Jialiang Hu

Matrix metalloproteinase inhibitors as therapy for inflammatory and vascular diseases

Matrix metalloproteinases (MMPs) have outgrown the field of extracellular-matrix biology and have progressed towards being important regulatory molecules in cancer and inflammation. This rise in status was accompanied by the development of various classes of inhibitors. Although clinical trials with synthetic inhibitors for the treatment of cancer were disappointing, recent data indicate that the use of selective inhibitors might lead to new therapies for acute and chronic inflammatory and vascular diseases. In this Review, we compare the major classes of MMP inhibitors and advocate that future drug discovery should be based on crucial insights into the differential roles of specific MMPs in pathophysiology obtained with animal models, including knockout studies.

Two Distinct Jacalin-Related Lectins with a Different Specificity and Subcellular Location Are Major Vegetative Storage Proteins in the Bark of the Black Mulberry Tree

Using a combination of protein isolation/characterization and molecular cloning, we have demonstrated that the bark of the black mulberry tree (Morus nigra) accumulates large quantities of a galactose-specific (MornigaG) and a mannose (Man)-specific (MornigaM) jacalin-related lectin. MornigaG resembles jacalin with respect to its molecular structure, specificity, and co- and posttranslational processing indicating that it follows the secretory pathway and eventually accumulates in the vacuolar compartment. In contrast, MornigaM represents a novel type of highly active Man-specific jacalin-related lectin that is synthesized without signal peptide or other vacuolar targeting sequences, and accordingly, accumulates in the cytoplasm. The isolation and cloning, and immunocytochemical localization of MornigaG and MornigaM not only demonstrates that jacalin-related lectins act as vegetative storage proteins in bark, but also allows a detailed comparison of a vacuolar galactose-specific and a cytoplasmic Man-specific jacalin-related lectin from a single species. Moreover, the identification of MornigaM provides the first evidence, to our knowledge, that bark cells accumulate large quantities of a cytoplasmic storage protein. In addition, due to its high activity, abundance, and ease of preparation, MornigaM is of great potential value for practical applications as a tool and bioactive protein in biological and biomedical research.

Interleukin-17 regulates chemokine and gelatinase B expression in fibroblasts to recruit both neutrophils and monocytes

Interleukin 17 (IL-17) is a proinflammatory cytokine, produced only by activated lymphocytes, but with a broad cellular host range. The effects of IL-17 on fibroblasts were investigated by analysis of the induction of chemokine and matrix metalloprotease (MMP) mRNA levels by RT-PCR. IL-17 stimulated CC chemokine (monocyte chemotactic protein-1; MCP-1/JE) and CXC (KC, MIP-2) chemokine and TIMP-1 mRNA expression in fibroblastoid L929 cells. In normal mouse embryonic fibroblasts (MEF) this induction profile by IL-17 was extended with the mRNAs encoding the chemokine granulocyte chemotactic protein-2 (GCP-2) and the proteases MMP-3, MMP-9 and MMP-13. The MMP-9 and GCP-2 induction by IL-17 in MEF, and the absence of induction in L929 cells, were corroborated by gelatin zymography and ELISA, respectively. The induction of MCP-1/CCL2 by IL-17 was confirmed in human diploid fibroblasts. We conclude that IL-17 regulates differentially chemokine and MMP expression by normal and transformed fibroblasts and is indirectly capable of attracting both monocytes and neutrophils to the inflammatory focus.

Inhibition of lethal endotoxin shock with an L-pyridylalanine containing metalloproteinase inhibitor selected by high-throughput screening of a new peptide library.

Gelatinase B/MMP-9 fulfills crucial regulator or effector functions in disease states and may be pharmacologically targeted by specific inhibitors. The characteristics of cleavages of a natural gelatinase B substrate were simulated and amino acids with zinc-ion chelating side-groups were employed to design a library of peptide-based inhibitors. Here, we extend previous findings of combinatorial chemical synthesis and subsequent library deconvolution with a recently established high-throughput technology. This enabled us to study MMP inhibitors with two zinc-binding groups and to identify a new L-pyridylalanine-containing gelatinase B inhibitor. The peptide analog was found to inhibit, almost to the same degree, the neutrophil enzymes collagenase 2/ MMP-8 and MMP-9 and the monocytic tumor necrosis factor-alpha (TNF-alpha) converting enzyme (TACE/ADAM-17) in vitro and to protect mice against lethal endotoxinemia in vivo. These data illustrate the usefulness of the screening platform for protective inhibitor discovery and complement recent insights in the pathogenesis and treatment of shock syndromes.

