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Biomolecules Apr 2023Ectopic calcification and dysregulated extracellular matrix remodeling are prominent hallmarks of the complex heterogenous pathobiochemistry of pseudoxanthoma elasticum...
Ectopic calcification and dysregulated extracellular matrix remodeling are prominent hallmarks of the complex heterogenous pathobiochemistry of pseudoxanthoma elasticum (PXE). The disease arises from mutations in , an ATP-binding cassette transporter expressed predominantly in the liver. Neither its substrate nor the mechanisms by which it contributes to PXE are completely understood. The fibroblasts isolated from PXE patients and mice were subjected to RNA sequencing. A group of matrix metalloproteinases (MMPs) clustering on human chromosome 11q21-23, respectively, murine chromosome 9, was found to be overexpressed. A real-time quantitative polymerase chain reaction, enzyme-linked immunosorbent assay and immunofluorescent staining confirmed these findings. The induction of calcification by CaCl resulted in the elevated expression of selected MMPs. On this basis, the influence of the MMP inhibitor Marimastat (BB-2516) on calcification was assessed. PXE fibroblasts (PXEFs) exhibited a pro-calcification phenotype basally. PXEF and normal human dermal fibroblasts responded with calcium deposit accumulation and the induced expression of osteopontin to the addition of Marimastat to the calcifying medium. The raised MMP expression in PXEFs and during cultivation with calcium indicates a correlation of ECM remodeling and ectopic calcification in PXE pathobiochemistry. We assume that MMPs make elastic fibers accessible to controlled, potentially osteopontin-dependent calcium deposition under calcifying conditions.
Topics: Humans; Mice; Animals; Pseudoxanthoma Elasticum; Osteopontin; Calcium; Calcinosis; Phenotype; Matrix Metalloproteinases; Multidrug Resistance-Associated Proteins
PubMed: 37189419
DOI: 10.3390/biom13040672 -
BioRxiv : the Preprint Server For... Sep 2023peptidoglycan (PGN) is a major component of the bacterial cell wall and a key pathogen-associated molecular pattern (PAMP) contributing to anthrax pathology, including...
peptidoglycan (PGN) is a major component of the bacterial cell wall and a key pathogen-associated molecular pattern (PAMP) contributing to anthrax pathology, including organ dysfunction and coagulopathy. Increases in apoptotic lymphocytes are a late-stage feature of anthrax and sepsis, suggesting there is a defect in apoptotic clearance. Here, we tested the hypothesis that PGN inhibits the capacity of human monocyte-derived macrophages (MΦ) to efferocytose apoptotic cells. Exposure of CD163CD206 MΦ to PGN for 24h impaired efferocytosis in a manner dependent on human serum opsonins but independent of complement component C3. PGN treatment reduced cell surface expression of the pro-efferocytic signaling receptors MERTK, TYRO3, AXL, integrin αVβ5, CD36 and TIM-3, whereas TIM-1, αVβ3, CD300b, CD300f, STABILIN-1 and STABILIN-2 were unaffected. ADAM17 is a major membrane-bound protease implicated in mediating efferocytotic receptor cleavage. We found multiple ADAM17-mediated substrates increased in PGN-treated supernatant suggesting involvement of membrane-bound proteases. ADAM17 inhibitors TAPI-0 and Marimastat prevented TNF release, indicating effective protease inhibition, and modestly increased cell-surface levels of MerTK and TIM-3 but only partially restored efferocytic capacity by PGN-treated MΦ. We conclude that human serum factors are required for optimal recognition of PGN by human MΦ and that PGN inhibits efferocytosis in part by reducing cell surface expression of MERTK and TIM-3.
PubMed: 37066181
DOI: 10.1101/2023.03.30.535001 -
Journal of Thrombosis and Haemostasis :... Jul 2023ADAMTS13 is a circulating metalloprotease that cleaves von Willebrand factor (VWF) in a shear-dependent manner. ADAMTS13 is secreted as an active protease but has a long...
BACKGROUND
ADAMTS13 is a circulating metalloprotease that cleaves von Willebrand factor (VWF) in a shear-dependent manner. ADAMTS13 is secreted as an active protease but has a long half-life, suggesting that it is resistant to circulating protease inhibitors. These zymogen-like properties indicate that ADAMTS13 exists as a latent protease that is activated by its substrate.
OBJECTIVES
To investigate the mechanism of ADAMTS13 latency and resistance to metalloprotease inhibitors.
