Endothelial MT 1‐ MMP targeting limits intussusceptive angiogenesis and colitis via TSP1/nitric oxide axis

Article3 December 2019Open Access Source DataTransparent process Endothelial MT1-MMP targeting limits intussusceptive angiogenesis and colitis via TSP1/nitric oxide axis Sergio Esteban Sergio Esteban Vascular Pathophysiology Area, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain Search for more papers by this author Cristina Clemente Cristina Clemente Vascular Pathophysiology Area, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain Centro de Investigaciones Biológicas (CIB-CSIC), Madrid, Spain Search for more papers by this author Agnieszka Koziol Agnieszka Koziol Vascular Pathophysiology Area, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain Search for more papers by this author Pilar Gonzalo Pilar Gonzalo Vascular Pathophysiology Area, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain Search for more papers by this author Cristina Rius Cristina Rius Vascular Pathophysiology Area, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain CIBER de Enfermedades Cardiovasculares (CIBER-CV), Madrid, Spain Search for more papers by this author Fernando Martínez Fernando Martínez Bioinformatics Unit, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain Search for more papers by this author Pablo M Linares Pablo M Linares Gastroenterology Unit, Hospital Universitario de La Princesa, Instituto de Investigación Sanitaria Princesa (IIS-IP), Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBER-EHD), Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author María Chaparro María Chaparro Gastroenterology Unit, Hospital Universitario de La Princesa, Instituto de Investigación Sanitaria Princesa (IIS-IP), Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBER-EHD), Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Ana Urzainqui Ana Urzainqui Immunology Department, FIB-Hospital Universitario de La Princesa, Instituto de Investigación Sanitaria Princesa (IIS-IP), Madrid, Spain Search for more papers by this author Vicente Andrés Vicente Andrés orcid.org/0000-0002-0125-7209 Vascular Pathophysiology Area, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain CIBER de Enfermedades Cardiovasculares (CIBER-CV), Madrid, Spain Search for more papers by this author Motoharu Seiki Motoharu Seiki Division of Cancer Cell Research, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Javier P Gisbert Javier P Gisbert Gastroenterology Unit, Hospital Universitario de La Princesa, Instituto de Investigación Sanitaria Princesa (IIS-IP), Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBER-EHD), Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Alicia G Arroyo Corresponding Author Alicia G Arroyo [email protected] orcid.org/0000-0002-1536-3846 Vascular Pathophysiology Area, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain Centro de Investigaciones Biológicas (CIB-CSIC), Madrid, Spain Search for more papers by this author Sergio Esteban Sergio Esteban Vascular Pathophysiology Area, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain Search for more papers by this author Cristina Clemente Cristina Clemente Vascular Pathophysiology Area, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain Centro de Investigaciones Biológicas (CIB-CSIC), Madrid, Spain Search for more papers by this author Agnieszka Koziol Agnieszka Koziol Vascular Pathophysiology Area, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain Search for more papers by this author Pilar Gonzalo Pilar Gonzalo Vascular Pathophysiology Area, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain Search for more papers by this author Cristina Rius Cristina Rius Vascular Pathophysiology Area, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain CIBER de Enfermedades Cardiovasculares (CIBER-CV), Madrid, Spain Search for more papers by this author Fernando Martínez Fernando Martínez Bioinformatics Unit, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain Search for more papers by this author Pablo M Linares Pablo M Linares