemic hind-limb model, intravascular delivery of primed (mob)PBSCs enhanced both engraftment and neovascularization. CONCLUSIONS: The short-term priming with COMP-Ang1 may be a feasible and promising option to activate (mob)PBSCs by enhancing differentiation and adhesiveness and to improve the efficacy of cell therapy for ischemic diseases.

New mechanism of rosiglitazone to reduce neointimal hyperplasia: activation of glycogen synthase kinase-3beta followed by inhibition of MMP-9.

OBJECTIVE: Mechanism of neointimal hyperplasia after vascular injury includes activation of signaling pathways and matrix metalloproteinases (MMPs) that are involved in cell proliferation and migration. Rosiglitazone, a synthetic peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonist, was reported to inhibit neointimal hyperplasia in diabetic animals and humans. But the underlying mechanism has not been clarified. In this study, we examined how rosiglitazone inhibited neointimal hyperplasia. METHODS AND RESULTS: The proliferation and survival of cultured rat VSMCs were reduced by rosiglitazone, which was mediated by inhibition of ERK and activation GSK-3beta, without change of Akt. The antiproliferative effect of rosiglitazone was reversed by GSK-3beta inactivation. The migration of VSMCs was also suppressed by rosiglitazone that inhibited the expression and activity MMP-9 through GSK-3beta activation. Thus migration of MMP-9(-/-) VSMCs from MMP-9 knockout mice was not affected by rosiglitazone. The underlying mechanism of MMP-9 suppression by rosiglitazone was that it inhibited NF-kappaB DNA binding activity, which was also dependent on GSK-3beta. In rat carotid artery, balloon injury significantly inactivated GSK-3beta with induction of MMP-9, which was effectively prevented by rosiglitazone. Thus, rosiglitazone significantly decreased the ratio of intima to media by reducing proliferation and inducing apoptosis of VSMCs at neointima, which was reversed by inactivation of GSK-3beta with adenoviral transfer of catalytically-inactive GSK-KM gene. CONCLUSIONS: Rosiglitazone activates GSK-3beta, which inhibits not only proliferation of VSMCs but also migration of VSMCs through blocking NF-kappaB-dependent MMP-9 activation.

Impact of myocardial infarct proteins and oscillating pressure on the differentiation of mesenchymal stem cells: effect of acute myocardial infarction on stem cell differentiation.

Stem cell transplantation in acute myocardial infarction (AMI) has emerged as a promising therapeutic option. We evaluated the impact of AMI on mesenchymal stem cell (MSC) differentiation into cardiomyocyte lineage. Cord blood-derived human MSCs were exposed to in vitro conditions simulating in vivo environments of the beating heart with acute ischemia, as follows: (a) myocardial proteins or serum obtained from sham-operated rats, and (b) myocardial proteins or serum from AMI rats, with or without application of oscillating pressure. Expression of cardiac-specific markers on MSCs was greatly induced by the infarcted myocardial proteins, compared with the normal proteins. It was also induced by application of oscillating pressure to MSCs. Treatment of MSCs with infarcted myocardial proteins and oscillating pressure greatly augmented expression of cardiac-specific genes. Such expression was blocked by inhibitor of transforming growth factor beta(1) (TGF-beta(1)) or bone morphogenetic protein-2 (BMP-2). In vitro cellular and electrophysiologic experiments showed that these differentiated MSCs expressing cardiomyocyte-specific markers were able to make a coupling with cardiomyocytes but not to selfbeat. The pathophysiologic significance of in vitro results was confirmed using the rat AMI model. The protein amount of TGF-beta(1) and BMP-2 in myocardium of AMI was significantly higher than that in normal myocardium. When MSCs were transplanted to the heart and analyzed 8 weeks later, they expressed cardiomyocyte-specific markers, leading to improved cardiac function. These in vitro and in vivo results suggest that infarct-related biological and physical factors in AMI induce commitment of MSCs to cardiomyocyte-like cells through TGF-beta/BMP-2 pathways.

Identification of a novel role of T cells in postnatal vasculogenesis: characterization of endothelial progenitor cell colonies.

