Silvia D'Alessio

uPAR-deficient mouse keratinocytes fail to produce EGFR-dependent laminin-5, affecting migration in vivo and in vitro

The urokinase receptor (uPAR) is involved in a series of pathological processes, from inflammation to cancer. We have analyzed in detail the role of uPAR and the mechanisms involved in keratinocyte behavior during wound healing by exploiting uPAR-knockout (KO) mice. In vivo, uPAR-KO mice showed delayed wound healing, with abnormal keratinocyte migration and proliferation. In vitro, unlike wild-type cells, primary uPAR-KO keratinocytes did not proliferate in response to epidermal growth factor (EGF), their growth and migration were not inhibited by EGF-receptor (EGFR) inhibitors, and they did not adhere to uncoated surfaces. Whereas EGFR levels in uPAR-KO keratinocytes were normal, there was no tyrosine phosphorylation upon addition of EGF, and its downstream targets, extracellular-signal-regulated kinases 1 and 2 (ERK1/2), were not activated. Re-introduction of mouse uPAR rescued all phenotypes. In vitro adhesion and migration defects were associated with the failure of uPAR-KO keratinocytes to normally produce and secrete laminin-5 (LN5), an event that requires EGFR signaling. These results were confirmed in vivo, with LN5 being upregulated during wound healing in wild-type but not in uPAR-KO epidermis.

Advances in therapeutic interventions targeting the vascular and lymphatic endothelium in inflammatory bowel disease

Purpose of review The review summarizes the current knowledge of the roles played by the vascular and lymphatic endothelium throughout the gut in the pathogenesis of inflammatory bowel disease (IBD) and gives an update on emerging strategies targeting both vasculatures. Recent findings Enormous efforts have been made to understand the mechanisms underlining the origin, development and maintenance of intestinal chronic inflammation. In particular, new studies focused their attention on the role played by the microvascular and lymphatic endothelium in the pathogenesis of IBD. During inflammation, whereas the microvasculature is responsible for the entry and distribution of immune cells in the mucosa, the lymphatic system controls leukocyte exit, bacterial clearance and edema absorption. The study of these events, which are aberrant during chronic inflammation, has resulted in the identification and validation of several targets for the treatment of experimental colitis, some of which have translated into effective treatments for patients with IBD. Summary Although much attention has been paid to the microvascular endothelium and to antiangiogenic therapies, specific studies on the lymphatic vasculature and its functions in IBD are still at the initial stage, and other molecular mechanisms, genes, molecules and new pathways must definitely be explored.

Full-length soluble urokinase plasminogen activator receptor down-modulates nephrin expression in podocytes

Increased plasma level of soluble urokinase-type plasminogen activator receptor (suPAR) was associated recently with focal segmental glomerulosclerosis (FSGS). In addition, different clinical studies observed increased concentration of suPAR in various glomerular diseases and in other human pathologies with nephrotic syndromes such as HIV and Hantavirus infection, diabetes and cardiovascular disorders. Here, we show that suPAR induces nephrin down-modulation in human podocytes. This phenomenon is mediated only by full-length suPAR, is time-and dose-dependent and is associated with the suppression of Wilms’ tumor 1 (WT-1) transcription factor expression. Moreover, an antagonist of αvβ3 integrin RGDfv blocked suPAR-induced suppression of nephrin. These in vitro data were confirmed in an in vivo uPAR knock out Plaur−/− mice model by demonstrating that the infusion of suPAR inhibits expression of nephrin and WT-1 in podocytes and induces proteinuria. This study unveiled that interaction of full-length suPAR with αvβ3 integrin expressed on podocytes results in down-modulation of nephrin that may affect kidney functionality in different human pathologies characterized by increased concentration of suPAR.

A direct link between expression of urokinase plasminogen activator receptor, growth rate and oncogenic transformation in mouse embryonic fibroblasts.

