Sang-Mo Kwon

Specific Jagged-1 Signal From Bone Marrow Microenvironment Is Required for Endothelial Progenitor Cell Development for Neovascularization

Background— Despite accumulating evidence that proves the pivotal role of endothelial progenitor cells (EPCs) in ischemic neovascularization, the key signaling cascade that regulates functional EPC kinetics remains unclear. Methods and Results— In this report, we show that inactivation of specific Jagged-1 (Jag-1)–mediated Notch signals leads to inhibition of postnatal vasculogenesis in hindlimb ischemia via impairment of proliferation, survival, differentiation, and mobilization of bone marrow–derived EPCs. Bone marrow–derived EPCs obtained from Jag-1−/− mice, but not Delta-like (Dll)-1−/− mice, demonstrated less therapeutic potential for ischemic neovascularization than EPCs from the wild type. In contrast, a gain-of-function study using 3T3 stromal cells overexpressing Notch ligand revealed that Jag-1–mediated Notch signals promoted EPC commitment, which resulted in enhanced neovascularization. The impaired neovascularization in Jag-1−/− mice was profoundly rescued by transplantation of Jag-1–stimulated EPCs. Conclusions— These data indicate that specific Jag-1–derived Notch signals from the bone marrow microenvironment are critical for EPC–mediated vasculogenesis, thus providing an important clue for modulation of strategies for therapeutic neovascularization.

CD34+ Cells Represent Highly Functional Endothelial Progenitor Cells in Murine Bone Marrow

Background Endothelial progenitor cells (EPCs) were shown to have angiogenic potential contributing to neovascularization. However, a clear definition of mouse EPCs by cell surface markers still remains elusive. We hypothesized that CD34 could be used for identification and isolation of functional EPCs from mouse bone marrow. Methodology/Principal Findings CD34+ cells, c-Kit+/Sca-1+/Lin− (KSL) cells, c-Kit+/Lin− (KL) cells and Sca-1+/Lin− (SL) cells were isolated from mouse bone marrow mononuclear cells (BMMNCs) using fluorescent activated cell sorting. EPC colony forming capacity and differentiation capacity into endothelial lineage were examined in the cells. Although CD34+ cells showed the lowest EPC colony forming activity, CD34+ cells exhibited under endothelial culture conditions a more adherent phenotype compared with the others, demonstrating the highest mRNA expression levels of endothelial markers vWF, VE-cadherin, and Flk-1. Furthermore, a dramatic increase in immediate recruitment of cells to the myocardium following myocardial infarction and systemic cell injection was observed for CD34+ cells comparing with others, which could be explained by the highest mRNA expression levels of key homing-related molecules Integrin β2 and CXCR4 in CD34+ cells. Cell retention and incorporation into the vasculature of the ischemic myocardium was also markedly increased in the CD34+ cell-injected group, giving a possible explanation for significant reduction in fibrosis area, significant increase in neovascularization and the best cardiac functional recovery in this group in comparison with the others. Conclusion These findings suggest that mouse CD34+ cells may represent a functional EPC population in bone marrow, which could benefit the investigation of therapeutic EPC biology.

Local transplantation of human multipotent adipose-derived stem cells accelerates fracture healing via enhanced osteogenesis and angiogenesis.

Adipose tissue is one of the promising sources of multipotent stem cells in human. Human multipotent adipose-derived stem (hMADS) cells have recently been isolated and showed differentiation potential into multiple mesenchymal lineages in vitro and in vivo. On the basis of these evidences, we examined the therapeutic efficacy of hMADS cells for fracture healing in an immunodeficient rat femur non-union fracture model. Local transplantation of hMADS cells radiographically and histologically promoted fracture healing with significant improvement of biomechanical function at the fracture sites compared with local transplantation of human fibroblasts (hFB) or PBS administration. Histological capillary density and physiological blood flow by laser Doppler perfusion imaging were significantly greater in hMADS group than hFB and PBS groups. Expressions of intrinsic (rat) bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF) and angiopoietin-1 in peri-fracture tissue were upregulated in hMADS group than other groups. In addition, presence of BMP-2 or VEGF activated the proliferation and migration of hMADS cells in vitro. These results indicate that hMADS cells stimulate the interaction between the transplanted cells and the resident cells stronger than other cells, and they promote fracture healing more effectively. Furthermore, immunohistochemistry for human-specific antibodies revealed direct differentiation of hMADS cells into osteoblasts or endothelial cells in newly formed callus or vasculature, respectively. RT-PCR for human-specific primers for osteogenic/endothelial markers also disclosed osteogenic and vasculogenic plasticity of the transplanted hMADS cells at the early stage of fracture healing. The present results suggest that transplantation of hMADS cells may become a useful strategy for cell-based bone regeneration in the future clinical setting.

