Lewins Walter

Constitutive expression of MMP9 in intestinal epithelium worsens murine acute colitis and is associated with increased levels of proinflammatory cytokine Kc

Inflammatory bowel disease (IBD), which includes ulcerative colitis and Crohns disease, is a chronic inflammatory disease associated with an increased risk for colon cancer. Matrix metalloproteinases (MMPs) are the predominant proteinases expressed in the gut mucosa during active IBD. Our laboratory has previously demonstrated that epithelial-derived MMP9 is absent in normal colonic tissue but is upregulated during IBD. In this study MMP9 transgenic mice (Tg-villin-MMP9) are generated specifically to overexpress MMP9 in intestinal epithelium to examine the role and underlying mechanism by which it modulates the pathogenesis of acute colitis. Dextran sodium sulfate (3% DSS)- and Salmonella typhimurium (S.T.)-induced colitis models were used to study gut inflammation in Tg-villin-MMP9 and wild-type littermates (WT). Colonic tissue was analyzed via Western blot, histology, myeloperoxidase (MPO) assay, and quantitative PCR. Tg-villin-MMP9 mice expressed significantly increased MMP9 mRNA and protein expression at basal level. There was a significant decrease in the goblet cells, but a significant increase in proliferation and apoptosis were observed among Tg-villin-MMP9 mice compared with WT mice. There was also a significant increase in the proinflammatory chemokine Kc among Tg-villin-MMP9 compared with WT mice. Tg-villin-MMP9 exhibited a severe inflammatory response than WT mice in both DSS- and S.T.-induced colitis models as evident by greater weight loss and higher clinical score, histological score, and MPO activity, which correlated with relative levels of Kc mRNA. MMP9 expressed by intestinal epithelial cells mediates inflammation in colitis with simultaneous increase in proinflammatory cytokine Kc.

PepT1 Expression Helps Maintain Intestinal Homeostasis by Mediating the Differential Expression of miRNAs along the Crypt-Villus Axis

In the jejunum, PepT1 is particularly enriched in the well-differentiated absorptive epithelial cells in the villi. Studies of expression and function of PepT1 along the crypt-villus axis demonstrated that this protein is crucial to the process of di/tripeptide absorption. We recently exhibited that PepT1 plays an important role in multiple biological functions, including the ability to regulate the expression/secretion of specific microRNAs (miRNAs) and the expression levels of multiple proteins. In this study, we observed that PepT1 knockout (KO) mice exhibited reduced body weight and shorten intestinal microvilli. We then examined the expression levels of various miRNAs and their target proteins along the crypt-villi axis in the jejunum of PepT1 KO mice. We found that PepT1 KO altered the distribution of miRNAs along the crypt-villus axis and changed the miRNA profiles of both villi and crypts. Using miRNA-target prediction and 2D-DIGE/mass spectrometry on villi and crypts samples, we found that ablation of PepT1 further directly or indirectly altered expression levels of certain protein targets. Collectively, our results suggest that PepT1 contributes to maintain balance of homeostasis and proper functions in the small intestine, and dysregulated miRNAs and proteins along the crypt-villus axis are highly related to this process.

Effects of tripolyphosphate on cellular uptake and RNA interference efficiency of chitosan-based nanoparticles in Raw 264.7 macrophages

Tumor necrosis factor-α (TNF-α) is a major pro-inflammatory cytokine that is mainly secreted by macrophages during inflammation. Here, we synthesized a series of N-(2-hydroxy)propyl-3-trimethyl ammonium chitosan chlorides (HTCCs), and then used a complex coacervation technique or tripolyphosphate (TPP)-assisted ionotropic gelation strategy to complex the HTCCs with TNF-α siRNA (siTNF) to form nanoparticles (NPs). The resultant NPs had a desirable particle size (210-279nm), a slightly positive zeta potential (14-22mV), and negligible cytotoxicity against Raw 264.7 macrophages and colon-26 cells. Subsequent cellular uptake tests demonstrated that the introduction of TPP to the NPs markedly increased their cellular uptake efficiency (to nearly 100%) compared with TPP-free NPs, and yielded a correspondingly higher intracellular concentration of siRNA. Critically, in vitro gene silencing experiments revealed that all of the TPP-containing NPs showed excellent efficiency in inhibiting the mRNA expression level of TNF-α (by approximately 85-92%, which was much higher than that obtained using Oligofectamine/siTNF complexes). Collectively, these results obviously suggest that our non-toxic TPP-containing chitosan-based NPs can be exploited as efficient siTNF carriers for the treatment of inflammatory diseases.

