Syotaro Obi

Shear Stress Increases Expression of the Arterial Endothelial Marker EphrinB2 in Murine ES Cells via the VEGF-Notch Signaling Pathways

Objective—Arterial-venous specification in the embryo has been assumed to depend on the influence of fluid mechanical forces, but its cellular and molecular mechanisms are still poorly understood. Our previous in vitro study revealed that fluid shear stress induces endothelial cell (EC) differentiation by murine embryonic stem (ES) cells. In the present study we investigated whether shear stress regulates the arterial-venous specification of ES-cell-derived ECs. Methods and Results—When murine ES cell–derived VEGFR2+ cells were exposed to shear stress, expression of the arterial EC marker protein ephrinB2 increased dose-dependently. The ephrinB2 mRNA levels also increased in response to shear stress, whereas the mRNA levels of the venous EC marker EphB4 decreased. Notch cleavage and translocation of the Notch intracellular domain (NICD) into the nucleus occurred as early as 30 minutes after the start of shear stress and increased with time. Gamma-Secretase inhibitors (DAPT and L685 458) and the recombinant extracellular domain of the Notch ligand DLL4 abolished the shear stress–induced NICD translocation, and that, in turn, blocked the shear stress–induced upregulation of ephrinB2 expression. In addition, the VEGF receptor kinase inhibitor SU1498 was found to suppress both the shear-stress-induced Notch cleavage and up-regulation of ephrinB2 expression. Conclusion—Exposure to shear stress induces an increase in expression of ephrinB2 in murine ES cells via VEGF-Notch signaling pathways.

Cyclic strain induces mouse embryonic stem cell differentiation into vascular smooth muscle cells by activating PDGF receptor β

Embryonic stem (ES) cells are exposed to fluid-mechanical forces, such as cyclic strain and shear stress, during the process of embryonic development but much remains to be elucidated concerning the role of fluid-mechanical forces in ES cell differentiation. Here, we show that cyclic strain induces vascular smooth muscle cell (VSMC) differentiation in murine ES cells. Flk-1-positive (Flk-1+) ES cells seeded on flexible silicone membranes were subjected to controlled levels of cyclic strain and examined for changes in cell proliferation and expression of various cell lineage markers. When exposed to cyclic strain (4-12% strain, 1 Hz, 24 h), the Flk-1+ ES cells significantly increased in cell number and became oriented perpendicular to the direction of strain. There were dose-dependent increases in the VSMC markers smooth muscle alpha-actin and smooth muscle-myosin heavy chain at both the protein and gene expression level in response to cyclic strain, whereas expression of the vascular endothelial cell marker Flk-1 decreased, and there were no changes in the other endothelial cell markers (Flt-1, VE-cadherin, and platelet endothelial cell adhesion molecule 1), the blood cell marker CD3, or the epithelial marker keratin. The PDGF receptor beta (PDGFR beta) kinase inhibitor AG-1296 completely blocked the cyclic strain-induced increase in cell number and VSMC marker expression. Cyclic strain immediately caused phosphorylation of PDGFR beta in a dose-dependent manner, but neutralizing antibody against PDGF-BB did not block the PDGFR beta phosphorylation. These results suggest that cyclic strain activates PDGFR beta in a ligand-independent manner and that the activation plays a critical role in VSMC differentiation from Flk-1+ ES cells.

Fluid shear stress induces differentiation of circulating phenotype endothelial progenitor cells

Endothelial progenitor cells (EPCs) are mobilized from bone marrow to peripheral blood, and contribute to angiogenesis in tissue. In the process, EPCs are exposed to shear stress generated by blood flow and tissue fluid flow. Our previous study showed that shear stress induces differentiation of mature EPCs in adhesive phenotype into mature endothelial cells and, moreover, arterial endothelial cells. In this study we investigated whether immature EPCs in a circulating phenotype differentiate into mature EPCs in response to shear stress. When floating-circulating phenotype EPCs derived from ex vivo expanded human cord blood were exposed to controlled levels of shear stress in a flow-loading device, the bioactivities of adhesion, migration, proliferation, antiapoptosis, tube formation, and differentiated type of EPC colony formation increased. The surface protein expression rate of the endothelial markers VEGF receptor 1 (VEGF-R1) and -2 (VEGF-R2), VE-cadherin, Tie2, VCAM1, integrin α(v)/β(3), and E-selectin increased in shear-stressed EPCs. The VEGF-R1, VEGF-R2, VE-cadherin, and Tie2 protein increases were dependent on the magnitude of shear stress. The mRNA levels of VEGF-R1, VEGF-R2, VE-cadherin, Tie2, endothelial nitric oxide synthase, matrix metalloproteinase 9, and VEGF increased in shear-stressed EPCs. Inhibitor analysis showed that the phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signal transduction pathway is a potent activator of adhesion, proliferation, tube formation, and differentiation in response to shear stress. Western blot analysis revealed that shear stress activated the VEGF-R2 phosphorylation in a ligand-independent manner. These results indicate that shear stress increases differentiation, adhesion, migration, proliferation, antiapoptosis, and vasculogenesis of circulating phenotype EPCs by activation of VEGF-R2 and the PI3K/Akt/mTOR signal transduction pathway.