In addition to its role in invasion and metastasis of several tumors, the multifunctional urokinase receptor uPAR (urokinase plasminogen activator receptor) is directly involved in the growth of several cancer cells in vitro and in vivo. We have compared growth rate and oncogenic transformation in wild-type (wt) or uPAR−/− mouse embryonic fibroblasts (MEFs). Surprisingly, uPAR−/− MEFs grew faster than wt MEFs. This agreed with elevated levels of cell cycle mediators like extracellular signal-regulated protein kinase, p38, AP1 and Cyclin D1. Infection with a uPAR retrovirus reverted the effect, decreasing the growth rate.When MEFs were transformed with H-RasV12 and E1A oncogenes, the efficiency of transformation in uPAR−/− MEFs was higher than in wt. UPAR−/− MEFs grew faster at low serum, produced more colonies in agar and produced tumors in vivo in nude mice with a lower latency period. The properties of the heterozygous uPAR+/− MEFs were always intermediate. We conclude therefore that in MEFs uPAR concentration controls cell proliferation and the transforming activity of some oncogenes.

Haematopoietic prolyl hydroxylase-1 deficiency promotes M2 macrophage polarization and is both necessary and sufficient to protect against experimental colitis

Prolyl hydroxylase domain‐containing proteins (PHDs) regulate the adaptation of cells to hypoxia. Pan‐hydroxylase inhibition is protective in experimental colitis, in which PHD1 plays a prominent role. However, it is currently unknown how PHD1 targeting regulates this protection and which cell type(s) are involved. Here, we demonstrated that Phd1 deletion in endothelial and haematopoietic cells (Phd1f/fTie2:cre) protected mice from dextran sulphate sodium (DSS)‐induced colitis, with reduced epithelial erosions, immune cell infiltration, and colonic microvascular dysfunction, whereas the response of Phd2f/+Tie2:cre and Phd3f/fTie2:cre mice to DSS was similar to that of their littermate controls. Using bone marrow chimeras and cell‐specific cre mice, we demonstrated that ablation of Phd1 in haematopoietic cells but not in endothelial cells was both necessary and sufficient to inhibit experimental colitis. This effect relied, at least in part, on skewing of Phd1‐deficient bone marrow‐derived macrophages towards an anti‐inflammatory M2 phenotype. These cells showed an attenuated nuclear factor‐κB‐dependent response to lipopolysaccharide (LPS), which in turn diminished endothelial chemokine expression. In addition, Phd1 deficiency in dendritic cells significantly reduced interleukin‐1β production in response to LPS. Taken together, our results further support the development of selective PHD1 inhibitors for ulcerative colitis, and identify haematopoietic cells as their primary target. Copyright

The urokinase plasminogen activator receptor (uPAR) controls macrophage phagocytosis in intestinal inflammation

Objective Inflammation plays crucial roles in the pathogenesis of several chronic inflammatory disorders, including Crohns disease (CD) and UC, the two major forms of IBD. The urokinase plasminogen activator receptor (uPAR) exerts pleiotropic functions over the course of both physiological and pathological processes. uPAR not only has a key role in fibrinolysis but also modulates the development of protective immunity. Additionally, uPAR supports extracellular matrix degradation and regulates cell migration, adhesion and proliferation, thus influencing the development of inflammatory and immune responses. This study aimed to evaluate the role of uPAR in the pathogenesis of IBD. Design The functional role of uPAR was assessed in established experimental models of colitis. uPAR deficiency effects on cytokine release, polarisation and bacterial phagocytosis were analysed in colonic macrophages. uPAR expression was analysed in surgical specimens collected from normal subjects and patients with IBD. Results In mice, uPAR expression is positively regulated as colitis progresses. uPAR-KO mice displayed severe inflammation compared with wild-type littermates, as indicated by clinical assessment, endoscopy and colon histology. The absence of uPAR led to an increased production of inflammatory cytokines by macrophages that showed an M1 polarisation and impaired phagocytosis. In human IBD, CD68+ macrophages derived from the inflamed mucosa expressed low levels of uPAR. Conclusions These findings point to uPAR as an essential component of intestinal macrophage functions and unravel a new potential target to control mucosal inflammation in IBD.