Targeting matrix metalloproteinases in acute inflammatory shock syndromes

The integrity of the vascular wall and its intrinsic basement membrane structures ensure that plasma and the corpuscular elements of the blood remain confined to the intravascular milieu and can enter into the extravascular compartment in a well controlled fashion in cases of tissue infection or inflammation. However, sometimes inflammatory stimuli act on blood leukocytes and on endothelial cells from within the blood vessels and in an overwhelming way, leading to inflammatory shock syndromes. These severe conditions with high mortality rates are characterized by intravascular neutrophil degranulation, permeability changes of endothelia and disintegration of basement membranes and lead to almost uncontrollable edema, coagulation changes and multi-organ failure. Matrix metalloproteinases (MMPs) have been functionally linked with septic and endotoxin shock, with cytokine release syndromes and with acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Here we review a number of association studies, compare inflammatory shock data from gene knockout studies in mice and provide some insights from recent investigations with inhibitors of MMPs. This evaluation strengthens the expectation that MMP inhibitors, in particular those blocking neutrophil proteases, may become useful in the early phase of acute inflammatory shock syndromes.

Inhibition of gelatinase B/MMP-9 does not attenuate colitis in murine models of inflammatory bowel disease

One third of patients with inflammatory bowel disease (IBD) inadequately respond to anti-TNF treatment and preclinical data suggest that matrix metalloproteinase-9 (MMP-9) is a novel therapeutic target. Here we show that IBD clinical and histopathological parameters found in wild type mice challenged with three different models of colitis, acute and chronic dextran sodium sulphate (DSS), and acute 2,4,6-trinitrobenzenesulfonic acid-induced colitis are not attenuated in MMP-9 knockout mice. We find similar colonic gene expression profiles in wild type and MMP-9 knockout mice in control and acute DSS conditions with the exception of eleven genes involved in antimicrobial response during colitis. Parameters of chronic colitis are similar in wild type and MMP-9 knockout mice. Pharmacological inhibition of MMP-9 with bio-active peptides does not improve DSS colitis. We suggest that MMP-9 upregulation is a consequence rather than a cause of intestinal inflammation and we question whether MMP-9 represents a disease target in IBD.

Reverse degradomics, monitoring of proteolytic trimming by multi-CE and confocal detection of fluorescent substrates and reaction products.

A platform for profiling of multiple proteolytic activities acting on one specific substrate, based on the use of a 96‐channel capillary DNA sequencer with CE‐LIF of labeled substrate peptides and reaction products is introduced. The approach consists of synthesis of a substrate peptide of interest, fluorescent labeling of the substrate, either aminoterminally by chemical coupling, or carboxyterminally by transglutaminase reaction, proteolysis by a biological mixture of proteases in the absence or presence of protease inhibitors, multi‐channel analysis of substrate and reaction products, and data collection and processing. Intact substrate and reaction products, even when varying by only one amino acid, can be relatively semi‐quantified in a high‐throughput manner, yielding information on proteases acting in complex biological mixtures and without prepurification. Monitoring, classification and inhibition of multiple proteolytic activities are demonstrated on a model substrate, the aminoterminus of the mouse granulocyte chemotactic protein‐2. In view of extensive processing of chemokines into various natural forms with different specific biological activities, and of the fragmentary knowledge of processing proteases, examples of processing by neutrophil degranulate, tumor cell culture fluids and plasma are provided. An example of selection and comparison of inhibitory mAbs illustrates that the platform is suitable for inhibitor screening. Whereas classical degradomics technologies analyze the substrate repertoire of one specific protease, here the complementary concept, namely the study of all proteases acting, in a biological context, on one specific substrate, is developed and tuned to identify key proteases and protease inhibitors for the processing of any biological substrate of interest.

Generation of antitumor peptides by connection of matrix metalloproteinase-9 peptide inhibitor to an endostatin fragment.