Gastroenterology Unit, Hospital Universitario de La Princesa, Instituto de Investigación Sanitaria Princesa (IIS-IP), Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBER-EHD), Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author María Chaparro María Chaparro Gastroenterology Unit, Hospital Universitario de La Princesa, Instituto de Investigación Sanitaria Princesa (IIS-IP), Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBER-EHD), Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Ana Urzainqui Ana Urzainqui Immunology Department, FIB-Hospital Universitario de La Princesa, Instituto de Investigación Sanitaria Princesa (IIS-IP), Madrid, Spain Search for more papers by this author Vicente Andrés Vicente Andrés orcid.org/0000-0002-0125-7209 Vascular Pathophysiology Area, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain CIBER de Enfermedades Cardiovasculares (CIBER-CV), Madrid, Spain Search for more papers by this author Motoharu Seiki Motoharu Seiki Division of Cancer Cell Research, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Javier P Gisbert Javier P Gisbert Gastroenterology Unit, Hospital Universitario de La Princesa, Instituto de Investigación Sanitaria Princesa (IIS-IP), Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBER-EHD), Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Alicia G Arroyo Corresponding Author Alicia G Arroyo [email protected] orcid.org/0000-0002-1536-3846 Vascular Pathophysiology Area, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain Centro de Investigaciones Biológicas (CIB-CSIC), Madrid, Spain Search for more papers by this author Author Information Sergio Esteban1, Cristina Clemente1,2, Agnieszka Koziol1, Pilar Gonzalo1, Cristina Rius1,3, Fernando Martínez4, Pablo M Linares5, María Chaparro5, Ana Urzainqui6, Vicente Andrés1,3, Motoharu Seiki7, Javier P Gisbert5 and Alicia G Arroyo *,1,2 1Vascular Pathophysiology Area, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain 2Centro de Investigaciones Biológicas (CIB-CSIC), Madrid, Spain 3CIBER de Enfermedades Cardiovasculares (CIBER-CV), Madrid, Spain 4Bioinformatics Unit, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain 5Gastroenterology Unit, Hospital Universitario de La Princesa, Instituto de Investigación Sanitaria Princesa (IIS-IP), Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBER-EHD), Universidad Autónoma de Madrid, Madrid, Spain 6Immunology Department, FIB-Hospital Universitario de La Princesa, Instituto de Investigación Sanitaria Princesa (IIS-IP), Madrid, Spain 7Division of Cancer Cell Research, Institute of Medical Science, University of Tokyo, Tokyo, Japan *Corresponding author. Tel: +34 91 837 31 12; Fax: +34 91 536 04 32; E-mail: [email protected] EMBO Mol Med (2020)12:e10862https://doi.org/10.15252/emmm.201910862 See also: G D'Amico et al (February 2020) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Pathological angiogenesis contributes to cancer progression and chronic inflammatory diseases. In inflammatory bowel disease, the microvasculature expands by intussusceptive angiogenesis (IA), a poorly characterized mechanism involving increased blood flow and splitting of pre-existing capillaries. In this report, mice lacking the protease MT1-MMP in endothelial cells (MT1iΔEC) presented limited IA in the capillary plexus of the colon mucosa assessed by 3D imaging during 1% DSS-induced colitis. This resulted in better tissue perfusion, preserved intestinal morphology, and milder disease activity index. Combined in vivo intravital microscopy and lentiviral rescue experiments with in vitro cell culture demonstrated that MT1-MMP activity in endothelial cells is required for vasodilation and IA, as well as for nitric oxide production via binding of the C-terminal fragment of MT1-MMP substrate thrombospondin-1 (TSP1) to CD47/αvβ3 integrin. Moreover, TSP1 levels were significantly higher in serum from IBD patients and in vivo administration of an anti-MT1-MMP inhibitory antibody or a nonamer peptide spanning the αvβ3 integrin binding site in TSP1 reduced IA during mouse