Abstract-Angiogenesis plays a critical role in wound repair. Endothelial cells present CXC receptor 3 (CXCR3) for chemokines expressed late in wound regeneration. To understand the physiological role CXCR3 plays in regulating endothelial function, we analyzed the ability of a CXCR3 ligand, IP-10 (CXCL10), to influence endothelial cell tube formation. Treatment of endothelial cells with IP-10 in the presence of vascular endothelial growth factor (VEGF) inhibited tube formation on growth factor-reduced Matrigel and in a subcutaneous Matrigel plug. Furthermore, IP-10 significantly inhibited VEGF-induced endothelial motility, a response critical for angiogenesis. Previous work showed that CXCR3 ligandation initiates protein kinase A (PKA) phosphorylation-dependent inhibition of m-calpain, required for induced cell motility, in fibroblasts but not epithelial cells. Here we show that CXCR3 activation in endothelial cells induces an increase in cAMP and PKA activation. Treatment of endothelial cells with Rp-8-Br-cAMP, an inhibitor of PKA, or small interference RNA to PKA was able to reverse the inhibitory effects of IP-10 on VEGF-mediated tube formation and motility. Importantly, treatment of endothelial cells with VEGF induced the activation of m-calpain, but costimulation with IP-10 significantly decreased this activity. Using Rp-8-Br-cAMP, we show blocking PKA reversed the IP-10 inhibition of VEGF-induced m-calpain activity. These data indicate that the activation of CXCR3 inhibits endothelial tube formation through a PKA mediated inhibition of m-calpain. This provides a means by which late wound repair signals limit the angiogenesis driven early in the wound response process. Key Words: CXCL10 Ⅲ CXCR3 Ⅲ angiogenesis Ⅲ signal transduction Ⅲ cAMP Ⅲ receptor tyrosine kinase W ound healing is a dynamic complex biological event. 1 During the regenerative phase of wound healing angiogenesis stops, followed by involution during the remodeling phase. Key to both the initial proliferation of these vessels and the subsequent stasis and then involution of the vascular network is the response of the endothelial cells to signals from the surrounding tissues. At present it is not known whether the termination of angiogenesis occurs because of depletion of the proangiogenic or induction of antiangiogenic signals. Recently, we have found that during the regenerative phase of wound repair, chemokines are expressed to limit fibroblast immigration and motility. 2 Thus, we asked whether the same signals stopped angiogenic ingrowth.Two of the ELR (glutamic acid-leucine-arginine)-negative CXC chemokines appear late in the regenerative phase. IP-10 (interferon ␥-inducible protein 10, CXCL10) is produced by endothelial cells themselves late in the regenerative phase 3 and IP-9 (I-TAC, CXCL11) derives from redifferentiating keratinocytes. 2 These ELR-negative chemokines are of particular interest, because they have been reported to be angiostatic. [3][4][5] These chemokines bind to the common CXC chemokine receptor 3 (CXCR3), 6 which has...

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How m-calpain is activated in cells has challenged investigators because in vitro activation requires nearmillimolar calcium. Previously, we demonstrated that m-calpain activation by growth factors requires extracellular signal-regulated kinase (ERK); this enables tail deadhesion and allows productive motility. We now show that ERK directly phosphorylates and activates m-calpain both in vitro and in vivo. We identified serine 50 as required for epidermal growth factor (EGF)-induced calpain activation in vitro and in vivo. Replacing the serine with alanine limits activation by EGF and subsequent cell deadhesion and motility. A construct with the serine converted to glutamic acid displays constitutive activity in vivo; expression of an estrogen receptor fusion construct produces a tamoxifen-sensitive enzyme. Interestingly, EGF-induced m-calpain activation occurs in the absence of increased intracellular calcium levels; EGF triggers calpain even in the presence of intracellular calcium chelators and in calcium-free media. These data provide evidence that m-calpain can be activated through the ERK cascade via direct phosphorylation and that this activation may occur in the absence of cytosolic calcium fluxes.The calpain family of intracellular cysteine proteinases includes 13 known members, of which at least 2 are ubiquitously expressed (47, 48). These, m-and -calpain (calpain II and calpain I, respectively), are involved in cell migration and adhesion, being regulated downstream of both integrin and growth factor receptor activation (19). -Calpain has been implicated strongly in cell motility and adhesion primarily driven by integrin-mediated signals: calpains have been shown to be required during both cell spreading and adhesion (3,4,41) and for the release of the rear of migrating cells (26). On the other hand, m-calpain has been observed to be activated downstream of the epidermal growth factor (EGF) receptor (EGFR) and is required for growth factor-induced motility and deadhesion (18,44). This effect is specific to m-calpain, as antisense down-regulation of -calpain did not appreciably affect growth factor-induced motility and as EGF-induced calpain activity and motility were dependent on m-calpain (18). m-Calpain affects the migration of EGF-induced fibroblasts by promoting rear release during active motility (2, 43, 44). In general, functions of calpains in motility and adhesion apparently derive from their ability to cleave components of adhesion complexes in a limited manner, altering their function and leading to increased adhesion turnover (19,29,39). However, the molecular mechanism by which calpain activities are regulated during these events is not understood.The two ubiquitous isoforms, -and m-calpain, are presumed to be activated by intracellular calcium fluxes, since these enzymes require this divalent cation in vitro. Indeed mand -calpain are named for their relative requirement for calcium, with -calpain requiring micromolar, and m-calpain requiring near-millimolar, concentrations of calcium (16)....