Matrix metalloproteinase MMP9 maintains epithelial barrier function and preserves mucosal lining in colitis associated cancer

In colitis associated cancer (CAC), chronic inflammation exposes the epithelial mucosal defensive lining to inflammatory mediators such as cytokines and anti-microbial peptides (AMPs) causing the dysbiosis of microbiota population and the dysregulation of immune response. Matrix Metalloproteinases (MMPs) are zinc dependent endopeptidases which mediate inflammation, tissue remodeling, and carcinogenesis. MMP9 is undetectable in healthy tissue, although highly upregulated during inflammation and cancer. We have previously shown that MMP9 plays a protective role in CAC opposite to its conventional role of acute inflammation and cancer mediator. In this study, we investigated the mechanistic role of MMP9 in preserving the epithelial mucosal integrity to suppress the progression of tumor microenvironment in CAC. We used transgenic mice constitutively expressing MMP9 in colonic epithelium (TgM9) as an in vivo model and intestinal cell line CaCo2BBE as an in vitro model. We induced CAC with three cycles of dextran sodium sulfate (DSS). We observed that MMP9 expression in colonic epithelium maintains the microbiota. We also observed that MMP9 mediates pro-inflammatory cytokine levels and AMPs but suppresses IL-22 resulting in lower levels of REG3-g and S100A8 AMPs. We also found that MMP9 maintains an efficient barrier function and the integrity of tight junctions. We also observed increased levels of mucin and intestinal trefoil factor among TgM9 mice in CAC. We also found that MMP9 expressing CaCo2BBE cells had increased expressions of EGFR and nuclear transcription factor- specificity protein 1 (Sp1). These data imply that MMP9 acts as a tumor suppressor in CAC by sustaining the epithelial mucosal integrity due to the activation of EGFR-Sp1 signaling pathway.

Epithelial derived-matrix metalloproteinase (MMP9) exhibits a novel defensive role of tumor suppressor in colitis associated cancer by activating MMP9-Notch1-ARF-p53 axis

Colitis associated cancer (CAC) is chronic inflammation driven colon cancer, prevalent among individuals with Inflammatory Bowel Disease. Matrix-metalloproteinase (MMP9) is one of the essential regulators of extra cellular matrix components. We have shown that MMP9 is protective in CAC contrary to its inflammatory role in acute-colitis. Aim of our study is to identify the mechanism of the protective role of epithelial derived-MMP9 in CAC. We used homozygous transgenic mice constitutively-expressing MMP9 in colonic-epithelium (TgM9) and wild-type (WT) littermates for in vivo experiments. Stably-transfected HCT116 with/without MMP9, and mouse embryonic-fibroblasts (WT and MMP9−/−, MEFs) were used for in vitro experiments. TgM9 mice exhibited less tumor burden, increased apoptosis, and increased expressions of active-Notch1, p53, p21WAF1/Cip1, caspase-3 and cyclin E in CAC compared to WTs. These results were supported by MEFs data. HCT116-cells overexpressing MMP9 indicated decreased cell proliferation, S-phase cell-cycle arrest and less DNA damage compared to vector. MMP9−/− mice showed attenuation of MMP9 was directly associated with p19ARF. Our study identifies the tumor suppressor role of epithelial derived-MMP9 in CAC via novel mechanistic pathway “MMP9-Notch1-ARF-p53 axis” regulating apoptosis, cell-cycle arrest and DNA damage implying, that MMP9 expression might be a natural/biological way to suppress colonic ulceration due to chronic inflammation.