colitis. Our results identify MT1-MMP as a new actor in inflammatory IA and a promising therapeutic target for inflammatory bowel disease. Synopsis Inflammatory bowel disease comprising ulcerative colitis and Crohn's disease does not have a universal efficient therapy. This study identifies the molecular axis MT1-MMP/thrombospondin-1/αvβ3 integrin/nitric oxide as a target to reduce inflammatory intussusceptive angiogenesis and improve colitis. Early expansion of the capillaries in the intestinal mucosa during colitis was caused by intussusceptive/splitting angiogenesis (IA). Deletion of the protease MT1-MMP from endothelial cells reduced IA and colitis severity. Thrombospondin-1 (TSP1) processing by MT1-MMP promoted nitric oxide production, vasodilation and IA. High levels of TSP1 were found in sera from patients suffering low active IBD. Targeting MT1-MMP/TSP1/αvβ3 integrin/nitric oxide pathway by monoclonal antibodies or minipump-delivered peptides decreased IA and ameliorates colitis. Introduction The vasculature delivers oxygen and nutrients to all tissues and must constantly and dynamically adapt to tissue needs. Angiogenesis, the formation of new capillaries from pre-existing vessels, is required to expand the vasculature not only during development and tissue repair, but also in pathological conditions such as cancer and chronic inflammatory disease (Potente et al, 2011). In these contexts, the newly formed vasculature is often marginally functional and leaky, contributing to disease progression (De Bock et al, 2011; Parma et al, 2017). Angiogenesis often occurs by capillary sprouting, which is mainly triggered by hypoxia and the subsequent production of vascular endothelial growth factor (VEGF). New therapeutic interventions in cancer and other diseases have therefore focused on inhibiting sprouting angiogenesis, mostly by blocking VEGF (Potente et al, 2011). However, the development of resistance to this approach and its overall limited success have shifted attention to the possible existence of alternative modes of capillary expansion (Ribatti & Djonov, 2012). Intussusceptive angiogenesis (IA) was formally recognized in the 1980s and involves the expansion of the microvasculature through the formation of intraluminal pillars, eventually resulting in capillary splitting (Burri et al, 2004). IA contributes to the physiological expansion of capillary beds during embryonic development and postnatal coronary vasculature remodeling (van Groningen et al, 1991; Djonov et al, 2000). IA also occurs in certain cancers and chronic diseases such as bronchopulmonary dysplasia, in which IA generates an aberrant and dysfunctional vasculature that may contribute to disease progression (Ribatti & Djonov, 2012; Giacomini et al, 2015; De Paepe et al, 2017). Sprouting and intussusception angiogenesis mechanisms can co-exist in pathophysiological settings (Konerding et al, 2012; Karthik et al, 2018). IA is a dynamic, fast, and metabolically undemanding process that barely involves proliferation but instead progresses through intraluminal endothelial cell rearrangements (Burri et al, 2004). IA is usually driven by a persistent increase in blood flow, and it aimed to restore shear stress in the split vessels (Styp-Rekowska et al, 2011). However, knowledge is scarce about the cellular and molecular mechanisms underlying IA. This is partly due to the absence of in vitro models of IA and the limited experimental techniques to identify and quantify genuine IA events in vivo, apart from scanning electron microscopy and corrosion casts (Nowak-Sliwinska et al, 2018). Nevertheless, genes whose expression is enriched during IA have been identified in skeletal muscle, in which the vasodilator prazosin induces IA and the excision of the agonist muscle, sprouting angiogenesis (Zhou et al, 1998; Egginton, 2011). Upregulated genes in skeletal muscle of prazosin-treated mice included endothelial nitric oxide synthase (eNOS) and neuropilin-1 (Nrp1), suggesting a role for these pathways in IA. Analysis of eNOS (Nos3)-deficient mice confirmed that