Several lines of evidence suggest that tumor growth, angiogenesis, and metastasis are dependent on matrix metalloproteinase (MMP) activity. However, the lack of inhibitors specific for the type IV collagenase/gelatinase family of MMPs has thus far prevented the selective targeting of MMP-2 (gelatinase A) and MMP-9 (gelatinase B) for therapeutic intervention in cancer. Here, we describe the isolation of specific gelatinase inhibitors from phage display peptide libraries. We show that cyclic peptides containing the sequence HWGF are potent and selective inhibitors of MMP-2 and MMP-9 but not of several other MMP family members. Our prototype synthetic peptide, CTTHWGFTLC, inhibits the migration of human endothelial cells and tumor cells. Moreover, it prevents tumor growth and invasion in animal models and improves survival of mice bearing human tumors. Finally, we show that CTTHWGFTLC-displaying phage specifically target angiogenic blood vessels in vivo. Selective gelatinase inhibitors may prove useful in tumor targeting and anticancer therapies.

Leukocyte recruitment to sites of infection or inflammation requires multiple adhesive events. While numerous players promoting leukocyte-endothelial interactions have been characterized, functionally important endogenous inhibitors of leukocyte adhesion have not been identified. Here, we describe the endothelial-derived secreted molecule, developmental endothelial locus-1 (Del-1), as an anti-adhesive factor that interferes with the integrin LFA-1-dependent leukocyte-endothelial adhesion. Endothelial Del-1-deficiency increased LFA-1-dependent leukocyte adhesion in vitro and in vivo. Del-1-/-mice displayed significantly higher neutrophil accumulation in LPS-induced lung inflammation in vivo, which was reversed in Del-1/LFA-1-double deficient mice. Thus, Del-1 is an endogenous inhibitor of inflammatory cell recruitment and could provide a basis for targeting leukocyte-endothelial interactions in disease.Leukocyte extravasation is integral to the response to infection or injury and to inflammation and autoimmunity. Leukocyte recruitment comprises a well coordinated cascade of adhesive events including selectin-mediated rolling, firm adhesion of leukocytes to endothelial cells and & This manuscript has been accepted for publication in Science. This version has not undergone final editing. Please refer to the complete version of record at http://www.sciencemag.org/. The manuscript may not be reproduced or used in any manner that does not fall within the fair use provisions of the Copyright Act without the prior, written permission of AAAS. †To whom correspondence should be addressed chavakist@mail.nih.gov. * EYC and EC contributed equally # MAC and HL contributed equally NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript their subsequent transendothelial migration. The interaction between LFA-1 (αLβ2, CD11a/ CD18) and endothelial ICAM-1 is crucial during firm endothelial adhesion of leukocytes (1-5). Whereas numerous adhesion receptors promoting inflammatory cell recruitment have been identified, very little information exists about endogenous inhibitors of the leukocyte adhesion cascade (1-7). Developmental endothelial locus-1 (Del-1) is a glycoprotein that is secreted by endothelial cells and can associate with the endothelial cell surface and the extracellular matrix (8-10). Del-1 is regulated upon hypoxia or vascular injury and has been implicated in vascular remodelling during angiogenesis (10-12). Here, we sought to determine whether endothelial-derived Del-1 participates in leukocyte-endothelial interactions. RT-PCR analysis revealed Del-1 mRNA predominantly in the brain and lung, with no expression in liver, spleen, or whole blood (Fig. 1A and fig. S1A). Del-1 was expressed in WT but not in Del-1-/-murine lung endothelial cells (Fig. 1B, 9). Immunohistochemistry of lung tissues demonstrated the presence of Del-1 in vessels, as observed by co-staining with the endothelial marker PECAM-1 ( fig. S1B).To determine whether Del-1 participates in leukocyte recruitmen...

