IP-10 induces dissociation of newly formed blood vessels

The signals that prune the exuberant vascular growth of tissue repair are still ill defined. We demonstrate that activation of CXC chemokine receptor 3 (CXCR3) mediates the regression of newly formed blood vessels. We present evidence that CXCR3 is expressed on newly formed vessels in vivo and in vitro. CXCR3 is expressed on vessels at days 7-21 post-wounding, and is undetectable in unwounded or healed skin. Treatment of endothelial cords with CXCL10 (IP-10), a CXCR3 ligand present during the resolving phase of wounds, either in vitro or in vivo caused dissociation even in the presence of angiogenic factors. Consistent with this, mice lacking CXCR3 express a greater number of vessels in wound tissue compared to wild-type mice. We then hypothesized that signaling from CXCR3 not only limits angiogenesis, but also compromises vessel integrity to induce regression. We found that activation of CXCR3 triggers μ-calpain activity, causing cleavage of the cytoplasmic tail of β3 integrins at the calpain cleavage sites c'754 and c'747. IP-10 stimulation also activated caspase 3, blockage of which prevented cell death but not cord dissociation. This is the first direct evidence for an extracellular signaling mechanism through CXCR3 that causes the dissociation of newly formed blood vessels followed by cell death.

[1]  R. Bodnar,et al.  Critical roles for the COOH-terminal NITY and RGT sequences of the integrin β3 cytoplasmic domain in inside-out and outside-in signaling , 2003, The Journal of cell biology.

[2]  T. Veikkola,et al.  Interaction of endostatin with integrins implicated in angiogenesis. , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[3]  P. Hebda,et al.  ELR-negative CXC chemokine CXCL11 (IP-9/I-TAC) facilitates dermal and epidermal maturation during wound repair. , 2008, The American journal of pathology.

[4]  P. Flevaris,et al.  Tyrosine Phosphorylation of the Integrin β3 Subunit Regulates β3 Cleavage by Calpain* , 2006, Journal of Biological Chemistry.

[5]  Sergio Romagnani,et al.  An Alternatively Spliced Variant of CXCR3 Mediates the Inhibition of Endothelial Cell Growth Induced by IP-10, Mig, and I-TAC, and Acts as Functional Receptor for Platelet Factor 4 , 2003, The Journal of experimental medicine.

[6]  M. Burdick,et al.  CXCL11 attenuates bleomycin-induced pulmonary fibrosis via inhibition of vascular remodeling. , 2005, American journal of respiratory and critical care medicine.

[7]  C. Ruhrberg,et al.  Endogenous inhibitors of angiogenesis. , 2001, Journal of cell science.

[8]  M. Nose,et al.  Accumulation of plasma cells expressing CXCR3 in the synovial sublining regions of early rheumatoid arthritis in association with production of Mig/CXCL9 by synovial fibroblasts , 2005, Clinical and experimental immunology.

[9]  K. Yamaguchi,et al.  Platelet factor 4 gene transfection into tumor cells inhibits angiogenesis, tumor growth and metastasis. , 2005, Anticancer research.

[10]  P. Romagnani,et al.  CXCR3-binding chemokines: novel multifunctional therapeutic targets. , 2005, Current drug targets. Immune, endocrine and metabolic disorders.

[11]  B. Sykes,et al.  The CXCR3 binding chemokine IP-10/CXCL10: structure and receptor interactions. , 2002, Biochemistry.

[12]  R. Bodnar,et al.  Delayed and deficient dermal maturation in mice lacking the CXCR3 ELR-negative CXC chemokine receptor. , 2007, The American journal of pathology.

[13]  D. Pode,et al.  Selective ablation of immature blood vessels in established human tumors follows vascular endothelial growth factor withdrawal. , 1999, The Journal of clinical investigation.

[14]  R. Bodnar,et al.  IP-10 Blocks Vascular Endothelial Growth Factor-Induced Endothelial Cell Motility and Tube Formation via Inhibition of Calpain , 2006, Circulation research.

[15]  M. Burdick,et al.  Platelet factor-4 variant chemokine CXCL4L1 inhibits melanoma and lung carcinoma growth and metastasis by preventing angiogenesis. , 2007, Cancer research.

[16]  A. Feldman,et al.  Interferon γ–Inducible Protein 10 Selectively Inhibits Proliferation and Induces Apoptosis in Endothelial Cells , 2006, Annals of Surgical Oncology.