BACKGROUND: The colony number of early endothelial progenitor cells (EPCs) has been used as a quantitative indicator of the number of EPCs in the blood or as a biological marker of cardiovascular diseases. In the present study, we found a subset of T cells that were localized at the center of the EPC colony and played a pivotal role in colony formation and differentiation of early EPCs. METHODS AND RESULTS: We found that CD3+ CD31+ CXCR4+ T cells (referred to as angiogenic T cells in the present study) constituted the center of EPC colonies during cultures of human peripheral blood mononuclear cells. These angiogenic T cells were required for colony formation and differentiation of early EPCs. They secreted high levels of angiogenic cytokines such as vascular endothelial growth factor, interleukin-8, and matrix metalloproteinases. Angiogenic T cells showed superior angiogenic potential to the other subset of T cells in the experiments with regard to Matrigel tube formation, adhesion, transendothelial migration, and collagen invasion assay, mainly through the stromal cell-derived factor 1/CXCR-4 axis. Furthermore, angiogenic T cells enhanced endothelial cell proliferation and function. In vivo study showed that angiogenic T cells play an important role in the process of vessel formation. Clinical study showed that the level of angiogenic T cells in the peripheral blood was well correlated with EPC colony numbers and had inverse relationships with age and the number of risk factors for coronary artery disease. CONCLUSIONS: These findings suggest that angiogenic T cells could be a potential therapeutic target for ischemic cardiovascular diseases.

Intercellular adhesion molecule-1 is upregulated in ischemic muscle, which mediates trafficking of endothelial progenitor cells.

BACKGROUND: Trafficking of transplanted endothelial progenitor cells (EPCs) to an ischemic organ is a critical step in neovascularization. This study was performed to elucidate the molecular mechanism of EPC trafficking in terms of adhesion molecules. METHODS AND RESULTS: Using murine hindlimb ischemia model, we examined expressions of E-selectin, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and platelet-endothelial cell adhesion molecule-1 (PECAM-1) in ischemic muscle by immunofluorescence. ICAM-1 was overexpressed in ischemic muscle compared with nonischemic muscle, whereas expressions of E-selectin, VCAM-1, and PECAM-1 did not show that much difference. ICAM-1 was also upregulated by hypoxia in murine endothelial cells (ECs) as assessed by immunoblot and flow cytometry. EPCs were attached to ECs specifically through ICAM-1/beta-2 integrin interaction in vitro. When EPCs were labeled with fluorescent dye or radioisotope (Tc-99m-HMPAO) and systemically administrated in vivo, EPCs preferentially homed to ischemic muscle. By blocking ICAM-1, EPCs entrapment to ischemic limb in vivo was significantly reduced and neovascularization induced by EPC transplantation was attenuated. CONCLUSIONS: ICAM-1 is upregulated by ischemia, and this is closely associated with EPCs entrapment to ischemic limb. Our findings suggest that ICAM-1 expression might be important in regulating the process of neovascularization through its ability to recruit EPCs.

Identification of the early and late responder genes during the generation of induced pluripotent stem cells from mouse fibroblasts.

BACKGROUND: The generation of induced pluripotent stem cell (iPSC), a substitute for embryonic stem cell (ESC), requires the proper orchestration of a transcription program at the chromatin level. Our recent approach for the induction of pluripotent stem cells from fibroblasts using protein extracts from mouse ESCs could overcome the potential tumorigenicity risks associated with random retroviral integration. Here, we examine the epigenetic modifications and the transcriptome of two types of iPSC and of partially reprogrammed iPSCs (iPSCp) generated independently from adult cardiac and skin fibroblasts to assess any perturbations of the transcription program during reprogramming. RESULTS: The comparative dissection of the transcription profiles and histone modification patterns at lysines 4 and 27 of histone H3 of the iPSC, iPSCp, ESC, and somatic cells revealed that the iPSC was almost completely comparable to the ESC, regardless of their origins, whereas the genes of the iPSCp were dysregulated to a larger extent. Regardless of the origins of the somatic cells, the fibroblasts induced using the ESC protein extracts appear to be completely reprogrammed into pluripotent cells, although they show unshared marginal differences in their gene expression programs, which may not affect the maintenance of stemness. A comparative investigation of the iPSCp generated by unwanted reprogramming showed that the two groups of genes on the pathway from somatic cells to iPSC might function as sequential reprogramming-competent early and late responders to the induction stimulus. Moreover, some of the divergent genes expressed only in the iPSCp were associated with many tumor-related pathways. CONCLUSIONS: Faithful transcriptional reprogramming should follow epigenetic alterations to generate induced pluripotent stem cells from somatic cells. This genome-wide comparison enabled us to define the early and late responder genes during the cell reprogramming process to iPSC. Our results indicate that the cellular responsiveness to external stimuli should be pre-determined and sequentially orchestrated through the tight modulation of the chromatin environment during cell reprogramming to prevent unexpected reprogramming.

