Matrix Metalloprotease Inhibitor Research Articles

Evaluation of Small Molecule Inhibitors of Pseudomonas Virulence factor LasB as Non‐Traditional Immunotherapeutics

Pseudomonas aeruginosa can cause severe opportunistic pulmonary infections associated with mechanical ventilation and the genetic disease cystic fibrosis. Due to its multidrug resistance, it is a priority pathogen for therapeutic development. Inflammation during such infections, which causes destruction of lung architecture and function, is activated in part through the maturation of IL‐1β by the virulence factor LasB. Previous results in mouse models suggest that matrix metalloprotease inhibitor drugs also inhibit LasB, preventing cell death and inflammation caused by caused by P. aeruginosa. We hypothesized that other inhibitors of matrix metalloproteases, including tetracycline‐family antibiotics, could be used to target LasB. We evaluated small molecule inhibitors against LasB activation during infections of macrophages, neutrophils, and monocytes and the synergy of these inhibitors with conventional antibiotics and anti‐inflammatories. We first optimized the drug concentration by testing the ability of the various drugs to inhibit LasB at various dilutions. Using an effective concentration, we then infected the various cell types with P. aeruginosa and analyzed the impact of the inhibitors on cell death and maturation of IL‐1β in infected cells. The results indicate that several tetracycline‐family compounds inhibit LasB during P. aeruginosa infections and decrease cell death. This was true for compounds that had no antimicrobial activity yet still inhibited LasB. In addition, the results suggest that the drugs target different cell types. Together, we find that tetracyclines may have therapeutic benefits, even against resistant bacteria, due to their ability to decrease inflammation. This may serve as an effective adjunctive therapy during infection to limit tissue damage and give the immune system and antibiotics more time to respond.

Identification of allosteric fingerprints of alpha-synuclein aggregates in matrix metalloprotease-1 and substrate-specific virtual screening with single molecule insights

Alpha-synuclein (aSyn) has implications in pathological protein aggregations in neurodegeneration. Matrix metalloproteases (MMPs) are broad-spectrum proteases and cleave aSyn, leading to aggregation. Previous reports showed that allosteric communications between the two domains of MMP1 on collagen fibril and fibrin depend on substrates, activity, and ligands. This paper reports quantification of allostery using single molecule measurements of MMP1 dynamics on aSyn-induced aggregates by calculating Forster Resonance Energy Transfer (FRET) between two dyes attached to the catalytic and hemopexin domains of MMP1. The two domains of MMP1 prefer open conformations that are inhibited by a single point mutation E219Q of MMP1 and tetracycline, an MMP inhibitor. A two-state Poisson process describes the interdomain dynamics, where the two states and kinetic rates of interconversion between them are obtained from histograms and autocorrelations of FRET values. Since a crystal structure of aSyn-bound MMP1 is unavailable, binding poses were predicted by molecular docking of MMP1 with aSyn using ClusPro. MMP1 dynamics were simulated using predicted binding poses and compared with the experimental interdomain dynamics to identify an appropriate pose. The selected aSyn-MMP1 binding pose near aSyn residue K45 was simulated and analyzed to define conformational changes at the catalytic site. Allosteric residues in aSyn-bound MMP1 exhibiting strong correlations with the catalytic motif residues were compared with allosteric residues in free MMP1, and aSyn-specific residues were identified. The allosteric residues in aSyn-bound MMP1 are K281, T283, G292, G327, L328, E329, R337, F343, G345, N346, Y348, G353, Q354, D363, Y365, S366, S367, F368, P371, R372, V374, K375, A379, F391, A394, R399, M414, F419, V426, and C466. Shannon entropy was defined to quantify MMP1 dynamics. Virtual screening was performed against a site on selected aSyn-MMP1 binding poses, which showed that lead molecules differ between free MMP1 and substrate-bound MMP1. Also, identifying aSyn-specific allosteric residues in MMP1 enabled further selection of lead molecules. In other words, virtual screening needs to take substrates into account for potential substrate-specific control of MMP1 activity in the future. Molecular understanding of interactions between MMP1 and aSyn-induced aggregates may open up the possibility of degrading aggregates by targeting MMPs.

Coagulation factor protein abundance in the pre-septic state predicts coagulopathic activities that arise during late-stage murine sepsis.

