Y-27632 Impairs Angiogenesis on Extra-Embryonic Vasculature in Post-Gastrulation Chick Embryos

Y-27632 inhibits Rho-associated coiled-coil-containing protein kinase (ROCK) signaling, which is involved in various embryonic developmental processes, including angiogenesis, by controlling actin cytoskeleton assembly and cell contractility. Administration of Y-27632 impairs cytoskeletal arrangements in post-gastrulation chick embryos, leading to ventral body wall defects (VBWDs). Impaired angiogenesis has been hypothesized to contribute to VBWDs. ROCK is essential in transmitting signals downstream of vascular endothelial growth factor (VEGF). VEGF-mediated angiogenesis induces gene expressions and alterations of the actin cytoskeleton upon binding to VEGF receptors (VEGFRs). The aim of this study was to investigate effects of Y-27632 on angiogenesis in post-gastrulation chick embryos during early embryogenesis. After 60 h incubation, embryos in shell-less culture were treated with Y-27632 or vehicle for controls. Y-27632-treated embryos showed reduced extra-embryonic blood vessel formation with impaired circulation of the yolk sac, confirmed by fractal analysis. Western blot confirmed impaired ROCK downstream signaling by decreased expression of phosphorylated myosin light chain. Interestingly, RT-PCR demonstrated increased gene expression of VEGF and VEGFR-2 1 h post-treatment. Protein levels of VEGF were higher in Y-27632-treated embryos at 8 h following treatment, whereas no difference was seen in membranes. We hypothesize that administration of Y-27632 impairs vessel formation during angiogenesis, which may contribute to failure of VWB closure, causing VBWDs.

[1]  F. Khan,et al.  Anatomy and embryology of abdominal wall defects. , 2022, Seminars in pediatric surgery.

[2]  R. Benndorf,et al.  A current overview of RhoA, RhoB, and RhoC functions in vascular biology and pathology. , 2022, Biochemical pharmacology.

[3]  E. Pechriggl,et al.  Embryology of the Abdominal Wall and Associated Malformations—A Review , 2022, Frontiers in Surgery.

[4]  Chien-Chih Chiu,et al.  Small GTPases and Their Regulators: A Leading Road toward Blood Vessel Development in Zebrafish , 2022, International journal of molecular sciences.

[5]  P. Puri,et al.  Teratogenesis in the chick embryo following post-gastrulation exposure to Y-27632 effect of Y-27632 on embryonic development. , 2020, Toxicology and applied pharmacology.

[6]  P. Carmeliet,et al.  Basic and Therapeutic Aspects of Angiogenesis Updated. , 2020, Circulation research.

[7]  D. Katz,et al.  An Algorithmic Approach to Complex Fetal Abdominal Wall Defects. , 2019, AJR. American journal of roentgenology.

[8]  Gang Bao,et al.  Rho-Associated Coiled-Coil Kinase (ROCK) in Molecular Regulation of Angiogenesis , 2018, Theranostics.

[9]  A. Fougerat,et al.  Small GTPases orchestrate cell-cell communication during collective cell movement , 2020, Small GTPases.

[10]  Jennifer Thompson,et al.  Delayed vasculogenesis and impaired angiogenesis due to altered Ang-2 and VE-cadherin levels in the chick embryo model following exposure to cadmium , 2016, Pediatric Surgery International.

[11]  N. Buchete,et al.  Exposure of chick embryos to cadmium changes the extra-embryonic vascular branching pattern and alters expression of VEGF-A and VEGF-R2. , 2015, Toxicology and applied pharmacology.

[12]  P. Puri,et al.  Impaired cytoskeletal arrangements and failure of ventral body wall closure in chick embryos treated with rock inhibitor (Y-27632) , 2015, Pediatric Surgery International.

[13]  Zhongmin Liu,et al.  The role of the RhoA/Rho kinase pathway in angiogenesis and its potential value in prostate cancer (Review) , 2014, Oncology letters.

[14]  Z. Uni,et al.  Changes in yolk sac membrane absorptive area and fat digestion during chick embryonic development. , 2013, Poultry science.

[15]  P. Ongusaha,et al.  Rock1 & 2 Perform Overlapping and Unique Roles in Angiogenesis and Angiosarcoma Tumor Progression , 2012, Current molecular medicine.

[16]  Chang-xin Wu,et al.  Identification of chicken eNOS gene and differential expression in highland versus lowland chicken breeds. , 2012, Poultry science.

[17]  S. Koch,et al.  Signal transduction by vascular endothelial growth factor receptors. , 2012, Cold Spring Harbor perspectives in medicine.

[18]  D. Ledbetter.  Congenital abdominal wall defects and reconstruction in pediatric surgery: gastroschisis and omphalocele. , 2012, The Surgical clinics of North America.