METHODS
Probe the active site of ADAMTS13 and variants using alpha-2 macroglobulin (A2M), tissue inhibitors of metalloproteases (TIMPs), and Marimastat.
RESULTS
ADAMTS13 and C-terminal deletion mutants are not inhibited by A2M, TIMPs, or Marimastat, but cleave FRETS-VWF73, suggesting that the metalloprotease domain is latent in the absence of substrate. Within the metalloprotease domain, mutating the gatekeeper triad (R193, D217, D252) or substituting the calcium-binding (R180-R193) or the variable (G236-S263) loops with corresponding features from ADAMTS5 did not sensitize MDTCS to inhibition. However, substituting the calcium-binding loop and an extended variable loop (G236-S263) corresponding to the S1-S1' pockets with those from ADAMTS5, resulted in MDTCS-GVC5 inhibition by Marimastat, but not by A2M or TIMP3. Substituting the MD domains of ADAMTS5 into full-length ADAMTS13 resulted in a 50-fold reduction in activity compared with the substitution into MDTCS. However, both chimeras were susceptible to inhibition, suggesting that the closed conformation does not contribute to the latency of the metalloprotease domain.
CONCLUSION
The metalloprotease domain protects ADAMTS13 from inhibitors and exists in a latent state that is partially maintained by loops flanking the S1 and S1' specificity pockets.
Topics: Humans; von Willebrand Factor; ADAM Proteins; Calcium; Hydroxamic Acids; ADAMTS13 Protein
PubMed: 36990157
DOI: 10.1016/j.jtha.2023.03.021 -
Nutrients Feb 2023High-grade adult-type diffuse gliomas are the most common and deadliest malignant adult tumors of the central nervous system. Despite the advancements in the... (Review)
Review
High-grade adult-type diffuse gliomas are the most common and deadliest malignant adult tumors of the central nervous system. Despite the advancements in the multimodality treatment of high-grade adult-type diffuse gliomas, the five-year survival rates still remain poor. The biggest challenge in treating high-grade adult-type diffuse gliomas is the intra-tumor heterogeneity feature of the glioma tumors. Introducing dietary flavonoids to the current high-grade adult-type diffuse glioma treatment strategies is crucial to overcome this challenge, as flavonoids can target several molecular targets. This review discusses the anticancer mechanism of flavonoids (quercetin, rutin, chrysin, apigenin, naringenin, silibinin, EGCG, genistein, biochanin A and C3G) through targeting molecules associated with high-grade adult-type diffuse glioma cell proliferation, apoptosis, oxidative stress, cell cycle arrest, migration, invasion, autophagy and DNA repair. In addition, the common molecules targeted by the flavonoids such as Bax, Bcl-2, MMP-2, MMP-9, caspase-8, caspase-3, p53, p38, Erk, JNK, p38, beclin-1 and LC3B were also discussed. Moreover, the clinical relevance of flavonoid molecular targets in high-grade adult-type diffuse gliomas is discussed with comparison to small molecules inhibitors: ralimetinib, AMG232, marimastat, hydroxychloroquine and chloroquine. Despite the positive pre-clinical results, further investigations in clinical studies are warranted to substantiate the efficacy and safety of the use of flavonoids on high-grade adult-type diffuse glioma patients.
Topics: Humans; Adult; Brain Neoplasms; Glioma; Flavonoids; Quercetin; Rutin
PubMed: 36839156
DOI: 10.3390/nu15040797 -
Journal of Translational Medicine Feb 2023In the past decades studies on anti-tumoral drugs inhibiting matrix metalloproteinase (MMPs) were disappointing. Recently, we demonstrated that mature endothelial cells...
BACKGROUND
In the past decades studies on anti-tumoral drugs inhibiting matrix metalloproteinase (MMPs) were disappointing. Recently, we demonstrated that mature endothelial cells (ECs) and endothelial colony forming cells (ECFCs) can switch between invasion modes to cope with challenging environments, performing the "amoeboid angiogenesis" in the absence of proteases activity.
METHODS
We first set out to investigate by ELISA if the inhibitors of the main protease family involved in angiogenesis were differently expressed during breast cancer progression. We used Marimastat, a broad-spectrum MMP inhibitor, as a means of inducing amoeboid characteristics and studied VEGF role in amoeboid angiogenesis. Thus, we performed invasion and capillary morphogenesis assay, morphological, cell signaling and in vivo mouse studies.
