LRP1 in the NVU to promote tPA-mediated activation of PDGF-CC. Mac-1-deficient mice (Mac-1 −/−) are protected from tPA-induced BBB permeability but not from perme-ability induced by intracerebroventricular injection of active PDGF-CC. Immunofluorescence analysis demonstrates that Mac-1, LRP1, and the PDGFRα all localize to the NVU of arterioles, and following middle cerebral artery occlusion (MCAO) Mac-1 −/− mice show significantly less PDGFRα phosphorylation, BBB permeability, and infarct volume compared to wild-type mice. Bone-marrow transplantation studies indicate that resident CD11b + cells, but not bone-marrow-derived leukocytes, mediate the early activation of PDGF-CC by tPA after MCAO. Finally, using a model of thrombotic stroke with late thrombolysis, we show that wild-type mice have an increased incidence of spontaneous ICH following thrombolysis with tPA 5 h after MCAO, whereas Mac-1 −/− mice are resistant to the development of ICH even with late tPA treatment. Together, these results indicate that Mac-1 and LRP1 act as co-factors for the activation of PDGF-CC by tPA in the NVU, and suggest a novel mechanism for tightly regulating PDGFRα signaling in the NVU and controlling BBB permeability. Abstract Treatment of acute ischemic stroke with the thrombolytic tissue plasminogen activator (tPA) can significantly improve neurological outcomes; however, throm-bolytic therapy is associated with an increased risk of intra-cerebral hemorrhage (ICH). Previously, we demonstrated that during stroke tPA acting on the parenchymal side of the neurovascular unit (NVU) can increase blood-brain barrier (BBB) permeability and ICH through activation of latent platelet-derived growth factor-CC (PDGF-CC) and signaling by the PDGF receptor-α (PDGFRα). However , in vitro, activation of PDGF-CC by tPA is very inefficient and the mechanism of PDGF-CC activation in the NVU is not known. Here, we show that the integrin Mac-1, expressed on brain microglia/macrophages (denoted micro-glia throughout), acts together with the endocytic receptor Enming Joseph Su and Chunzhang Cao contributed equally to this work. Daniel A. Lawrence and Li Zhang contributed equally to this work. Electronic supplementary material The online version of this article (

Cortactin, a prominent substrate for pp60 c-src , is a filamentous actin (F-actin) binding protein. We show here that cortactin can promote sedimentation of Factin at centrifugation forces under which F-actin is otherwise not able to be precipitated. Electron microscopic analysis after negative staining further revealed that actin filaments in the presence of cortactin are cross-linked into bundles of various degrees of thickness. Hence, cortactin is also an F-actin cross-linking protein. We also demonstrate that the optimal F-actin cross-linking activity of cortactin requires a physiological pH in a range of 7.3-7.5. Furthermore, pp60 c-src phosphorylates cortactin in vitro, resulting in a dramatic reduction of its F-actin cross-linking activity in a manner depending on levels of tyrosine phosphorylation. In addition, pp60 c-src moderately inhibits the F-actin binding activity of cortactin. This study presents the first evidence that pp60 c-src can directly regulate the activity of its substrate toward the cytoskeleton and implies a role of cortactin as an F-actin modulator in tyrosine kinase-regulated cytoskeleton reorganization.

Bisphosphonate-associated osteonecrosis of the jaw (BONJ) is a morbid bone disease linked to long-term bisphosphonate use. Despite its broad health impact, mechanistic study is lacking. In this study, we have established a mouse model of BONJ-like disease based on the equivalent clinical regimen in myeloma patients, a group associated with high risk of BONJ. We demonstrate that the murine BONJ-like disease recapitulates major clinical and radiographical manifestations of the human disease, including characteristic features of osseous sclerosis, sequestra, avascular, and radiopaque alveolar bone in the jaw that persists beyond a normal course of wound healing following tooth extraction. We find that long-term administration of bisphosphonates results in an increase in the size and number of osteoclasts and the formation of giant osteoclast-like cells within the alveolar bone. We show that the development of necrotic bone and impaired soft tissue healing in our mouse model is dependent on long-term use of high-dose bisphosphonates, immunosuppressive and chemotherapy drugs, as well as mechanical trauma.Most importantly, we demonstrate that bisphosphonate is the major cause of BONJ-like disease in mice , mediated in part by its ability to suppress osseous angiogenesis and bone remodeling. The availability of this novel mouse model of BONJ-like disease will help elucidate the pathophysiology of BONJ and ultimately develop novel approaches for prevention and treatment of human BONJ. (Am J Pathol

