Effects of exogenous sonic hedgehog peptide on proliferation, adhesion, migration of endothelial progenitor cells from peripheral blood

Abstract

In order to study the effects of exogenous sonic hedgehog (shh) peptide on proliferation, adhesion, migration of endothelial progenitor cells (EPCs) from rat peripheral blood, the mononuclear cells were collected from rat peripheral blood by Ficoll density gradient centrifugation. EPCs were isolated with adherence screening method and cultured in M199 culture medium with the supplement of VEGF and bFGF. The immunohistochemical staining was used to identify cell markers such as CDl33 and VEGFR-2. EPCs were stimulated with exogenous shh peptide of different final concentrations (0.01, 0.1, 1, 10 μg/mL). The proliferation, adhesion and migration of EPCs were detected by MTT chromometry, adhesion test and transwell system, respectively. The results of this study showed that, after 7 days of culture, cells formed clusters, assuming typical cobbles-tone pattern under microscope. After 2 weeks of culture, cells were arranged in cord-like fashion and sometimes grew like “micro-vessels”. Immunohistochemical staining showed that the cultured cells were positive for both CD133 and VEGFR-2. The proliferation, adhesion and migration of EPCs could be promoted by endogenous shh peptide at concentrations from 0.1 μg/mL to 10 μg/mL in a concentration-dependent manner. The findings indicate that exogenous shh peptide can enhance EPCs proliferation, adhesion, and migration, which may have a potential value for clinical application.

Keywords

endothelial progenitor cell / shh / proliferation / adhesion / migration

References

[1] NishiwakiY, YoshidaM, IwaguroH, et al.. Endothelial E-selectin potentiates neovascularization via endothelial progenitor cell-dependent and -independent mechanisms. Arterioscler Thromb Vasc Biol, 2007, 27(3): 512-518

[2] BalaskasN, RibeiroA, PanovskaJ, et al.. Gene regulatory logic for reading the sonic hedgehog signaling gradient in the vertebrate neural tube. Cell, 2012, 148(1–2): 273-284

[3] BahramiSB, VeisehM, BoudreauNJ. Isolation and expansion of endothelial progenitor cells derived from mouse embryonic stem cells. Methods Mol Biol, 2012, 916: 81-96

[4] HuangPH, ChenJW, LinCP, et al.. Far infra-red therapy promotes ischemia-induced angiogenesis in diabetic mice and restores high glucose-suppressed endothelial progenitor cell functions. Cardiovasc Diabetol, 2012, 11(1): 99

[5] IgrejaC, CourinhaM, CachaçoAS, et al.. Characterization and clinical relevance of circulating and biopsy-derived endothelial progenitor cells in lymphoma patients. Haematologica, 2007, 92(4): 469-477

[6] SmadjaDM, BiècheI, HelleyD, et al.. Increased VEGFR2 expression during human late endothelial progenitor cells expansion enhances in vitro angiogenesis with up-regulation of integrin alpha(6). J Cell Mol Med, 2007, 11(5): 1149-1161

[7] YinAH, MiragliaS, ZanjaniE e a1. ACl33, a novel marker for human hematopoietic stem and progenitor cells. Blood, 1997, 90(12): 5002-5012

[8] ZhuZ, ChenG, LiX e a1. Endothelial progenitor cells homing to the orthotopic implanted liver tumor of nude mice. J Huazhong Univ Sci Technol Med Sci, 2012, 32(5): 675-679

[9] VasaM, FichtlschererS, AicherA, et al.. Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease. Circ Res, 2001, 89(1): E1-E7

[10] HillJM, ZalosG, HalcoxJP, et al.. Circulating endothelial progenitor cells, vascular function, and cardiovascular risk. N Engl J Med, 2003, 348(7): 593-600

[11] de GrootK, BahlmannFH, SowaJ, et al.. Uremia causes endothelial progenitor cell deficiency. Kidney Int, 2004, 66(2): 641-646

[12] SangidorjO, YangSH, JangHR, et al.. Bone marrow-derived endothelial progenitor cells confer renal protection in a murine chronic renal failure model. Am J Physiol Renal Physiol, 2010, 299(2): F325-F335

[13] HasebeT, KajitaM, FuL, et al.. Thyroid hormone-induced sonic hedgehog signal up-regulates its own pathway in a paracrine manner in the Xenopus laevis intestine during metamorphosis. Dev Dyn, 2012, 241(2): 403-414

[14] LeeJM, MiyazawaS, ShinJO, et al.. Shh signaling is essential for rugae morphogenesis in mice. Histochem Cell Biol, 2011, 136(6): 663-675

[15] AmirpourN, KaramaliF, RabieeF, et al.. Differentiation of human embryonic stem cell-derived retinal progenitors into retinal cells by Sonichedgehog and/or retinal pigmented epithelium and transplantation into the subretinal space of sodium iodate-injected rabbits. Stem Cells Dev, 2012, 21(1): 42-53

[16] ZhouK, HuC, ZhouZ, et al.. Fetal liver stromal cells promote hematopoietic cell expansion. Biochem Biophys Res Commun, 2009, 387(3): 596-601

[17] LawsonND, VogelAM, WeinsteinBM. Sonic hedgehog and vascular endothelial growth factor act upstream of the Notch pathway during arterial endothelial differentiation. Dev Cell, 2002, 3(1): 127-136