There is mounting evidence that perturbations in endoplasmic reticulum (ER) function play a key role in the pathogenesis of a broad range of diseases. We have examined the ability of ER stress to modulate leukocyte binding to colonic and aortic smooth muscle cells. In vitro, control smooth muscle cells bind few leukocytes, but treatment with compounds that induce ER stress, including tunicamycin, A23187, and thapsigargin, promotes leukocyte binding. Likewise, dextran sulfate, another agent capable of inducing ER stress and promoting inflammation in vivo, strongly induces leukocyte adhesion. The bound leukocytes are released by hyaluronidase treatment, indicating a critical role for hyaluronan-containing structures in mediating leukocyte binding. Affinity histochemistry demonstrated that hyaluronan accumulates and is present in cable-like structures in the treated, but not the untreated, cultures and that these structures serve as attachment sites for leukocytes. Hyaluronan-rich regions of both murine and human inflamed colon contain numerous cells that stain intensely for ER-resident chaperones containing the KDEL sequence, demonstrating a relationship between ER stress and hyaluronan deposition in vivo. These results indicate that ER stress may contribute to chronic inflammation by forming a hyaluronan-rich extracellular matrix that is conducive to leukocyte binding.

In addition to mesenchymal cells, endothelial cells may contribute to fibrosis through the process of endothelial-to-mesenchymal transition (EndoMT). We investigated whether human intestinal microvascular endothelial cells (HIMEC) undergo EndoMT and contribute to fibrosis in human and experimental inflammatory bowel disease (IBD). HIMEC were exposed to TGF-β1, IL-1β, and TNF-α or supernatants of lamina propria mononuclear cells (LPMC) and evaluated for morphological, phenotypic, and functional changes compatible with EndoMT. Genomic analysis was used to identify transcription factors involved in the transformation process. Evidence of in situ and in vivo EndoMT was sought in inflamed human and murine intestine. The combination of TGF-β1, IL-1β and TNF-α, or activated LPMC supernatants induced morphological and phenotypic changes consistent with EndoMT with a dominant effect by IL-1. These changes persisted after removal of the inducing agents and were accompanied by functional loss of acetylated LDL-uptake and migratory capacity, and acquisition of de novo collagen synthesis capacity. Sp1 appeared to be the main transcriptional regulator of EndoMT. EndoMT was detected in microvessels of inflammatory bowel disease (IBD) mucosa and experimental colonic fibrosis of Tie2-green fluorescent protein (GFP) reporter-expressing mice. In conclusion, chronic inflammation induces transdifferentiation of intestinal mucosal microvascular cells into mesenchymal cells, suggesting that the intestinal microvasculature contributes to IBD-associated fibrosis through the novel process of EndoMT.

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Mice supplied with dextran sulfate sodium (DSS) in their drinking water reproducibly develop colitis in a concentrationand time-dependent manner.11 DSS compromises the epithelial barrier and the pathological effects are first observed in the distal colon in this model, where the bacterial burden is highest. This is important because of the well-established notion that bacteria play a key role in initiating and perpetuating inflammation in human and experimental IBD. [12][13][14][15] HA has been implicated as a key player in leukocyte recruitment during inflammation both directly and indirectly through its association with the CD44 receptor. In vivo, CD44-HA interactions have been shown to be crucial for inflammatory lymphocyte infiltration, and inflammation can be abrogated by either CD44 antibody or hyaluronidase treatment in a variety of experimental models. [16][17][18] In vitro, inflammatory cytokines and LPS have been shown to induce leukocyte-adhesive HA production specifically by small vessel endothelium. 7 In this article we demonstrate that HA deposition is dramatically altered in gut inflammation and, more important, precedes the influx of inflammatory cells. Additionally, we identify HA in intestinal small vessels and demonstrate the ability of human mucosal endothelium to make leukocyte-adhesive HA in response to TNF-a, a key cytokine in the pathogenesis of IBD. We also show that IBD mucosal endothelium produces more leukocyteadhesive HA than control cells. Materials and Methods DSS murine model of colitisMale C57/Bl6 mice were purchased from Jackson Lab and conventionally housed. All husbandry and treatments were conducted according to Institutional Animal Care and Use Committee (IACUC)-approved protocols. To induce colitis, mice were allowed to drink sterile tap water with or without the addition of 2.5% dextran sodium sulfate (MP Biomedicals, Solon, OH, USA) ad libitum. Animals were euthanized on days 0, 3, 7, 10, and 13. The large intestine from the cecum to the rectum was