Local Delivery of Granulocyte Colony Stimulating Factor-Mobilized CD34-Positive Progenitor Cells Using Bioscaffold for Modality of Unhealing Bone Fracture

We recently reported that i.v. transplantation of adult human circulating CD34+ cells, an endothelial/hematopoietic progenitor‐enriched cell population, contributes to fracture healing through the enhancement of vasculogenesis and osteogenesis. However, the scarcity of CD34+ cells in the adult human is a critical issue for the future clinical application of this method. To overcome this issue, we assessed in vitro and in vivo capacity of granulocyte colony‐stimulating factor‐mobilized peripheral blood (GM‐PB) human CD34+ cells for vasculogenesis and osteogenesis. First, we confirmed the differentiation capability of GM‐PB CD34+ cells into osteoblasts in vitro. Second, local transplantation of GM‐PB CD34+ cells on atelocollagen scaffold was performed in nude rats in a model of unhealing fractures. Immunostaining for human leukocyte antigen‐ABC of tissue samples 1 week after fracture and cell therapy showed the superior incorporation after local transplantation compared with systemic infusion. Third, the effects of local transplantation of 105 (Hi), 104 (Mid), or 103 (Lo) doses of GM‐PB CD34+ cells or phosphate‐buffered saline (PBS) on fracture healing were compared. Extrinsic vasculogenic and osteogenic differentiation of GM‐PB CD34+ cells, enhancement of the intrinsic angio‐osteogenesis by recipient cells, augmentation of blood flow recovery at the fracture sites, and radiological and histological confirmation of fracture healing were observed only in the Hi and Mid groups but not in the Lo and PBS groups. These results strongly suggest that local transplantation of GM‐PB CD34+ cells with atelocollagen scaffold is a feasible strategy for therapeutic vasculogenesis and osteogenesis needed for fracture healing.

Pivotal Role of Lnk Adaptor Protein in Endothelial Progenitor Cell Biology for Vascular Regeneration

Despite the fact that endothelial progenitor cells (EPCs) are important for postnatal neovascularization, their origins, differentiation, and modulators are not clear. Here, we demonstrate that Lnk, a negative regulator of hematopoietic stem cell proliferation, controls endothelial commitment of c-kit+/Sca-1+/Lineage− (KSL) subpopulations of bone marrow cells. The results of EPC colony–forming assays reveal that small (primitive) EPC colony formation by CD34− KSLs and large (definitive) EPC colony formation by CD34(dim) KSLs are more robust in lnk−/− mice. In hindlimb ischemia, perfusion recovery is augmented in lnk−/− mice through enhanced proliferation and mobilization of EPCs via c-Kit/stem cell factor. We found that Lnk-deficient EPCs are more potent actors than resident cells in hindlimb perfusion recovery and ischemic neovascularization, mainly via the activity of bone marrow-EPCs. Similarly, lnk−/− mice show augmented retinal neovascularization and astrocyte network maturation without an increase in indicators of pathogenic angiogenesis in an in vivo model of retinopathy. Taken together, our results provide strong evidence that Lnk regulates bone marrow-EPC kinetics in vascular regeneration. Selective targeting of Lnk may be a safe and effective strategy to augment therapeutic neovascularization by EPC transplantation.