eNOS is required for IA but not for sprouting angiogenesis in skeletal muscle (Williams et al, 2006); more recently, nitric oxide (NO) has been shown to contribute to pathological IA in tumors (Vimalraj et al, 2018). Advanced microscopy techniques and increasing knowledge about endothelial cell responses to blood flow have together favored the recent characterization of the IA modulators endoglin and ephrinB2/EphB4 (Hlushchuk et al, 2017; Groppa et al, 2018). However, it remains unclear how these pathways are regulated and contribute to IA, particularly during disease. IA is the mechanism of capillary expansion during intestinal inflammation, and analysis of chemically induced murine colitis (e.g., with dextran sodium sulfate; DSS) has advanced knowledge about the morphogenesis and hemodynamics underlying inflammatory IA (Mori et al, 2005; Ravnic et al, 2007; Filipovic et al, 2009; Konerding et al, 2010). These studies show that mechanical forces and changes in intraluminal blood flow drive IA and that a marked vasodilation occurs during the first stages of IA during colitis, before complete duplication of the mucosal plexus (Filipovic et al, 2009; Konerding et al, 2010). The DSS mouse model of colitis recapitulates some of the features of human inflammatory bowel disease (IBD), a chronic inflammatory disease of the intestine comprising ulcerative colitis and Crohn's disease and characterized by phases of remission and relapse (Podolsky, 2002). IBD is a multifactorial disease featuring a primary defect in intestinal epithelial barrier integrity and an exacerbated immune response to the microbiota and for which there is as yet no universal and efficient therapy (Gyires et al, 2014). As in other chronic inflammatory diseases, colitis progression is believed to involve angiogenesis, which is therefore regarded as a potential treatment target for human IBD (Chidlow et al, 2006; Koutroubakis et al, 2006; Danese, 2007). Attempts have been made to reduce angiogenesis and reduce colitis symptoms by targeting diverse molecular pathways, such as VEGF, TSP1/CD36, and CD40-CD40L, with limited success (Danese et al, 2007a,b; Punekar et al, 2008; Scaldaferri et al, 2009). There is therefore a need to decipher the molecular pathways involved in colitis-associated IA in order to design more rational therapeutic strategies. During colitis, epithelial and mesenchymal cells, as well as other cell types, increase expression of MT1-MMP (Pender et al, 2000; te Velde et al, 2007; Alvarado et al, 2008), a membrane-anchored matrix metalloproteinase whose activity contributes to sprouting angiogenesis in vitro and in vivo through the combined processing of substrates such as TSP1, NID1, and CYR61 (Galvez et al, 2001, 2005; Koziol et al, 2012a). MT1-MMP also has non-proteolytic actions that contribute to the regulation of Rac1 or HIF1α signaling (Koziol et al, 2012b). MT1-MMP is expressed in endothelial cells (ECs) at low levels in homeostatic conditions, but it is upregulated by the pro-inflammatory cytokines TNFα and interleukin-1 (Rajavashisth et al, 1999; Koziol et al, 2012a). Despite the important role of MT1-MMP in sprouting angiogenesis, the potential contribution of endothelial MT1-MMP to IA, particularly in the context of inflammation and IBD, has not been explored previously. In the present study, the analysis of mice specifically lacking MT1-MMP in ECs identified this protease as an actor in inflammation-driven IA whose endothelial targeting results in preserved vasculature and amelioration of colitis. Deciphering the underlying MT1-MMP/TSP1/αvβ3 integrin/NO molecular axis opens avenues for the development of new diagnostic and therapeutic interventions in IBD. Results MT1-MMP is required for intussusceptive angiogenesis (IA) in DSS-induced colitis To investigate the possible role of MT1-MMP (gene name MMP14) in inflammation-driven IA, we used the DSS-induced colitis model, a widely recognized model of IA (Konerding et al, 2010). We first assessed MT1-MMP expression in ECs of the mucosa vasculature in the colon by tracking β-gal