Synergistic neovascularization by mixed transplantation of early endothelial progenitor cells and late outgrowth endothelial cells: the role of angiogenic cytokines and matrix metalloproteinases.

BACKGROUND: Two types of cells are cultured from the human peripheral blood, early endothelial progenitor cells (EPCs) and outgrowth endothelial cells (OECs), as previously reported. Here, we further characterize these cells, especially with respect to their different origins and functions both in vitro and in vivo. We also investigated whether the combination of these different cell types shows synergism during neovascularization. METHODS AND RESULTS: Early EPCs were heterogeneously made up of both CD14+ monocyte-derived cells, which secrete cytokines, and CD14(-)-derived cells, which contain high levels of (CD34+)KDR+ cells. OECs were cultured almost exclusively from CD14- cells, not CD14+ cells, and were distinct from mature endothelial cells in terms of proliferation potential, KDR+ expression level, and telomerase activity. A portion of cells from CD14- cells and early EPCs produced rapidly proliferating, capillary-forming cells in both the Matrigel plug and the ischemic hind limb similar to OECs. Early EPCs and OECs expressed receptors for vascular endothelial growth factor and interleukin-8, cytokines secreted by early EPCs. There was a differential increase in matrix metalloproteinases (MMPs): MMP-9 in early EPCs and MMP-2 in OECs. In vitro, the angiogenic capability of the 2 cell types was augmented by mutual interaction through cytokines and MMPs. Injection of a mixture of the 2 cells resulted in superior neovascularization in vivo to any single-cell-type transplantation. CONCLUSIONS: Distinct origins of the different types of EPCs exist that have different functions in neovascularization. Mixed transplantation of these cells results in synergistic neovascularization through cytokines and MMPs.

Dipeptidyl Peptidase-4 Inhibitor Increases Vascular Leakage in Retina through VE-cadherin Phosphorylation.

The inhibitors of CD26 (dipeptidyl peptidase-4; DPP4) have been widely prescribed to control glucose level in diabetic patients. DPP4-inhibitors, however, accumulate stromal cell-derived factor-1α (SDF-1α), a well-known inducer of vascular leakage and angiogenesis both of which are fundamental pathophysiology of diabetic retinopathy. The aim of this study was to investigate the effects of DPP4-inhibitors on vascular permeability and diabetic retinopathy. DPP4-inhibitor (diprotin A or sitagliptin) increased the phosphorylation of Src and vascular endothelial-cadherin (VE-cadherin) in human endothelial cells and disrupted endothelial cell-to-cell junctions, which were attenuated by CXCR4 (receptor of SDF-1α)-blocker or Src-inhibitor. Disruption of endothelial cell-to-cell junctions in the immuno-fluorescence images correlated with the actual leakage of the endothelial monolayer in the transwell endothelial permeability assay. In the Miles assay, vascular leakage was observed in the ears into which SDF-1α was injected, and this effect was aggravated by DPP4-inhibitor. In the model of retinopathy of prematurity, DPP4-inhibitor increased not only retinal vascularity but also leakage. Additionally, in the murine diabetic retinopathy model, DPP4-inhibitor increased the phosphorylation of Src and VE-cadherin and aggravated vascular leakage in the retinas. Collectively, DPP4-inhibitor induced vascular leakage by augmenting the SDF-1α/CXCR4/Src/VE-cadherin signaling pathway. These data highlight safety issues associated with the use of DPP4-inhibitors.

Seborrheic dermatitis treatment with stellate ganglion block: a case report.

Seborrheic dermatitis is a chronic recurrent inflammatory disorder presumed to be caused by increased sebaceous gland secretion, metabolic changes in the cutaneous microflora, and changes in the host immune function. Stellate ganglion block (SGB) is known to increase the blood flow rate without altering the blood pressure, heart rate, or cardiac output, to stabilize hypertonic conditions of the sympathetic nerves, and to affect the endocrine and immune systems. It is used in the differential diagnosis and treatment of autonomic nervous system disorders of the head, neck, and upper limbs. The authors report the first case of successful treatment of a patient with seborrheic dermatitis through repeated SGB trials.