PLA2-MjTX-II from Bothrops moojeni snake venom exhibits antimetastatic and antiangiogenic effects on human lung cancer cells.

Phospholipases A2 (PLA2s) from snake venom possess antitumor and antiangiogenic properties. In this study, we evaluated the antimetastatic and antiangiogenic effects of MjTX-II, a Lys49 PLA2 isolated from Bothrops moojeni venom, on lung cancer and endothelial cells. Using in vitro and ex vivo approaches, we demonstrated that MjTX-II reduced cell proliferation and inhibited fundamental processes for lung cancer cells (A549) growth and metastasis, such as adhesion, migration, invasion, and actin cytoskeleton decrease, without significantly interfering with non-tumorigenic lung cells (BEAS-2B). Furthermore, MjTX-II caused cell cycle alterations, increased reactive oxygen species production, modulated the expression of pro- and antiangiogenic genes, and decreased vascular endothelial growth factor (VEGF) expression in HUVECs. Finally, MjTX-II inhibited ex vivo angiogenesis processes in an aortic ring model. Therefore, we conclude that MjTX-II exhibits antimetastatic and antiangiogenic effects in vitro and ex vivo and represents a molecule that hold promise as a pharmacological model for antitumor therapy.

Antiangiogenic properties of BthMP, a P-I metalloproteinase from Bothrops moojeni snake venom by VEGF pathway in endothelial cells.

Angiogenesis is a process that is controlled by a delicate combination of proangiogenic and antiangiogenic molecules and can be disrupted in various illnesses, including cancer. Non-cancerous diseases can also have an abnormal or insufficient vascular growth, inflammation and hypoxia, which exacerbate angiogenesis. These conditions include atherosclerosis, psoriasis, endometriosis, asthma, obesity and AIDS. Based on that, the present work assessed the in vitro and ex vivo antiangiogenic properties stemming from BthMP, a P-I metalloproteinase from Bothrops moojeni snake venom, via the VEGF pathway. BthMP at a concentration of 5 and 40 µg/mL showed no toxicity to endothelial cells (HUVEC) in the MTT assay and was not able to induce necrosis and colony proliferation. Interestingly, BthMP inhibited adhesion, migration and invasion of HUVECs in Matrigel and arrested in vitro angiogenesis by reducing the average number of nodules in toxin-treated cells by 9.6 and 17.32 at 5 and 40 µg/mL, respectively, and the number of tubules by 15.9 at 5 µg/mL and 21.6 at 40 µg/mL in a VEGF-dependent way, an essential proangiogenic property. Furthermore, BthMP inhibited the occurrence of the angiogenic process in an ex vivo aortic ring test by decreasing new vessel formation by 52% at 5 µg/mL and by 66% at 40 µg/mL and by increasing the expression of an antiangiogenic gene, SFLT-1, and decreasing the expression of the proangiogenic genes VEGFA and ANGPT-1. Finally, this toxin reduces the production of nitric oxide, a marker that promotes angiogenesis and VEGF modulation, and decreases the protein expression of VEGFA in the supernatant of the HUVEC culture by about 30 %. These results suggest that BthMP has a promising antiangiogenic property and proves to be a biotechnological mechanism for understanding the antiangiogenic responses induced by snake venom metalloproteinases, which could be applied to a variety of diseases that exhibit an imbalance of angiogenesis mechanisms.

The Role of Snake Venom Disintegrins in Angiogenesis.

Angiogenesis, the formation of new blood vessels, plays a critical role in various physiological and pathological conditions. Snake venom disintegrins (SVDs) have been identified as significant regulators of this process. In this review, we explore the dual roles of SVD in angiogenesis, both as antiangiogenic agents by inhibiting integrin binding and interfering with vascular endothelial growth factors and as proangiogenic agents by enhancing integrin binding, stimulating cell migration and proliferation, and inducing neoangiogenesis. Studies in vitro and in animal models have demonstrated these effects and offer significant therapeutic opportunities. The potential applications of SVD in diseases related to angiogenesis, such as cancer, ocular diseases, tissue regeneration, wound healing, and cardiovascular diseases, are also discussed. Overall, SVDs are promising potential therapeutics, and further advances in this field could lead to innovative treatments for diseases related to angiogenesis.