Activated protein C (APC), the only FDA-approved biotherapeutic drug for sepsis, possesses anticoagulant, antiinflammatory, and barrier-protective activities. However, the mechanisms underlying its antiinflammatory functions are not well defined. Here, we report that the antiinflammatory activity of APC on macrophages is dependent on integrin CD11b/CD18, but not on endothelial protein C receptor (EPCR). We showed that CD11b/CD18 bound APC within specialized membrane microdomains/lipid rafts and facilitated APC cleavage and activation of protease-activated receptor-1 (PAR1), leading to enhanced production of sphingosine-1-phosphate (S1P) and suppression of the proinflammatory response of activated macrophages. Deletion of the γ-carboxyglutamic acid domain of APC, a region critical for its anticoagulant activity and EPCR-dependent barrier protection, had no effect on its antiinflammatory function. Genetic inactivation of CD11b, PAR1, or sphingosine kinase-1, but not EPCR, abolished the ability of APC to suppress the macrophage inflammatory response in vitro. Using an LPS-induced mouse model of lethal endotoxemia, we showed that APC administration reduced the mortality of wild-type mice, but not CD11b-deficient mice. These data establish what we believe to be a novel mechanism underlying the antiinflammatory activity of APC in the setting of endotoxemia and provide clear evidence that the antiinflammatory function of APC is distinct from its barrier-protective function and anticoagulant activities.

Summary Background The interaction of fibrin βN-domain with VE-cadherin on endothelial cells was implicated in transendothelial migration of leukocytes and theβ15-42 fragment representing part of this domain was shown to inhibit this process. However, our previous study revealed that only a dimeric (β15-66)2 fragment corresponding to the full-length βN-domain and mimicking its dimeric arrangement in fibrin bound to VE-cadherin. Objective To test our hypothesis that dimerization of β15-42-containing fragments increases their affinity to VE-cadherin and ability to inhibit transendothelial migration of leukocytes. Methods Interaction of β15-42-containing fragments with VE-cadherin was characterized by ELISA and surface plasmon resonance. Inhibitory effect of such fragments was tested in vitro using leukocyte transendothelial migration assay and in vivo using mouse models of peritonitis and myocardial ischemia-reperfusion injury. Results First, we prepared the monomeric β15-42 and β15-64 fragments and their dimeric forms, (β15-44)2 and (β15-66)2, and studied their interaction with fibrin-binding domain of VE-cadherin, VE-cad(3). The experiments revealed that both dimeric fragments bound to VE-cad(3) with high affinity while the affinities of monomeric β15-42 and β15-64 were significantly lower. Next, we tested the ability of these fragments to inhibit leukocyte transmigration in vitro and infiltration into the inflamed peritoneum in vivo and found that the inhibitory effects of the dimers on these processes were also superior. Furthermore, dimeric (β15-44)2 significantly reduced myocardial injury induced by ischemia-reperfusion. Conclusion The results confirm our hypotheses and indicate that dimeric (β15-66)2 and (β15-44)2, which exhibited much higher affinity to VE-cadherin, are highly effective in suppressing inflammation by inhibiting leukocyte transmigration.

Activation of the platelet-derived growth factor receptor-␤ (PDGFR-␤) leads to tyrosine phosphorylation of the cytoplasmic domain of LRP and alters its association with adaptor and signaling proteins, such as Shc. The mechanism of the PDGF-induced LRP tyrosine phosphorylation is not well understood, especially since PDGF not only activates PDGF receptor but also binds directly to LRP. To gain insight into this mechanism, we used a chimeric receptor in which the ligand binding domain of the PDGFR-␤ was replaced with that from the macrophage colony-stimulating factor (M-CSF) receptor, a highly related receptor tyrosine kinase of the same subfamily, but with different ligand specificity. Activation of the chimeric receptor upon the addition of M-CSF readily mediated the tyrosine phosphorylation of LRP. Since M-CSF is not recognized by LRP, these results indicated that growth factor binding to LRP is not necessary for this phosphorylation event. Using a panel of cytoplasmic domain mutants of the chimeric M-CSF/PDGFR-␤, we confirmed that the kinase domain of PDGFR-␤ is absolutely required for LRP tyrosine phosphorylation but that PDGFR-␤-mediated activation of phosphatidylinositol 3-kinase, RasGAP, SHP-2, phospholipase C-␥, and Src are not necessary for LRP tyrosine phosphorylation. To identify the cellular compartment where LRP and the PDGFR-␤ may interact, we employed immunofluorescence and immunogold electron microscopy. In WI-38 fibroblasts, these two receptors co-localized in coated pits and endosomal compartments following PDGF stimulation. Further, phosphorylated forms of the PDGFR-␤ coimmunoprecipitated with LRP following PDGF treatment. Together, these studies revealed close association between activated PDGFR-␤ and LRP, suggesting that LRP functions as a co-receptor capable of modulating the signal transduction pathways initiated by the PDGF receptor from endosomes.The low density lipoprotein receptor-related protein (LRP) 1 is a large endocytic receptor composed of a 515-kDa heavy chain to which ligands bind and a non-covalently associated 85-kDa light chain containing a transmembrane and cytoplasmic domain. LRP is a member of the LDL receptor family and is required for embryonic development in mice (1). Although LRP was originally identified as an endocytic receptor for ␣-2-macroglobulin-protease complexes (1, 2) and apoE-enriched lipoprotein particles (3, 4), this receptor is now known to recognize more than 30 distinct ligands including lipoproteins, proteases, proteinase inhibitor complexes, matrix proteins, bacterial toxins, viruses, intracellular proteins, and growth factors (for a review, see Ref. 5).In addition to its prominent role in mediating the cellular uptake of a variety of ligands, recent work has implicated LRP in several signal transduction pathways and has expanded its realm to include regulation of cell migration (6 -8) and modulation of the integrity of the blood brain barrier (9). In the vasculature, LRP is expressed in smooth muscle cells, and a tissue-specific deletion of the LRP ge...

