Hui-Chin Peng

Disintegrin causes proteolysis of β-catenin and apoptosis of endothelial cells: Involvement of cell—cell and cell—ECM interactions in regulating cell viability

Abstract Disintegrins, the snake venom-derived arginine-glycine-aspartic acid-containing peptides, have been demonstrated to inhibit angiogenesis through induction of endothelial cell apoptosis. However, it is not clear how a disintegrin causes endothelial apoptosis. In this study, we elucidated the action mechanism of disintegrin in causing endothelial apoptosis by using rhodostomin as a tool. We showed that cell detachment was observed at the early stage of rhodostomin treatment. It was initiated through the blockade by integrin ανβ3 and was accelerated by a mechanical stretch from neighboring cells. Both rhodostomin and poly(HEME) induced a higher percentage of cells at G2-M phase, the cleavage of β-catenin and poly(ADP-ribose) polymerase during apoptosis, indicating that cell detachment is a prerequisite for rhodostomin-induced apoptosis. Moreover, pp125FAK phosphorylation and actin cytoskeleton were affected upon rhodostomin treatment. The activation of caspase-3 but not that of caspase-9 was detected after rhodostomin treatment. In addition, general caspase inhibitors inhibited the cleavage of β-catenin and poly(ADP-ribose) polymerase, and DNA fragmentation, whereas they did not prevent cell shape change or detachment. According to these results, we concluded that disintegrin-induced endothelial apoptosis is a complex process, not merely caused by a blockade of endothelial integrin ανβ3 but also by an accompanied shape change and mechanical stretches among cells.

Triflavin, an Arg-Gly-Asp-Containing Peptide, Inhibits the Adhesion of Tumor Cells to Matrix Proteins via Binding to Multiple Integrin Receptors Expressed on Human Hepatoma Cells

Abstract Integrins are a superfamily of cell surface glycoproteins that promote cellular adhesion. The interaction of integrins with extracellular matrices such as fibronectin and vitronectin has been shown to be mediated through an arginine-glycine-aspartic acid (RGD) sequence within adhesive proteins. Triflavin, a 7.5-kDa cysteine-rich polypeptide purified from Trimeresurus flavoviridis snake venom, belongs to a family of RGD-containing peptides, termed disintegrins. Disintegrins have been isolated from the venom of various vipers and have been shown to be potent inhibitors of platelet aggregation. In this study, we found that human hepatoma cell adhesion to immobilized matrix proteins (i.e, fibronectin, collagen, laminin, and vitronectin) was differentially affected by various anti-integrin monoclonal antibodies (mAbs) (i.e., α3β1, α5β1, α6β1, and αvβ3 as well as by the peptide GRGDS. Indirect flow cytometric analysis of hepatoma cells with anti-integrin mAbs demonstrated that α6β1 was uniformly expressed at a high density, while α3β1 and α5β1 were moderately expressed and αvβ3 was expressed in small amounts on hepatoma cells, consistent with the results obtained from immunofluorescence microscopic analysis. When immobilized on plastic wells, triflavin promoted hepatoma cell attachment; this cell attachment was inhibited by either GRGDS or mAbs against integrins (α3β1, α5β1 and αvβ3). In addition, the binding of FITC-conjugated triflavin to hepatoma cells was inhibited by GRGDS, anti-α3β1, anti-α5β1, and anti-αvβ3 mAbs. Among these mAbs, anti-α5β1 exerted the most pronounced inhibitory effect (>70%) on the binding of triflavin to hepatoma cells. Taken together, these results suggest that triflavin binds via its RGD sequence to multiple integrin receptors (i.e., α5β1, α3β1, and αvβ3) expressed on the surface of hepatoma cells, resulting in inhibition of hepatoma cell adhesion to extracellular matrices (i.e., fibronectin and vitronectin).