Identification of miRnas with possible prognostic roles for HAM/TSP.

Human T-cell lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropic spastic paraparesis (HAM/TSP) is an insidiously progressive spinal cord disease for which there is no effective treatment. There is great interest in developing potential biomarkers to predict the pathogenesis of HAM/TSP disease. In this study, Illumina Massive Parallel Sequencing (MPS) technology was used to investigate the cellular global noncoding RNAome expression profile in HAM/TSP patients (n = 10), asymptomatic HTLV-1-infected carriers (ASP, n = 8), and a second group of healthy controls (n = 5). Various bioinformatics tools were used to align, annotate, and profile the sRNA-MPS reads. Among the 402 sRNAs detected, 251 were known and 50 were potentially novel sRNAs in the HAM and ASP groups compared with the HC group. Sixty-eight known sRNAs were significantly different between the ASP and HAM groups. Eighty-eight mature miRNAs were downregulated in subjects from HAM compared with ASP. Three of these miRs (hsa-miR-185-5p, 32-5p, and 192-5p) have the potential to be used as biomarkers for predicting the pathogenesis of HAM/TSP. The seven most deregulated miRs target genes have been associated with a variety of biological processes and molecular functions. The reactome pathways relevant to our findings provide a rich source of data and offer the opportunity to better understand sRNA regulation and function in HTLV-1 pathophysiology. To the best of our knowledge, this study is the first to demonstrate evaluates sRNAs in HTLV-1 patients with HAM/TSP.

Antileishmanial effects of γCdcPLI, a phospholipase A2 inhibitor from Crotalus durissus collilineatus snake serum, on Leishmania (Leishmania) amazonensis

BACKGROUND Leishmaniasis, a neglected disease caused by the parasite Leishmania, is treated with drugs associated with high toxicity and limited efficacy, in addition to constant reports of the emergence of resistant parasites. In this context, snake serums emerge as good candidates since they are natural sources with the potential to yield novel drugs. OBJECTIVES We aimed to show the antileishmanial effects of γCdcPLI, a phospholipase A2 inhibitor from Crotalus durissus collilineatus snake serum, against Leishmania (Leishmania) amazonensis. METHODS Promastigotes forms were exposed to γCdcPLI, and we assessed the parasite viability and cell cycle, as well as invasion and proliferation assays. FINDINGS Despite the low cytotoxicity effect on macrophages, our data indicate that γCdcPLI has a direct effect on parasites promoting an arrest in the G1 phase and reduction in the G2/M phase at the highest dose tested. Moreover, this PLA2 inhibitor reduced the parasite infectivity when promastigotes were pre-treated. Also, we demonstrated that the γCdcPLI treatment modulated the host cell environment impairing early and late steps of the parasitism. MAIN CONCLUSIONS γCdcPLI is an interesting tool for the discovery of new essential targets on the parasite, as well as an alternative compound to improve the effectiveness of the leishmaniasis treatment.

Jararhagin-C, a disintegrin-like protein, improves wound healing in mice through stimulation of M2-like macrophage, angiogenesis and collagen deposition.

Jararhagin-C (Jar-C) is a disintegrin-like protein, isolated from the venom of B. jararaca, with affinity for α2ß1 integrin and the ability to incite processes such as angiogenesis and collagen deposition in vivo. Thus, we raised the hypothesis that this protein could be used as a therapeutic strategy for stimulating the healing of excisional wounds in mice. Four wounds were made on the back of Swiss mice, treated with daily intradermal injections of PBS (control group) or Jar-C (200 ng). Ten animals from each experimental group were euthanized and the tissue from the wounds and skin around them were collected for further biochemical, histological and molecular analysis. Wounds treated with Jar-C showed a faster closure rate, accompanied by a reduction in neutrophil infiltrate (MPO), pro-inflammatory cytokine levels (TNF, CXCL1 and CCL2) and an accumulation of macrophages in the analyzed tissues. It was also observed a greater expression of genes associated with the phenotype of alternatively activated macrophages (M2). Concomitantly, the administration of Jar-C holds an angiogenic potential, increasing the density of blood vessels and the synthesis of pro-angiogenic cytokines (VEGF and FGF). We also observed an increase in collagen deposition, accompanied by higher levels of the pro-fibrogenic cytokine TGF-ß1. Our data suggests Jar-C stimulates wound healing through stimulation of M2-like macrophage, angiogenesis and collagen deposition. Jar-C may be explored as a therapeutic strategy for wound healing, including the treatment of chronic wounds, where processes such as inflammation, angiogenesis and the deposition / remodeling of the matrix constituents are unregulated.