[17]  E. Jaffe,et al.  Anti-tumor activities of the angiogenesis inhibitors interferon-inducible protein-10 and the calreticulin fragment vasostatin , 2002, Cancer Immunology, Immunotherapy.

[18]  R. Bodnar,et al.  Epidermal Growth Factor Activates m-Calpain (Calpain II), at Least in Part, by Extracellular Signal-Regulated Kinase-Mediated Phosphorylation , 2004, Molecular and Cellular Biology.

[19]  R. Swerlick,et al.  HMEC-1: establishment of an immortalized human microvascular endothelial cell line. , 1992, The Journal of investigative dermatology.

[20]  Andrew P. McMahon,et al.  WNT7b mediates macrophage-induced programmed cell death in patterning of the vasculature , 2005, Nature.

[21]  C. Tait,et al.  Angiopoietins in tumours: the angiogenic switch , 2004, The Journal of pathology.

[22]  G. Davis,et al.  Molecular balance of capillary tube formation versus regression in wound repair: role of matrix metalloproteinases and their inhibitors. , 2006, The journal of investigative dermatology. Symposium proceedings.

[23]  W. Hancock,et al.  Requirement of the Chemokine Receptor CXCR3 for Acute Allograft Rejection , 2000, The Journal of experimental medicine.

[24]  A. Richmond,et al.  The angiostatic activity of interferon-inducible protein-10/CXCL10 in human melanoma depends on binding to CXCR3 but not to glycosaminoglycan. , 2004, Molecular therapy : the journal of the American Society of Gene Therapy.

[25]  M. Serio,et al.  Cell cycle-dependent expression of CXC chemokine receptor 3 by endothelial cells mediates angiostatic activity. , 2001, The Journal of clinical investigation.

[26]  T. Byzova,et al.  Mechanisms of Integrin–Vascular Endothelial Growth Factor Receptor Cross-Activation in Angiogenesis , 2007, Circulation research.

[27]  Xiaoping Du,et al.  Cleavage of the Cytoplasmic Domain of the Integrin β3 Subunit during Endothelial Cell Apoptosis* , 1998, Journal of Biological Chemistry.

[28]  George Kolios,et al.  The Chemokines CXCL9, CXCL10, and CXCL11 Differentially Stimulate Gαi-Independent Signaling and Actin Responses in Human Intestinal Myofibroblasts1 , 2005, The Journal of Immunology.

[29]  E. Hudson,et al.  Differential expression and responsiveness of chemokine receptors (CXCR1–3) by human microvascular endothelial cells and umbilical vein endothelial cells , 2000, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[30]  P. Carmeliet,et al.  Targeted Deficiency or Cytosolic Truncation of the VE-cadherin Gene in Mice Impairs VEGF-Mediated Endothelial Survival and Angiogenesis , 1999, Cell.

[31]  C. Craddock,et al.  CXCL10-CXCR3 interactions play an important role in the pathogenesis of acute graft-versus-host disease in the skin following allogeneic stem-cell transplantation. , 2007, Blood.

[32]  Jianhua Huang,et al.  A Role for VEGF as a Negative Regulator of Pericyte Function and Vessel Maturation , 2008, Nature.

[33]  O. Volpert,et al.  Signals leading to apoptosis-dependent inhibition of neovascularization by thrombospondin-1 , 2000, Nature Medicine.

[34]  P. Flevaris,et al.  A molecular switch that controls cell spreading and retraction , 2007, The Journal of cell biology.

[35]  G. Brunner,et al.  Extracellular regulation of TGF-β activity in wound repair: growth factor latency as a sensor mechanism for injury , 2004, Thrombosis and Haemostasis.

[36]  P. Flevaris,et al.  Tyrosine phosphorylation of the integrin beta 3 subunit regulates beta 3 cleavage by calpain. , 2006, The Journal of biological chemistry.

[37]  Z. Jia,et al.  Activation of m-Calpain (Calpain II) by Epidermal Growth Factor Is Limited by Protein Kinase A Phosphorylation of m-Calpain , 2002, Molecular and Cellular Biology.

[38]  N. Giese,et al.  Suppression of metastatic hemangiosarcoma by a parvovirus MVMp vector transducing the IP-10 chemokine into immunocompetent mice , 2002, Cancer Gene Therapy.

[39]  A. Wells,et al.  Interferon-Inducible Protein 9 (CXCL11)-Induced Cell Motility in Keratinocytes Requires Calcium Flux-Dependent Activation of μ-Calpain , 2005, Molecular and Cellular Biology.