expression in MT1lacZ/+ reporter mice. In non-treated mice, nuclear β-gal expression was present in ECs in the vessels nearby the muscularis mucosa but was barely detected in those of the mucosal plexus, the polygonal capillary network around the colonic crypts. Treatment of mice with 1% DSS significantly increased the proportion of endothelial cells expressing β-gal in the mucosal plexus after 3 days with a remaining augmented trend at 7 days (Appendix Fig S1A and B). Patches of β-gal-positive ECs were frequently detected near the Y-junctions (tri-corners) in the mucosal vascular plexus (Appendix Fig S1C). MT1-MMP was deleted in the ECs of MT1-MMPf/f;Cdh5CreERT2 mice (MT1iΔEC) mice by daily injections of 4-hydroxy-tamoxifen (4-OHT) for 5 days; 4-OHT injections began 3 days before DSS treatment. Isolated lung ECs were examined to confirm efficient recombination of the floxed Mmp14 allele and the absence of MT1-MMP mRNA (Fig EV1A–C). MT1-MMP expression was also reduced in the colonic capillaries from MT1iΔEC mice examined at 7 days post-DSS (Fig EV1D). Click here to expand this figure. Figure EV1. Strategy and validation of endothelial cell-type-specific deletion of MT1-MMP in mice A. Strategy for obtaining endothelial cell-specific MT1-MMP (Mmp14)-null mice by crossing MT1-MMPf/f mice (Gutierrez-Fernandez et al, 2015) with Cdh5-CreERT2/+ mice (Wang et al, 2010). The floxed and targeted Mmp14 alleles are illustrated. B. PCR of lung DNA from MT1f/f and MT1iΔEC mice detecting the targeted (floxed-out) Mmp14 allele (810 bp). C. qPCR analysis of relative Mmp14 and Cdh5 mRNA levels in sorted lung endothelial cells from MT1f/f and MT1iΔEC mice. n = 3 mice per genotype. Data are shown as mean ± SEM. D. Representative maximum-intensity projection images of staining for MT1-MMP (red) and CD31 (green) in colon sections from MT1f/f and MT1iΔEC mice. Scale bar, 10 μm. Data information: Please see Appendix Table S3 for exact P-values. Source data are available online for this figure. Download figure Download PowerPoint To test the effect of prophylactic endothelial MT1-MMP deletion on DSS-induced IA, we implemented a 3D imaging method based on high-resolution confocal microscopy and Imaris® image analysis of whole-mount CD31-stained mouse intestine; this method allowed us to quantify holes in the vascular tri-corners indicating intraluminal pillars as well as capillary loops and duplications in the mucosal plexus, all hallmarks of IA (Fig EV2A). We also measured capillary bifurcation angles in the mucosal plexus, which decrease during IA (Fig EV2B; Ackermann et al, 2013). DSS-induced mild colitis in MT1f/f;Cdh5CreERT2-negative (MT1f/f) control mice was associated with significantly increased IA events (holes, loops, and duplications) and smaller capillary bifurcation angles at 3 days compared with non-treated mice (Fig 1A–C). Of note, MT1iΔEC mice showed significantly reduced numbers of DSS-induced IA capillary events (holes, loops, and duplications) at 3 and 7 days compared with MT1f/f mice (Fig 1A and B); intercapillary angles were also preserved in the colon mucosal plexus of MT1iΔEC mice at 3 days post-DSS treatment (Fig 1C). These findings identify MT1-MMP as a novel endothelial actor in inflammatory IA. Click here to expand this figure. Figure EV2. Visualization of intussusceptive angiogenesis hallmarks in mouse colitis by confocal microscopy and 3D-image rendering A. Representative maximum-intensity projection (MIP) images of mucosal plexus in whole-mount distal colon stained for CD31 (green) from a MT1f/f mouse treated with 1% DSS for 3 days (upper row). 