The antitumor efficacy of monomeric disintegrin obtustatin in S-180 sarcoma mouse model.

Obtustatin, isolated from the Levantine Viper snake venom (Macrovipera lebetina obtusa -MLO), is the shortest known monomeric disintegrin shown to specifically inhibit the binding of the α1ß1 integrin to collagen IV. Its oncostatic effect is due to the inhibition of angiogenesis, likely through α1ß1 integrin inhibition in endothelial cells. To explore the therapeutic potential of obtustatin, we studied its effect in S-180 sarcoma-bearing mice model in vivo as well as in human dermal microvascular endothelial cells (HMVEC-D) in vitro, and tested anti-angiogenic activity in vivo using the chick embryo chorioallantoic membrane assay (CAM assay). Our in vivo results show that obtustatin inhibits tumour growth by 33%. The expression of vascular endothelial growth factor (VEGF) increased after treatment with obtustatin, but the level of expression of caspase 8 did not change. In addition, our results demonstrate that obtustatin inhibits FGF2-induced angiogenesis in the CAM assay. Our in vitro results show that obtustatin does not exhibit cytotoxic activity in HMVEC-D cells in comparison to in vivo results. Thus, our findings disclose that obtustatin might be a potential candidate for the treatment of sarcoma in vivo with low toxicity.

Effects of VLA-1 Blockade on Experimental Inflammation in Mice.

VLA-1 (very late antigen-1) is implicated in recruitment, retention and activation of leukocytes and its blockade has been referred as a potential target of new drug discovery to address unmet medical needs in inflammatory disease area. In the present study, we investigate the effects of an anti-murine CD49a (integrin α subunit of VLA-1) monoclonal antibody (Ha31/8) on various experimental models of inflammatory diseases in mice. Pretreatment with Ha31/8 at an intraperitoneal dose of 250 µg significantly (P<0.01) reduced arthritic symptoms and joint tissue damage in mice with type II collagen-induced arthritis. In addition, Ha31/8 at an intraperitoneal dose of 100 µg significantly (P<0.01) inhibited airway inflammatory cell infiltration induced by repeated exposure to cigarette smoke. In contrast, Ha31/8 failed to inhibit oxazolone-induced chronic dermatitis and OVA-induced airway hyperresponsiveness at an intraperitoneal dose of 100 µg. These results show that VLA-1 is involved, at least partly, in the pathogenesis of type II collagen-induced arthritis and cigarette smoke-induced airway inflammatory cell infiltration in mice, indicating the therapeutic potential of VLA-1 blockade against rheumatoid arthritis and chronic occlusive pulmonary disease.

Alpha1beta1 and integrin-linked kinase interact and modulate angiotensin II effects in vascular smooth muscle cells.

The effects of angiotensin II (Ang II) on vascular smooth muscle cells (VSMC) are modulated by reactive oxygen species (ROS) and also involve integrin engagement. However, the potential link between alpha1beta1 integrin signaling with NOX system and their combined contribution to Ang II effects on VSMC have not been investigated. We aimed to elucidate the moslecular mechanisms underlying the activation of these two pathways in Ang II effects on VSMC. Ang II-induced VSMC migration (2-fold increase) and proliferation (2.5-fold increase) is modulated by alpha1beta1 integrin, being inhibited by obtustatin, a specific alpha1beta1 integrin blocker. Ang II also stimulates ROS production in VSMC (140%) that is NOX1 dependent, being completely inhibited in NOX1 silenced cells. The ROS production develops in two peaks, and the second peak is maintained by NOX2 activation. Apocynin and obtustatin inhibit the NOX2-associated second peak, but not the first peak of ROS production, which is related to NOX1 activation. Corroborating the involvement of alpha1beta1 integrin, the pretreatment of VSMC with obtustatin impaired Ang II-induced FAK phosphorylation, AKT activation, p21 degradation and the increase of ILK expression. Silencing of ILK blocked cell migration, AKT phosphorylation and the second peak of ROS, but partially inhibits (70%) VSMC proliferation induced by Ang II. The data demonstrate a novel role for NOX2 in Ang II effects on VSMC, and suggest alpha1beta1 integrin and ILK as target molecules to the development of more effective therapeutic interventions in cardiovascular diseases.