SummaryBackgroundAlthough sepsis accounts for 1 in 5 deaths globally, few molecular therapies exist for this condition. The development of effective biomarkers and treatments for sepsis requires a more complete understanding of host responses and pathogenic mechanisms at early stages of disease to minimize host-driven pathology.MethodsAn alternative to the current symptom-based approach used to diagnose sepsis is a precise assessment of blood proteomic changes during the onset and progression of Salmonella Typhimurium (ST) murine sepsis.FindingsA distinct pattern of coagulation factor protein abundance was identified in the pre-septic state– prior to overt disease symptoms or bacteremia– that was predictive of the dysregulation of fibrinolytic and anti-coagulant activities and resultant consumptive coagulopathy during ST murine sepsis. Moreover, the changes in protein abundance observed generally have the same directionality (increased or decreased abundance) reported for human sepsis. Significant overlap of ST coagulopathic activities was observed in Gram-negative Escherichia coli– but not in Gram-positive staphylococcal or pneumococcal murine sepsis models. Treatment with matrix metalloprotease inhibitors prevented aberrant inflammatory and coagulopathic activities post-ST infection and increased survival. Antibiotic treatment regimens initiated after specific changes arise in the plasma proteome post-ST infection were predictive of an increase in disease relapse and death after cessation of antibiotic treatment.InterpretationAltered blood proteomics provides a platform to develop rapid and easy-to-perform tests to predict sepsis for early intervention via biomarker incorporation into existing blood tests prompted by patient presentation with general malaise, and to stratify Gram-negative and Gram-positive infections for appropriate treatment. Antibiotics are less effective in microbial clearance when initiated after the onset of altered blood proteomics as evidenced by increased disease relapse and death after termination of antibiotic therapy. Treatment failure is potentially due to altered bacterial / host-responses and associated increased host-driven pathology, providing insight into why delays in antibiotic administration in human sepsis are associated with increased risk for death. Delayed treatment may thus require prolonged therapy for microbial clearance despite the prevailing notion of antibiotic de-escalation and shortened courses of antibiotics to improve drug stewardship.FundingNational Institutes of Health, U.S. Army.

The Transactivation of the Erbb Family of Receptor Tyrosine Kinases Is Regulated by Neurotensin Receptors in Cancer

Neurotensin (NTS)-like peptides are autocrine growth factors for lung cancer. NTS is present in and secreted from lung cancer cells and binds to G protein-coupled receptors causing signal transduction and proliferation. The growth of non-small cell lung cancer (NSCLC) cells is stimulated by NTS and inhibited by SR48692, a small molecule NTSR1 antagonist. Adding NTS to NSCLC cells increases the tyrosine phosphorylation of ErbB receptor tyrosine kinases EGFR, HER2, and HER3 by transactivation. The NTSR1 regulation of EGFR, HER2, and HER3 transactivation is blocked by SR48692, specific tyrosine kinase inhibitors, and certain monoclonal antibodies. Additional agents which impair the transactivation process include PP2 (Src inhibitor), GM6001 (matrix metalloprotease inhibitor), and N-acetyl-cysteine (antioxidant). The results indicate growth stimulation caused by the adding NTS to NSCLC cells may be mediated by transactivation of ErbB RTKs.

Matrix Metalloproteinases in Human Decidualized Endometrial Stromal Cells.

Cyclic changes, such as growth, decidualization, shedding, and regeneration, in the human endometrium are regulated by the reciprocal action of female hormones, such as estradiol (E2), and progesterone (P4). Matrix metalloproteases (MMPs) and tissue inhibitors of MMPs (TIMPs) control the invasion of extravillous trophoblast cells after implantation. Several MMPs and TIMPs function in the decidua and endometrial stromal cells (ESCs). Here, we aimed to systematically investigate the changes in MMPs and TIMPs associated with ESC decidualization. We evaluated the expression of 23 MMPs, four TIMPs, and four anti-sense non-coding RNAs from MMP loci. Primary ESC cultures treated with E2 + medroxyprogesterone acetate (MPA), a potent P4 receptor agonist, showed significant down-regulation of MMP3, MMP10, MMP11, MMP12, MMP20, and MMP27 in decidualized ESCs, as assessed by quantitative reverse transcription PCR. Further, MMP15 and MMP19 were significantly upregulated in decidualized ESCs. siRNA-mediated silencing of Heart and Neural Crest Derivatives Expressed 2 (HAND2), a master transcriptional regulator in ESC decidualization, significantly increased MMP15 expression in untreated human ESCs. These results collectively indicate the importance of MMP15 and MMP19 in ESC decidualization and highlight the role of HAND2 in repressing MMP15 transcription, thereby regulating decidualization.

