Novel treatment with double scope technique for disconnected pancreatic duct syndrome with external pancreatic fistula.

Background and study aims External pancreatic fistula in association with disconnected pancreatic duct syndrome is a common sequelae of the percutaneous step-up approach for infected pancreatic necrosis and is associated with significant morbidity. The present study aimed to report the initial outcome of a novel technique of two-scope guided tractogastrostomy for management of this condition. Patients and methods The present study was a retrospective analysis of data from patients with external pancreatic fistula and disconnected pancreatic duct syndrome, who underwent two-scope-guided tractogastrostomy. All the patients had a 24F or larger drain placed in the left retroperitoneum. Transgastric echo endoscopy and sinus tract endoscopy were performed simultaneously to place a stent between the gastric lumen and the sinus tract. Technical success was defined as placement of the stent between the tract and the stomach. Clinical success was defined as successful removal of the percutaneous drain without the occurrence of pancreatic fluid collection, ascites, external fistula, or another intervention 12 weeks after the procedure. Results Three patients underwent two scope-guided tractogastrostomy. Technical and clinical success were achieved in all the patients. No procedure-related side effects or recurrence occurred in any of the patients. Conclusions Two-scope-guided tractogastrostomy for treatment of external pancreatic fistula due to disconnected pancreatic duct syndrome is a feasible technique and can be further evaluated.

Cavity Catalysis of an Enantioselective Reaction under Vibrational Strong Coupling.

Strong light-matter interaction is emerging as an exciting tool for controlling chemical reactions. Here, we demonstrate an L-proline-catalyzed direct asymmetric Aldol reaction under vibrational strong coupling. Both the reactants (4-nitrobenzaldehyde and acetone) carbonyl bands are coupled to an infrared photon and react in the presence of L-proline. The reaction mixture is eluted from the cavity, and the conversion yields and enantiomeric excess are quantified using NMR and chiral HPLC. The conversion yields increase by up to 90 % in ON-resonance conditions. Interestingly, a large increase in the conversion yield does not affect the enantiomeric excess. Further control experiments were carried out by varying the temperature, and we propose that the rate-limiting step may not be the deciding factor in enantioselectivity. Whereas the formation of the enamine intermediate is modified by cavity coupling experiments. For this class of enantioselective reactions, strong coupling does not change the enantiomeric excess, possibly due to the large energy difference in chiral transition states. Strong coupling can boost the formation of enamine intermediate, thereby favouring the product yield. This gives more hope to test polaritonic chemistry based on enantioselective reactions in which the branching ratios can be controlled.

Safety and efficacy of the novel motorized power spiral enteroscopy: A single-center experience.

Objective: Evaluation and management of small bowel disorders have always been challenging due to the limitations of the existing technology. Motorized power spiral enteroscopy (PSE) is an innovative new technique that offers easier, faster, and more complete small bowel evaluation with the ability to perform therapeutics. We aimed to evaluate the safety and efficacy of PSE in various indications. Methods: Retrospective analysis of prospectively collected data of consecutive patients, who underwent PSE at a tertiary care center. Primary outcome measures were technical success rate, pan-enteroscopy rate, diagnostic and therapeutic yield, and the secondary outcomes measures were the depth of maximal insertion, median insertion time, and adverse events. Results: Fifty-four patients (mean age of 49.38 ± 16.26 years) underwent PSE for small bowel evaluation. Technical success rate was 95.55% (antegrade route) and 93.10% (retrograde route).  Pan-enteroscopy rate is 46.29% and antegrade enteroscopy to the cecum was achieved in eight patients.  Overall diagnostic and therapeutic yields were 85.18% and 30.76%, respectively. The most common findings were ulcero-stricturing lesions (51.92%) followed by vascular lesions (9.61%). The most common histopathologic diagnosis was Crohn's disease in 29.62%. Median depth of maximal insertion was 400 cm (range 150-550 cm; antegrade route) and 180 cm (range 50-350 cm; retrograde route). The median insertion time to depth of maximal insertion was 70 min (range 30-110 min; antegrade route) and 45 min (range 20-70 min; retrograde route).  PSE-associated major adverse events occurred in one patient and minor adverse events were seen in 48.14%. Conclusion: PSE is a safe and effective modality for the evaluation of small bowel disorders with a high diagnostic yield.

Is Fatty Liver Associated with Increased Mortality and Morbidity in Coronavirus Disease 2019 (COVID-19) Pneumonia?

