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.

Objective To understand the role, if any, played by pericytes in the regulation of newly formed vessels during angiogenesis. In this study, we investigate whether pericytes regulate the number of nascent endothelial tubes. Approach and Results Using an in vitro angiogenesis assay (Matrigel assay), we demonstrate that pericytes can inhibit vessel formation and induce vessel dissociation via CXCR3-induced involution of the endothelial cells. In a coculture Matrigel assay for cord formation, pericytes prevented endothelial cord formation of human dermal microvascular endothelial cells but not umbilical vein endothelial cells. Blockade of endothelial CXCR3 function or expression inhibited the repressing effect of the pericytes. We further show that pericytes are also able to induce regression of newly formed microvascular cords through CXCR3 activation of calpain. When CXCR3 function was inhibited by a neutralizing antibody or downregulated by siRNA, cord regression mediated by pericytes was abolished. Conclusions We show for the first time that pericytes regulate angiogenic vessel formation, and that this is mediated through CXCR3 expressed on endothelial cells. This suggests a role for pericytes in the pruning of immature vessels overproduced during wound repair.

Angiogenesis plays a critical role in processes such as organ development, wound healing, and tumor growth. It requires well-orchestrated integration of soluble and matrix factors and timely recognition of such signals to regulate this process. Previous work has shown that newly forming vessels express the chemokine receptor CXC receptor 3 (CXCR3) and, activation by its ligand IP-10 (CXCL10), both inhibits development of new vasculature and causes regression of newly formed vessels. To identify and develop new therapeutic agents to limit or reverse pathological angiogenesis, we identified a 21 amino acid fragment of IP-10, spanning the α-helical domain residues 77–98, that mimic the actions of the whole IP-10 molecule on endothelial cells. Treatment of the endothelial cells with the 22 amino acid fragment referred to as IP-10p significantly inhibited VEGF-induced endothelial motility and tube formation in vitro , properties critical for angiogenesis. Using a Matrigel plug assay in vivo , we demonstrate that IP-10p both prevented vessel formation and induced involution of nascent vessels. CXCR3 neutralizing antibody was able to block the inhibitory effects of the IP-10p, demonstrating specificity of the peptide. Inhibition of endothelial function by IP-10p was similar to that described for IP-10, secondary to CXCR3-mediated increase in cAMP production, activation of PKA inhibiting cell migration, and inhibition of VEGF-mediated m-calpain activation. IP-10p provides a novel therapeutic agent that inhibits endothelial cell function thus, allowing for the modulation of angiogenesis.

No abstract

The giving of consent by the patient before medical treatment is a central tenet of medical law. Although it may be fairly clear when an adult patient has given consent, the situation can be less clear, and more complex, when a child patient is involved. This article seeks to explore the legal issues and concepts that arise when treating a minor, and to show how the courts and legal process may become involved.