Proteomics and Risk of Atrial Fibrillation in Older Adults (From the Atherosclerosis Risk in Communities [ARIC] Study)

Plasma proteomic profiling may aid in the discovery of novel biomarkers upstream of the development of atrial fibrillation (AF). We used data from the Atherosclerosis Risk in Communities study to examine the relation between large-scale proteomics and incident AF in a cohort of older-aged adults in the United States. We quantified 4,877 plasma proteins in Atherosclerosis Risk in Communities participants at visit 5 (2011-2013) using an aptamer-based proteomic profiling platform. We used Cox proportional hazards models to assess the association between protein levels and incident AF, and explored relation of selected protein biomarkers using annotated pathway analysis. Our study included 4,668 AF-free participants (mean age 75 ± 5 years; 59% female; 20% Black race) with proteomic measures. A total of 585 participants developed AF over a mean follow-up of 5.7 ± 1.7 years. After adjustment for clinical factors associated with AF, N-terminal pro-B-type natriuretic peptide (NT-proBNP) was associated with the risk of incident AF (hazard ratio, 1.82; 95% CI, 1.68 to 1.98; p, 2.91×10-45 per doubling of NT-proBNP). In addition, 36 other proteins were also significantly associated with incident AF after Bonferroni correction. We further adjusted for medication use and estimated glomerular filtration rate and found 17 proteins, including angiopoietin-2 and transgelin, that remained significantly associated with incident AF. Pathway analyses implicated the inhibition of matrix metalloproteases as the top canonical pathway in AF pathogenesis. In conclusion, using a large-scale proteomic platform, we identified both novel and established proteins associated with incident AF and explored mechanistic pathways of AF development.

TGF-beta pathway inhibition as the therapeutic acceleration of diabetic bone regeneration.

Bone regeneration and fracture healing are impaired in diabetic patients due to defective functions of associated cells. Thus, the search for molecular causes and new treatment strategies are of particular clinical relevance. We investigated the gene expression profile of bones from type 2 diabetic (db- /db- ) mice and wild-type (wt) mice by comparative microarray analyses before and after placing tibial defects and examined the expression of several osteogenesis- and osteoclastogenesis-related markers by quantitative real-time polymerase chain reaction. In regenerating wt bones, pathways related to, for example, inhibition of matrix metalloproteases were activated, whereas in db- /db- bones activation of pathways related to, for example, osteoarthritis, transforming growth factor-beta (Tgfb), or hypoxia-inducible factor 1a were detected during regeneration. We defined the Tgfb pathway as a potential therapeutic target and locally applied a single dose (0.5 µg) of the Tgfb 1, 2, and 3 neutralizing antibody 1D11 on tibial defects in db- /db- mice (n = 7). Seven days postoperation, histological and immunohistochemical stainings were performed. Decreased bone regeneration, osteogenic differentiation, osteoclast invasion, and angiogenesis in db- /db- mice were significantly restored by local 1D11 application in comparison to the phosphate-buffered saline controls. Thus, local treatment of db- /db- bony defects with Tgfb neutralizing antibody 1D11 might be considered a good candidate for the successful acceleration of bone regeneration.

Modulation of gene expression on a transcriptome-wide level following human neural stem cell transplantation in aged mouse stroke brains

IntroductionNeural stem cell (NSC) transplantation offers great potential for treating ischemic stroke. Clinically, ischemia followed by reperfusion results in robust cerebrovascular injury that upregulates proinflammatory factors, disrupts neurovascular units, and causes brain cell death. NSCs possess multiple actions that can be exploited for reducing the severity of neurovascular injury. Our previous studies in young adult mice showed that human NSC transplantation during the subacute stage diminishes stroke pathophysiology and improves behavioral outcome. MethodsWe employed a well-established and commonly used stroke model, middle cerebral artery occlusion with subsequent reperfusion (MCAO/R). Here, we assessed the outcomes of hNSC transplantation 48 h post-MCAO (24 h post-transplant) in aged mouse brains in response to stroke because aging is a crucial risk factor for cerebral ischemia. Next, we tested whether administration of the integrin α5β1 inhibitor, ATN-161, prior to hNSC transplantation further affects stoke outcome as compared with NSCs alone. RNA sequencing (RNA-seq) was used to assess the impact of hNSC transplantation on differentially expressed genes (DEGs) on a transcriptome-wide level. ResultsHere, we report that hNSC-engrafted brains with or without ATN-161 showed significantly reduced infarct size, and attenuated the induction of proinflammatory factors and matrix metalloproteases. RNA-seq analysis revealed DEGs and molecular pathways by which hNSCs induce a beneficial post-stroke outcome in aged stroke brains. 811 genes were differentially expressed (651 downregulated and 160 upregulated) in hNSC-engrafted stroke brains. Functional pathway analysis identified enriched and depleted pathways in hNSC-engrafted aged mouse stroke brains. Depletion of pathways following hNSC-engraftment included signaling involving neuroinflammation, acute phase response, leukocyte extravasation, and phagosome formation. On the other hand, enrichment of pathways in hNSC-engrafted brains was associated with PPAR signaling, LXR/RXR activation, and inhibition of matrix metalloproteases. Hierarchical cluster analysis of DEGs in hNSC-engrafted brains indicate decreased expression of genes encoding TNF receptors, proinflammatory factors, apoptosis factors, adhesion and leukocyte extravasation, and Toll-like receptors. ConclusionsOur study is the first to show global transcripts differentially expressed following hNSC transplantation in the subacute phase of stroke in aged mice. The outcome of our transcriptome study would be useful to develop new therapies ameliorating early-stage stroke injury.