Pharmacological characterization and antithrombotic effect of agkistin, a platelet glycoprotein Ib antagonist

Agkistin, purified from the snake venom of Formosan Agkistrodon acutus, belongs to the family of C‐type lectin GPIb binding proteins. It is a heterodimeric molecule, consisting of α‐ (16.5 kDa) and β‐ (15.5 kDa) subunits with a molecular mass of 32,512 Daltons examined by SDS – PAGE and mass spectrometry. In vitro, agkistin concentration‐dependently inhibited ristocetin‐induced human platelet agglutination and aggregation in the presence of vWF. It also inhibited TXA2 formation and prolonged the latent period in triggering aggregation by a low concentration of thrombin (0.03 u ml−1). 125I‐agkistin specifically bound to unactivated human platelets in a saturable manner with a KD value of 223±10.6 nM. This binding reaction was rapid and reversible. Monoclonal antibodies, AP1 and 6D1 raised against platelet GPIb, almost completely blocked 125I‐agkistin binding to platelets. However, monoclonal antibody 7E3 raised against GPIIb/IIIa complex, trigramin, a GPIIb/IIIa antagonist, ADP and EDTA did not affect 125I‐agkistin binding reaction. Agkistin (250 μg kg−1) significantly prolonged the bleeding time and induced transient thrombocytopenia of mice when given intravenously. Furthermore, it markedly inhibited platelet plug formation in irradiated mesenteric venules of fluorescein‐treated mice in vivo. In conclusion, agkistin inhibits ristocetin induced platelet aggregation mainly through its specific binding to platelet GPIb, thereby blocking the interaction between GPIb and vWF. In addition, agkistin exhibits antithrombotic activity in vivo.

The integrin α2β1 agonist, aggretin, promotes proliferation and migration of VSMC through NF-kB translocation and PDGF production.

Background and purpose:  During the development of atherosclerotic plaques, vascular smooth muscle cells (VSMCs) migrate from the media to the intima through the basement membrane and interstitial collagenous matrix, and proliferate to form neointima. Here, we investigate the mechanism of VSMC migration and proliferation caused by aggretin, a snake venom integrin α2β1 agonist.

A new short chain RGD-containing disintegrin, accutin, inhibits the common pathway of human platelet aggregation.

A new short-chain disintegrin, accutin, was purified from the Formosan Agkistrodon acutus venom by using of gel filtration, ion exchanger and reverse phase HPLC. The homogeneous protein is a 47-residue polypeptide with a molecular mass of 5241 Da containing an Arg-Gly-Asp sequence and seven cysteinyl residues at positions highly homologous to other disintegrins. Accutin dose-dependently inhibited human platelet aggregation stimulated by ADP, collagen, thrombin or the thromboxane analogue U46619 in platelet suspension with IC50 values of 66-267 nM. It was also active in inhibiting platelet aggregation of platelet-rich plasma. However, accutin apparently did not affect the shape change caused by these agonists. Accutin also inhibited fibrinogen-induced aggregation of human elastase-treated platelets in a dose-dependent manner. Furthermore, accutin dose-dependently inhibited the binding reaction of fluorescein isothiocyanate (FITC)-conjugated arietin, a member of the disintegrin family, to human platelets. In addition, the binding of FITC-conjugated accutin to platelets was almost completely blocked by a monoclonal antibody, 7E3, raised against the platelet glycoprotein IIb/IIIa complex. On the other hand, accutin as well as other disintegrins, rhodostomin and arietin, exhibited an inhibitory effect on 7E3 binding toward platelets and endothelial cells in a dose-dependent manner. It is concluded that accutin, a new platelet aggregation inhibitor belonging to the short-chain disintegrin family, acts specifically on a binding epitope of GPIIb/IIIa overlapping with that of 7E3, leading to the blockade of fibrinogen binding to its receptor.