Dextran induces differentiation of circulating endothelial progenitor cells

Endothelial progenitor cells (EPCs) have been demonstrated to be effective for the treatment of cardiovascular diseases. However, the differentiation process from circulation to adhesion has not been clarified because circulating EPCs rarely attached to dishes in EPC cultures previously. Here we investigated whether immature circulating EPCs differentiate into mature adhesive EPCs in response to dextran. When floating‐circulating EPCs derived from ex vivo expanded human cord blood were cultured with 5% and 10% dextran, they attached to fibronectin‐coated dishes and grew exponentially. The bioactivities of adhesion, proliferation, migration, tube formation, and differentiated type of EPC colony formation increased in EPCs exposed to dextran. The surface protein expression rate of the endothelial markers vascular endothelial growth factor (VEGF)‐R1/2, VE‐cadherin, Tie2, ICAM1, VCAM1, and integrin αv/β3 increased in EPCs exposed to dextran. The mRNA levels of VEGF‐R1/2, VE‐cadherin, Tie2, endothelial nitric oxide synthase, MMP9, and VEGF increased in EPCs treated with dextran. Those of endothelium‐related transcription factors ID1/2, FOXM1, HEY1, SMAD1, FOSL1, NFkB1, NRF2, HIF1A, EPAS1 increased in dextran‐treated EPCs; however, those of hematopoietic‐ and antiangiogenic‐related transcription factors TAL1, RUNX1, c‐MYB, GATA1/2, ERG, FOXH1, HHEX, SMAD2/3 decreased in dextran‐exposed EPCs. Inhibitor analysis showed that PI3K/Akt, ERK1/2, JNK, and p38 signal transduction pathways are involved in the differentiation in response to dextran. In conclusion, dextran induces differentiation of circulating EPCs in terms of adhesion, migration, proliferation, and vasculogenesis. The differentiation mechanism in response to dextran is regulated by multiple signal transductions including PI3K/Akt, ERK1/2, JNK, and p38. These findings indicate that dextran is an effective treatment for EPCs in regenerative medicines.

Differentiation of circulating endothelial progenitor cells induced by shear stress

Endothelial cells have the ability of cell division and migration not only in embryos but also in adult life. When part of the endothelium is injured and detached, neighboring endothelial cells proliferate, migrate, and cover the exposed surface. In addition endothelial cells always regenerate and new blood vessels are made in hypoxic lesions. Endothelial progenitor cells (EPCs) are also demonstrated to play an important role in vascular regeneration [1]. EPCs are mobilized from bone marrow to peripheral blood, attach to existing endothelial cells in nearby hypoxic lesions, transmigrate into tissue, proliferate, differentiate, secrete angiogenic factors, and induce neovascularization.

Shear Stress Induces Differentiation of Arterial Endothelial Cells From Murine Embryonic Stem Cells

The development of vasculature in the embryo has been assumed to depend on the influence of fluid mechanical forces, but the cellular and molecular mechanisms of its development are still poorly understood. The aim of the present study was to investigate whether shear stress affects embryonic stem (ES) cell differentiation. When VEGF receptor 2 (VEGF2)-positive murine ES cells were exposed to shear stress in a flow-loading device, expression of the endothelial cell (EC) markers VEGFR2, Flt-1, VE-cadherin, and PECAM-1 increased at both the protein level and the mRNA level, but expression of the mural cell marker SM-α-actin, blood cell marker CD3, or the epithelial cell marker keratin remained unchanged. These findings indicate that shear stress selectively promote the differentiation of VEGFR2-positive ES cells into the EC lineage. Shear stress also increased expression of the arterial EC marker ephrinB2, whereas it decreased expression of the venous EC marker EphB4. Shear stress induced tyrosine phosphorylation of VEGFR and caused Notch cleavage in VEGFR2-positive ES cells. The VEGFR kinase inhibitor SU1498 abolished shear stress-induced VEGFR phosphorylation, Notch cleavage, and increase in ephrinB2 expression. The γ-secretase inhibitors DAPT and L685.458 also blocked shear-stress induced Notch cleavage and up-regulation of ephrinB2 expression. Taken together, these findings suggest that shear stress induces differentiation of ES cells into arterial ECs through the VEGF-Notch signaling pathways.