Urokinase receptor promotes skin tumor formation by preventing epithelial cell activation of Notch1

The urokinase-type plasminogen activator receptor (uPAR) has a well-established role in cancer progression, but it has been little studied at earlier stages of cancer initiation. Here, we show that uPAR deficiency in the mouse dramatically reduces susceptibility to the classical two-stage protocol of inflammatory skin carcinogenesis. uPAR genetic deficiency decreased papilloma formation and accelerated keratinocyte differentiation, effects mediated by Notch1 hyperactivation. Notably, Notch1 inhibition in uPAR-deficient mice rescued their susceptibility to skin carcinogenesis. Clinically, we found that human differentiated keratoacanthomas expressed low levels of uPAR and high levels of activated Notch1, with opposite effects in proliferating tumors, confirming the relevance of the observations in mice. Furthermore, we found that TACE-dependent activation of Notch1 in basal kerantinocytes was modulated by uPAR. Mechanistically, uPAR sequestered TACE within lipid rafts to prevent Notch1 activation, thereby promoting cell proliferation and tumor formation. Given that uPAR signaling is nonessential for normal epidermal homeostasis, our results argue that uPAR may present a promising disease-specific target for preventing skin cancer development.

Targeting lymphatics in inflammatory bowel disease.

The inflammatory bowel diseases (IBDs), consisting of ulcerative colitis (UC) and Crohns disease (CD), are characterized by chronic inflammation of the gastrointestinal tract in genetically susceptible individuals exposed to environmental risk factors. Although the precise pathophysiology of IBD is unknown, alterations in the intestinal lymphatic network such as lymphangiogenesis and lymphatic vessel (LV) dysfunction, are well-established features of human and experimental IBD (1). Such lymphangiogenic expansion might enhance classic intestinal lymphatic transport, eliminating excess accumulations of fluids, inflammatory cells and mediators, and could therefore be interpreted as an “adaptive” response to acute and chronic inflammatory processes. However, whether these new LVs are functional, dysregulated or immature is currently an area under investigation. Together with LV dysfunction, macrophages (MΦs) have also a fundamental contribution to IBD pathogenesis. They have been found to play opposing roles in mouse models of intestinal inflammation. In fact, while classically activated M1 MΦs contribute to the exacerbation of the inflammation in mouse models of colitis (2), alternatively activated M2 MΦs contribute to the resolution of the disease (3). Thus, the factors that modulate MΦ polarization could affect the severity of human and experimental colitis. These observations suggest that resolution of chronic inflammation may require restoration of proper lymphatic function and proper MΦ activation, thus maintaining normal flow balance and helping the removal of inflammatory cells, mediators, and bacterial antigens away from inflamed sites. Lymphangiogenesis is mediated by binding of the lymphatic vascular endothelial selective growth factors VEGF-C and VEGF-D to VEGFR3. Anti-lymphatic treatment with anti-VEGFR3 antibodies in an animal model of IBD has been shown to aggravate inflammation and submucosal edema, increase leukocyte infiltration, and to cause tortuous LVs (4). Besides, VEGF-C is also chemotactic for MΦs during pathological conditions, with its receptor VEGFR3 expressed by a substantial fraction of peripheral blood monocytes and activated tissue MΦs (5). Overall, these studies have established a direct role for VEGF-C/VEGFR3 signaling in both inflammation-induced lymphangiogenesis and immune response and suggest that therapies aimed at promoting lymphatic function, e.g., with prolymphangiogenic factors, such as VEGF-C, may provide a novel strategy for the treatment of inflammatory conditions, including IBD. Recently, we examined the effect of stimulating lymphatic function and adaptive immune response via VEGF-C/VEGFR3 signaling on the severity of intestinal inflammation, on lymphatic drainage, as well as on bacterial antigen clearance and MΦ activation during inflammatory conditions. Furthermore, we evaluated the mechanism through which this pathway acts in experimental disease progression. We provided evidence, for the first time, that the specific promotion of LV function limits experimental chronic intestinal inflammation in mice; this is mediated by a unique MΦ polarization and activation, accompanied by modification of the tissue cytokine milieu (6). We reported that systemic inhibition of VEGFR3 blocked lymphangiogenesis, reducing both area density and LV dimension and growth, while significantly increasing inflammatory edema formation and impeding disease resolution. In contrast, lymphatic drainage was enhanced by systemic delivery of VEGF-C, which in turn significantly induced LV density and proliferation, improving intestinal inflammation. Interestingly, the enhanced lymphatic drainage by VEGF-C was observed in combination with increased inflammatory cell mobilization and bacterial antigen clearance, all LV functions that we found to be inhibited by VEGFR3 blockade. VEGF-C–dependent antigen clearance was a MΦ-specific effect, thus demonstrating a potential role for VEGFR3 signaling in immunity by mediating antigen-presenting cell (APC) trafficking through MΦ recruitment. The most intriguing and novel finding in this study is that the protection we observed in VEGF-C–treated mice during disease progression was not only a consequence of increased lymphangiogenesis and enhanced lymphatic flow and function, but was also a result of a previously unknown direct VEGF-C–induced MΦ activation through STAT6 signaling. This is intriguing, because some reports suggest that there may be a dysregulation of STAT6 signaling in the ungoverned immune response associated with colitis, and this transcription factor plays a regulatory role in the pathogenesis of IBD (7). In conclusion, our study provides the first proof of concept to our knowledge that it may be possible to treat chronic gastrointestinal inflammatory disorders by stimulating LV functions to promote bacterial antigen clearance, drainage of fluids and inflammatory cells, together with adaptive immunity, effects achievable through modulation of the VEGF-C/VEGFR3 pathway, as shown schematically in Figure ​Figure1.1. These findings are important because support the potential use of lymphangiogenic growth factors as a novel therapeutic approach for the treatment of IBD and other chronic inflammatory diseases. Figure 1 Potential use of lymphangiogenic factors (VEGF-C) as a novel therapeutic approach for the treatment of IBD

