Interstitial fluid flow intensity modulates endothelial sprouting in restricted Src-activated cell clusters during capillary morphogenesis.

Development of tissues in vitro with dimensions larger than 150 to 200 microm requires the presence of a functional vascular network. Therefore, we have studied capillary morphogenesis under controlled biological and biophysical conditions with the aim of promoting vascular structures in tissue constructs. We and others have previously demonstrated that physiological values of interstitial fluid flow normal to an endothelial monolayer in combination with vascular endothelial growth factor play a critical role during capillary morphogenesis by promoting cell sprouting. In the present work, we studied the effect that a range of interstitial flow velocities (0-50 microm/min) has in promoting the amount, length, and branching of developing sprouts during capillary morphogenesis. The number of capillary-like structures developed from human umbilical vein endothelial cell monolayers across the interstitial flow values tested was not significantly affected. Instead, the length and branching degree of the sprouts presented a significant maximum at flow velocities of 10 to 20 microm/min. More-over, at these same flow values, the phosphorylation level of Src also showed its peak. We discovered that capillary morphogenesis is restricted to patches of Src-activated cells (phosphorylated Src (pSrc)) at the monolayer, suggesting that the transduction pathway in charge of sensing the mechanical stimulus induced by flow is promoting predetermined mechanically sensitive areas (pSrc) to undergo capillary morphogenesis

[1]  I. Zachary.  VEGF signalling: integration and multi-tasking in endothelial cell biology. , 2003, Biochemical Society transactions.

[2]  L. Claesson‐Welsh,et al.  FGF and VEGF function in angiogenesis: signalling pathways, biological responses and therapeutic inhibition. , 2001, Trends in pharmacological sciences.

[3]  V. Vogel,et al.  The tissue engineeting puzzle: a molecular perspective. , 2003, Annual review of biomedical engineering.

[4]  G. Davis,et al.  An alpha 2 beta 1 integrin-dependent pinocytic mechanism involving intracellular vacuole formation and coalescence regulates capillary lumen and tube formation in three-dimensional collagen matrix. , 1996, Experimental cell research.

[5]  Signaling in morphogenesis: transport cues in morphogenesis. , 2003, Current opinion in biotechnology.

[6]  Elliot L. Botvinick,et al.  Visualizing the mechanical activation of Src , 2005, Nature.

[7]  M. Rothmund,et al.  Targeting the EGF/VEGF-R system by tyrosine-kinase inhibitors—a novel antiproliferative/antiangiogenic strategy in thyroid cancer , 2006, Langenbeck's Archives of Surgery.

[8]  Melody A. Swartz,et al.  Interstitial Flow as a Guide for Lymphangiogenesis , 2003, Circulation research.

[9]  R. Nagai,et al.  Shear stress increases heparin-binding epidermal growth factor-like growth factor mRNA levels in human vascular endothelial cells. , 1993, Biochemical and biophysical research communications.

[10]  Melody A Swartz,et al.  A driving force for change: interstitial flow as a morphoregulator. , 2007, Trends in cell biology.

[11]  S. Chien.  Mechanotransduction and endothelial cell homeostasis: the wisdom of the cell. , 2007, American journal of physiology. Heart and circulatory physiology.

[12]  D. Lauffenburger,et al.  Autocrine EGF receptor activation mediates endothelial cell migration and vascular morphogenesis induced by VEGF under interstitial flow. , 2005, Experimental cell research.

[13]  M. Shibuya,et al.  Nucleotide sequence and expression of a novel human receptor-type tyrosine kinase gene (flt) closely related to the fms family. , 1990, Oncogene.

[14]  E. Haber,et al.  Vascular Endothelial Growth Factor Induces Heparin-binding Epidermal Growth Factor-like Growth Factor in Vascular Endothelial Cells* , 1998, The Journal of Biological Chemistry.

[15]  M. Rusnati,et al.  Fibroblast growth factors/fibroblast growth factor receptors as targets for the development of anti-angiogenesis strategies. , 2007, Current pharmaceutical design.