3D rendering of the raw images was performed with Imaris® (lower panels). Holes, loops, and duplications are visible and indicated by arrowhead, asterisk, and arrow, respectively. Scale bar, 10 μm. B. Magnified view of a tri-corner in the colon mucosal plexus (left) and a splitting corner after 3 days of 1% DSS (right), illustrating quantification of the intercapillary angles (Ackermann et al, 2013). Scale bar, 10 μm. Download figure Download PowerPoint Figure 1. MT1-MMP expression in endothelial cells contributes to intussusceptive angiogenesis in colitis A. Representative maximum-intensity projection images of colon mucosal plexus in CD31-stained (green) whole-mount distal colon from MT1f/f and MT1iΔEC mice left untreated (basal) or treated with 1% DSS for 3 or 7 days. Scale bar, 40 μm. Arrows, arrowheads, and asterisks indicate duplications, loops, and pillars, respectively. B. Quantification of IA events in the colon mucosal plexus of mice treated as in (A), including vascular holes, duplications, and loops; n = 9–15 mice per genotype and condition. Data are shown as mean ± SEM and were tested by one-way ANOVA with Benjamini and Hochberg post-test; *P < 0.05, **P < 0.01. C. Quantification of vascular angles at the Y-junctions in the colon mucosal plexus of mice left untreated or treated with 1% DSS for 3 days; n = 6 mice per genotype and condition. Data are shown as mean ± SEM and additionally as individual animal values and were tested by one-way ANOVA with Benjamini and Hochberg post-test; **P < 0.01. Data information: Please see Appendix Table S3 for exact P-values. Source data are available online for this figure. Source Data for Figure 1 [emmm201910862-sup-0005-SDataFig1.xlsx] Download figure Download PowerPoint Loss of endothelial MT1-MMP preserves intestinal vascular perfusion and ameliorates colitis To investigate the impact of inflammatory IA on vascular function, we analyzed vessel perfusion by intravascular injection of isolectin B4 (IB4). Perfusion decreased during colitis progression in the mucosa vascular plexus of both MT1f/f and MT1iΔEC mice, but it was significantly better preserved in the latter at 7 days post-1% DSS treatment (Fig 2A and B). Furthermore, 1% DSS induced vascular leakage at days 3 and 7 in the colonic mucosa of MT1f/f mice and at a lower extent in MT1iΔEC mice (Appendix Fig S2A). Figure 2. MT1-MMP absence from endothelial cells limits deterioration of vascular perfusion and impedes colitis progression A. Representative maximum-intensity projection images of whole-mount distal colons stained for CD31 (green) and IB4 (red) in MT1f/f and MT1iΔEC mice left untreated (basal) or treated with 1% DSS for 3 or 7 days. Scale bar, 40 μm. B. Perfusion decreased during 1% DSS-induced colitis in MT1f/f and MT1iΔEC mice; n = 8–12 mice per genotype and condition. Data are shown as mean ± SEM and were tested by one-way ANOVA with Benjamini and Hochberg post-test; **P < 0.01. C. Representative H&E-stained colon sections from MT1f/f and MT1iΔEC mice left untreated (basal) or treated with 1% DSS for 3 or 7 days. Arrows indicate crypt destruction. Scale bar, 50 μm. D. Representative second-harmonic generation (SHG) microscopy images of mucosal plexus in whole-mount colons from MT1f/f or MT1iΔEC mice left untreated (basal) or treated with 1% DSS for 3 or 7 days. Scale bar, 40 μm. E. Disease activity index (DAI, a composite of weight change, stool consistency, and presence of fecal blood) during 1% DSS-induced colitis in MT1f/f and MT1iΔEC mice. Untreated mice were included as a control. n = 19–24 per genotype and condition. Data are shown as mean ± SEM and were tested by two-way ANOVA with Benjamini and Hochberg post-test; ***P < 0.001. Data information: Please see Appendix Table S3 for exact P-values. Source data are available online for this figure. Source Data for Figure 2 [emmm201910862-sup-0006-SDataFig2.xlsx] Download figure Download PowerPoint Treatment with 1% DSS produced only subtle alterations in the intestinal mucosa after 3 days, but at 7 days, hematoxylin and eosin histology revealed better preservation of colon morphology in MT1iΔEC mice, showing fewer areas of crypt destruction than control mice (Fig 2C). Second-harmonic generation (SHG) microscopy confirmed the presence of well-structured collagen fibers surrounding crypts in MT1iΔEC mice at 7 days post-DSS, contrasting with abundant and disorganized collagen fibers in control mice (Fig 2D), an additional sign of enhanced tissue damage and fibrosis. To estimate disease severity over the 7 days of 