Decoding Cytoskeleton-Anchored and Non-Anchored Receptors from Single-Cell Adhesion Force Data.

Complementary to parameters established for cell-adhesion force curve analysis, we evaluated the slope before a force step together with the distance from the surface at which the step occurs and visualized the result in a two-dimensional density plot. This new tool allows detachment steps of long membrane tethers to be distinguished from shorter jumplike force steps, which are typical for cytoskeleton-anchored bonds. A prostate cancer cell line (PC3) immobilized on an atomic-force-microscopy sensor interacted with three different substrates: collagen-I (Col-I), bovine serum albumin, and a monolayer of bone marrow-derived stem cells (SCP1). To address PC3 cells' predominant Col-I binding molecules, an antibody-blocking ß1-integrin was used. Untreated PC3 cells on Col-I or SCP1 cells, which express Col-I, predominantly showed jumps in their force curves, while PC3 cells on bovine-serum-albumin- and antibody-treated PC3 cells showed long membrane tethers. The probability density plots thus revealed that ß1-integrin-specific interactions are predominately anchored to the cytoskeleton, while the nonspecific interactions are mainly membrane-anchored. Experiments with latrunculin-A-treated PC3 cells corroborated these observations. The plots thus reveal details of the anchoring of bonds to the cell and provide a better understanding of receptor-ligand interactions.

Blocking of α1ß1 and α2ß1 adhesion molecules inhibits eosinophil migration through human lung microvascular endothelial cell monolayer.

INTRODUCTION: In cell trafficking to the airways in asthma, among integrins the most important are those containing α4 and ß2 subunits. We have previously shown that also blocking of collagen receptors, α1ß1 and α2ß1 integrins, inhibits transmigration of eosinophils of asthmatic subjects through a monolayer of skin microvascular endothelial cells seeded on collagen IV coated inserts. However, it was not clear whether this observation was limited to asthma or depended on the type of microvascular cell and collagen IV used as a base. MATERIALS & METHODS: In the current study we performed a transmigration assay using human lung microvascular endothelial cells seeded directly on a plastic surface as a base and blood cells isolated from 12 representatives of each of two groups, asthmatics and healthy donors, by gradient centrifugation, followed by immunomagnetic negative separation of eosinophils. Isolated eosinophils and peripheral blood mononuclear cells (PBMC) were inhibited by snake venom-derived integrin antagonists including viperistatin and VP12, as inhibitors of α1ß1 and α2ß1 integrin, respectively, and VLO5 and VLO4, as inhibitors of α4ß1 and α5ß1 integrin, respectively. RESULTS: All snake venom-derived anti-adhesive proteins were effective in inhibiting eosinophil transmigration, whilst only VLO5 and VLO4 reduced PBMC mobility in this assay. This observation was similar in both groups of subjects studied. DISCUSSION: α1ß1 and α2ß1 integrins could be involved in transmigration of eosinophil to the inflammatory site. Migratory inhibition was observed in asthma subjects as well as in healthy donors, and did not depend on origin of endothelial cells or the extracellular matrix component used as a base.

Vimocin and vidapin, cyclic KTS peptides, are dual antagonists of α1ß1/α2ß1 integrins with antiangiogenic activity.

Obtustatin and viperistatin, members of the disintegrin protein family, served as lead compounds for the synthesis of linear and cyclic peptides containing the KTS binding motif. The most active linear peptide, a viperistatin analog, indicated the importance of Cys(19) and Cys(29), as well as the presence of Arg at position 24 for their biologic activity, and was used as the basic sequence for the synthesis of cyclic peptides. Vimocin (compound 6) and vidapin (compound 10) showed a high potency (IC50 = 0.17 nM) and intermediate efficacy (20 and 40%) in inhibition of adhesion of α1/α2 integrin overexpressor cells to respective collagens. Vimocin was more active in inhibition of the wound healing (53%) and corneal micropocket (17%) vascularization, whereas vidapin was more potent in inhibition of migration in the Matrigel tube formation assay (90%). Both compounds similarly inhibited proliferation (50-90%) of endothelial cells, and angiogenesis induced by vascular endothelial growth factor (80%) and glioma (55%) in the chorioallantoic membrane assay. These peptides were not toxic to endothelial cell cultures and caused no acute toxicity upon intravenous injection in mice, and were stable for 10-30 hours in human serum. The in vitro and in vivo potency of the peptides are consistent with conformational ensembles and "bioactive" space shared by obtustatin and viperistatin. These findings suggest that vimocin and vidapin can serve as dual α1ß1/α2ß1 integrin antagonists in antiangiogenesis and cancer therapy.