Characterization of integrin expression and regulation on SW-480 human colon adenocarcinoma cells and the effect of rhodostomin on basal and upregulated tumor cell adhesion

Integrins are a superfamily of cell surface glycoproteins that mediate cell-extracellular matrix (ECM) and cell-cell adhesion. Immunofluorescence microscopy and flow cytometric analysis using anti-integrin mAbs as the primary binding ligands demonstrated that the platelet integrin receptor alpha IIb beta 3, as well as alpha v beta 3, alpha 5 beta 1 and alpha 6 beta 1, are present on the surface of SW-480 human colon adenocarcinoma cells. Monoclonal antibodies (mAbs) against alpha IIb beta 3 and alpha 5 beta 1 inhibited unstimulated basal adhesion to fibronectin by approximately 30% and 40%, respectively. The surface immunoreactivity of tumor cells for alpha IIb beta 3 was enhanced by pretreatment (5 min) with a phorbol ester (12-O-tetradecanoylphorbol-13-acetate (TPA)) or a lipoxygenase metabolite of arachidonic acid, 12-hydroxyeicosatetraenoic acid (12-HETE) in a dose- and time-dependent manner. SW-480 cells possess a large intracellular pool of alpha IIb beta 3, from which the receptor complex translocates to the cell surface following pretreatment with TPA or 12(S)-HETE. This pretreatment enhances adhesion to fibronectin, which is mediated exclusively by alpha IIb beta 3 integrins. Staurosporine was found to block alpha IIb beta 3 up-regulation and enhanced-adhesion. TPA and 12(S)-HETE also facilitated the redistribution of alpha IIb beta 3 during the enhanced-spreading process. Rhodostomin, an Arg-Gly-Asp- (RGD) containing antiplatelet snake venom peptide, was about 400-times more potent than RGDS at inhibiting control, TPA- or 12(S)-HETE-enhanced adhesion of SW-480 cells to fibronectin. The binding of mAbs against alpha IIb beta 3, alpha v beta 3 and alpha 5 beta 1 was inhibited by pretreatment with rhodostomin, suggesting that rhodostomin binds via its RGD sequence to multiple integrin receptors (i.e., alpha IIb beta 3, alpha v beta 3, alpha 5 beta 1) expressed on the SW-480 cell surface, inhibiting cell adhesion to ECM.

Edema-producing proteins isolated from Trimeresurus mucrosquamatus snake venom.

Seven edema-producing fractions were isolated from Trimeresurus mucrosquamatus snake venom by CM-Sephadex C-50 chromatography and further purified by gel filtration on a Sephadex G-75 column. They were homogeneous as judged by electrophoresis on polyacrylamide gels. The mol. wts were estimated to be 26,900 (Fr. IV-2), 21,500 (Fr. X-2), 23,000 (Fr. XII-2), 21,800 (Fr. XIII-2), 24,600 (Fr. XIV-2), 80,000 (Fr. XVIII-1) and 22,500 (Fr. XXII-2). Fraction IV-2 had weak esterase activity, fractions X-2, XII-2, XIII-2 and XIV-2 possessed proteolytic activity toward casein and fibrinogen, while fractions XVIII-1 and XXII-2 possessed phospholipase A2 activity. Fractions with phospholipase A2 activity had greater edema-producing activity than those with protease and/or esterase activity. It is concluded that the edema caused by T. mucrosquamatus venom may be due to phospholipases A2, proteases and esterases.

Inhibition of neuropathic pain by a potent disintegrin—triflavin

Injury to peripheral nerves may result in severe and intractable neuropathic pain. Many efforts have been focused on the elucidation of the mechanisms of neuropathic pain. It was found here that integrin plays an important role in the induction of neuropathic pain and treatment of disintegrin is able to attenuate neuropathic pain. The rats were induced hyperalgesia by tightly ligating the L5 spinal nerve and cut just distal to the ligature on one side. Mechanical and thermal stimuli were applied in the middle dermatome of the hind paw. Epidural administration of triflavin (TFV), an arginine-glycine-aspartic acid (RGD) containing disintegrin, inhibited hyperalgesia induced by either mechanical or thermal stimulation. Immunohistochemistry showed that the sprouting of sympathetic nerves into DRG by neuropathic surgery was markedly inhibited by TFV. Beta 1 integrin mRNA of L5 DRG increased immediately 1 day after tight ligation and cut of L5 spinal nerve. However, beta 1 integrin mRNA in uninjured L4 DRG increased later on Day 3 after surgery. On the other hand, alpha-CGRP precursor mRNA decreased in ipsilateral L5 DRG but increased in L4 DRG after neuropathic surgery. Immunohistochemistry shows that beta 3 integrins of L5 as well as L4 increased in response to neuropathic surgery and administration of triflavin antagonized the increasing action. These results suggest that there is interaction between injured and uninjured neurons and the induction of neuropathic pain is related to neuronal sprouting. Disintegrin is able to inhibit neuronal sprouting and the induction of hyperalgesia induced by peripheral nerve injury and may thus be a new category of drugs to be developed for the treatment of neuropathic pain.