Abstract 3670: Matrix metalloproteinase 9 has a novel tumor suppressive role in inflammation associated colorectal cancer by modulating intrinsic tumor microenvironment

Colitis associated cancer (CAC) is a deadly complication of inflammatory bowel disease, and a subtype of colon cancer. CAC malignancy progresses through enhanced DNA damage. Observance of microsatellite instability and chromosomal instability in non-cancer related inflammatory conditions, supports the fact that inflammation could contribute to genomic destabilization by generating reactive oxygen nitrogen species, producing double strand breaks leading to DNA damage. Thus chronic colitis can induce DNA damage and modulate DNA repair systems- components of the intrinsic pathway to modulate tumor microenvironment. Matrix metalloproteinases (MMPs) are the most prominent family of proteinases associated with almost all the junctures of inflammation and tumorigenesis. They mediate inflammation, tissue remodeling, invasion, metastasis and tumor growth. Among the 24 known human MMPs, MMP9 is very unique as it is undetectable in normal tissues but highly expressed in inflamed or ulcerated tissues. We have observed that MMP9 has a protective role in CAC, which is exclusive and contrary to its traditional role of a facilitator in acute inflammation and sporadic colon cancer. In this study we explore the molecular mechanism by which MMP9 prevents DNA damage by activating tumor suppressors and DNA repair pathways in CAC. We generated transgenic mice overexpressing MMP9 in intestinal epithelium (TgM9) and to mimic human CAC, 3 cycles of 3% DSS (dextran sodium sulfate, a colonic inflammation inducer) for 5 days, followed by two weeks of recovery was used for in vivo model. Stably-transfected HCT116 cells with/without p-EGFP-MMP9 plasmid were used for in vitro experiments. We have observed that in CAC, TgM9 mice had significantly decreased tumor incidence and dysplastic lesions and a lower histological score compared to their WT littermates. TgM9 mice exhibited increased apoptosis, protein expressions of active-Notch1, p19ARF, p53, p21WAF1/Cip1, caspase-3 and cyclinE in CAC compared to WTs. HCT116-cells overexpressing MMP9 indicated decreased cell proliferation. FACS analysis indicated S-phase cell-cycle arrest and WB analysis of phosphorylated gamma-H2AX expressions indicated less DSBs compared to vector. HCT116 cells overexpressing MMP9 indicated increased expression of MDC1 and mis-match repair proteins MLH1. Our study highlights two novel findings i) MMP9 being a secretory protein activates transcellular protein Notch1 which translocates to nucleus and activates wildtype p53 via ARF and ii) MMP9 suppresses DNA double strand breaks by activating MMR gene- MLH1 and removing the damaged cells by apoptosis and or cell cycle arrest. Our study highlights the paradox of using MMP9 inhibitors in current therapies to treat CAC patients, implying that MMP9 expression might be a natural/ biological way to suppress inflammation associated ulceration. Citation Format: Lewins Walter, Adani Pujada, Sayeed Mohammadian, Agnieszka Bialkowska, Vincent Yang, Pallavi Garg. Matrix metalloproteinase 9 has a novel tumor suppressive role in inflammation associated colorectal cancer by modulating intrinsic tumor microenvironment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3670.

Abstract 2675: MMP9 modulates ROS levels and DNA repair pathway to maintain genomic stability in colitis associated cancer

Introduction: Chronic inflammation predisposes tissues to oncogenic, and in the colon this condition is termed as colitis associated cancer (CAC). CAC is the inflammation-dysplasia-carcinoma pathway which is significantly different compared to the adenoma-carcinoma pathway of sporadic colon cancer (CRC). In CAC, chronic inflammation causes accumulation of reactive oxygen species (ROS) resulting in DNA damage that nurtures tumor microenvironment and accelerates cancer cell growth. Gut microbiota is important in regulating ROS levels, and DNA repair pathways are critical in restoring the genomic instability. Matrix Metalloproteinases (MMPs) are a family of zinc dependent endopeptidases which mediate inflammation, tissue remodeling, and tumorigenesis. MMP9 is the most unique MMP because it is undetectable in healthy tissue but highly upregulated during inflammation and cancer. We have previously shown that MMP9 plays a protective role in CAC which is opposite to its conventional role of mediator in acute inflammation and cancer. Aim: In this study, we examined that if MMP9 acts as a tumor suppressor by modulating gut microbiota and mismatch repair (MMR) genes to reinstate genomic stability in CAC. Methods: We used C57/B6 transgenic MMP9 mice which can express MMP9 under villin promoter (TgM9) and their wild type (WT) littermates. CAC was induced by azoxymethane (AOM) and dextran sodium sulfate (DSS, inflammation inducer) to assess the microbiota population. As a proof of principal model, we used MMP9 small interfering RNA (siRNA) encoded within nanoparticles and gavaged WT mice to inhibit MMP9 in colonic epithelium. Results: QPCR data showed that mRNA levels of total microbiota population were more among TgM9 mice compared to WTs in CAC. QPCR analysis also indicated increased mRNA levels for the phylum Bacteroidetes sp., Akkermansia muciniphila but decreased mRNA levels for the phylum Firmicutes sp. TgM9 mice had lower levels of ROS compared to WTs in CAC. WB data showed decrease in protein expressions of γH2AX and MDC1 while an increase in protein expressions of MLH1 and pCNA compared to WTs in CAC. WT mice treated with MMP9 siRNA loaded nanoparticles showed worsened CAC condition compared to WT mice given scrambled siRNA loaded nanoparticles as reflected by significant bodyweight loss and higher clinical score. We also observed that WT mice treated with MMP9 siRNA loaded nanoparticles showed increase in protein expression of γH2AX while decrease in MLH1 protein levels compared to control group. Conclusion: Our study has two clinically relevant outcomes that MMP9 expression in CAC: i) promotes the beneficial microbiota population and controls the ROS production, ii) restores the DNA damage via activation of the MMR pathway. This implies the contradiction of the use of targeted MMP9 inhibitors in CAC treatment therapies by showing that MMP9 expression may be a natural way to suppress CAC. Citation Format: Lewins Walter, Adani Pujada, Brandon Canup, Hamed Laroui, Pallavi Garg. MMP9 modulates ROS levels and DNA repair pathway to maintain genomic stability in colitis associated cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2675. doi:10.1158/1538-7445.AM2017-2675