DOP008 The absence of the Triggering Receptor Expressed on Myeloid Cells type-2 (TREM-2) induces a transmissible and protective intestinal microbiota for colitis and colitis associated cancer

Results: Villin-Cre Sphk1flox/flox mice had significant higher colitis with longer segment of ulceration compared to Sphk1flox/flox mice (p 3mm were significantly decreased in villin-Cre Sphk1flox/flox Apcmin/+ mice compared with littermates. Histologic grade was more severe in Sphk1flox/flox Apcmin/+ mice compared with villin-Cre Sphk1flox/flox Apcmin/+ mice (invasive caricinoma, 71% vs 13%, p < 0.05). The expression of Sphk2 and the b-catenin target genes cyclin D1/c-myc were decreased in Sphk1-deficient tumors. The activities of b-catenin (nucleus), cyclin D1 and c-myc and were significantly decreased in villin-Cre Sphk1flox/flox Apcmin/+ mice compared with littermates in both IHC and Western blot analyses. In AOM/DSS CAC model, tumor number and size were not significantly different between Sphk1flox/flox and villin-Cre Sphk1flox/flox mice but there was a change of tumor size distribution between two groups. Conclusions: Epithelial deletion of Sphk1 inhibits CAC in Apcmin/+-DSS model in mice by the modulation of b-catenin and its target signal pathway.

P020 Silencing of prolyl hydroxylase 1 in intestinal microvascular endothelial cells prevents inflammation-induced endothelial dysfunction and dampens murine colitis

P019 T cell specific loss of PTPN2 results in aggravated colitis and pronounced intestinal dysbiosis as observed in Crohn’s disease patients M. Spalinger1 *, C. Chassard2, S. Kasper1, L. Biedermann1, S. Vavricka3, I. Frey-Wagner1, K. Atrott1, C. Lacroix2, M. Fried1,4, G. Rogler1,4, M. Scharl1,4. 1University Hospital Zurich, Gastroenterology & Hepatology, Zurich, Switzerland, 2ETH Zurich, Institute of Food, Nutrition and Health, Zurich, Switzerland, 3Hospital Triemli, Gastroenterology & Hepatology, Zurich, Switzerland, 4Zurich Institute for Human Physiology, University Zurich, Zurich, Switzerland