Replacement of wounded skin requires the initially florid cellular response to abate and even regress as the dermal layer returns to a relatively paucicellular state. The signals that direct this "stop and return" process have yet to be deciphered. CXCR3 chemokine receptor and its ligand CXCL11/IP-9/I-TAC are expressed by basal keratinocytes and CXCL10/IP-10 by keratinocytes and endothelial cells during wound healing in mice and humans. In vitro, these ligands limit motility in dermal fibroblasts and endothelial cells. To examine whether this signaling pathway contributes to wound healing in vivo, full-thickness excisional wounds were created on CXCR3 wild-type (؉/؉) or knockout (؊/؊) mice. Even at 90 days, long after wound closure, wounds in the CXCR3 ؊/؊ mice remained hypercellular and presented immature matrix components. The CXCR3 ؊/؊ mice also presented poor remodeling and reorganization of collagen, which resulted in a weakened healed dermis. This in vivo model substantiates our in vitro findings that CXCR3 signaling is necessary for inhibition of fibroblast and endothelial cell migration and subsequent redifferentiation of the fibroblasts to a contractile state. These studies establish a pathophysiologic role for CXCR3 and its ligand during wound repair. Skin wound repair is a complex, highly orchestrated event consisting of an early hypercellular infiltrate that resolves over time, with loss of most of the regenerativephase dermal fibroblasts and vascular conduits.1 This reversion of the dermal cellularity is necessary for the maturation and strengthening of the matrix, which when lacking, leads to chronic wounds.2 This leaves open the question of which signals define both the transition from regeneration to resolution and the cellular involution that accompanies these changes.Wound repair requires the ordered immigration of fibroblasts into the provisional matrix and keratinocytes over this matrix. This immigration and replacement of the tissue appears to be under the influence of both soluble factors secreted first by platelets and then by inflammatory cell infiltrates, and also matrix components produced by these cells and the immigrated fibroblasts and endothelial cells. Among the latter, tenascin-C and thrombospondins seem to play a major role and thereby mark the immature, regenerative phase of wound healing. [3][4][5] These influence the functionality of the vasculogenesis by acting, directly or indirectly, through growth factor receptors.6,7 These events involve a degree of cellular dedifferentiation to enable migration and proliferation. During the remodeling phase, sufficient cells have migrated into the provisional dermal matrix to mature this structure and across the missing epidermal gap to re-establish a keratinocyte covering. These cells then differentiate into synthetic fibroblasts to produce a mature collagen I-rich dermis or basal keratinocytes primed to differentiate vertically. Interestingly, a fully repaired dermis is paucicellular compared with the regenerative phase, implying a sig...

The platelet receptor for von Willebrand factor (VWF), glycoprotein (GP) Ib-IX, mediates platelet adhesion and activation. The cytoplasmic domains of the GPIb ␣ and ␤ subunits contain binding sites for the phosphorylation-dependent signaling molecule, 14-3-3. Here we show that a novel membrane-permeable inhibitor of 14-3-3-GPIb␣ interaction, MP␣C, potently inhibited VWF binding to platelets and VWF-mediated platelet adhesion under flow conditions. MP␣C also inhibited VWF-dependent platelet agglutination induced by ristocetin. Furthermore, activation of the VWF binding function of GPIb-IX induced by GPIb␤ dephosphorylation is diminished by mutagenic disruption of the 14-3-3 binding site in the C-terminal domain of GPIb␣, mimicking MP␣C-induced inhibition, indicating that the inhibitory effect of MP␣C is likely to be caused by disruption of 14-3-3 binding to GPIb␣. These data suggest a novel 14-3-3-dependent regulatory mechanism that controls the VWF binding function of GPIb-IX, and also suggest a new type of antiplatelet agent that may be potentially useful in preventing or treating thrombosis. ( IntroductionPlatelet adhesion plays an important role in hemostasis and thrombosis. Under high shear rate flow conditions such as in arteries and capillaries, platelet adhesion to the subendothelium is dependent on interaction between subendothelial-bound von Willebrand factor (VWF) and its receptor, the platelet glycoprotein Ib-IX-V complex (GPIb-IX-V). 1 VWF-GPIb-IX-V interaction also mediates the formation of platelet microaggregates in patients with abnormally large VWF multimers, 2 resulting in thrombotic thrombocytopenic purpura (TTP). Interaction of VWF with GPIb-IX-V triggers intracellular signaling events such as elevation of intracellular calcium 3 and cyclic guanosine monophosphate (cGMP) levels, 4 activation of phosphoinositide 3-kinase, 5 and activation of multiple protein kinase pathways. 4,[6][7][8] These events lead to the activation of the ligand binding function of the integrin ␣ IIb ␤ 3 , 9-11 which mediates platelet spreading and aggregation.GPIb-IX-V consists of 4 different transmembrane subunits. GPIb, which consists of disulfide-linked GPIb␣ and GPIb␤ subunits, forms a 1:1 complex with GPIX. The GPIb-IX complex (GPIb-IX) is sufficient for ligand binding and signaling function. 11,12 GPIb-IX forms a 2:1 complex with GPV. 12 The N-terminal region of GPIb␣ contains binding sites for VWF and thrombin. Binding of VWF to GPIb-IX is tightly controlled, and normally occurs only at sites of vascular injury or under pathologically high shear stress. Shear stress may induce changes in both VWF and platelets. In vitro, ristocetin and botrocetin are used to induce VWF binding to GPIb-IX by mimicking the effects of the subendothelial matrix and shear stress on VWF and/or GPIb-IX. Increasing evidence indicates that VWF binding to GPIb-IX is regulated by intraplatelet signals such as cyclic adenosine monophosphate (cAMP) levels. [13][14][15] Elevation of intracellular cAMP activates the cAMP-dependent protein kin...

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.

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