Background: Fatty liver has been shown to be associated with severe COVID-19 disease without any impact on mortality. This is based on heterogenous criteria for defining both fatty liver as well as the severity parameters. This study aimed to study the impact of fatty liver on the mortality and severity of disease in patients with COVID-19 pneumonia. Methods: In a case control study design, patients with COVID-19 pneumonia (COVID-19 computed tomography severity index [CTSI] on high-resolution computed tomography chest of ≥1) with fatty liver (defined as liver to spleen attenuation index ≤5 on noncontrast computed tomography cuts of upper abdomen) were compared with those without fatty liver. The primary outcome measure was in-hospital mortality, and the secondary outcome measures were CTSI score, need for intensive care unit (ICU) care, need for ventilatory support, duration of ICU stay, and duration of hospital stay. Results: Of 446 patients with COVID-19 pneumonia, 289 (64.7%)admitted to Max Hospital, Saket, India, between January 1, 2021, and October 30, 2021, had fatty liver. Fifty-nine of 446 patients died during the index admission. In-hospital mortality was not different between patients with fatty liver (38 [13.24%]) or without fatty liver (21 [13.81%]). COVID-19 CTSI score was found to be significantly higher among patients who had fatty liver (13.40 [5.16] vs 11.81 [5.50]; P = 0.003). There was no difference in the requirement of ICU (94 [32%] vs 62 [39.49%]; P = 0.752), requirement of ventilatory support (27 [9.34%] vs 14 [8.91%]; P = 0.385), duration of ICU stay (8.29 [6.87] vs 7.07 [5.71] days; P = 0.208), and duration of hospital stay (10.10 [7.14] vs 10.69 [8.13] days; P = 0.430) between the groups with fatty liver or no fatty liver. Similarly, no difference was found in primary or secondary outcomes measure between the group with severe fatty liver vs mild/moderate or no fatty liver. High total leucocyte count and Fibrosis-4 (FIB-4) index were independently associated with mortality. Conclusions: Fatty liver may not be associated with increased mortality or clinical morbidity in patients who have COVID-19 pneumonia.

Matrix metalloproteinase 9 (MMP9) limits reactive oxygen species (ROS) accumulation and DNA damage in colitis-associated cancer.

Colitis-associated cancer (CAC) is a subtype of colon cancer that is driven by chronic inflammation and is prevalent in chronic ulcerative colitis patients. The development of CAC is associated with the inflammation-dysplasia-carcinoma pathway which is significantly different than adenoma-carcinoma pathway of sporadic colon cancer (CRC). Matrix Metalloproteinase 9 (MMP9) is a zinc-dependent endopeptidase against extracellular matrix (ECM) proteins expressed in the gastrointestinal tract during inflammation. We have previously shown that MMP9 plays a tumor suppressor role in CAC via "MMP9-Notch1-ARF-p53 axis" pathway. The aim of this study is to determine the role of MMP9 in maintaining genomic stability in CAC. Homozygous transgenic mice with constitutive-expression of MMP9 in the colonic epithelium (TgM9) with their wild-type littermates (WT) and stably transfected HCT116 cells with/without MMP9 were used for in vivo and in vitro experiments, respectively. As 'proof of concept' model, nanoparticles (NPs) loaded with MMP9 siRNA were used to examine the effect of MMP9 silencing in the colonic epithelium. In CAC, colonic epithelium of TgM9 mice exhibited lower amounts of reactive oxygen species (ROS), less DNA damage, and increased expression of mismatch repair genes compared to WTs. Our study showed that MMP9 expression correlates with the reduced ROS levels, decreased DNA damage, and upregulated mismatch repair pathway. This suggests that MMP9 expression is a natural biological way to suppress CAC by limiting ROS accumulation and DNA damage in the colon. Therefore, MMP9 inhibition could be deleterious for CAC patient.

Internalization of Garlic-Derived Nanovesicles on Liver Cells is Triggered by Interaction With CD98.

The mechanism of how plant-derived nanovesicles are uptaken by cells remains unknown. In this study, the garlic-derived nanovesicles (GDVs) were isolated and digested with trypsin to remove all surface proteins. Digested GDVs showed less uptake compared to undigested GDVs, confirming that the surface proteins played a role in the endocytosis. On the cell side (HepG2), interestingly, blocking the CD98 receptors significantly reduced the uptake of GDVs. During the cellular internalization of GDVs, we observed that some surface proteins of GDVs were co-localized with CD98. A total lysate of the GDV surface showed a high presence of a mannose-specific binding protein, II lectin. Blocking GDV II lectin (using mannose preincubation) highly reduced the GDV internalization, which supports that direct interaction between II lectin and CD98 plays an important role in internalization. The GDVs also exhibited in vitro anti-inflammatory effect by downregulating proinflammatory factors on the HepG2 cells. This work contributes to understanding a part of the GDV internalization process and the cellular anti-inflammatory effects of garlic.