Articular Cartilage Fragmentation Improves Chondrocyte Migration by Upregulating Membrane Type 1 Matrix Metalloprotease.

This study was undertaken to elucidate the mechanism of improved chondrocyte migration after juvenile articular cartilage fragmentation. In vitro organ culture with rabbit cartilage fragments and cell culture with rabbit chondrocytes were performed. In part A, minced juvenile cartilage fragments (~0.5 × 0.5 × 0.5 mm) from rabbits, planted in gelatin sponge and fibrin glue, were cultured for 2, 4, or 6 weeks in vitro and compared with the cartilage chunks (~4 × 4 × 1 mm) and membrane type 1 matrix metalloprotease (MT1-MMP) inhibitor groups. Chondrocyte outgrowth was evaluated on histology and confocal laser scanning microscopy. MT1-MMP expression was compared between the cartilage fragment group and the cartilage chunks group. In part B, articular chondrocytes were harvested from juvenile rabbits, MT1-MMP was transfected into the cells, and cell migration was evaluated using the Transwell and wound healing tests. The histology and confocal microscopy results revealed that cell accumulation occurred at the edge of cartilage fragments, and outgrowth was better in the cartilage fragment group than those in the cartilage chunks group. Similar results were observed for MT1-MMP expression. After MT1-MMP inhibition, cells did not accumulate at the edge of the cartilage fragments, and chondrocyte outgrowth did not occur. Furthermore, overexpression of MT1-MMP enhanced the migration of articular chondrocytes. Juvenile articular cartilage fragmentation improved chondrocyte migration by upregulating MT1-MMP.

Extended Cheese Whey Fermentation Produces a Novel Casein-Derived Antibacterial Polypeptide That Also Inhibits Gelatinases MMP-2 and MMP-9.

Our previous works produced a whey fermentation methodology that yielded antibacterial activity and potential inhibition of matrix metalloproteases (MMP)-2 and -9. Here, we evaluated if these activities were due to fermentation-produced peptides. Prolonged fermentation was carried out in the presence of our specific lactic acid bacteria (LAB) consortium. LAB fermentation yielded a total of 11 polypeptides, which were predominantly produced after 6 days of fermentation. One which was derived from beat casein presented a particularly high antibacterial activity against food pathogenic bacteria and was more effective than standard food disinfectants. This polypeptide was further studied and was also found to be active against several strains of pathogenic bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), in a dose-dependent manner. It also inhibited MMP-2 and MMP-9 whilst reducing HT29 cancer cell migration in vitro. Overall, this novel whey-derived polypeptide presents dual antibacterial and anti-inflammatory activity, revealing a strong potential to be used in functional foods or as a nutraceutical. Its identification and further characterization can open novel perspectives in the field of preventive/curative diets related to gut microbiota, gut inflammation, and cancer prevention, particularly if used in in vivo studies.

Bio-O1-09 - Secretomic and proteomic analysis of cutaneous T cell lymphoma-associated fibroblasts

<h3>Introduction:</h3> Cancer-associated fibroblasts (CAFs), a component of tumor microenvironment, play a key role in oncogenesis and promote growth, invasion, metastasis and chemo-resistance of tumor cells. Our previous RNAseq results brought evidence of the contribution of dermal fibroblasts obtained from cutaneous T-cell lymphoma (CTCL) lesions to support tumor growth. Here, we further go on by analyzing the alteration of protein production by dermal fibroblasts obtained from CTCL lesions versus control fibroblasts. <h3>Materials and methods:</h3> Secretome and proteome was performed on fibroblast primary cultures skin biopsies of 6 cutaneous T-cell lymphoma (CTCL[GD1]) versus fibroblasts from healthy mammary skin controls (age-paired). The fibroblasts of the two groups were studied at the 3rd passage, after stimulation or not with 5 ng/ml TGF-β for 24 h. The supernatants and lysates collected underwent enzymatic digestion with trypsin before peptide analysis by HLPC/Mass spectrometry <h3>Results:</h3> The comparison of the secretomes from the CTCL groups (± TGF-ß) and the control donors showed a global significant differential expression (fold >2, p-value <0.05) of 55 proteins. Concerning the proteome, a differential expression of 92 proteins was quantified in control condition and of 146 proteins in stimulated condition (fold >2, p-value <0.05). The majority of proteins differing between the CTCL patient and control groups were linked to components of the cytoskeleton of fibroblasts. Some sets of molecules were associated with the regeneration of the extracellular matrix and the maintenance of tissue inflammation. The global analysis highlighted 12 main networks and as in previous RNAseq analysis, several pathway-associated sets including "Inhibition of Matrix Metalloproteases" or "Leukocyte Extravasation Signaling" were detected. <h3>Conclusion:</h3> The present data demonstrate the potent proteomic alteration of CTCL-associated dermal fibroblasts, confirming the involvement of mesenchymal cells on the behavior of CTCL.