Inhibitory effects of recombinant RTS-jerdostatin on integrin α1ß1 function during adhesion, migration and proliferation of rat aortic smooth muscle cells and angiogenesis.

Jerdostatin, a short RTS-disintegrin cloned from venom gland mRNA of Protobothrops jerdonii, selectively blocks the adhesion of α1ß1 integrin to collagen IV. Integrin α1ß1 is highly expressed in smooth muscle cells (SMC) surrounding small blood vessels and vascular endothelial cells. Vascular SMC adhesion, migration and proliferation are important processes during normal vascular development. Using recombinant jerdostatin we have investigated the role of the α1ß1 integrin on the adhesion of vascular SMC to collagen IV, and the potential relevance of blocking this crucial component of focal adhesions as an anti-angiogenic strategy. Our results show that jerdostatin does not interact with canonical collagen-binding site on the isolated A-domain of the α1 integrin subunit. r-Jerdostatin inhibited the adhesion of RASMCs to immobilized CB3 fragment in a dose-dependent manner, triggering to round-up, retraction, and finally detachment of the cells. r-Jerdostatin did not affect the adhesion of human SMCs to CB3, presumably because the high expression of α2ß1 integrin compensated for α1ß1 integrin blockage by jerdostatin. r-Jerdostatin dose-dependently inhibited α1ß1 integrin-dependent HUVEC tube formation. However, VEGF-driven tube formation in the matrigel assay was only completely abolished when binding of integrin α2ß1 to collagen was also inhibited by the C-type lectin-like rhodocetin. As a whole, our work emphasizes the relevance of using specific inhibitors for dissecting the role of α1ß1 integrin in physiological and pathological conditions.

Collagen receptors α(1)ß(1) and α(2)ß(1) integrins are involved in transmigration of peripheral blood eosinophils, but not mononuclear cells through human microvascular endothelial cells monolayer.

UNLABELLED: Asthma development may be driven by T helper lymphocytes with eosinophils playing the role of major effector cells. Recruitment of the inflammatory cells from blood to the airways is mediated by adhesive molecules, e.g. selectins and integrins. The most important in cell trafficking are integrins containing α(4) and ß(2) subunits. We hypothesized that also collagen receptors: α(1)ß(1) and α(2)ß(1), may be involved in cell migration to the inflammatory site in asthma. The aim of the study was to determine whether the inhibition of α(1)ß(1) or α(2)ß(1) integrins, affects transmigration of eosinophils and peripheral blood mononuclear cells (PBMC) through human microvascular endothelial cells monolayer (HMVEC) seeded on collagen IV coated wells in moderate persistent atopic asthmatics. METHODS: PBMC from 9 asthmatics were separated by gradient centrifugation followed by negative magnetic separation of eosinophils. Snake venom derived anti-adhesive proteins: viperistatin and VP12 (potent and selective inhibitors of α(1)ß(1) and α(2)ß(1) integrins, respectively) as well as VLO4 (a non-selective inhibitor of α(4)ß(1), α(5)ß(1) and α(v)ß(3) - used as a positive control), were used for inhibition studies. All anti-adhesive proteins studied, inhibited eosinophils, but only VLO4 affected PBMC transmigration through HMVEC. In bronchial asthma both collagen receptors α(1)ß(1) and α(2)ß(1) are likely to be involved in eosinophil transmigration to the inflammatory site. The role of α(2)ß(1) on lymphocytes is probably different. As the α(2)ß(1) integrin has been described as a stimulator of collagen accumulation, it might be, at least in part, responsible for asthma airway remodelling.

Combined blockade of VEGFR-3 and VLA-1 markedly promotes high-risk corneal transplant survival.