High‐mobility group box 1 (HMGB1) is released extracellularly upon cell necrosis acting as a mediator in tissue injury and inflammation. However, the molecular mechanisms for the proinflammatory effect of HMGB1 are poorly understood. Here, we define a novel function of HMGB1 in promoting Mac‐1‐dependent neutrophil recruitment. HMGB1 administration induced rapid neutrophil recruitment in vivo. HMGB1‐mediated recruitment was prevented in mice deficient in the β2‐integrin Mac‐1 but not in those deficient in LFA‐1. As observed by bone marrow chimera experiments, Mac‐1‐dependent neutrophil recruitment induced by HMGB1 required the presence of receptor for advanced glycation end products (RAGE) on neutrophils but not on endothelial cells. In vitro, HMGB1 enhanced the interaction between Mac‐1 and RAGE. Consistently, HMGB1 activated Mac‐1 as well as Mac‐1‐mediated adhesive and migratory functions of neutrophils in a RAGE‐dependent manner. Moreover, HMGB1‐induced activation of nuclear factor‐κB in neutrophils required both Mac‐1 and RAGE. Together, a novel HMGB1‐dependent pathway for inflammatory cell recruitment and activation that requires the functional interplay between Mac‐1 and RAGE is described here.

Leukocyte integrins of the ␤2 family are essential for immune cell-cell adhesion. In activated cells, ␤2 integrins are phosphorylated on the cytoplasmic Thr758, leading to 14-3-3 protein recruitment to the ␤2 integrin. The mutation of this phosphorylation site impairs cell adhesion, actin reorganization, and cell spreading. Thr758 is contained in a Thr triplet of ␤2 that also mediates binding to filamin. Here, we investigated the binding of filamin, talin, and 14-3-3 proteins to phosphorylated and unphosphorylated ␤2 integrins by biochemical methods and x-ray crystallography. 14-3-3 proteins bound only to the phosphorylated integrin cytoplasmic peptide, with a high affinity (K d , 261 nM), whereas filamin bound only the unphosphorylated integrin cytoplasmic peptide (K d , 0.5 mM). Phosphorylation did not regulate talin binding to ␤2 directly, but 14-3-3 was able to outcompete talin for the binding to phosphorylated ␤2 integrin. X-ray crystallographic data clearly IntroductionIntegrins are heterodimeric plasma membrane receptors that mediate binding to the extracellular matrix and to ligands present on the surface of other cells. Their function is tightly regulated; they bind ligands only after activation. Modulation of integrin activity occurs through tightly regulated interactions between cytoplasmic molecules and integrin intracellular tails. Factors binding to integrin cytoplasmic domains regulating integrin adhesiveness include the cytoskeletal proteins talin 1,2 and filamin, 3 and the 14-3-3 proteins, which are molecular adaptors that bind to phosphorylated serine or threonine (pSer/ pThr) containing polypeptide sequences. 4 The ␤2 integrins are expressed exclusively on leukocytes and bind ICAM molecules on other leukocytes and endothelial cells after cell activation. 5,6 Talin binds to ␤2 integrins in vitro and in cells and is involved in activating the ␤2 integrins, resulting in binding to ICAMs. 1,4,[7][8][9] The ␤2 integrin polypeptide chain is phosphorylated on the intracellular domain on several residues after cell stimulation with various agents. 10 Thr758 is a physiologically important amino acid residue in the ␤2 cytoplasmic tail, and becomes phosphorylated after T-cell stimulation with T-cell receptor (TCR) antibodies or with phorbol esters. [11][12][13] After its phosphorylation, ␤2 binds to 14-3-3 proteins both in vitro and in cells. 4 Blocking of this interaction with a ␤2 Thr758 to Ala mutation, or by expression of constructs that bind to 14-3-3 proteins and block their interactions with target proteins, leads to abrogation of actin cytoskeleton rearrangements, cell spreading, and adhesion to ICAM ligands. 4 ␤2-Thr758 phosphorylation leads to the activation of the actin cytoskeleton modulators, Rac1/Cdc42, in cells. 13 The region in the ␤2 cytoplasmic tail that binds 14-3-3 proteins has been reported to interact with filamin in other integrins, 14 and for the strong filamin-binder ␤7 integrin, phosphorylation mimicking substitutions of 3 threonine residues (TTT) reduces filamin affinity. 3 Fi...

Rosetting is a virulent Plasmodium falciparum phenomenon associated with severe malaria. Here we demonstrate that P. falciparum-encoded repetitive interspersed families of polypeptides (RIFINs) are expressed on the surface of infected red blood cells (iRBCs), where they bind to RBCs--preferentially of blood group A--to form large rosettes and mediate microvascular binding of iRBCs. We suggest that RIFINs have a fundamental role in the development of severe malaria and thereby contribute to the varying global distribution of ABO blood groups in the human population.