Research Article| Volume 73, ISSUE 3, P628-639, September 2020

Download started.


Paneth cells promote angiogenesis and regulate portal hypertension in response to microbial signals

Published:March 20, 2020DOI:


      • Paneth cells synthesize and secret anti-bacterial peptides to prevent dysbiosis.
      • In response to microbial stimuli, Paneth cells also secrete pro-angiogenic signalling molecules.
      • Secretory pro-angiogenic signaling molecules promote intestinal and mesenteric angiogenesis, regulating portal hypertension.
      • In the absence of Paneth cells, portal hypertension, intestinal and mesenteric angiogenesis are significantly decreased.

      Background & Aims

      Paneth cells (PCs) synthesize and secrete antimicrobial peptides that are key mediators of host-microbe interactions, establishing a balance between intestinal microflora and enteric pathogens. We observed that their number increases in experimental portal hypertension and aimed to investigate the mechanisms by which these cells can contribute to the regulation of portal pressure.


      We first treated Math1Lox/LoxVilcreERT2 mice with tamoxifen to induce the complete depletion of intestinal PCs. Subsequently, we performed partial portal vein or bile duct ligation. We then studied the effects of these interventions on hemodynamic parameters, proliferation of blood vessels and the expression of genes regulating angiogenesis. Intestinal organoids were cultured and exposed to different microbial products to study the composition of their secreted products (by proteomics) and their effects on the proliferation and tube formation of endothelial cells (ECs). In vivo confocal laser endomicroscopy was used to confirm the findings on blood vessel proliferation.


      Portal hypertension was significantly attenuated in PC-depleted mice compared to control mice and was associated with a decrease in portosystemic shunts. Depletion of PCs also resulted in a significantly decreased density of blood vessels in the intestinal wall and mesentery. Furthermore, we observed reduced expression of intestinal genes regulating angiogenesis in Paneth cell depleted mice using arrays and next generation sequencing. Tube formation and wound healing responses were significantly decreased in ECs treated with conditioned media from PC-depleted intestinal organoids exposed to intestinal microbiota-derived products. Proteomic analysis of conditioned media in the presence of PCs revealed an increase in factors regulating angiogenesis and additional metabolic processes. In vivo endomicroscopy showed decreased vascular proliferation in the absence of PCs.


      These results suggest that in response to intestinal flora and microbiota-derived factors, PCs secrete not only antimicrobial peptides, but also pro-angiogenic signaling molecules, thereby promoting intestinal and mesenteric angiogenesis and regulating portal hypertension.

      Lay summary

      Paneth cells are present in the lining of the small intestine. They prevent the passage of bacteria from the intestine into the blood circulation by secreting substances to fight bacteria. In this paper, we discovered that these substances not only act against bacteria, but also increase the quantity of blood vessels in the intestine and blood pressure in the portal vein. This is important, because high blood pressure in the portal vein may result in several complications which could be targeted with novel approaches.

      Graphical abstract


      To read this article in full you will need to make a payment

      Purchase one-time access:

      Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'


      Subscribe to Journal of Hepatology
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect


        • Bosch J.
        • Groszmann R.J.
        • Shah V.H.
        Evolution in the understanding of the pathophysiological basis of portal hypertension: how changes in paradigm are leading to successful new treatments.
        J Hepatol. 2015; 62: S121-S130
        • Gracia-Sancho J.
        • Marrone G.
        • Fernandez-Iglesias A.
        Hepatic microcirculation and mechanisms of portal hypertension.
        Nat Rev Gastroenterol Hepatol. 2019; 16: 221-234
        • Fernandez M.
        • Semela D.
        • Bruix J.
        • Colle I.
        • Pinzani M.
        • Bosch J.
        Angiogenesis in liver disease.
        J Hepatol. 2009; 50: 604-620
        • Carmeliet P.
        • Jain R.K.
        Molecular mechanisms and clinical applications of angiogenesis.
        Nature. 2011; 473: 298-307
        • Geerts A.M.
        • Vanheule E.
        • Van Vlierberghe H.
        • Leybaert L.
        • Van Steenkiste C.
        • De Vos M.
        • et al.
        Rapamycin prevents mesenteric neo-angiogenesis and reduces splanchnic blood flow in portal hypertensive mice.
        Hepatol Res. 2008; 38: 1130-1139
        • Fernandez M.
        • Vizzutti F.
        • Garcia-Pagan J.C.
        • Rodes J.
        • Bosch J.
        Anti-VEGF receptor-2 monoclonal antibody prevents portal-systemic collateral vessel formation in portal hypertensive mice.
        Gastroenterology. 2004; 126: 886-894
        • Reiberger T.
        • Angermayr B.
        • Schwabl P.
        • Rohr-Udilova N.
        • Mitterhauser M.
        • Gangl A.
        • et al.
        Sorafenib attenuates the portal hypertensive syndrome in partial portal vein ligated rats.
        J Hepatol. 2009; 51: 865-873
        • Fernandez M.
        • Mejias M.
        • Angermayr B.
        • Garcia-Pagan J.C.
        • Rodes J.
        • Bosch J.
        Inhibition of VEGF receptor-2 decreases the development of hyperdynamic splanchnic circulation and portal-systemic collateral vessels in portal hypertensive rats.
        J Hepatol. 2005; 43: 98-103
        • Moghadamrad S.
        • McCoy K.D.
        • Geuking M.B.
        • Sagesser H.
        • Kirundi J.
        • Macpherson A.J.
        • et al.
        Attenuated portal hypertension in germ-free mice: function of bacterial flora on the development of mesenteric lymphatic and blood vessels.
        Hepatology. 2015; 61: 1685-1695
        • Misra V.
        • Misra S.P.
        • Dwivedi M.
        • Gupta S.C.
        Histomorphometric study of portal hypertensive enteropathy.
        Am J Clin Pathol. 1997; 108: 652-657
        • Hooper L.V.
        • Stappenbeck T.S.
        • Hong C.V.
        • Gordon J.I.
        Angiogenins: a new class of microbicidal proteins involved in innate immunity.
        Nat Immunol. 2003; 4: 269-273
        • Allaire J.M.
        • Crowley S.M.
        • Law H.T.
        • Chang S.Y.
        • Ko H.J.
        • Vallance B.A.
        The intestinal epithelium: central coordinator of mucosal immunity.
        Trends Immunol. 2018; 39: 677-696
        • Bevins C.L.
        • Salzman N.H.
        Paneth cells, antimicrobial peptides and maintenance of intestinal homeostasis.
        Nat Rev Microbiol. 2011; 9: 356-368
        • Salzman N.H.
        • Hung K.
        • Haribhai D.
        • Chu H.
        • Karlsson-Sjoberg J.
        • Amir E.
        • et al.
        Enteric defensins are essential regulators of intestinal microbial ecology.
        Nat Immunol. 2010; 11: 76-83
        • Ayabe T.
        • Satchell D.P.
        • Wilson C.L.
        • Parks W.C.
        • Selsted M.E.
        • Ouellette A.J.
        Secretion of microbicidal alpha-defensins by intestinal Paneth cells in response to bacteria.
        Nat Immunol. 2000; 1: 113-118
        • Lala S.
        • Ogura Y.
        • Osborne C.
        • Hor S.Y.
        • Bromfield A.