The HIV-1 accessory protein Nef increases surface expression of the checkpoint receptor Tim-3 in infected CD4+ T cells

Prolonged immune activation drives the upregulation of multiple checkpoint receptors on the surface of virus-specific T cells, inducing their exhaustion. Reversing HIV-1-induced T cell exhaustion is imperative for efficient virus clearance; however, viral mediators of checkpoint receptor upregulation remain largely unknown. The enrichment of checkpoint receptors on T cells upon HIV-1 infection severely constrains the generation of an efficient immune response. Herein, we examined the role of HIV-1 Nef in mediating the upregulation of checkpoint receptors on peripheral blood mononuclear cells. We demonstrate that the HIV-1 accessory protein Nef upregulates cell surface levels of the checkpoint receptor T-cell immunoglobulin mucin domain-3 (Tim-3) and that this is dependent on Nef's dileucine motif LL164/165. Furthermore, we used a bimolecular fluorescence complementation assay to demonstrate that Nef and Tim-3 form a complex within cells that is abrogated upon mutation of the Nef dileucine motif. We also provide evidence that Nef moderately promotes Tim-3 shedding from the cell surface in a dileucine motif–dependent manner. Treating HIV-1-infected CD4+ T cells with a matrix metalloprotease inhibitor enhanced cell surface Tim-3 levels and reduced Tim-3 shedding. Finally, Tim-3-expressing CD4+ T cells displayed a higher propensity to release the proinflammatory cytokine interferon-gamma. Collectively, our findings uncover a novel mechanism by which HIV-1 directly increases the levels of a checkpoint receptor on the surface of infected CD4+ T cells.

Expression of cardiac copper chaperone encoding genes and their correlation with cardiac function parameters in goats

Copper deficiency (CuD) is a common cause of oxidative cardiac tissue damage in ruminants. The expression of copper chaperone (Cu-Ch) encoding genes enables an in-depth understanding of copper-associated disorders, but no previous studies have been undertaken to highlight Cu-Ch disturbances in heart tissue in ruminants due to CuD. The current study aimed to investigate the Cu-Ch mRNA expression in the heart of goats after experimental CuD and highlight their relationship with the cardiac measurements. Eleven male goats were enrolledin this study and divided into the control group (n = 4) and CuD group (n = 7), which received copper-reducing dietary regimes for 7months. Heart function was evaluated by electrocardiography and echocardiography, and at the end of the experiment, all animals were sacrificed and the cardiac tissues were collected for histopathology and quantitative mRNA expression by real-time PCR. In the treatment group, cardiac measurements revealed increased preload and the existence of cardiac dilatation, and significant cardiac tissue damage by histopathology. Also, the relative mRNA expression of Cu-Ch encoding genes; ATP7A, CTr1, LOX, COX17, as well as ceruloplasmin (CP), troponin I3 (TNNI3), glutathione peroxidase (GPX1), and matrix metalloprotease inhibitor (MMPI1) genes were significantly down-regulated in CuD group. There was a significant correlation between investigated genes and some cardiac function measurements; meanwhile, a significant inverse correlation was observed between histopathological score and ATP7B, CTr1, LOX, and COX17. In conclusion, this study revealed that CuD induces cardiac dilatation and alters the mRNA expression of Cu-Ch genes, in addition to TNNI3, GPX1, and MMPI1 that are considered key factors in clinically undetectable CuD-induced cardiac damage in goats which necessitate further studies for feasibility as biomarkers.

Abstract 3159: Targeting the TGFΒ/TIMP-1/2 axes to re-educate pro-inflammatory/pro angiogenic NK cells in cancer patients