Global expression of noncoding RNome reveals dysregulation of small RNAs in patients with HTLV-1-associated adult T-cell leukemia: a pilot study.

BACKGROUND: Adult T cell lymphoma/leukemia (ATLL) is a peripheral T-cell neoplasm caused by human T-cell lymphotropic virus-1 (HTLV-1). Small RNAs (sRNAs), including microRNAs (miRNAs), play a pivotal role in the initiation and development of hematological malignancies and may represent potential therapeutic target molecules. However, little is known about how these molecules impact the pathogenesis of ATLL. In this study, we aimed to identify sRNA expression signatures associated with ATLL and to investigate their potential implication in the pathophysiology of the disease. METHODS: Small-RNAseq analysis was performed in peripheral blood mononuclear cells from HTLV-1- associated ATLL (n = 10) in comparison to asymptomatic carriers (n = 8) and healthy controls (n = 5). Sequencing was carried out using the Illumina MiSeq platform, and the deregulation of selected miRNAs was validated by real-time PCR. Pathway analyses of most deregulated miRNA were performed and their global profiling was combined with transcriptome data in ATLL. RESULTS: The sequencing identified specific sRNAs signatures associated with ATLL patients that target pathways relevant in ATLL, such as the transforming growth factor-(ßTGF-ß), Wnt, p53, apoptosis, and mitogen-activated protein kinase (MAPK) signaling cascades. Network analysis revealed several miRNAs regulating highly connected genes within the ATLL transcriptome. miR-451-3p was the most downregulated miRNA in active patients. CONCLUSIONS: Our findings shed light on the expression of specific sRNAs in HTLV-1 associated ATLL, which may represent promising candidates as biomarkers that help monitor the disease activity.

Small RNA profiles of HTLV-1 asymptomatic carriers with monoclonal and polyclonal rearrangement of the T-cell antigen receptor γ-chain using massively parallel sequencing: A pilot study.

In the present pilot study, massively parallel sequencing (MPS) technology was used to investigate cellular small RNA (sRNA) levels in the peripheral blood mononuclear cells (PBMCs) of human T-lymphotropic virus type I (HTLV-I) infected asymptomatic carriers with monoclonal (ASM) and polyclonal (ASP) T cell receptor (TCR) γ gene. Blood samples from 15 HTLV-I asymptomatic carriers (seven ASM and eight ASP) were tested for the clonal TCR-γ gene and submitted for sRNA library construction together with blood samples of five healthy controls (HCs) using Illumina sequencing platform. The sRNA-sequencing reads were aligned, annotated and profiled using various bioinformatics tools. Based on these results, possible markers were validated in the study samples by performing reverse transcription-quantitative (RT-q)PCR analysis. A total of 76 known sRNAs and 52 putative novel sRNAs were identified. Among them, 44 known and 34 potential novel sRNAs were differentially expressed in the ASM and ASP libraries compared with HCs. In addition, 10 known sRNAs were exclusively dysregulated in the ASM group and one (transfer RNA 65) was significantly upregulated in the ASP group. Homo sapiens (hsa) microRNA (miRNA/mir)-23a-3p, -28-5p, hsa-let-7e-5p and hsa-mir-28-3p and -361-5p were the most abundantly upregulated mature miRNAs and hsa-mir-363-3p, -532-5p, -106a-5p, -25-3p and -30e-5p were significantly downregulated miRNAs (P<0.05) with a >2-fold difference between the ASM and ASP groups compared with HCs. Based on these results, hsa-mir-23a-3p and -363-3p were selected for additional validation. However, the quantification of these two miRNAs using RT-qPCR did not provide any significant differences. While the present study failed to identify predictive sRNA markers to distinguish between ASM and ASP, the MPS results revealed differential sRNA expression profiles in the PBMCs of HTLV-1 asymptomatic carriers (ASM and ASP) compared with HCs.