PURPOSE. High-risk corneal transplantation refers to grafting performed on inflamed and highly vascularized host beds. It represents a clinical dilemma because the rejection rate can be as high as 90%, irrespective of current treatment modalities. This study was conducted to investigate whether combined blockade of VEGFR-3 (vascular endothelial growth factor receptor-3) and VLA-1 (very late antigen-1) promotes high-risk transplant survival and how it correlates with corneal lymphangiogenesis and hemangiogenesis before and after transplantation. METHODS. High-risk corneal transplantation was performed between normal C57BL/6 (donor) and inflamed BALB/c (recipient) mice. The recipients were randomized to receive intraperitoneal injections of VEGFR-3 and VLA-1-neutralizing antibodies or their controls twice a week for up to 8 weeks after transplantation. Corneal grafts were evaluated by ophthalmic slit-lamp biomicroscopy and analyzed by Kaplan-Meier survival curve. Additionally, whole-mount corneas before and after transplantation were examined by immunofluorescent microscopic assays, and the correlation between lymphatic or blood vessel distribution and transplant outcome was analyzed. RESULTS. The combined blockade markedly promotes 90% survival of high-risk transplants. This strategy specifically modified host beds by selective inhibition of lymphangiogenesis but not hemangiogenesis. A strong correlation was also identified between high-risk transplant rejection and severe lymphatic invasion reaching the donor-graft border. CONCLUSIONS. These novel findings not only provide a new and potentially powerful strategy to promote high-risk transplant survival, they also confirm a critical role of high-degree lymphangiogenesis in mediating high-risk transplant rejection. Results from this study may also shed new light on our understanding and management of other lymphatic- and immune-related diseases in general.

NMR structure and dynamics of recombinant wild type and mutated jerdostatin, a selective inhibitor of integrin α1ß1.

NMR analysis of four recombinant jerdostatin molecules was assessed to define the structural basis of two naturally occurring gain-of-function events: C-terminal dipeptide processing and mutation of the active residue K21 to arginine. Removal of the highly mobile and a bulky C-terminal dipeptide produced pronounced chemical shift changes in the sequentially unconnected but spatially nearby α(1)ß(1) inhibitory loop. Analysis of chemical shift divergence and (15)N backbone relaxation dynamics indicated differences in motions in the picosecond to nanosecond time scale, and the higher T(2) rate of S25, S26, and H27 of rJerK21 point to a slowdown in the microsecond to millisecond motions of these residues when compared with rJerR21. The evidence presented in this article converges on the hypothesis that dynamic differences between the α(1)ß(1) recognition loops of rJerR21 and rJerK21 may influence the thermodynamics of their receptor recognition and binding. A decrease in the µs-ms time scale may impair the binding affinity by reducing the rate of possible conformations that the rJerK21 can adopt in this time scale.

Recombinant expression in human cells of active integrin alpha 1 beta 1-blocking RTS-disintegrin jerdostatin.

Jerdostatin, an RTS short disintegrin cloned from Protobothrops jerdonii and recombinantly produced in Escherichia coli, is a potent and specific antagonist of the alpha(1)beta(1) integrin. Jerdostatin selectively blocked the adhesion of alpha(1)beta(1)-K562 cell to collagens I and IV in vitro and angiogenesis in vivo. Here we report the recombinant production of jerdostatin in a mammalian cell system, a prerequisite for developing a conditional transgenic mouse to investigate the effect of systemic expression of jerdostatin on tumor development. For proper export of jerdostatin, a secretion leader sequence was engineered at the protein's N-terminus. A FLAG epitope was also included at the N-terminus of the mature disintegrin to facilitate its isolation and characterization of recombinant jerdostatin (rJerd). This pRc-CMV/FLAG-rJerd construct was transiently expressed in HEK-293 cells and was efficiently secreted into the culture medium. rJerd bound to recombinant soluble alpha(1)beta(1) integrin in a saturable and cation-independent manner. Soluble rJerd also inhibited the binding of alpha(1)beta(1) integrin to the CB3 fragment of collagen IV in a dose-dependent manner (IC(50) 570 nM). Mammalian cell-expressed jerdostatin disrupted the adhesion of RuGli cells to collagen IV. Our results highlight pRc-CMV/FLAG-rJerd as a suitable construct for expressing soluble active alpha(1)beta(1)-blocking jerdostatin in a mammalian cell system.

A horsefly saliva antigen 5-like protein containing RTS motif is an angiogenesis inhibitor.