Abstract Tissue inhibitors of metalloproteases (TIMPs) have been reported to exert dual roles in cancer, based on the different cancer type and interactions with the tumor microenvironments. As inhibitors of Matrix metalloproteases (MMP) they downregulate invasion, metastasis and angiogenesis. However, TIMP1 levels have been found increased in patients with different types of cancer. Natural Killer cells have been found to be phenotypically and functionally compromised in several solid and hematological cancers. We were the first in characterizing NK pro-angiogenic phenotype in the peripheral blood and tumor tissues of patients with Non-small Cell Lung cancer (NSCLC), colorectal cancers (CRC) and in pleural effusion (PE) of patients with metastatic cancers. Also, we found that pro-angiogenic NK cells in the peripheral blood of colorectal cancer patients release of TIMP-1 and TIMP-2, in a STAT3/STAT5-independent manner. Here, we investigated the effects of TIMP-1 and TIMP-2 recombinant proteins, using a TGFβ-mediated NK cell polarization model, in vitro. The effects of TGFβ, alone or in combination with TIMP-1 or TIMP-2 peptides, was determined by multicolor flow cytometry for surface antigens and cytokine productions, on cytolytic NK cells isolated from healthy subjects. Commercially available antibody membrane arrays was used to determine the modulation of the secretome, on FACS-sorted NK cells, from healthy subjects, following 72 hours of exposure to TGFβ, alone or in combination with TIMP-1 or TIMP-2 peptide. We found that NK cell from healthy donors, exposed to TGFβ, acquire the CD56brightCD9+CD49a+ decidual-like phenotype, together with decreased levels of the activation marker NKG2D. Administration of TIMP1 or TIMP2 peptides was effective in interfering with TGFβ-induced NK cell polarization towards the CD56brightCD9+CD49a+ decidual-like phenotype and in restoration of the levels of the NKG2D activation marker. We also observed that TGFβ-polarized NK cell, when co-treated with TIMP1 or TIMP2, exhibit a trend toward decreased production of VEGF and increased production of the anti-tumor cytokines IFNγ and TNFα Our results suggest a potential role of TIMPs in contrasting the angiogenic switch in NK cells and suggest a possible use of TIMPs peptides in the re-education of anergic/pro-angiogenic NK cells in cancer patients. Citation Format: Matteo Gallazzi, Antonino Bruno, Douglas M. Noonan, William G. Stetler-Stevenson, Adriana Albini. Targeting the TGFΒ/TIMP-1/2 axes to re-educate pro-inflammatory/pro angiogenic NK cells in cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3159.

Differential Proinflammatory Responses to Aspergillus fumigatus by Airway Epithelial Cells In Vitro Are Protease Dependent.

Aspergillus fumigatus is an important human respiratory mould pathogen. In addition to a barrier function, airway epithelium elicits a robust defence against inhaled A. fumigatus by initiating an immune response. The manner by which A. fumigatus initiates this response and the reasons for the immunological heterogeneity with different isolates are unclear. Both direct fungal cell wall–epithelial cell interaction and secretion of soluble proteases have been proposed as possible mechanisms. Our aim was to determine the contribution of fungal proteases to the induction of epithelial IL-6 and IL-8 in response to different A. fumigatus isolates. Airway epithelial cells were exposed to conidia from a low or high protease-producing strain of A. fumigatus, and IL-6 and IL-8 gene expression and protein production were quantified. The role of proteases in cytokine production was further determined using specific protease inhibitors. The proinflammatory cytokine response correlated with conidia germination and hyphal extension. IL-8 induction was significantly reduced in the presence of matrix metalloprotease or cysteine protease inhibitors. With a high protease-producing strain of A. fumigatus, IL-6 release was metalloprotease dependent. Dectin-1 antagonism also inhibited the production of both cytokines. In conclusion, A. fumigatus-secreted proteases mediate a proinflammatory response by airway epithelial cells in a strain-dependent manner.

An Integrated Analysis of the Response of Colorectal Adenocarcinoma Caco-2 Cells to X-Ray Exposure.

Colorectal cancer is among the three top cancer types for incidence and the second in terms of mortality, usually managed with surgery, chemotherapy and radiotherapy. In particular, radiotherapeutic concepts are crucial for the management of advanced rectal cancer, but patients’ survival remains poor, despite advances in treatment modalities. The use of well-characterized in vitro cell culture systems offers an important preclinical strategy to study mechanisms at the basis of cell response to therapeutic agents, including ionizing radiation, possibly leading to a better understanding of the in vivo response to the treatment. In this context, we present an integrated analysis of results obtained in an extensive measurement campaign of radiation effects on Caco-2 cells, derived from human colorectal adenocarcinoma. Cells were exposed to X-rays with doses up to 10 Gy from a radiotherapy accelerator. We measured a variety of endpoints at different post-irradiation times: clonogenic survival after ~ 2 weeks; cell cycle distribution, cell death, frequency of micronucleated cells and atypical mitoses, activation of matrix metalloproteases (MMPs) and of different proteins involved in DNA damage response and cell cycle regulation at earlier time points, up to 48 h post-exposure. Combined techniques of flow cytometry, immunofluorescence microscopy, gelatin zymography and western blotting were used. For selected endpoints, we also addressed the impact of the irradiation protocol, comparing results obtained when cells are plated before irradiation or first-irradiated and then re-plated. Caco-2 resistance to radiation, previously assessed up to 72 h post exposure in terms of cell viability, does not translate into a high clonogenic survival. Survival is not affected by the irradiation protocol, while endpoints measured on a shorter time frame are. Radiation mainly induces a G2-phase arrest, confirmed by associated molecular markers. The activation of death pathways is dose- and time-dependent, and correlates with a dose-dependent inhibition of MMPs. Genomic aberrations are also found to be dose-dependent. The phosphorylated forms of several proteins involved in cell cycle regulation increase following exposure; the key regulator FoxM1 appears to be downregulated, also leading to inhibition of MMP-2. A unified molecular model of the chain of events initiated by radiation is proposed to interpret all experimental results.

