TOLGA ERTEKİN, ERDOĞAN UNUR, HARUN ÜLGER, Mehtap NİSARİ, ÖMÜR KARACA SAYGILI, UĞUR ŞAHİN, FERHAN ELMALI

Effects of Angiostatin on in vitro Embryonic Rat Development

Anjiogenezis süreci anjiogenez inhibitör ve aktivatörleri arasındaki denge ile kontrol edilmektedir. Anjiostatin endotel hücrelerin çoğalmasını, farklılaşmasını ve diğer fonksiyonlarını etkileyerek endotel hücreleri üzerinde antianjiogenik etki gösterir. Bu çalışmada, anjiostatin’in sıçan embriyolarında in vitro total embriyonik gelişim süreci üzerine etkileri araştırıldı. 9.5 günlük embriyolar normal sıçan serumunda (kontrol grubu) ve normal sıçan serumuna 0.5, 2.5 ve 5 μg/ml anjiostatin eklenerek (deney grupları) kültüre edildi. 48 saatlik kültür periyodu sonunda tüm gruplardaki embriyolar morfolojik olarak değerlendirildi. Deney grupları kontrol grubu ile karşılaştırıldığında organagenezis süresince embriyonik gelişim ve büyümede bir gerilemenin varlığı gözlemlendi. Sıçan serumu ve sıçan serumu+0.5, 2.5 ve 5 μg/ml anjiostatin’de kültür edilen embriyolarda ortalama morfolojik skor sırasıyla 59.8±2.02, 34.8±8.52, 22.4±6.41 ve 17.8±6.19, yolk sac çapı 6.0±0.2, 4.8±0.55, 4.5±057 ve 4.2±0.39 mm, baş-kıç uzunluğu 5.4±0.23, 4.4±0.42, 4.1±0.87 ve 3.7±0.51 mm ve ortalama protein içeriği 140.76±12.21, 112.66±8.67, 89.85±10.89 ve 53.77±8.15 μg/ml olarak belirlendi. Ortalama somit sayısı kontrol grubunda 24 (24-25) iken deney gruplarında azalmaktaydı ve sırasıyla 14 (12-14), 12 (11-17) ve 13 (8-17) olarak hesaplandı. Sonuçlar, anjiostatin’in antianjiojenik etkisine bağlı olarak embriyonik gelişimde bir gerilemenin olabileceğini ortaya koymaktadır.

Anahtar Kelimeler:

embriyo, embriyo kültürü, sıçan, in vitro kültür, embriyonik gelişim

Anjiostatinin in vitro Embriyonik Rat Gelişimi Üzerine Etkileri

The progression of angiogenesis is controlled by a delicate balance between the positive and negative regulators. Angiostatin has potent antiangiogenic effect on endothelial cells, influencing their proliferation, differentiation and other functions. In this study, in vitro effects of angiostatin on total embryonic growth were investigated in rat embryos. The rat embryos were explanted on day 9.5 and cultured in whole rat serum (WRS) (for control) and adding 0.5, 2.5 and 5 μg/ml angiostatin (for experimentals) in WRS. After 48 hours culture period, the embryos from each group were harvested and analysed morphologically. The results showed that the embryonic growth and development during organogenesis decreased in the presence of angiostatin when compared to embryos grown in WRS. Mean morphological scores for the embryos grown in WRS and in the presence of 0.5, 2.5 and 5 μg/ml angiostatin were 59.8±2.02, 34.8±8.52, 22.4±6.41 and 17.8±6.19, yolk sac diameters were 6.0±0.2, 4.8±0.55, 4.5±057 and 4.2±0.39 mm, crown-rump lengths were 5.4±0.23, 4.4±0.42, 4.1±0.87 and 3.7±0.51 mm and mean protein contents of embryos were 140.76±12.21, 112.66±8.67, 89.85±10.89 and 53.77±8.15 μg/ml respectively. Median value of the somite numbers was 24 (24-25) in control group and it was diminished 14 (12-14), 12 (11-17) and 13 (8-17) respectively in experimental groups. As a result the angiostatin could cause developmental retardation of embryo because of its antiangiogenic effect.

Keywords:

embryos, embryo culture, rats, in vitro culture, embryonic development,

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Prandini MH, Desroches-Castan A, Feraud O, Vittet D: No evidence for vasculogenesis regulation by angiostatin during mouse embryonic stem cell differentiation. J Cell Physiol, 213 (1): 27-35, 2007.
O’Reilly MS, Holmgren L, Shing Y, Chen C, Rosenthal RA, Moses M, Lane WS, Cao Y, Sage EH, Folkman J: Angiostatin: A novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma. Cell, 79 (2): 315-328, 1994.
Cao Y, Ji RW, Davidson D, Schaller J, Marti D, Söhndel S, McCance SG, O’Reilly MS, Llinás M, Folkman J: Kringle domains of human angiostatin. Characterization of the anti-proliferative activity on endothelial cells. J Biol Chem, 271 (46): 29461-29467, 1996.
Cao Y: Therapeutic potentials of angiostatin in the treatment of cancer. Haematologica, 84 (7): 643-50, 1999.
Kim JH, Kim JC, Shin SH, Chang SI, Lee HS, Chung SI: The inhibitory effects of recombinant plasminogen kringle 1-3 on the neovascularization of rabbit cornea induced by angiogenin, bFGF, VEGF. Exp Mol Med, 31 (4): 203-209, 1999.
Lucas R, Holmgren L, Garcia I, Jimenez B, Mandriota SJ, Borlat F, Sim BK, Wu Z, Grau GE, Shing Y, Soff GA, Bouck N, Pepper MS: Multiple forms of angiostatin induce apoptosis in endothelial cells. Blood, 92 (12): 4730-4741, 1998.
Yokoyama Y, Dhanabal M, Griffioen AW, Sukhatme VP, Ramakrishnan S: Synergy between angiostatin and endostatin: Inhibition of ovarian cancer growth. Cancer Res, 60 (8): 2190-2196, 2000.
New DAT: Whole embryo culture and the study of mammalian embryos during organogenesis. Biol Rev Camb Philos Soc, 53 (1): 81-122, 1978.
New DAT, Coppola PT, Cockroft DL: Comparison of growth in vitro and in vivo of post-implantation rat embryos. J Embryol Exp Morphol, 36 (1): 133-144, 1976.
Van Maele-Fabry G, Delhaise F, Picard JJ: Morphogenesis and quantification of the development of post-implantation mouse embryos in vitro. Toxicol In Vitro, 4 (2): 149-156, 1990.
Lowry DH, Rosebrough NJ, Farr AL, Randal RJ: Protein measurements with Folin Phenol reagents. J Biol Chem, 193 (1): 265-275, 1951.
Jin SW, Beis D, Mitchell T, Chen JN, Stainier DY: Cellular and molecular analyses of vascular tube and lumen formation in zebrafish. Development , 132 (23): 5199-5209, 2005.
Patan S: Vasculogenesis and angiogenesis as mechanisms of vascular network formation, growth and remodeling. J Neurooncol, 50 (1-2): 1-15, 2000.
Schmidt A, Brixius K, Bloch W: Endothelial precursor cell migration during vasculogenesis. Circ Res, 101 (2): 125-136, 2007.
Urbich C, Dimmeler S: Endothelial progenitor cells: Characterization and role in vascular biology. Circ Res, 95 (4): 343-353, 2004.
Ranieri G, Gasparini G: Angiogenesis and angiogenesis inhibitors: A new potential anticancer therapeutic strategy. Curr Drug Targets Immune Endocr Metabol Disord, 1 (3): 241-253, 2001.
Velasco P, Lange-Asschenfeldt B: Dermatological aspects of angio-genesis. Br J Dermatol, 147 (5): 841-852, 2002.
Ulger H, Ozdamar S, Unur E, Pratten MK: The effect of vascular endothelial growth factor on in vitro embryonic heart development in rats. Anat Histol Embryol, 33 (6): 334-338, 2004.
Ulger H, Karabulut AK, Pratten MK: The growth promoting effects of bFGF, PD-ECGF and VEGF on cultured postimplantation rat embryos deprived of serum fractions. J Anat, 197 (2): 207-219, 2000.
Ulger H, Ozdamar S, Unur E, Pratten M: The effect of basic fibroblast growth factor (bFGF) on in vitro embryonic growth, heart and neural tube development in rat. Kafkas Univ Vet Fak Derg, 15 (5): 673-679, 2009.
O’Reilly MS, Boehm T, Shing Y, Fukai N, Vasios G, Lane WS, Flynn E, Birkhead JR, Olsen BR, Folkman J: Endostatin: An endogenous inhibitor of angiogenesis and tumor growth. Cell, 88 (2): 277-285, 1997.
Sim BK, O’Reilly MS, Liang H, Fortier AH, He W, Madsen JW, Lapcevich R, Nacy CA: A recombinant human angiostatin protein inhibits experimental primary and metastatic cancer. Cancer Res, 57 (7): 1329-1334, 1997.
Claesson-Welsh L, Welsh M, Ito N: Angiostatin induces endothelial cell apoptosis and activation of focal adhesion kinase independently of the integrin-binding motif RGD. Proc Natl Acad Sci USA, 95 (10): 5579-5583, 1998.
Hermant B, Desroches-Castan A, Dubessay ML, Prandini MH, Huber P, Vittet D: Development of a one-step embryonic stem cell-based assay for the screening of sprouting angiogenesis. BMC Biotechnol, 16 (7): 20, 2007.
Moser TL, Stack MS, Asplin I, Enghild JJ, Højrup P, Everitt L, Hubchak S, Schnaper HW, Pizzo SV: Angiostatin binds ATP synthase on the surface of human endothelial cells. Proc Natl Acad Sci USA, 96 (6): 2811-2816, 1999.
Feraud O, Cao Y, Vittet D: Embryonic stem cell-derived embryoid bodies development in collagen gels recapitulates sprouting angio-genesis. Lab Invest, 81 (12): 1669-1681, 2001.
Unur E, Ulger H, Ekinci N, Hacialiogullari M, Ertekin T, Kilic E: Effect of anti-basic fibroblast growth factor (Anti-bFGF) on in vitro embryonic development in rat. Anat Histol Embryol, 38 (4): 241-245, 2009.
Hacıaliogulları M: Effect of interleukin 12 (Il-12) on embryonic yolk sac vascularisation. Yüksek Lisans Tezi, Erciyes Üniv Sağlık Bil Enst, 2005.