RESULTS
Our data showed that TIMP1, TIMP2, alpha2-antiplasmin, PAI-1 and cystatin increase in breast cancer serum of patients with primary cancer and lymph node positive compared to healthy women. In vitro results revealed that the most high-powered protease inhibitors able to induce amoeboid invasion of ECFCs were TIMP1, 2 and 3. Surprisingly, Marimastat promotes ECFC invasion and tubular formation in vitro and in vivo, inducing amoeboid characteristics. We observed that the combination of Marimastat plus VEGF doesn't boost neither cell invasion nor vessel formation capacity. Moreover, inhibition of VEGF activity with Bevacizumab in the presence of Marimastat confirmed that amoeboid angiogenesis is independent from the stimulus of the main vascular growth factor, VEGF.
CONCLUSIONS
We underline the importance to consider the amoeboid mechanism of endothelial and cancer cell invasion, probably responsible for the failure of synthetic metalloproteinase inhibitors as cancer therapy and tumor resistance to VEGF-targeted therapies, to set-up new drugs to be used in cancer therapy.
Topics: Animals; Female; Mice; Amoeba; Angiogenesis Inhibitors; Endothelial Cells; Matrix Metalloproteinases; Morphogenesis; Neoplasms; Neovascularization, Pathologic; Signal Transduction; Vascular Endothelial Growth Factor A; MAP Kinase Signaling System
PubMed: 36759828
DOI: 10.1186/s12967-023-03954-6 -
Toxins Dec 2022The African viperid snake genera , and are closely related, similar in size, but occupy extremely divergent ecological niches (arboreal in tropical rainforests,...
The African viperid snake genera , and are closely related, similar in size, but occupy extremely divergent ecological niches (arboreal in tropical rainforests, fossorial in deserts, and swamp-dwelling, respectively). Their venoms have not previously been subjected to comparative analyses for their action upon the coagulation of blood, most notably with significant data deficiencies from and In contrast, the closely related genus is well-documented as capable of producing potent procoagulant effects. In light of this, we set out to compare the coagulotoxic actions of , , , , , , , , and and explore potential pharmacological interventions to reestablish normal blood coagulation. All venoms displayed extremely potent procoagulant effects, over twice as fast as the most potent reported to date. Although is used in the immunising mixture of two different regionally available antivenoms (Inoserp-MENA with , , and Saudi Arabian polyvalent with ), none of the other species in this study are included in the immunising mixture of any antivenom. Notably, all the species were only neutralised by the Inoserp-MENA antivenom. venom was not neutralised well by the Saudi Arabian antivenom, with the low levels of recognition for any of the venoms suggesting a strong regional variation in the venom of this species, as the venom tested was of African (Tunisian) origin versus Saudi locality used in that antivenom's production. The other antivenoms (Micropharm EchiTAbG, ICP EchiTAb-Plus-ICP, Inosan Inoserp Pan-Africa, Premium Serums PANAF Sub-Sahara Africa, South African Vaccine Producers , South African Vaccine Producers Polyvalent) all displayed trivial-to-no ability to neutralise the procoagulant toxicity of any of the , or venoms. Comparative testing of the enzyme inhibitors DMPS, marimastat, and prinomastat, revealed a very potent neutralising capacity of marimastat, with prinomastat showing lower but still significant potency at the same molar concentration, while a 5× molar concentration of DMPS had no apparent effect on procoagulant venom effects normalized by the other inhibitors. These results and methods contribute to the body of knowledge of potential clinical effects and data necessary for evidence-based advancement of clinical management strategies.
Topics: Animals; Humans; Antivenins; Viperidae; Saudi Arabia; Viper Venoms; Africa South of the Sahara; Enzyme Inhibitors; African People; Snake Bites
PubMed: 36548733
DOI: 10.3390/toxins14120836 -
Toxins Oct 2022Envenomation by elapid snakes primarily results in neurotoxic symptoms and, consequently, are the primary focus of therapeutic research concerning such venoms. However,...