Impaired Lymphopoiesis and Altered B Cell Subpopulations in Mice Overexpressing Lnk Adaptor Protein

Lnk is an adaptor protein expressed primarily in lymphocytes and hemopoietic precursor cells. Marked expansion of B lineage cells occurs in lnk−/− mice, indicating that Lnk regulates B cell production by negatively controlling pro-B cell expansion. In addition, lnk−/− hemopoietic precursors have an advantage in repopulating the hemopoietic system of irradiated host animals. In this study, we show that Lnk overexpression results in impaired expansion of lymphoid precursor cells and altered mature B cell subpopulations. The representation of both B lineage and T lineage cells was reduced in transgenic mice overexpressing Lnk under the control of a lymphocyte-specific expression vector. Whereas the overall number of B and T cells was correlated with Lnk protein expression levels, marginal zone B cells in spleen and B1 cells in the peritoneal cavity were relatively resistant to Lnk overexpression. The C-terminal tyrosine residue, conserved among Lnk family adaptor proteins, was dispensable for the negative regulatory roles of Lnk in lymphocyte development. Our results illuminate the novel negative regulatory mechanism mediated by the Lnk adaptor protein in controlling lymphocyte production and function.

Ginsenoside Rg3 attenuates tumor angiogenesis via inhibiting bioactivities of endothelial progenitor cells.

Accumulating evidence suggests that Ginsenoside Rg3 appears to inhibit tumor growth including Lewis lung carcinoma, intestinal adenocarcinomas or B16 melanoma by inhibiting cell proliferation, tumor cell invasion and metastasis. Endothelial progenitor cells (EPCs) appear to play a key role in the growth of early tumors by intervening with the angiogenic switch promoting tumor neovessel formation by producing angiogenic cytokines during tumor progression. This paper reports a novel mechanism of Ginsenoside Rg3, a candidate anticancer bio-molecule, on tumor angiogenesis by inhibiting the multiple bioactivities of EPCs. When Ginsenoside Rg3 was applied to the ex vivo cultured outgrowth ECs, a type of EPCs, it inhibited the cell proliferation, cell migration and tubular formation of EPCs. Importantly, Ginsenoside Rg3 attenuated the phosphorylation cascade of the VEGF dependent p38/ERK signaling in vitro. The xenograft tumor model clearly showed that Ginsenoside Rg3 suppresses tumor growth and tumor angiogenesis by inhibiting the mobilization of EPCs from the bone marrow microenvironment to the peripheral circulation and modulates VEGF-dependent tumor angiogenesis. In conclusion, this study provides a potential therapeutic molecule, Ginsenoside Rg3, as an anticancer drug by inhibiting the EPC bioactivities.

Differential activity of bone marrow hematopoietic stem cell subpopulations for EPC development and ischemic neovascularization

Although endothelial progenitor cells (EPCs) differentiate from minor populations of stem cells in bone marrow (BM), the differential role of hematopoietic stem cell (HSC) subpopulations in EPC development is largely unclear. Morphological characterization of EPC colonies has revealed that c-kit+/Sca-1+/lineage (Lin)-(KSL) cells mainly develop small EPC-colony forming units (CFUs) not large EPC-CFUs. In contrast, c-kit+/Sca-1-/Lin- (KL) cells develop large EPC-CFUs not small EPC-CFUs. Neither c-kit-/Sca-1+/Lin- (SL) cells nor c-kit-/Sca-1-/Lin- (L) cells develop EPC-CFUs to an appreciable extent. Hindlimb ischemia enhances formation of large EPC-CFUs from all HSC subpopulations, suggesting an important role for ischemia in functional EPC development. Real time RT-PCR analysis shows that KSL, KL and SL cells but not L cells express various factors at high levels, maintaining a BM-EPC pool. In hindlimb ischemia, transplanted KSL, KL and SL cells efficiently differentiate into endothelial lineage cells in situ and augment capillary density. The percentage of Ki-67+ cycling cells among transplanted cells in ischemic tissue was also greater for KSL, KL and SL cells than L cells. Moreover, the frequency of VEGF- or SDF-1-expressing cells was higher transplanted KSL, KL or SL cells than L cells. Thus, KSL, KL and SL cells are not different in their angiogenic competence under ischemic conditions. In conclusion, although KSL cells are clearly the most potent contributors to EPC development, KL and SL cells may also contribute to neovascularization via both autocrine and paracrine mechanisms in response to ischemic signals.