[19]  M. Felcht,et al.  Angiopoietin-2 differentially regulates angiogenesis through TIE2 and integrin signaling. , 2012, The Journal of clinical investigation.

[20]  A. Eichmann,et al.  VEGF signaling inside vascular endothelial cells and beyond. , 2012, Current opinion in cell biology.

[21]  P. Puri,et al.  ROCK inhibitor (Y-27632) disrupts somitogenesis in chick embryos , 2012, Pediatric Surgery International.

[22]  M. Masu,et al.  Impaired vascular remodeling in the yolk sac of embryos deficient in ROCK‐I and ROCK‐II , 2011, Genes to cells : devoted to molecular & cellular mechanisms.

[23]  L. Claesson‐Welsh,et al.  Signal transduction by vascular endothelial growth factor receptors. , 2011, The Biochemical journal.

[24]  Tien Yin Wong,et al.  Fractal analysis of retinal microvasculature and coronary heart disease mortality. , 2011, European heart journal.

[25]  Z. Uni,et al.  The chick embryo yolk sac membrane expresses nutrient transporter and digestive enzyme genes. , 2011, Poultry science.

[26]  J. Liao,et al.  RhoA/ROCK signaling is essential for multiple aspects of VEGF‐mediated angiogenesis , 2010, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[27]  T. Sadler.  The embryologic origin of ventral body wall defects. , 2010, Seminars in pediatric surgery.

[28]  T. Wieland,et al.  Inhibition of Rho-dependent Kinases Rock I/ii Activates Vegf-driven Retinal Neovascularization and Sprouting Angiogenesis Sion Silencing of Rock I/ii by Specific Small-interfering Rnas (sirnas) in Endothelial Cells Activates Vegf-driven In-gel Sprouting Angiogenesis. Molecularly, Rock I/ii Inhibitio , 2022 .

[29]  V. V. van Hinsbergh,et al.  Role of ROCK I/II in vascular branching. , 2009, American journal of physiology. Heart and circulatory physiology.

[30]  P. Puri,et al.  Downregulation of ROCK-I and ROCK-II gene expression in the cadmium-induced ventral body wall defect chick model , 2008, Pediatric Surgery International.

[31]  G. Shaw,et al.  Gastroschisis: A gene–environment model involving the VEGF–NOS3 pathway , 2008, American journal of medical genetics. Part C, Seminars in medical genetics.

[32]  T. Williams.  Animal models of ventral body wall closure defects: A personal perspective on gastroschisis , 2008, American journal of medical genetics. Part C, Seminars in medical genetics.

[33]  T. Sadler,et al.  The embryology of body wall closure: Relevance to gastroschisis and other ventral body wall defects , 2008, American journal of medical genetics. Part C, Seminars in medical genetics.

[34]  W. Bloch,et al.  Endothelial precursor cell migration during vasculogenesis. , 2007, Circulation research.

[35]  B. Bryan,et al.  What tangled webs they weave: Rho-GTPase control of angiogenesis , 2007, Cellular and Molecular Life Sciences.

[36]  Kazuhiro Takahashi,et al.  Fasudil inhibits vascular endothelial growth factor–induced angiogenesis in vitro and in vivo , 2007, Molecular Cancer Therapeutics.

[37]  T. Sadler,et al.  Development of gastroschisis: Review of hypotheses, a novel hypothesis, and implications for research , 2007, American journal of medical genetics. Part A.

[38]  J. Huot,et al.  Endothelial cell migration during angiogenesis. , 2007, Circulation research.

[39]  R. Scott,et al.  LIM kinases: function, regulation and association with human disease , 2007, Journal of Molecular Medicine.

[40]  E. Génot,et al.  TGFbeta1-induced aortic endothelial morphogenesis requires signaling by small GTPases Rac1 and RhoA. , 2006, Experimental cell research.

[41]  K. Aldape,et al.  Sustained angiopoietin-2 expression disrupts vessel formation and inhibits glioma growth. , 2006, Neoplasia.

[42]  L. Claesson‐Welsh,et al.  VEGF receptor signalling ? in control of vascular function , 2006, Nature Reviews Molecular Cell Biology.

[43]  S. Yuan,et al.  Rho and ROCK Signaling in VEGF‐Induced Microvascular Endothelial Hyperpermeability , 2006, Microcirculation.

[44]  B. Stosic,et al.  Multifractal analysis of human retinal vessels , 2004, IEEE Transactions on Medical Imaging.

[45]  K. Ballmer-Hofer,et al.  The role of VEGF receptors in angiogenesis; complex partnerships , 2006, Cellular and Molecular Life Sciences.