Matrix metalloproteinases as potential fecal biomarkers for ulcerative colitis – a function beyond their proteolytic activity

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Tu1653 MMP9 Expression in Colonic Epithelium Is Associated With Altered Mucin Level and Immune/Chemokine Response

Background: Dendritic cells (DC) play an important role in maintaining the balance between immune activation and tolerance. We have previously demonstrated that TGF β signaling in DCs is critical for maintenance of self-tolerance and that TGF βR2 deficient DCs have increased pro-inflammatory potential. To understand the contribution of T cells in the development of autoimmunity in these mice, we used the model of adoptive T cell transfer into DC-Tgfbr2 KO on Rag-deficient backgorund and followed disease progression. Methods: DC-Tgfbr2 KO mice were bred onto a Rag1-/background and then transferred with total CD3 T cells isolated from Naive wild-type mice. Cremice transferred with CD3 T cells served as controls. Baselines were established with Cre+/Cremice on Rag1-/background injected with PBS. Mice were monitored every week for body weight changes. At the end of 8 weeks, mice were sacrificed and tissues were harvested for histological and proinflammatory cytokine analyses. Spleens and mesenteric lymph nodes (MLNs) were also harvested and analyzed for DC and T cell specific markers by flow cytometry. Results: DCTgfbr2 KO/Rag mice transferred with CD3 T cells were normal until about 5 weeks, after which they showed significant body weight loss as compared to control mice. Flow cytometry analysis of spleen and MLN cells revealed increased expression of MHCII, CD80, CD86 and CD40 on the surface of DCs from DC-Tgfbr2 KO/Rag mice suggesting the presence of more mature and activatedDCs. However the basal expression of these markers was not significantly different between control and KO mice injected with PBS. The proportion of activated T cells based on CD62L and CD44 expression was also elevated in DC-Tgfbr2 KO/Rag mice as compared to Cremice transferred with T cells. Histological analysis revealed increased monocytic and lymphocytic infiltration of the colon, liver and pancreas of DC-Tgfbr2 KO/ Rag mice transferred with T cells whereas control mice showed no significant changes. However, in contrast to DC-Tgfbr2 KO mice which spontaneously developed severe gastritis, DC-Tgfbr2 KO/Rag mice transferred with CD3 T cells did not demonstrate histological symptoms of gastritis. These changes were confirmed with qRT-PCR with increased expression of pro-inflammatory cytokines such as TNFα, IFNγ, IL1β, IL6 and IL12 only in the colon, but not the stomach of DC-Tgfbr2 KO/Ragmice transferred with T cells. Conclusions: Our studies with DC-Tgfbr2 KO/Rag mice demonstrate that maturation and increased stimulation of T cells by TGFβR deficient DCs is an early event that then contributes to the development of autoimmunity. Lack of autoimmune gastritis symptoms indicates that T cells may not be the primary contributing factor and that the role of B cells and autoantibodies in the process cannot be excluded.