Oral Targeted Delivery by Nanoparticles Enhances Efficacy of an Hsp90 Inhibitor by Reducing Systemic Exposure in Murine Models of Colitis and Colitis-Associated Cancer.

BACKGROUND AND AIMS: Heat shock protein 90 [Hsp90]-targeted therapy has been proposed as a promising strategy for the treatment of ulcerative colitis [UC] and colitis-associated cancer [CAC]. Systemic administration of the Hsp90 inhibitor, 17-AAG, was found to be profoundly protective in preclinical mouse models of inflammatory bowel disease [IBD]. However, the therapeutic potential of 17-AAG is limited by potential side effects associated with its systemic exposure and the modest bioavailability afforded by its oral administration. METHODS: To address these issues, we used a versatile single-step surface-functionalizing technique to prepare a 17-AAG oral delivery system using PLGA/PLA-PEG-FA nanoparticles [NP-PEG-FA/17-AAG]. RESULTS: NP-PEG-FA could be efficiently taken up by mouse Colon-26 cells and activated Raw 264.7 cells in vitro and by inflamed mouse colitis tissues in vivo. The therapeutic efficacy of orally administrated NP-PEG-FA/17-AAG was evaluated in in vivo models using dextran sulphate sodium [DSS]-induced UC and azoxymethane [AOM]/DSS-induced CAC, and the results indicated that NP-PEG-FA/17-AAG significantly alleviated the symptoms of UC and CAC. More importantly, our inflamed colitis-targeted 17-AAG nano-formulation reduced systemic exposure and provided a degree of therapeutic response similar to that obtained by systemic administration [intraperitoneal] of 17-AAG, but at a ten-fold lower dose. CONCLUSIONS: We describe a convenient, orally administrated 17-AAG delivery system that exhibits enhanced efficacy in UC and CAC therapy while reducing systemic exposure. This system may represent a promising therapeutic approach for treating UC and CAC.

Highly Biocompatible Functionalized Layer-by-Layer Ginger Lipid Nano Vectors Targeting P-selectin for Delivery of Doxorubicin to Treat Colon Cancer.

A biocompatible natural nanoparticle drug delivery system that has specific cancer-targeting function holds vast promise for cancer therapy. Here, a fucoidan/poly-lysine-functionalized layer-by-layer ginger-derived lipid vector (LbL-GDLV) was designed to target P-selectin (overexpressed by endothelial cells) and deliver a loaded drug into vascularized colon cancer. In vitro, LbL-GDLVs selectively bound to P-selectin, and the degradation of the fucoidan outer layer in a milieu similar to the cancer microenvironment resulted in rapid attachment of the cancer cell and internalization of the remaining positively charged poly-lysine coated-GDLVs. Upon enzymolysis of the poly-lysine layer inside the cancer cell, the GDLV core released loaded doxorubicin (Dox) which had the expected effects. In vivo bio-distribution studies showed that intravenously injected LbL-GDLVs exhibited enhanced accumulation at the vascularized tumor site (~ 4.4-fold higher than control vesicles), presumably due to P-selectin-mediated targeting plus the enhanced permeability and retention effect (EPR). In two animal models used to screen anti-cancer efficacy (Luc-HT-29 and HCT-116 xenografts), Dox-loaded LbL-GDLVs (LbL-GDLVs/Dox) significantly inhibited tumor growth and demonstrated much better therapeutic efficiency than free Dox. More importantly, LbL-GDLVs/Dox exhibited excellent biocompatibility, and LbL-GDLVs encapsulation largely reduced the cardiotoxicity of free Dox and avoided the notorious drug resistance of colon cells against free Dox. Together, these findings demonstrate the potential of our newly designed and highly biocompatible plant-derived LbL nanoparticles and their precise colon cancer drug delivery function.

Overexpression of CD98 in intestinal epithelium dysregulates miRNAs and their targeted proteins along the ileal villus-crypt axis.