The Ag5 proteins are the most abundant and immunogenic proteins in the venom secretory ducts of stinging insects. An antigen 5-like protein (named tabRTS) composed of 221 amino acid residues was purified and characterized from the salivary glands of the horsefly, Tabanus yao (Diptera, Tabanidae). Its cDNA was cloned from the cDNA library of the horsefly's salivary gland. TabRTS containing the SCP domain (Sc7 family of extracellular protein domain) was found in insect antigen 5 proteins. More interestingly, there is an Arg-Thr-Ser (RTS) disintegrin motif at the C-terminus of tabRTS. The RTS motif is positioned in a loop bracketed by cysteine residues as those found in RTS-disintegrins of Crotalidae and Viperidae snake venoms, which act as angiogenesis inhibitors. Endothelial Cell Tube formation assay in vitro and chicken chorioallantoic membrane (CAM) angiogenesis assay in vivo were performed as to investigate the effect of tabRTS on angiogenesis. It was found that tabRTS could significantly inhibit angiogenesis in vitro and in vivo. Anti-alpha(1)beta(1) monoclonal antibody could dose-dependently inhibit the anti-angiogenic activity of tabRTS. This result indicated that tabRTS possibly targets the alpha(1)beta(1) integrin to exert the anti-angiogenic activity as snake venom RTS-/KTS-disintegrins do. The current work revealed the first angiogenesis inhibitor protein containing RTS motif from invertebrates, a possible novel type of RTS-disintegrin.

Effect of VP12 and viperistatin on inhibition of collagen-receptor-dependent melanoma metastasis.

Viperistatin and VP12 isolated from Vipera paleastinae venom showed a potent inhibitory activity against collagen receptors, alpha1beta1 and alpha2beta1 integrins, respectively. Structurally, viperistatin belongs to the disintegrin family of proteins, whereas VP12 is composed of two subunits VP12A and VP12B displaying amino acid sequence homology with heterodimeric C-lectin type proteins. Viperistatin and VP12 used separately and simultaneously inhibited pro-metastatic activities of melanoma cells lines. The level of inhibition of MV3 and HS.939T human cell lines in cell adhesion and migration assays by both compounds was correlated with expression of alpha1beta1 and alpha2beta1 integrins on the cell surface. MV3 cells express collagen receptors to much higher extent than HS.939T and required the application of higher concentrations of inhibitors to block their adhesion to collagen types I and IV. A melanoma cell transmigration assay through a dHMVEC layer revealed that alpha1beta1 integrin plays a significant role in invasion of HS.939T cells, while alpha2beta1 integrin appears to be more important for MV3 cells. In an animal model of hematogenous metastasis of the mouse B16F10 cell line, the inhibitory effect of viperistatin and VP12 was only partial. These data suggest that collagen receptors may be an interesting target for development of new anti-metastatic therapies.

Structural requirements of KTS-disintegrins for inhibition of alpha(1)beta(1) integrin.

Obtustatin and viperistatin represent the shortest known snake venom monomeric disintegrins. In the present study, we have produced recombinant full-length wild-type and site-directed mutants of obtustatin to assess the role of the K(21)TS(23) tripeptide and C-terminal residues for specific inhibition of the alpha(1)beta(1) integrin. Thr(22) appeared to be the most critical residue for disintegrin activity, whereas substitution of the flanking lysine or serine residues for alanine resulted in a less pronounced decrease in the anti-alpha(1)beta(1) integrin activity of the disintegrin. The triple mutant A(21)AA(23) was devoid of blocking activity towards alpha(1)beta(1) integrin-mediated cell adhesion. The potency of recombinant KTS-disintegrins also depended on the residue C-terminally adjacent to the active motif. Substitution of Leu(24) of wild-type obtustatin for an alanine residue slightly decreased the inhibitory activity of the mutant, whereas an arginine residue in this position enhanced the potency of the mutant over wild-type obtustatin by 6-fold. In addition, the replacements L38V and P40Q may account for a further 25-fold increase in alpha(1)beta(1) inhibitory potency of viperistatin over KTSR-obtustatin.

Angiostatic activity of obtustatin as alpha1beta1 integrin inhibitor in experimental melanoma growth.