Improvements in endotoxemic syndromes using a disintegrin, rhodostomin, through integrin αvβ3‐dependent pathway

Summary.  Background and objectives: Septic shock is a major cause of morbidity and mortality in intensive care units, but there is still no effective therapy for the patients. We evaluated the effects of rhodostomin (Rn), an Arg‐Gly‐Asp‐containing snake venom disintegrin, on lipopolysaccharide (LPS)‐activated phagocytes in vitro and LPS‐induced endotoxemia in vivo. Methods and results: Rn inhibited adhesion, migration, cytokine production and mitogen‐activated protein kinase (MAPK) activation of macrophage induced by LPS. Flow cytometric analysis revealed that Rn specifically blocked anti‐αv mAb binding to RAW264.7. Besides inhibiting MAPK activation of THP‐1, Rn bound to LPS‐activated THP‐1 and specifically blocked anti‐αvβ3 mAb binding to THP‐1. Binding assays proved that integrin αvβ3 was the binding site for rhodostomin on phagocytes. Rn reversed the enhancement of fibronectin and vitronectin on LPS‐induced monocyte adhesion and cytokine release. Transfection of integrin αv siRNA also inhibited LPS‐induced activation of monocyte, and Rn exerted no further inhibitory effect. Furthermore, Rn significantly decreased the production of tumor necrosis factor‐α (TNF‐a), interleukin (IL)‐6, ‐1β and ‐10 and attenuated cardiovascular dysfunction, including blood pressure and heart pulse, and thrombocytopenia in LPS‐induced endotoxemic mice. Rn also protected against tissue inflammation as evidenced by histological examination. Conclusions: Rn may interact with αvβ3 integrin of monocytes/macrophages leading to interfere with the activation of phagocytes triggered by LPS. These results suggest that the protective function of Rn in LPS‐induced endotoxemia may be attributed to its anti‐inflammation activities in vivo.

Rhodostomin, a Disintegrin, Inhibits Adhesion of Neutrophils to Fibrinogen and Attenuates Superoxide Production

Disintegrins are a group of Arg (or Lys)-Gly-Asp-containing snake venom proteins which inhibit platelet aggregation via the blockade of αIIbβ3 integrin. Here, we studied the effect of rhodostomin, a disintegrin purified from the venom of Calloselasma rhodostoma, on the functions of neutrophils. By flow cytometric analysis of whole blood, we found that rhodostomin interacted with leukocytes of the myeloid and monocytic lineage as well as with platelets. The binding of rhodostomin to neutrophils could reach saturation in a dose-dependent manner, and its binding was increased in neutrophils stimulated with phorbol 12-myristate 13-acetate (PMA) and N-formyl-Met-Leu-Phe. EDTA did not inhibit the binding of rhodostomin. In addition, bound rhodostomin was not internalized. Soluble fibrinogen, a natural ligand of Mac-1 (CD11b/CD18, αMβ2), and the peptide, GRGDS, inhibited the binding of rhodostomin to PMA-activated neutrophils, while 7E3, a monoclonal antibody (mAb) raised against β3 integrin, or mAbs raised against αM and β2 integrin did not. Rhodostomin blocked the Mac-1-dependent adhesion of neutrophils to immobilized fibrinogen, in parallel with decreasing the production of superoxide from adherent neutrophils. Taken together, our results indicate that rhodostomin binds to activated neutrophils in an RGD-dependent manner, blocks the adhesion of activated neutrophils to fibrinogen and attenuates superoxide production, suggesting that rhodostomin may have anti-inflammatory properties.