CD98 has been implicated in the experimental model of inflammatory bowel disease. We have previously shown that IEC-specific overexpression of CD98 mediates intestinal inflammation and intestinal epithelial barrier dysfunction. Mice overexpressing CD98 exhibited severe colitis and a greater susceptibility to CAC. Here we demonstrated CD98 overexpression to dysregulate homeostatic gradient profile of miRNA and protein expression along the ileal villus-crypt axis. Using miRNA-target gene prediction module, we observed differentially expressed miRNAs to target proteins of villus and crypt profoundly affected by CD98 overexpression. We have utilized online bioinformatics as methods to further scrutinize the biological meanings of miRNA-target data. We identified significant interactions among the differentially regulated proteins targeted by altered miRNAs in Tg mice. The biological processes affected by the predicted targets of miRNAs deviate from the homeostatic functions of the miRNA-gene-protein axis of the wildtype mice. Our results emphasize a dynamic perturbation of miRNA and protein expression in villus-crypt axis contributing to potential biological consequences of altering CD98 expression. Our findings also suggest the need for a consideration of arrays of interacting biological entities (i.e. miRNAs-mRNAs, protein-protein interaction) or a combination comparison for a better understanding of the disease pathology which is necessary for an effective therapeutic target development.

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.

CD98 siRNA-loaded nanoparticles decrease hepatic steatosis in mice.

Non-alcoholic fatty liver disease (NAFLD) is characterized by excessive lipid hepatic accumulation. Here, we investigated whether a reduction of CD98 expression mediated by CD98 siRNA-loaded nanoparticles (NPs) could attenuate liver disease markers in a mouse model of NAFLD. NPs were generated using a double emulsion/solvent evaporation technique. Mice fed a high fat diet for 8 weeks to induce fatty liver were treated with vein tail injections of CD98 siRNA-loaded NPs. In vitro, HepG2 treated with CD98 siRNA-loaded NPs showed significant downregulation of CD98 leading to a significant decrease of major pro-inflammatory cytokines and markers. In vivo, CD98 siRNA-loaded NPs strongly decreased all markers of NAFLD, including the blood levels of ALT and lipids accumulation, fibrosis evidence and pro-inflammatory cytokines. In conclusion, our results indicate that CD98 appears to function as a key actor/inducer in NAFLD, and that our NPs approach may offer a new targeted therapeutic for this disease.

Critical role of PepT1 in promoting colitis-associated cancer and therapeutic benefits of the anti-inflammatory PepT1-mediated tripeptide KPV in a murine model.

BACKGROUND AND AIMS: The human intestinal peptide transporter 1, hPepT1, is expressed in the small intestine at low levels in the healthy colon and upregulated during inflammatory bowel disease. hPepT1 plays a role in mouse colitis and human studies have demonstrated that chronic intestinal inflammation leads to colorectal cancer (colitis-associated cancer; CAC). Hence, we assessed here the role of PepT1 in CAC. METHODS: Mice with hPepT1 overexpression in intestinal epithelial cells (TG) or PepT1 (PepT1-KO) deletion were used and CAC was induced by AOM/DSS. RESULTS: TG mice had larger tumor sizes, increased tumor burdens, and increased intestinal inflammation compared to WT mice. Conversely, tumor number and size and intestinal inflammation were significantly decreased in PepT1-KO mice. Proliferating crypt cells were increased in TG mice and decreased in PepT1-KO mice. Analysis of human colonic biopsies revealed an increased expression of PepT1 in patients with colorectal cancer, suggesting that PepT1 might be targeted for the treatment of CAC. The use of an anti-inflammatory tripeptide KPV (Lys-Pro-Val) transported by PepT1 was able to prevent carcinogenesis in WT mice. When administered to PepT1-KO mice, KPV did not trigger any of the inhibitory effect on tumorigenesis observed in WT mice. CONCLUSIONS: The observations that pepT1 was highly expressed in human colorectal tumor and that its overexpression and deletion in mice increased and decreased colitis associated tumorigenesis, respectively, suggest that PepT1 is a potential therapeutic target for the treatment of colitis associated tumorigenesis.

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.

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 Crohn's 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.

A(2B)AR expression in non-immune cells plays an important role in the development of murine colitis.

