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Endothelial progenitor cells (EPCs) are a promising cell source for the treatment of several ischemic diseases for their potentials in neovascularization. However, the application of EPCs in cell-based therapy has shown low therapeutic efficacy due to hostile tissue conditions after ischemia. In this study, a bio-blood-vessel (BBV) is developed, which is produced using a novel hybrid bioink (a mixture of vascular-tissue-derived decellularized extracellular matrix (VdECM) and alginate) and a versatile 3D coaxial cell printing method for delivering EPC and proangiogenic drugs (atorvastatin) to the ischemic injury sites. The hybrid bioink not only provides a favorable environment to promote the proliferation, differentiation, and neovascularization of EPCs but also enables a direct fabrication of tubular BBV. By controlling the printing parameters, the printing method allows to construct BBVs in desired dimensions, carrying both EPCs and atorvastatin-loaded poly(lactic-co-glycolic) acid microspheres. The therapeutic efficacy of cell/drug-laden BBVs is evaluated in an ischemia model at nude mouse hind limb, which exhibits enhanced survival and differentiation of EPCs, increased rate of neovascularization, and remarkable salvage of ischemic limbs. These outcomes suggest that the 3D-printed ECM-mediated cell/drug implantation can be a new therapeutic approach for the treatment of various ischemic diseases.

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 Ϫ/Ϫ mice, but not Delta-like (Dll)-1 Ϫ/Ϫ 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 Ϫ/Ϫ 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. Key Words: angiogenesis Ⅲ progenitor cells Ⅲ ischemia Ⅲ signal transduction G rowing evidence indicates that the perturbation of Notch signaling leads to dysfunctional behavior of the vascular system. 1 A human degenerative vascular disease, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), is related to mutations in the Notch3 receptor. Alagille syndrome, caused by mutation of the Jagged-1 (Jag-1) gene, is a pleiotropic developmental disease that is accompanied by features of congenital heart defects with cardiovascular anomalies. 2 Murine genetic studies that generate loss or gain of function of Notch receptors or ligands have exhibited abnormalities in blood vessel formation, such as impaired proliferation and migration of endothelial cells (ECs) 3 and arterial-venous identification. 4 -7 These findings indicate the involvement of Notch1, 7 Notch3, 8 and Notch4 7 receptors, as well as Deltalike ligand (Dll)-4 4,5 and Jag-1 9 ligands, in vascular formation. Recently, Notch ligand, especially Dll-4, has been focused on as an essential regulator for tumor angiogenesis and vascular development in terms of ligand signal control from tissue environment for EC bioactivity through Notch receptors. 10,11 Clinical Perspective p 165Although pioneered in the field of vascular biology, especially in terms of EC morphogenesis for blood vessel development and EC determination of arterial-venous specification, the role of Notch signal in stem cell-related postnatal vasculogenesis has not been investigated. Endothelial progenitor cells (EPCs) derived from bone marrow (BM) play an important role in the promotion of vascular and tissue repair in physiological and pathological conditions, such as coronary or periphe...

ABSTRACT

BackgroundEndothelial 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 FindingsCD34+ 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.ConclusionThese findings suggest that mouse CD34+ cells may represent a functional EPC population in bone marrow, which could benefit the investigation of therapeutic EPC biology.

Peroxisome proliferator-activated receptors (PPARs), members of the nuclear receptor superfamily, are important in whole-body energy metabolism. PPARs are classified into three isoforms, namely, PPARα, β/δ, and γ. They are collectively involved in fatty acid oxidation, as well as glucose and lipid metabolism throughout the body. Importantly, the three isoforms of PPARs have complementary and distinct metabolic activities for energy balance at a cellular and whole-body level. PPARs also act with other co-regulators to maintain energy homeostasis. When endogenous ligands bind with these receptors, they regulate the transcription of genes involved in energy homeostasis. However, the exact molecular mechanism of PPARs in energy metabolism remains unclear. In this review, we summarize the importance of PPAR signals in multiple organs and focus on the pivotal roles of PPAR signals in cellular and whole-body energy homeostasis.

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.