Leukocyte recruitment induced by snake venom metalloproteinases: Role of the catalytic domain.

Snake venom metalloproteinases (SVMPs) are key toxins involved in local inflammatory reactions after snakebites. This study aimed to investigate the effect of SVMP domains on the alterations in leukocyte-endothelium interactions in the microcirculation of mouse cremaster muscle. We studied three toxins: BnP1, a PI-toxin isolated from Bothrops neuwiedi venom, which only bears a catalytic domain; Jararhagin (Jar), a PIII-toxin isolated from Bothrops jararaca venom with a catalytic domain, as well as ECD-disintegrin and cysteine-rich domains; and Jar-C, which is produced from the autolysis of Jar and devoid of a catalytic domain. All these toxins induced an increase in the adhesion and migration of leukocytes. By inhibiting the catalytic activity of Jar and BnP1 with 1.10-phenanthroline (oPhe), leukocytes were no longer recruited. Circular dichroism analysis showed structural changes in oPhe-treated Jar, but these changes were not enough to prevent the binding of Jar to collagen, which occurred through the ECD-disintegrin domain. The results showed that the catalytic domain of SVMPs is the principal domain responsible for the induction of leukocyte recruitment and suggest that the other domains could also present inflammatory potential only when devoid of the catalytic domain, as with Jar-C.

Inflammation, angiogenesis and fibrogenesis are differentially modulated by distinct domains of the snake venom metalloproteinase jararhagin.

Jararhagin, a metalloprotease from Bothrops jararaca snake venom, is a toxin containing the metalloproteinase, disintegrin-like and cysteine-rich domains; it causes acute inflammation and damage to vascular tissue. However, the actions of these domains on key components of chronic inflammation have not been determined. Our aim was to investigate the effects of jararhagin (Jar), jararhagin-C (Jar-C) and o-phenantrolin-treated jararhagin (Jar-Phe), on inflammatory response, blood vessel formation and extracellular matrix deposition in the murine sponge model. The polyether-polyurethane sponge matrix was implanted into Balb/c mice and injected daily with Jar (400 ng), Jar-Phe (400 ng), Jar-C (200 ng) or saline (control). Nine days after implantation, the sponge discs were removed and processed. In the Jar-treated implants, some of inflammatory markers (N-acetyl-ß-d-glucosaminidase activity, CCL2 and TNF-α) and TGF-ß1 levels were higher compared with the control group. In the Jar-C group, the inflammatory markers myeloperoxidase activity and CXCL1 were higher compared with the control. In this group, VEGF levels and collagen deposition were also higher. Jar-Phe treatment was able to inhibit the activity and/or production of MPO, CXCL1, CCL2 and TGF-ß. The differential effects of these proteins in modulating the main components of fibrovascular tissue may be exploited in the management fibroproliferative diseases.

Bothrops jararaca accessory venom gland is an ancillary source of toxins to the snake.