SDF‐1/CXCR4 Axis in Tie2‐Lineage Cells Including Endothelial Progenitor Cells Contributes to Bone Fracture Healing

CXC chemokine receptor 4 (CXCR4) is a specific receptor for stromal‐derived‐factor 1 (SDF‐1). SDF‐1/CXCR4 interaction is reported to play an important role in vascular development. On the other hand, the therapeutic potential of endothelial progenitor cells (EPCs) in fracture healing has been demonstrated with mechanistic insight of vasculogenesis/angiogenesis and osteogenesis enhancement at sites of fracture. The purpose of this study was to investigate the influence of the SDF‐1/CXCR4 pathway in Tie2‐lineage cells (including EPCs) in bone formation. We created CXCR4 gene conditional knockout mice using the Cre/loxP system and set two groups of mice: Tie2‐CreER CXCR4 knockout mice (CXCR4−/−) and wild‐type mice (WT). We report here that in vitro, EPCs derived from of CXCR4−/− mouse bone marrow demonstrated severe reduction of migration activity and EPC colony‐forming activity when compared with those derived from WT mouse bone marrow. In vivo, radiological and morphological examinations showed fracture healing delayed in the CXCR4−/− group and the relative callus area at weeks 2 and 3 was significantly smaller in CXCR4−/− group mice. Quantitative analysis of capillary density at perifracture sites also showed a significant decrease in the CXCR4−/− group. Especially, CXCR4−/−group mice demonstrated significant early reduction of blood flow recovery at fracture sites compared with the WT group in laser Doppler perfusion imaging analysis. Real‐time RT‐PCR analysis showed that the gene expressions of angiogenic markers (CD31, VE‐cadherin, vascular endothelial growth factor [VEGF]) and osteogenic markers (osteocalcin, collagen 1A1, bone morphogenetic protein 2 [BMP2]) were lower in the CXCR4−/− group. In the gain‐of‐function study, the fracture in the SDF‐1 intraperitoneally injected WT group healed significantly faster with enough callus formation compared with the SDF‐1 injected CXCR4−/− group. We demonstrated that an EPC SDF‐1/CXCR4 axis plays an important role in bone fracture healing using Tie2‐CreER CXCR4 conditional knockout mice.

The Sulfated Polysaccharide Fucoidan Rescues Senescence of Endothelial Colony‐Forming Cells for Ischemic Repair

The efficacy of cell therapy using endothelial colony‐forming cells (ECFCs) in the treatment of ischemia is limited by the replicative senescence of isolated ECFCs in vitro. Such senescence must therefore be overcome in order for such cell therapies to be clinically applicable. This study aimed to investigate the potential of sulfated polysaccharide fucoidan to rescue ECFCs from cellular senescence and to improve in vivo vascular repair by ECFCs. Fucoidan‐preconditioning of senescent ECFCs was shown by flow cytometry to restore the expression of functional ECFC surface markers (CD34, c‐Kit, VEGFR2, and CXCR4) and stimulate the in vitro tube formation capacity of ECFCs. Fucoidan also promoted the expression of cell cycle‐associated proteins (cyclin E, Cdk2, cyclin D1, and Cdk4) in senescent ECFCs, significantly reversed cellular senescence, and increased the proliferation of ECFCs via the FAK, Akt, and ERK signaling pathways. Fucoidan was found to enhance the survival, proliferation, incorporation, and endothelial differentiation of senescent ECFCs transplanted in ischemic tissues in a murine hind limb ischemia model. Moreover, ECFC‐induced functional recovery and limb salvage were markedly improved by fucoidan pretreatment of ECFCs. To our knowledge, the findings of our study are the first to demonstrate that fucoidan enhances the neovasculogenic potential of ECFCs by rescuing them from replicative cellular senescence. Pretreatment of ECFCs with fucoidan may thus provide a novel strategy for the application of senescent stem cells to therapeutic neovascularization. Stem Cells 2015;33:1939–1951