        • Davies S.
        • et al.
        Crohn's disease and the NOD2 gene: a role for paneth cells.
        Gastroenterology. 2003; 125: 47-57
        • Riba A.
        • Olier M.
        • Lacroix-Lamande S.
        • Lencina C.
        • Bacquie V.
        • Harkat C.
        • et al.
        Paneth cell defects induce microbiota dysbiosis in mice and promote visceral hypersensitivity.
        Gastroenterology. 2017; 153: 1594-1606.e2
        • Coutinho H.B.
        • da Mota H.C.
        • Coutinho V.B.
        • Robalinho T.I.
        • Furtado A.F.
        • Walker E.
        • et al.
        Absence of lysozyme (muramidase) in the intestinal Paneth cells of newborn infants with necrotising enterocolitis.
        J Clin Pathol. 1998; 51: 512-514
        • Wehkamp J.
        • Salzman N.H.
        • Porter E.
        • Nuding S.
        • Weichenthal M.
        • Petras R.E.
        • et al.
        Reduced Paneth cell alpha-defensins in ileal Crohn's disease.
        Proc Natl Acad Sci U S A. 2005; 102: 18129-18134
        • Shroyer N.F.
        • Helmrath M.A.
        • Wang V.Y.
        • Antalffy B.
        • Henning S.J.
        • Zoghbi H.Y.
        Intestine-specific ablation of mouse atonal homolog 1 (Math1) reveals a role in cellular homeostasis.
        Gastroenterology. 2007; 132: 2478-2488
        • Durand A.
        • Donahue B.
        • Peignon G.
        • Letourneur F.
        • Cagnard N.
        • Slomianny C.
        • et al.
        Functional intestinal stem cells after Paneth cell ablation induced by the loss of transcription factor Math1 (Atoh1).
        Proc Natl Acad Sci U S A. 2012; 109: 8965-8970
        • Moghadamrad S.
        • Hassan M.
        • McCoy K.D.
        • Kirundi J.
        • Kellmann P.
        • De Gottardi A.
        Attenuated fibrosis in specific pathogen-free microbiota in experimental cholestasis- and toxin-induced liver injury.
        FASEB J. 2019; 33: 12464-12476
        • Bialkowska A.B.
        • Ghaleb A.M.
        • Nandan M.O.
        • Yang V.W.
        Improved swiss-rolling technique for intestinal tissue preparation for immunohistochemical and immunofluorescent analyses.
        J Vis Exp. 2016; : 54161
        • Moolenbeek C.
        • Ruitenberg E.J.
        The ‘Swiss roll’: a simple technique for histological studies of the rodent intestine.
        Lab Anim. 1981; 15: 57-59
        • Wang L.
        • Wang S.
        • Li W.
        RSeQC: quality control of RNA-seq experiments.
        Bioinformatics. 2012; 28: 2184-2185
        • Kim D.
        • Langmead B.
        • Salzberg S.L.
        HISAT: a fast spliced aligner with low memory requirements.
        Nat Methods. 2015; 12: 357-360
        • Liao Y.
        • Smyth G.K.
        • Shi W.
        featureCounts: an efficient general purpose program for assigning sequence reads to genomic features.
        Bioinformatics. 2014; 30: 923-930
        • Love M.I.
        • Huber W.
        • Anders S.
        Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.
        Genome Biol. 2014; 15: 550
        • Mi H.
        • Muruganujan A.
        • Huang X.
        • Ebert D.
        • Mills C.
        • Guo X.
        • et al.
        Protocol Update for large-scale genome and gene function analysis with the PANTHER classification system (v.14.0).
        Nat Protoc. 2019; 14: 703-721
        • Sorribas M.
        • Jakob M.O.
        • Yilmaz B.
        • Li H.
        • Stutz D.
        • Noser Y.
        • et al.
        FxR-modulates the gut-vascular barrier by regulating the entry sites for bacterial translocation in experimental cirrhosis.
        J Hepatol. 2019; 71: 1126-1140
        • Sato T.
        • Vries R.G.
        • Snippert H.J.
        • van de Wetering M.
        • Barker N.
        • Stange D.E.
        • et al.