The serine proteinase inhibitor, plasminogen activator inhibitor type-1 (PAI-1), binds to the adhesion protein vitronectin with high affinity at a site that is located directly adjacent to the vitronectin RGD integrin binding sequence. The binding of PAI-1 to vitronectin sterically blocks integrin access to this site and completely inhibits the binding of purified integrins to vitronectin; however, its inhibition of endothelial and smooth muscle cell adhesion to vitronectin is at most 50 -75%. Because PAI-1 binds vitronectin with ϳ10 -100-fold higher affinity than purified integrins, we have analyzed the mechanism whereby these cells are able to overcome this obstacle. Our studies exclude proteolytic removal of PAI-1 from vitronectin as the mechanism, and show instead that cell adhesion in the presence of PAI-1 is dependent on integrin-cytoskeleton engagement. Disrupting endothelial or smooth muscle cell actin polymerization and/or focal adhesion assembly reduces cell adhesion to vitronectin in the presence of PAI-1 to levels similar to that observed for the binding of purified integrins to vitronectin. Furthermore, endothelial cell, but not smooth muscle cell adhesion to vitronectin in the presence of PAI-1 requires both polymerized microtubules and actin, further demonstrating the importance of the cytoskeleton for integrin-mediated adhesion. Finally, we show that cell adhesion in the presence of PAI-1 leads to colocalization of PAI-1 with the integrins ␣v␤3 and ␣v␤5 at the cell-matrix interface.Cell adhesion receptors play important roles in maintaining cell anchorage and polarity in addition to promoting migration and differentiation. Many of the known adhesion receptors belong to the integrin family of noncovalent heterodimeric transmembrane proteins that are expressed by most cell types. The integrin family consists of at least 18 ␣-and 8 ␤-subunits that combine to form as many as 24 different ␣␤ dimers (1). The extracellular domains of both integrin subunits are required for binding to adhesion proteins. In addition, integrins need physiological concentrations of divalent cations such as calcium or magnesium. Integrins have relatively short cytoplasmic tails that interact with an intracellular protein complex, termed focal contacts, which include adaptor proteins and kinases. The focal contacts control the linkage between the cytoskeleton and integrins and mediate intracellular signaling. Integrins direct forces generated by the cytoskeleton onto the extracellular matrix (ECM) 2 to produce the traction necessary for cell adhesion and migration (2). Understanding the mechanism and control of integrin-mediated cell adhesion and migration is of special interest because of their importance in processes such as wound healing and angiogenesis as well as pathologies associated with these processes.In general, integrins recognize short linear peptide sequences on adhesion proteins, the most prevalent being arginine-glycine-aspartic acid (RGD), which is found in many ECM proteins such as fibronectin, collagens, ...

Abstract-Myxomatous tissue is a characteristic component of human coronary artery lesions, found more often in restenotic lesions. It represents a bulky accumulation of stellate-shaped cells of unknown histogenesis that are embedded in a loose stroma. We analyzed 64 atherectomy specimens containing substantial amounts of myxomatous tissue by using immunohistochemistry, in situ hybridization, and electron microscopy techniques. Stellate cells represented a heterogeneous population, sharing features of smooth muscle cells (SMCs), macrophages, as well as antigen-presenting dendritic cells. Like quiescent medial SMCs, the stellate cells in all specimens expressed high levels of SM ␣-actin message and protein and showed heterogeneity with respect to heavy-chain myosin, SM22, desmin, and vimentin. Ultrastructurally, stellate cells resembled SMCs, with some peculiarities that distinguish them from both differentiated and dedifferentiated SMCs. In contrast to quiescent SMCs, the stellate cells expressed high levels of acidic fibroblast growth factor mRNA and protein similar to cells of monocyte/macrophage lineage. However, stellate cells did not express the marker of mature macrophages, HAM56, and were heterogeneous with respect to CD68. Moreover, unlike SMCs, the stellate cells bore some of the major phenotypic markers of dendritic cells: they were S100-positive and showed various reactivity with respect to CD1a and human leukocyte antigen (HLA)-DR. Invasion of myxomatous tissue with CD45RO-positive T lymphocytes was correlated with strong expression of CD1a in these specimens. Stellate cells also expressed a pericyte marker, high-molecular-weight melanoma-associated antigen. We conclude that stellate cells of myxomatous tissue represent a specific phenotype of mesenchymal cells (possibly pericytes), which is activated to express some markers of antigen-presenting cells.