Glucocorticoid Mediated Transcriptional Activity in Human Corneal Epithelial Cells Lacking the Glucocorticoid Receptor

The cornea is the dome-shaped transparent outermost layer of the eye, forming a physical barrier to protect the internal structures of the eye. Glucocorticoids are a mainstay in the treatment of ophthalmic diseases for their anti-inflammatory and anti-angiogenic properties. However, high doses or chronic use of glucocorticoid therapy can lead to vision-impairing effects such as increase in intraocular pressure and the formation of cataracts. The exact signaling pathways responsible for these undesirable effects of glucocorticoid use is poorly understood. One of the major molecular actions of glucocorticoids is to regulate transcription through its cognate nuclear receptor, the glucocorticoid receptor. We have previously reported the effect of glucocorticoids on global gene expression and their role in wound healing and barrier function in immortalized human corneal epithelial cells (HCE-T). In the current study, we knocked down glucocorticoid receptor using siRNA (GRKD) to determine the function of the glucocorticoid receptor in HCE-T cells. Successful knockdown of glucocorticoid receptor was confirmed by RT-PCR and immunoblot experiments. Genome-wide microarray analysis was performed and an FDR adjusted p value less than 0.01 was considered the cut off to create the list of differentially expressed genes (DEGs). Comparison of GRKD cells to HCE-T cells expressing endogenous glucocorticoid receptor (referred as NTC for No Target Control siRNA) revealed that expression of 2150 genes was altered in HCE-T cells when glucocorticoid receptor was knocked down, indicating that glucocorticoid receptor in corneal epithelial cells regulates a large cohort of genes. Inhibition of matrix metalloproteases, granulocyte adhesion and diapedesis, cyclins and cell cycle regulation were the top canonical pathways predicted by Ingenuity Pathway Analysis (IPA) to be altered in GRKD cells. In a 6-hour treatment with dexamethasone (Dex), a synthetic glucocorticoid, or with vehicle as a control, NTC cells had 2039 Dex-regulated genes, while Dex was still able to regulate 1087 genes in GRKD cells. Of these 1087 genes, 895 genes were uniquely regulated by Dex in GRKD cells suggesting that glucocorticoids might be signaling through another receptor in corneal epithelial cells. The top canonical pathways predicted to be altered by Dex in GRKD cells included PI3K/ATK Signaling, ERK5 Signaling, Prostrate Cancer Signaling, Aldosterone Signaling in Epithelial Cells, and PPAR signaling. These findings suggest that Dex could regulate large cohorts of genes through other nuclear receptors in corneal epithelial cells. Given the wide use of ophthalmic Dex in forms including eyedrops, ointments, gels, and implants, it is of clinical significance to understand the molecular actions of synthetic glucocorticoids since they appear to be ligands for multiple nuclear receptors in ocular cells and tissues.

The mast cell activator Compound 48/80 increases bladder compliance through matrix metalloprotease release

Bladder wall stiffness and elasticity is essential to its storage and voiding functions. In addition to smooth muscle contractility, the integrity of the extracellular matrix plays a crucial role in maintaining the optimal balance between wall stiffness and compliance as the bladder distends. Lower urinary tract symptoms (e.g., bladder overactivity and fibrosis) can be caused by mast cell activation and inflammation, which increases wall stiffness and matrix remodeling. Thus, we hypothesize that mast cell activation increases muscle contractility and decreases compliance. Whole mouse bladders were dissected and mounted in a custom-designed pentaplanar reflected image macroscopy (PRIM) system for simultaneous measurement of the following parameters during ex vivo bladder filling: intravesical pressure, bladder volume, wall deformation, mechanical stress, and mechanical stretch. Bladders were filled at a constant rate (30 µl/min) until intravesical pressure reached 25 mmHg, at which point bladders were emptied and allowed to recover for approximately 10 minutes prior to the next fill cycle. During filling, baseline bladder pressure remained relatively low over a large infused volume (~350 µL), after which pressure increased rapidly. Transient contractions began on the lateral sides of the bladder wall near the trigone, expanded upwards as the bladder filled, and corresponded to transient increases in intravesical pressure (0-2 mmHg). Using the pressure-volume curves and associated image data, we next calculated the bladder wall compliance (defined as the stress vs stretch relationship of the bladder wall). As with pressure, wall stress was relatively low over a large change in stretch, implying that bladder wall was highly compliant until larger infused volumes. Subsequent exposure to the mast cell activator Compound 48/80 (10 µg/ml) significantly increased the amplitude of transient pressure events but decreased wall stress per change in stretch, indicating a paradoxical increase in compliance. Incubation with the matrix metalloprotease inhibitor doxycycline (10 µM) prior to addition of compound 48/80 prevented this increase in compliance without preventing the increased amplitude of transient pressure events. These findings suggest that Compound 48/80 activates bladder mast cells to release at least two things: (1) matrix metalloproteases that rapidly degrade the extracellular matrix and increase bladder wall compliance; and (2) compounds that increase smooth muscle excitability. Thus, both decreases in stiffness and increases in smooth muscle excitability may accompany inflammatory bladder pathologies such as diabetes and interstitial cystitis.