Envenomation by elapid snakes primarily results in neurotoxic symptoms and, consequently, are the primary focus of therapeutic research concerning such venoms. However, mounting evidence suggests these venoms can additionally cause coagulopathic symptoms, as demonstrated by some Asian elapids and African spitting cobras. This study sought to investigate the coagulopathic potential of venoms from medically important elapids of the genera (true cobras), (rinkhals), and (mambas). Crude venoms were bioassayed for coagulant effects using a plasma coagulation assay before RPLC/MS was used to separate and identify venom toxins in parallel with a nanofractionation module. Subsequently, coagulation bioassays were performed on the nanofractionated toxins, along with in-solution tryptic digestion and proteomics analysis. These experiments were then repeated on both crude venoms and on the nanofractionated venom toxins with the addition of either the phospholipase A (PLA) inhibitor varespladib or the snake venom metalloproteinase (SVMP) inhibitor marimastat. Our results demonstrate that various African elapid venoms have an anticoagulant effect, and that this activity is significantly reduced for cobra venoms by the addition of varespladib, though this inhibitor had no effect against anticoagulation caused by mamba venoms. Marimastat showed limited capacity to reduce anticoagulation in elapids, affecting only and venom at higher doses. Proteomic analysis of nanofractionated toxins revealed that the anticoagulant toxins in cobra venoms were both acidic and basic PLAs, while the causative toxins in mamba venoms remain uncertain. This implies that while PLA inhibitors such as varespladib and metalloproteinase inhibitors such as marimastat are viable candidates for novel snakebite treatments, they are not likely to be effective against mamba envenomings.
Topics: Animals; Dendroaspis; Anticoagulants; Proteomics; Elapid Venoms; Elapidae; Snake Venoms; Phospholipases A2; Biological Assay; Metalloproteases; Antivenins
PubMed: 36355986
DOI: 10.3390/toxins14110736 -
Biosensors Aug 2022We have previously shown that human melanoma cells rapidly decrease human brain endothelial barrier strength. Our findings showed a fast mechanism of melanoma mediated...
We have previously shown that human melanoma cells rapidly decrease human brain endothelial barrier strength. Our findings showed a fast mechanism of melanoma mediated barrier disruption, which was localised to the paracellular junctions of the brain endothelial cells. Melanoma cells are known to release molecules which cleave the surrounding matrix and allow traversal within and out of their metastatic niche. Enzymatic families, such as matrix metalloproteinases (MMPs) and proteases are heavily implicated in this process and their complex nature in vivo makes them an intriguing family to assess in melanoma metastasis. Herein, we assessed the expression of MMPs and other proteases in melanoma conditioned media. Our results showed evidence of a high expression of MMP-2, but not MMP-1, -3 or -9. Other proteases including Cathepsins D and B were also detected. Recombinant MMP-2 was added to the apical face of brain endothelial cells (hCMVECs), to measure the change in barrier integrity using biosensor technology. Surprisingly, this showed no decrease in barrier strength. The addition of potent MMP inhibitors (batimastat, marimastat, ONO4817) and other protease inhibitors (such as aprotinin, Pefabloc SC and bestatin) to the brain endothelial cells, in the presence of various melanoma lines, showed no reduction in the melanoma mediated barrier disruption. The inhibitors batimastat, Pefabloc SC, antipain and bestatin alone decreased the barrier strength. These results suggest that although some MMPs and proteases are released by melanoma cells, there is no direct evidence that they are substantially involved in the melanoma-mediated disruption of the brain endothelium.
Topics: Blood-Brain Barrier; Brain; Endothelial Cells; Endothelium; Humans; Matrix Metalloproteinase 2; Melanoma; Peptide Hydrolases
PubMed: 36005056
DOI: 10.3390/bios12080660 -
Toxins Jul 2022Within Neotropical pit-vipers, the Mexican/Central-American clade consisting of , , , and is a wide-ranging, morphologically and ecologically diverse group of snakes....