[46]  P. McLoughlin,et al.  Inhibition of Rho-Kinase Attenuates Hypoxia-Induced Angiogenesis in the Pulmonary Circulation , 2005, Circulation research.

[47]  T. Sadler,et al.  Embryology of neural tube development , 2005, American journal of medical genetics. Part C, Seminars in medical genetics.

[48]  T. Williams,et al.  Finally, a sense of closure? Animal models of human ventral body wall defects , 2004, BioEssays : news and reviews in molecular, cellular and developmental biology.

[49]  B. Masters,et al.  Fractal analysis of the vascular tree in the human retina. , 2004, Annual review of biomedical engineering.

[50]  D. Senger,et al.  Rho activity critically and selectively regulates endothelial cell organization during angiogenesis , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[51]  T. Jaffredo,et al.  From mesoderm to blood islands: patterns of key molecules during yolk sac erythropoiesis. , 2003, Gene expression patterns : GEP.

[52]  Anne J. Ridley,et al.  ROCKs: multifunctional kinases in cell behaviour , 2003, Nature Reviews Molecular Cell Biology.

[53]  N. Ferrara,et al.  The biology of VEGF and its receptors , 2003, Nature Medicine.

[54]  V. V. van Hinsbergh,et al.  Involvement of RhoA/Rho Kinase Signaling in VEGF-Induced Endothelial Cell Migration and Angiogenesis In Vitro , 2003, Arteriosclerosis, thrombosis, and vascular biology.

[55]  Ho-Jin Park,et al.  3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Inhibitors Interfere With Angiogenesis by Inhibiting the Geranylgeranylation of RhoA , 2002, Circulation research.

[56]  I. Herman,et al.  Mechanisms of normal and tumor-derived angiogenesis. , 2002, American journal of physiology. Cell physiology.

[57]  R K Jain,et al.  Predominant role of endothelial nitric oxide synthase in vascular endothelial growth factor-induced angiogenesis and vascular permeability , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[58]  John I. Clark,et al.  Fibroblast growth factor-2 selectively stimulates angiogenesis of small vessels in arterial tree. , 2000, Arteriosclerosis, thrombosis, and vascular biology.

[59]  S. Arii,et al.  The suppression of small GTPase rho signal transduction pathway inhibits angiogenesis in vitro and in vivo. , 2000, Biochemical and biophysical research communications.

[60]  K. Haustein.  Cigarette smoking, nicotine and pregnancy. , 1999, International journal of clinical pharmacology and therapeutics.

[61]  G. Yancopoulos,et al.  The angiopoietins: Yin and Yang in angiogenesis. , 1999, Current topics in microbiology and immunology.

[62]  L. Claesson-Welsh.  Vascular Growth Factors and Angiogenesis , 1999, Current Topics in Microbiology and Immunology.

[63]  J. Waltenberger,et al.  VEGF-A induces expression of eNOS and iNOS in endothelial cells via VEGF receptor-2 (KDR). , 1998, Biochemical and biophysical research communications.

[64]  Thomas N. Sato,et al.  Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis. , 1997, Science.

[65]  Janet Rossant,et al.  A Requirement for Flk1 in Primitive and Definitive Hematopoiesis and Vasculogenesis , 1997, Cell.

[66]  M. Moses.  The Regulation of Neovascularization by Matrix Metalloproteinases and Their Inhibitors , 1997 .

[67]  W. Risau,et al.  Mechanisms of angiogenesis , 1997, Nature.

[68]  C. Curry,et al.  Maternal medications and environmental exposures as risk factors for gastroschisis. , 1996, Teratology.

[69]  Lieve Moons,et al.  Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele , 1996, Nature.

[70]  Kenneth J. Hillan,et al.  Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene , 1996, Nature.

[71]  Janet Rossant,et al.  Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice , 1995, Nature.

[72]  C. Curry,et al.  A population-based study of gastroschisis: demographic, pregnancy, and lifestyle risk factors. , 1994, Teratology.

[73]  J. Haddow,et al.  Young maternal age and smoking during pregnancy as risk factors for gastroschisis. , 1993, Teratology.

[74]  V. Hamburger,et al.  A series of normal stages in the development of the chick embryo. 1951. , 2012, Developmental dynamics : an official publication of the American Association of Anatomists.

[75]  M. Lawton,et al.  A new technique for explantation and in vitro cultivation of chicken embryos , 1991, The Anatomical record.

[76]  H. Hoyme,et al.  Gastroschisis: abdominal wall disruption secondary to early gestational interruption of the omphalomesenteric artery. , 1983, Seminars in perinatology.

[77]  P. Devries.  The pathogenesis of gastroschisis and omphalocele. , 1980, Journal of pediatric surgery.

[78]  Viktor Hamburger,et al.  A series of normal stages in the development of the chick embryo , 1992, Journal of morphology.