        Single Lgr5 stem cells build crypt–villus structures in vitro without a mesenchymal niche.
        Nature. 2009; 459: 262
        • Mahe M.M.
        • Aihara E.
        • Schumacher M.A.
        • Zavros Y.
        • Montrose M.H.
        • Helmrath M.A.
        • et al.
        Establishment of gastrointestinal epithelial organoids.
        Curr Protoc Mouse Biol. 2013; 3: 217-240
        • Fuller M.K.
        • Faulk D.M.
        • Sundaram N.
        • Shroyer N.F.
        • Henning S.J.
        • Helmrath M.A.
        Intestinal crypts reproducibly expand in culture.
        J Surg Res. 2012; 178: 48-54
        • Farin H.F.
        • Karthaus W.R.
        • Kujala P.
        • Rakhshandehroo M.
        • Schwank G.
        • Vries R.G.J.
        • et al.
        Paneth cell extrusion and release of antimicrobial products is directly controlled by immune cell-derived IFN-γ.
        J Exp Med. 2014; 211: 1393-1405
        • Balmer M.L.
        • Slack E.
        • de Gottardi A.
        • Lawson M.A.
        • Hapfelmeier S.
        • Miele L.
        • et al.
        The liver may act as a firewall mediating mutualism between the host and its gut commensal microbiota.
        Sci Transl Med. 2014; 6: 237ra66
        • Arnaoutova I.
        • Kleinman H.K.
        In vitro angiogenesis: endothelial cell tube formation on gelled basement membrane extract.
        Nat Protoc. 2010; 5: 628
        • Wolski W.
        • Grossmann J.
        • Panse C.
        SRMServices - R-Package to Report Quantitative Mass Spectometry Data.
        2018 (Available at)
        • Smyth G.K.
        Linear models and empirical bayes methods for assessing differential expression in microarray experiments.
        Stat Appl Genet Mol Biol. 2004; 3 (Article 3)
        • Wang J.
        • Vasaikar S.
        • Shi Z.
        • Greer M.
        • Zhang B.
        WebGestalt 2017: a more comprehensive, powerful, flexible and interactive gene set enrichment analysis toolkit.
        Nucleic Acids Res. 2017; 45: W130-W137
        • Szklarczyk D.
        • Morris J.H.
        • Cook H.
        • Kuhn M.
        • Wyder S.
        • Simonovic M.
        • et al.
        The STRING database in 2017: quality-controlled protein-protein association networks, made broadly accessible.
        Nucleic Acids Res. 2017; 45: D362-D368
        • Bosch J.
        • Abraldes J.G.
        • Fernández M.
        • García-Pagán J.C.
        Hepatic endothelial dysfunction and abnormal angiogenesis: new targets in the treatment of portal hypertension.
        J Hepatol. 2010; 53: 558-567
        • Angermayr B.
        • Fernandez M.
        • Mejias M.
        • Gracia-Sancho J.
        • Garcia-Pagan J.C.
        • Bosch J.
        NAD(P)H oxidase modulates angiogenesis and the development of portosystemic collaterals and splanchnic hyperaemia in portal hypertensive rats.
        Gut. 2007; 56: 560-564
        • Mejias M.
        • Garcia-Pras E.
        • Tiani C.
        • Miquel R.
        • Bosch J.
        • Fernandez M.
        Beneficial effects of sorafenib on splanchnic, intrahepatic, and portocollateral circulations in portal hypertensive and cirrhotic rats.
        Hepatology. 2009; 49: 1245-1256
        • Gao J.-H.
        • Wen S.-L.
        • Feng S.
        • Yang W.-J.
        • Lu Y.-Y.
        • Tong H.
        • et al.
        Celecoxib and octreotide synergistically ameliorate portal hypertension via inhibition of angiogenesis in cirrhotic rats.
        Angiogenesis. 2016; 19: 501-511
        • Stappenbeck T.S.
        • Hooper L.V.
        • Gordon J.I.
        Developmental regulation of intestinal angiogenesis by indigenous microbes via Paneth cells.
        Proc Natl Acad Sci U S A. 2002; 99: 15451-15455
        • Clevers H.C.
        • Bevins C.L.
        PCs: maestros of the small intestinal crypts.
        Annu Rev Physiol. 2013; 75: 289-311