BACKGROUND: Adenosine, an endogenous purine nucleoside, is involved in several physiological functions. We have previously shown that A(2B)AR plays a pro-inflammatory role during colitis. AIMS: Our goals were to determine if A(2B)AR expression was necessary on immune cells/non-immune cells during colitis and if A(2B)AR was a suitable target for treating intestinal inflammation. METHODS: Wild-type and A(2B)AR knockout mice were utilized in bone marrow transplants to explore the importance of immune/non-immune A(2B)AR expression during the development of colitis. Additionally, a T-cell transfer model of colitis was used in Rag1 knockout or A(2B)AR/RAG1 double knockout recipients. Finally, A(2B)AR small interfering RNA nanoparticles were administered to dextran sodium sulphate-treated mice. RESULTS: Wild-type mice receiving wild-type or knockout bone marrow developed severe colitis after dextran sodium sulphate treatment, whereas colitis was significantly attenuated in knockout mice receiving wild-type or knockout bone marrow. Colitis induced in Rag1 knockout animals was attenuated in A(2B)AR/RAG1 double knockout recipients. Animals receiving nanoparticles exhibited attenuated parameters of colitis severity compared to mice receiving control nanoparticles. CONCLUSIONS: Our results suggest that A(2B)AR on non-immune cells plays an important role for the induction of colitis and targeting A(2B)AR expression during colitis may be useful for alleviating symptoms of intestinal inflammation.

Notch1 regulates the effects of matrix metalloproteinase-9 on colitis-associated cancer in mice.

BACKGROUND & AIMS: Inflammatory bowel disease increases the risks of colon cancer and colitis-associated cancer (CAC). Epithelial cell-derived matrix metalloproteinase (MMP)-9 mediates inflammation during acute colitis and the cleavage and activation of the transcription factor Notch1, which prevents differentiation of progenitor cells into goblet cells. However, MMP-9 also protects against the development of CAC and acts as a tumor suppressor. We investigated the mechanisms by which MMP-9 protects against CAC in mice. METHODS: C57/B6 wild-type mice were given a single dose of azoxymethane and 2 cycles of dextran sulfate sodium (DSS). Mice were also given the γ-secretase inhibitor difluorophenacetyl-l-alanyl-S-phenylglycine t-butyl ester (DAPT) or dimethyl sulfoxide (control) during each DSS cycle; they were killed on day 56. We analyzed embryonic fibroblasts isolated from wild-type and MMP-9-/- mice and HCT116 cells that were stably transfected with MMP-9. RESULTS: Wild-type mice were more susceptible to CAC following inhibition of Notch1 by DAPT, shown by increased numbers of tumors and level of dysplasia compared with controls. Inhibition of Notch1 signaling significantly reduced protein levels of active Notch1, p53, p21WAF1/Cip1, Bax-1, active caspase-3, as well as apoptosis, compared with controls. Similar results were observed in transgenic HCT116 cells and embryonic fibroblasts from MMP-9-/- mice on γ-radiation-induced damage of DNA. CONCLUSIONS: MMP-9 mediates Notch1 signaling via p53 to regulate apoptosis, cell cycle arrest, and inflammation. By these mechanisms, it might prevent CAC.

Thrombospondin-1 opens the paracellular pathway in pulmonary microvascular endothelia through EGFR/ErbB2 activation.

Thrombospondin-1 (TSP1) is a multidomain protein that contains epidermal growth factor (EGF)-like repeats that indirectly activate the EGF receptor (EGFR) and selected downstream signaling pathways. In these studies, we show that TSP1 opens the paracellular pathway in human lung microvascular endothelial cells (HMVEC-Ls) in a dose-, time-, and protein tyrosine kinase (PTK)-dependent manner. TSP1 increased tyrosine phosphorylation of proteins enriched to intercellular boundaries including the zonula adherens (ZA) proteins, vascular endothelial-cadherin, γ-catenin, and p120 catenin. In HMVEC-Ls, EGFR and ErbB2 are expressed at low levels, and both heterodimerize and tyrosine autophosphorylate in response to TSP1. Prior EGFR-selective PTK inhibition with AG1478 or ErbB2-selective PTK inhibition with AG825 protected against TSP1-induced tyrosine phosphorylation of ZA proteins and barrier disruption. Preincubation of HMVEC-Ls with an EGFR ectodomain-blocking antibody also prevented TSP1-induced opening of the paracellular pathway. Therefore, in HMVEC-Ls, TSP1 increases tyrosine phosphorylation of ZA proteins and opens the paracellular pathway, in part, through EGFR/ErbB2 activation. Surprisingly, recombinant TSP1 EGF-like repeats 1-3 and the high-affinity EGFR ligands, EGF, TGF-α, and amphiregulin, each failed to increase paracellular permeability. However, HMVEC-Ls in which EGFR was overexpressed became responsive to the EGF-like repeats of TSP1 as well as to EGF. These studies indicate that TSP1 disrupts the endothelial barrier through EGFR/ErbB2 activation although additional signals are necessary in cells with low receptor expression.