Mechanism of hepatocyte mitochondrial NDUFA13 deficiency-induced liver fibrogenesis: the role of abnormal hepatic stellate cell activation

To investigate the role of hepatocyte mitochondrial NDUFA13 loss in the liver fibrogenesis in mice and explore the possible mechanisms. We used liver-specific NDUFA13 heterozygous knockout mouse models (NDUFA13fl/-; Alb-Cre) established previously by intercrossing NDUFA13fl/fl and Alb-Cre mice, with their littermate control NDUFA13fl/fl mice as the control (n=8). The mice were euthanized at the age of 4 weeks and 2 years, and the liver tissues were collected for HE and Masson staining to observe the pathological changes and fibrosis phenotypes. Western blotting was performed to detect the expression of NDUFA13 protein in the liver tissues, and the infiltration of F4/80+ macrophages and the expressions of TGF-β1, TNF-α and IL-1β were analyzed by immunofluorescence assay. The expression levels of α-SMA, matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of matrix metalloproteases 1 (TIMP-1), collagen-Ⅰ and collagen-Ⅲ were assayed by immunohistochemistry. HE and Masson staining showed obvious inflammatory infiltration but no significant fibrosis in the liver tissues of 4-week-old NDUFA13fl/- mice, but severe liver damage with massive fibrosis was observed in 2-year-old NDUFA13fl/- mice. NDUFA13 expression in 2-year-old NDUFA13fl/- mice markedly decreased compared with that in the control NDUFA13fl/fl mice as shown by Western blotting (P < 0.05). Immunohistochemistry showed obvious infiltration of F4/80+ macrophages in the liver tissue with a large amount of TGF-β1 production (P < 0.05) and TNF-α and IL-1β secretions in NDUFA13fl/- mice (P < 0.05). NDUFA13 knockout obviously promoted α-SMA expression (P < 0.05) and collagen-Ⅰ and collagen-Ⅲ deposition (P < 0.05) while significantly decreased MMP-9 and increased TIMP-1 expression in the liver (P < 0.05). Hepatocytes-specific NDUFA13 deficiency can trigger spontaneous and chronic liver fibrosis phenotypes in mice probably in association with abnormal activation of hepatic stellate cells induced by macrophages and inflammatory factors.

Multilevel proteomics reveals host perturbations by SARS-CoV-2 and SARS-CoV.

The emergence and global spread of SARS-CoV-2 has resulted in the urgent need for an in-depth understanding of molecular functions of viral proteins and their interactions with the host proteome. Several individual omics studies have extended our knowledge of COVID-19 pathophysiology1-10. Integration of such datasets to obtain a holistic view of virus-host interactions and to define the pathogenic properties of SARS-CoV-2 is limited by the heterogeneity of the experimental systems. Here we report a concurrent multi-omics study of SARS-CoV-2 and SARS-CoV. Using state-of-the-art proteomics, we profiled the interactomes of both viruses, as well as their influence on the transcriptome, proteome, ubiquitinome and phosphoproteome of a lung-derived human cell line. Projecting these data onto the global network of cellular interactions revealed crosstalk between the perturbations taking place upon infection with SARS-CoV-2 and SARS-CoV at different levels and enabled identification of distinct and common molecular mechanisms of these closely related coronaviruses. The TGF-β pathway, known for its involvement in tissue fibrosis, was specifically dysregulated by SARS-CoV-2 ORF8 and autophagy was specifically dysregulated by SARS-CoV-2 ORF3. The extensive dataset (available at https://covinet.innatelab.org ) highlights many hotspots that could be targeted by existing drugs and may be used to guide rational design of virus- and host-directed therapies, which we exemplify by identifying inhibitors of kinases and matrix metalloproteases with potent antiviral effects against SARS-CoV-2.