The presented results show the effect of targeting of collagen receptor, alpha1beta1 integrin expressed on the endothelial cells on the development of experimental melanoma and pathological angiogenesis. Obtustatin, a snake venom KTS-disintegrin, was applied as a specific inhibitor of this integrin. This low molecular weight peptide revealed a potent therapeutic effect on melanoma progression in 2 animal systems, mouse and quail. Its oncostatic effect was related to the inhibition of angiogenesis. Obtustatin inhibited the neovascularization ratio on the CAM embryo of quail, which was pathologically induced by the developing tumor. The i.v. administration of obtustatin completely blocked cancer growth of MV3 human melanoma in nude mice. In B16F10 syngeneic mouse model treatment with the disintegrin revealed a lower effect, although the development of the tumor was significantly reduced for both dosages. The mechanism of obtustatin action is related to the blocking of microvascular endothelial cell proliferation, which undergoes apoptosis in caspase-dependent manner. Summarizing, we present studies of low molecular weight disintegrin, obtustatin as a potential therapeutic compound for treatment of melanoma that contain a high level of vascularization.

KTS and RTS-disintegrins: anti-angiogenic viper venom peptides specifically targeting the alpha 1 beta 1 integrin.

Integrins alpha(1)beta(1) and alpha(2)beta(1) are highly expressed on the microvascular endothelial cells, and blocking of their adhesive properties significantly reduced the VEGF-driven neovascularization ratio and tumor growth in animal models. Hence, inhibitors of the alpha(1)beta(1) and alpha(2) beta(1) integrins, alone or in combination with antagonists of other integrins involved in angiogenesis (eg. alpha (v)beta(3), alpha(v)beta(5), and alpha(6)beta(4)), may prove beneficial in the control of tumor neovascularization. Viperidae snakes have developed in their venoms an efficient arsenal of integrin receptor antagonists. KTS-(obtustatin, viperistatin, lebestatin) and RTS- (jerdostatin) disintegrins represent viper venom peptides that specifically block the interaction of the alpha(1)beta (1) integrin with collagens IV and I in vitro and angiogenesis in vivo. The possible therapeutic approach towards tumor neovascularization by targeting the alpha (5)beta (1), alpha(v)beta(5) and alpha (v)beta(3) integrins with RGD-bearing disintegrins has been explored in a number of laboratories. Here we discuss structure-function correlations of the novel group of specific (K/R)TS-disintegrins targeting the alpha(1)beta(1) integrin.

Alpha1beta1 integrin is crucial for accumulation of epidermal T cells and the development of psoriasis.

Psoriasis is a common T cell-mediated autoimmune inflammatory disease. We show that blocking the interaction of alpha1beta1 integrin (VLA-1) with collagen prevented accumulation of epidermal T cells and immunopathology of psoriasis. Alpha1beta1 integrin, a major collagen-binding surface receptor, was exclusively expressed by epidermal but not dermal T cells. Alpha1beta1-positive T cells showed characteristic surface markers of effector memory cells and contained high levels of interferon-gamma but not interleukin-4. Blockade of alpha1beta1 inhibited migration of T cells into the epidermis in a clinically relevant xenotransplantation model. This was paralleled by a complete inhibition of psoriasis development, comparable to that caused by tumor necrosis factor-alpha blockers. These results define a crucial role for alpha1beta1 in controlling the accumulation of epidermal type 1 polarized effector memory T cells in a common human immunopathology and provide the basis for new strategies in psoriasis treatment focusing on T cell-extracellular matrix interactions.

Lebestatin, a disintegrin from Macrovipera venom, inhibits integrin-mediated cell adhesion, migration and angiogenesis.

Lebestatin, a new member of the lysine-threonine-serine (KTS)-disintegrin family, was purified to homogeneity from Tunisian snake (Macrovipera lebetina) venom. It is a single-chain polypeptide composed of 41 amino acids. The amino-acid sequence of lebestatin shows that it displays a pattern of cysteines similar to other short disintegrins, but contains the sequence KTS rather than RGD in its integrin-binding loop. Lebestatin presents a high homology with obtustatin and viperistatin. Lebestatin interacts specifically with the alpha1beta1 integrin. It was thus able to inhibit both adhesion and migration of PC12 and alpha1beta1 integrin-expressing CHO cells (CHO-alpha1) to type I and IV collagens. This disintegrin also affected adhesion and migration of endothelial cells and exhibited an anti-angiogenic effect in vivo when using the 8-day-old embryo chick chorioallantoic membrane model.