In Viperidae snakes, it has been attributed to the main venom gland, a component of the venom gland apparatus, the function of synthesizing all venom toxins and storing them inside a basal-central lumen. However, the role of the accessory gland is still unknown. Here, we analyzed the proteome and the transcriptome of the accessory gland during venom production and secretion cycle. We showed that the accessory gland expresses and synthesizes toxins that are similar to those produced by the main venom gland such as C-type lectin/C-type lectin-like proteins, metalloproteinase, phospholipase A2, cysteine rich secretory protein, nerve growth factor, vascular endothelial growth factor, serine proteinase, and l-amino acid oxidase. Our data have shown that toxin synthesis in the accessory gland is asynchronous when compared to the same process in the venom gland. Moreover, this gland also expresses inhibitors of venom phospholipases A2 and metalloproteinases. Transcriptome analysis showed that the transcripts that correspond to toxins in the accessory gland have a good correlation to the main venom gland transcripts. Therefore, it is proposed that the accessory gland is an ancillary source of toxins to the snake, and provides inhibitors that could control venom toxicity (and integrity) during storage. SIGNIFICANCE: In this study, we propose that the accessory venom gland acts as an important ancillary source of toxins to the snake, in lieu of a depleted main venom gland, and provides inhibiting agents that control venom toxicity (and integrity) during its storage.

Engineered mammalian vector to express EGFP-tagged proteins as biomarkers.

Due to its specialized post-translational machinery, mammalian cells represent an interesting and not fully explored system to express snake toxins. Therefore, in this work, we built up a new mammalian expression vector that enhances the feasibility to use mammalian cells to express proteins as biomarkers. Among the modifications, an Igκ signal peptide and a 6xHis tag were inserted into this vector in order to drive the protein to the supernatant and simplify its purification, respectively. In addition, to facilitate selection of high producing clones and also tag proteins which may function as a biomarker, the sequence of enhanced green fluorescent protein (EGFP) was added. The efficiency of the resulting vector (pToxEGFP) was tested by cloning and expressing the viper venom disintegrin echistatin (Ech) that due to its affinity to integrin αvß3 was tested as a molecular marker. Expression of EGFP-Ech was achieved in CHO-DXB11 cells resulting in a yield of 22 mg/L. The binding activity of this chimera protein was successfully achieved on human umbilical vein endothelial cells which highly express αvß3. The results indicate that pToxEGFP may constitute an efficient and versatile expression vector to express tagged proteins with potential biomarker activity.

"Insularin, a disintegrin from Bothrops insularis venom: inhibition of platelet aggregation and endothelial cell adhesion by the native and recombinant GST-insularin proteins".

Insularin (INS) was obtained from Bothrops insularis venom by reversed-phase high-performance liquid chromatography using a C(18) column and characterized as a disintegrin by peptide mass fingerprint and inhibition of ADP-induced platelet aggregation. A cDNA coding for P-II a metalloproteinase/disintegrin was cloned from a cDNA library from B. insularis venom glands. The deduced protein sequence possesses 73 amino acid residues, including the N-terminal, internal peptides of native insularin, the ARGDNP-sequence and 12 cysteines in a conserved alignment. This cDNA fragment was subcloned in the pGEX-4T-1 vector and expressed in a prokaryotic expression system as a fusion protein with glutathione S-transferase (GST-INS). Both native and recombinant insularin inhibited ADP-induced platelet aggregation and endothelial cells (HUVEC) adhesion with similar activities indicating that GST-INS folded correctly and preserved the integrin-binding loop. Insularin may be a tool in studies that involve platelets and endothelial cell adhesion dependent on alphaIIbeta3 and alphavbeta3 integrins.

Inflammatory pathogenesis of snake venom metalloproteinase-induced skin necrosis.

Local tissue damage, characterized by edema, hemorrhage and necrosis, is a common consequence of envenoming by many vipers. We have investigated the contribution of inflammatory responses induced by the venom metalloproteinase jararhagin (isolated from Bothrops jararaca venom) in the development of these lesions. Local venom effects (edema, hemorrhage and necrosis) were induced experimentally in knockout mice deficient in the TNF receptors TNFR1 or TNFR2, IL-1betaR, IL-6 and iNOS. Jararhagin-induced dermal necrosis was abolished in mice deficient in the TNF receptors TNFR1 and TNFR2, and the same activity was significantly reduced in IL-6(-/-) mice. There was no significant difference in edema and hemorrhage activities following jararhagin insult between knockout and WT strains, indicating that these local venom metalloproteinase-induced effects are independent of these pro-inflammatory mediators. The contribution of both TNF receptors and IL-6 in local tissue necrosis raises important therapeutic issues regarding the treatment of local envenoming.