Within Neotropical pit-vipers, the Mexican/Central-American clade consisting of , , , and is a wide-ranging, morphologically and ecologically diverse group of snakes. Despite their prevalence, little is known of the functional aspects of their venoms. This study aimed to fill the knowledge gap regarding coagulotoxic effects and to examine the potential of different therapeutic approaches. As a general trait, the venoms were shown to be anticoagulant but were underpinned by diverse biochemical actions. Pseudo-procoagulant activity (i.e., thrombin-like), characterized by the direct cleavage of fibrinogen to form weak fibrin clots, was evident for , , and In contrast, other venoms cleaved fibrinogen in a destructive (non-clotting) manner, with and being the most potent. In addition to actions on fibrinogen, clotting enzymes were also inhibited. FXa was only weakly inhibited by most species, but and were extremely strong in their inhibitory action. Other clotting enzymes were more widely inhibited by diverse species spanning the full taxonomical range, but in each case, there were species that had these traits notably amplified relatively to the others. and were the most potent amongst those that inhibited the formation of the prothrombinase complex and were also amongst the most potent inhibitors of Factor XIa. While most species displayed only low levels of thrombin inhibition, potently inhibited this clotting factor. The regional polyvalent antivenom produced by Instituto Picado Clodomiro was tested and was shown to be effective against the diverse anticoagulant pathophysiological effects. In contrast to the anticoagulant activities of the other species, was uniquely procoagulant through the activation of Factor VII and Factor XII. This viperid species is the first snake outside of the elapid snake clade to be shown to activate FVII and the first snake venom of any kind to activate FXII. Interestingly, while small-molecule metalloprotease inhibitors prinomastat and marimastat demonstrated the ability to prevent the procoagulant toxicity of , neither ICP antivenom nor inhibitor DMPS showed this effect. The extreme variation among the snakes here studied underscores how venom is a dynamic trait and how this can shape clinical outcomes and influence evolving treatment strategies.
Topics: Animals; Anticoagulants; Antivenins; Crotalid Venoms; Crotalinae; Elapid Venoms; Elapidae; Fibrinogen; Snake Venoms; Thrombin; Viperidae
PubMed: 35893753
DOI: 10.3390/toxins14080511 -
MBio Aug 2022The ongoing global vaccination program to prevent SARS-CoV-2 infection, the causative agent of COVID-19, has had significant success. However, recently, virus variants...
The ongoing global vaccination program to prevent SARS-CoV-2 infection, the causative agent of COVID-19, has had significant success. However, recently, virus variants that can evade the immunity in a host achieved through vaccination have emerged. Consequently, new therapeutic agents that can efficiently prevent infection from these new variants, and hence COVID-19 spread, are urgently required. To achieve this, extensive characterization of virus-host cell interactions to identify effective therapeutic targets is warranted. Here, we report a cell surface entry pathway of SARS-CoV-2 that exists in a cell type-dependent manner and is TMPRSS2 independent but sensitive to various broad-spectrum metalloproteinase inhibitors such as marimastat and prinomastat. Experiments with selective metalloproteinase inhibitors and gene-specific small interfering RNAS (siRNAs) revealed that a disintegrin and metalloproteinase 10 (ADAM10) is partially involved in the metalloproteinase pathway. Consistent with our finding that the pathway is unique to SARS-CoV-2 among highly pathogenic human coronaviruses, both the furin cleavage motif in the S1/S2 boundary and the S2 domain of SARS-CoV-2 spike protein are essential for metalloproteinase-dependent entry. In contrast, the two elements of SARS-CoV-2 independently contributed to TMPRSS2-dependent S2 priming. The metalloproteinase pathway is involved in SARS-CoV-2-induced syncytium formation and cytopathicity, leading us to theorize that it is also involved in the rapid spread of SARS-CoV-2 and the pathogenesis of COVID-19. Thus, targeting the metalloproteinase pathway in addition to the TMPRSS2 and endosomal pathways could be an effective strategy by which to cure COVID-19 in the future. To develop effective therapeutics against COVID-19, it is necessary to elucidate in detail the infection mechanism of the causative agent, SARS-CoV-2. SARS-CoV-2 binds to the cell surface receptor ACE2 via the spike protein, and then the spike protein is cleaved by host proteases to enable entry. Here, we found that the metalloproteinase-mediated pathway is important for SARS-CoV-2 infection in addition to the TMPRSS2-mediated pathway and the endosomal pathway. The metalloproteinase-mediated pathway requires both the prior cleavage of spike into two domains and a specific sequence in the second domain, S2, conditions met by SARS-CoV-2 but lacking in the related human coronavirus SARS-CoV. Besides the contribution of metalloproteinases to SARS-CoV-2 infection, inhibition of metalloproteinases was important in preventing cell death, which may cause organ damage. Our study provides new insights into the complex pathogenesis unique to COVID-19 and relevant to the development of effective therapies.
Topics: COVID-19; Humans; Metalloproteases; SARS-CoV-2; Serine Endopeptidases; Spike Glycoprotein, Coronavirus; Virus Internalization
PubMed: 35708281
DOI: 10.1128/mbio.00519-22