1% DSS treatment, we calculated the disease activity index (DAI), based on a composite of weight loss, stool consistency, and hemorrhage (see Methods). The DAI was significantly lower in MT1iΔEC mice than in MT1f/f controls from DSS day 5 onward, indicating milder disease in the absence of endothelial MT1-MMP (Fig 2E). Numbers of CD11b+ leukocytes (mostly neutrophils and monocytes) did not differ between genotypes at DSS day 3, when IA was already decreased in MT1iΔEC mice; in contrast, at DSS day 7, CD11b+ leukocyte numbers in MT1iΔEC mice showed a slight but significant reduction compared with MT1f/f controls (Appendix Fig S2B and C). Thus, endothelium-specific MT1-MMP deletion improves 1% DSS-induced colitis progression primarily through an early impact on IA events at 3 days rather than on vascular perfusion, leukocyte traffic, and tissue alterations modulated at 7 days. In light of these results, we next tested the therapeutic action of endothelial MT1-MMP deletion on colitis progression. Mice received five daily 4-OHT injections beginning 4 days after the initiation of 1% DSS treatment, and mice were sacrificed at DSS day 15. Therapeutic endothelial MT1-MMP deletion during established colitis significantly reduced weight loss and DAI from day 9 onward compared with MT1f/f control mice (Fig EV3). Click here to expand this figure. Figure EV3. Therapeutic deletion of MT1-MMP in endothelial cells hampers colitis progression A. Experimental design for therapeutic deletion of MT1-MMP in endothelial cells during 1% DSS-induced colitis progression. B, C. Graphs show daily monitoring of body weight (% variation in B) and disease activity index (C) in MT1f/f and MT1iΔEC mice during the 15 days of 1% DSS treatment; n = 11–12 mice per genotype and condition. Data are shown as mean ± SEM and were tested by two-way ANOVA with Benjamini and Hochberg post-test; *P < 0.05, ****P < 0.0001. Data information: Please see Appendix Table S3 for exact P-values. Source data are available online for this figure. Download figure Download PowerPoint MT1-MMP expression in ECs is required for vasodilation and NO production The cellular and molecular mechanisms underlying IA are poorly defined, but there is a general consensus that it is initiated by changes in hemodynamic forces and increased blood flow (Filipovic et al, 2009). To investigate the role of endothelial MT1-MMP in this early step of IA that involves vasodilation (Fig 3A), we relied on intravital microscopy of the cremaster muscle and monitored arteriole vasodilation by injecting acetylcholine (ACh), which triggers EC NO secretion (Rius & Sanz, 2015; Fig 3B–D). No differences between genotypes were observed in arteriole diameter (~ 30 μm) at baseline (Fig 3C, and Movies EV1 and EV2); in contrast, maximal Ach-induced vasodilation (reached 3 min after injection) was significantly impaired in arterioles of MT1iΔEC mice compared with MT1f/f controls (Fig 3D, and Movies EV1 and EV2). Given the observation of IA events in colon mucosal plexus capillaries, we also analyzed the capillaries in the cremaster muscle. The diameter of cremaster capillaries did not increase in MT1iΔEC mice after Ach injection; however, this impaired response was rescued by i.v. injection of the NO donor DEANO (Fig 3E and F). These results suggest that impaired vasodilation in MT1-MMP-null ECs may be due to decreased NO production. Low NO production was confirmed by DAF-FM analysis of aortic endothelial cells (MAEC) isolated from MT1iΔEC mice (Appendix Fig S3). Furthermore, human umbilical vein endothelial cells (HUVEC) expressing MT1-MMP-targeting siRNA (siMT1) not only produced significantly lower amounts of NO (Fig 4A) but also expressed lower amounts of eNOS protein and eNOS (Nos3) mRNA than corresponding control ECs (Fig 4B–D). Figure 3. Endothelial cell MT1-MMP expression is required for nitric oxide-dependent vasodilation in vivo A. Scheme showing IA process including vasodilatation, pillar formation, and vascular splitting. EC, endothelial cell. B. Representative intravital microscopy images of cr