Cyclic strain induces ES cell differentiation into vascular smooth muscle cells via PDGF receptor ß activation

Embryonic stem (ES) cells are exposed to fluid-mechanical forces, such as cyclic strain and shear stress, during the process of embryonic development, but much remains to be elucidated concerning the role of fluid-mechanical forces in ES cell differentiation. Here we show that cyclic strain induces vascular smooth muscle cell (VSMC) differentiation in murine ES cells. Flk-1- positive (Flk -1+) ES cells seeded on flexible silicone membranes were subjected to controlled levels of cyclic strain, and examined for changes in cell proliferation and expression of various cell lineage markers. When exposed to cyclic strain (4 -12% strain, 1 Hz, 24 hours), the Flk -1+ ES cells significantly increased in cell number and became oriented perpendicular to the direction of strain. There were dose-dependent increases in the VSMC markers SM a-actin and SM-myosin heavy chain (SM-MHC) at both the protein and gene expression level in response to cyclic strain, whereas expression of the vascular endothelial cell (EC) marker Flk -1 decreased, and there were no changes in the other EC markers Flt -1, VE-cadherin, and PECAM-1, the blood cell marker CD3, or the epithelial marker keratin. The PDGF receptor szlig (PDGFRszlig) kinase inhibitor AG1296 completely blocked the cyclic-strain-induced increase in cell number and VSMC marker expression. Cyclic strain immediately caused phosphorylation of PDGFRszlig in a dose-dependent manner, but neutralizing antibody against PDGF-BB did not block the PDGFRszlig phosphorylation. These results suggest that cyclic strain activates PDGFRszlig in a ligand-independent manner, and that the activation plays a critical role in VSMC differentiation from Flk-1+ ES cells.

Caveola ATP synthase mediates ATP release in vascular endothelial cells exposed to shear stress

Endothelial cells (ECs) release ATP in response to shear stress, a mechanical force generated by blood flow, and the ATP released modulates EC functions through activation of purinoceptors. The molecular mechanism of the shear-stress-induced ATP release, however, has not been fully understood. In this study, we have demonstrated that cell-surface ATP synthase is involved in shear stress-induced ATP release. Immunofluorescence staining of human pulmonary artery ECs (HPAECs) showed that cell-surface ATP synthase is distributed in lipid rafts and co-localized with caveolin-1. When exposed to shear stress, HPAECs released ATP in a dose-dependent manner, and the ATP release was markedly suppressed by a membrane-impermeable ATP synthase inhibitor, angiostatin, and by an anti-ATP synthase antibody. Depletion of plasma membrane cholesterol with methyl-beta cyclo-dextrin (MbetaCD) disrupted lipid rafts and abolished co-localization of ATP synthase with caveolin-1, which resulted in a marked reduction in shear-stress-induced ATP release. Down-regulation of caveolin-1 expression by transfection of caveolin-1 siRNA also markedly suppressed ATP-releasing responses to shear stress. These results suggest that the localization and targeting of ATP synthase to caveolae/lipid rafts, is critical for shear stress-induced ATP release by HPAECs.

Shear stress-mediated differentiation of vascular progenitors

Embryonic stem (ES) cells have the potential to differentiate into every cell type in the body, but the molecular mechanisms that regulate ES cell differentiation have not been sufficiently explored. Here, we report that shear stress, a mechanical force generated by fluid flow, can induce ES cell differentiation. When Flk-1-positive (Flk-1/sup +/) mouse ES cells were exposed to shear stress, their cell density increased markedly, and a larger percentage of the cells were in the S phase and G2-M phase of the cell cycle than Flk-1/sup +/ ES cells cultured under static conditions. Shear stress significantly increased the expression of the vascular endothelial cell-specific markers Flk-1, Flt-1, VE-cadherin, and PECAM-1, at both the protein level and the mRNA level, but it had no effect on expression of the mural cell marker SM-/spl alpha/-actin, the blood cell marker CD3, or the epithelial cell marker keratin. These findings indicate that shear stress selectively promotes the differentiation of Flk-1/sup +/ ES cells into the endothelial cell lineage rather than into other cell lineages.