[16]  Napoleone Ferrara,et al.  VEGF and the quest for tumour angiogenesis factors , 2002, Nature Reviews Cancer.

[17]  R K Jain,et al.  Direct measurement of interstitial convection and diffusion of albumin in normal and neoplastic tissues by fluorescence photobleaching. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[18]  David A. Schultz,et al.  A mechanosensory complex that mediates the endothelial cell response to fluid shear stress , 2005, Nature.

[19]  Richard T. Lee,et al.  Cell mechanics and mechanotransduction: pathways, probes, and physiology. , 2004, American journal of physiology. Cell physiology.

[20]  Michael P. Sheetz,et al.  Selective regulation of integrin–cytoskeleton interactions by the tyrosine kinase Src , 1999, Nature Cell Biology.

[21]  M. Swartz,et al.  Fibroblast alignment under interstitial fluid flow using a novel 3-D tissue culture model. , 2003, American journal of physiology. Heart and circulatory physiology.

[22]  D. Donner,et al.  Vascular Endothelial Cell Growth Factor Promotes Tyrosine Phosphorylation of Mediators of Signal Transduction That Contain SH2 Domains , 1995, The Journal of Biological Chemistry.

[23]  M. Gerritsen,et al.  Functional roles for PECAM-1 (CD31) and VE-cadherin (CD144) in tube assembly and lumen formation in three-dimensional collagen gels. , 1999, The American journal of pathology.

[24]  J. Brugge,et al.  Src kinase activation by direct interaction with the integrin β cytoplasmic domain , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[25]  Federica Boschetti,et al.  Synergy between interstitial flow and VEGF directs capillary morphogenesis in vitro through a gradient amplification mechanism. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[26]  G. Giaccone,et al.  Epidermal growth factor receptor and angiogenesis: Opportunities for combined anticancer strategies , 2005, International journal of cancer.

[27]  Melody A Swartz,et al.  Interstitial flow differentially stimulates blood and lymphatic endothelial cell morphogenesis in vitro. , 2004, Microvascular research.

[28]  W. Kilarski,et al.  Inactivation of Src family kinases inhibits angiogenesis in vivo: implications for a mechanism involving organization of the actin cytoskeleton. , 2003, Experimental cell research.

[29]  B. Sipos,et al.  Vascular endothelial growth factor mediated angiogenic potential of pancreatic ductal carcinomas enhanced by hypoxia: An in vitro and in vivo study , 2002, International journal of cancer.

[30]  Kayla J Bayless,et al.  Sphingosine-1-phosphate markedly induces matrix metalloproteinase and integrin-dependent human endothelial cell invasion and lumen formation in three-dimensional collagen and fibrin matrices. , 2003, Biochemical and biophysical research communications.

[31]  Song Li,et al.  Mechanotransduction in endothelial cell migration , 2005, Journal of cellular biochemistry.

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

[33]  Melody A Swartz,et al.  Engineered blood and lymphatic capillaries in 3‐D VEGF‐fibrin‐collagen matrices with interstitial flow , 2007, Biotechnology and bioengineering.

[34]  M. Swartz,et al.  The Role of Interstitial Stress in Lymphatic Function and Lymphangiogenesis , 2002, Annals of the New York Academy of Sciences.

[35]  D. Senger,et al.  Matrix‐specific activation of Src and Rho initiates capillary morphogenesis of endothelial cells , 2004, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[36]  Stanley J. Wiegand,et al.  Vascular-specific growth factors and blood vessel formation , 2000, Nature.

[37]  D. Mukhopadhyay,et al.  Complexity in the vascular permeability factor/vascular endothelial growth factor (VPF/VEGF)-receptors signaling , 2004, Molecular and Cellular Biochemistry.

[38]  S. Mullens,et al.  Permeability of porous gelcast scaffolds for bone tissue engineering , 2010 .

[39]  D. Lauffenburger,et al.  Determining Cell Fate Transition Probabilities to VEGF/Ang 1 Levels: Relating Computational Modeling to Microfluidic Angiogenesis Studies , 2010 .