Previous applications of matrix metalloproteinase (MMP) inhibitors in cancer treatment have resulted in disappointing outcomes. Therefore, it is necessary to develop more active or better targeted MMP inhibitors. In this study, Inhibitor2, a heptapeptide MMP inhibitor, was connected to the N-terminus or C-terminus of ES-2, an 11-amino-acid antiangiogenic peptide, and two designed peptides, P2 and P4, were generated. P2 inhibited MMP-2, MMP-8, MMP-9, and tumor necrosis factor-&agr; converting enzyme (TACE) activity with IC50 values of 1.40, 0.35, 1.36, and 1.95 &mgr;mol/l, whereas those for P4 were 19, 20, 18, and 18 &mgr;mol/l. P2 showed a higher affinity with integrin &agr;5&bgr;1 in a human umbilical vein endothelial cell (HUVEC) adhesion assay than P4. In the HUVEC migration assay, P2 showed a better inhibitory effect on HUVEC migration than P4 and Inhibitor2 and the inhibitory ratio at 2 &mgr;g/ml was 77%. In a chorioallantoic membrane assay, at a concentration of 3.28 &mgr;g/ml, P2 and P4 showed 69.8 and 56.8% inhibition of formation of new blood vessels on the embryo membrane. Furthermore, P2 significantly inhibited B16F10 growth in a syngeneic mouse model with an inhibition ratio of 57.92% by tumor weight at a dose of 20 mg/kg/day, whereas P4 and Inhibitor2 exerted no such in-vivo effect. The antiangiogenic effect of P2 was confirmed by CD31 staining of the tumor tissue sections. The Inhibitor2 part of P2 may function both as an MMP inhibitor and as a targeting motif. These studies represented an example of how to better apply the potent and peptidomimetic MMP inhibitors in cancer treatment.

RGD-modified endostatin fragments showed an antitumor effect through antiangiogenesis.

EDSM, an endostatin-derived synthetic polypeptide, contains the amino acids 6–48 of endostatin from its N-terminus, which could inhibit human umbilical vein endothelial cell (HUVEC) migration and tumor growth. To increase the targeted delivery of EDSM to tumors and further enhance its antiangiogenic activity, the RGD sequence (Arg–Gly–Asp) was introduced into EDSM and two peptides were obtained: EDSM-X with RGD on the N-terminus of EDSM and EDSM-Y with RGD on the C-terminus. Both modified peptides showed a significant antiangiogenic activity in the HUVEC migration assay, the HUVEC tube formation assay, and the murine aortic ring formation assay in vitro. In agreement with the in-vitro data, EDSM-X and EDSM-Y also showed a significant antitumor activity in vivo. From the cell adhesion assay, it was confirmed that the molecular target of the modified peptides on HUVECs was integrin &agr;v&bgr;3, rather than integrin &agr;5&bgr;1. Furthermore, EDSM-Y exhibited more potent antiangiogenic activity and antitumor activity than EDSM-X in vitro and in vivo, and this phenomenon was attributed to the difference in the two modified peptides in their three-dimensional structure modeling and their molecular dockings with integrin &agr;v&bgr;3.

Inhibition of Neutrophil Collagenase/MMP-8 and Gelatinase B/MMP-9 and Protection against Endotoxin Shock

Endotoxin shock is a life-threatening disorder, associated with the rapid release of neutrophil enzymes, including neutrophil collagenase/matrix metalloproteinase-8 (MMP-8) and gelatinase B/matrix metalloproteinase-9 (MMP-9). After activation, these enzymes cleave extracellular matrix components and cytokines and thus may contribute to shock syndrome development. MMP inhibitors have been suggested as immunotherapy of endotoxin shock. However, little is known about the therapeutic time window of MMP inhibition. Here, a sublethal endotoxin shock mouse model was used to evaluate the effect of an MMP inhibiting peptide (P2) after intravenous or intraperitoneal injection and to study the time window between LPS and inhibitor injections. With the use of a specific ELISA the plasma P2 concentrations were monitored. Whereas we corroborated the treatment strategy of MMP targeting in endotoxin shock with a new inhibitor, we also demonstrated that the time window, within which effective MMP inhibition increased the survival rates, is rather limited.