Inhibition of HIF-prolyl hydroxylases improves healing of intestinal anastomoses
Moritz J. Strowitzki, Gwendolyn Kimmer, Julian Wehrmann, Alina S. Ritter, Praveen Radhakrishnan, Vanessa M. Opitz, Christopher Tuffs, Marvin Biller, Julia Kugler, Ulrich Keppler, Jonathan M. Harnoss, Johannes Klose, Thomas Schmidt, Alfonso Blanco, Cormac T. Taylor, Martin Schneider
Moritz J. Strowitzki, Gwendolyn Kimmer, Julian Wehrmann, Alina S. Ritter, Praveen Radhakrishnan, Vanessa M. Opitz, Christopher Tuffs, Marvin Biller, Julia Kugler, Ulrich Keppler, Jonathan M. Harnoss, Johannes Klose, Thomas Schmidt, Alfonso Blanco, Cormac T. Taylor, Martin Schneider
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Research Article Gastroenterology Inflammation

Inhibition of HIF-prolyl hydroxylases improves healing of intestinal anastomoses

Abstract

Anastomotic leakage (AL) accounts for a major part of in-house mortality in patients undergoing colorectal surgery. Local ischemia and abdominal sepsis are common risk factors contributing to AL and are characterized by upregulation of the hypoxia-inducible factor (HIF) pathway. The HIF pathway is critically regulated by HIF-prolyl hydroxylases (PHDs). Here, we investigated the significance of PHDs and the effects of pharmacologic PHD inhibition (PHI) during anastomotic healing. Ischemic or septic colonic anastomoses were created in mice by ligation of mesenteric vessels or lipopolysaccharide-induced abdominal sepsis, respectively. Genetic PHD deficiency (Phd1–/–, Phd2+/–, and Phd3–/–) or PHI were applied to manipulate PHD activity. Pharmacologic PHI and genetic PHD2 haplodeficiency (Phd2+/–) significantly improved healing of ischemic or septic colonic anastomoses, as indicated by increased bursting pressure and reduced AL rates. Only Phd2+/– (but not PHI or Phd1–/–) protected from sepsis-related mortality. Mechanistically, PHI and Phd2+/– induced immunomodulatory (M2) polarization of macrophages, resulting in increased collagen content and attenuated inflammation-driven immune cell recruitment. We conclude that PHI improves healing of colonic anastomoses in ischemic or septic conditions by Phd2+/–-mediated M2 polarization of macrophages, conferring a favorable microenvironment for anastomotic healing. Patients with critically perfused colorectal anastomosis or abdominal sepsis could benefit from pharmacologic PHI.

Authors

Moritz J. Strowitzki, Gwendolyn Kimmer, Julian Wehrmann, Alina S. Ritter, Praveen Radhakrishnan, Vanessa M. Opitz, Christopher Tuffs, Marvin Biller, Julia Kugler, Ulrich Keppler, Jonathan M. Harnoss, Johannes Klose, Thomas Schmidt, Alfonso Blanco, Cormac T. Taylor, Martin Schneider

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Figure 4

Phd2 haplodeficiency reduces recruitment of inflammatory cells and enhances M2 macrophages during healing of septic colonic anastomoses.

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Phd2 haplodeficiency reduces recruitment of inflammatory cells and enhan...
(A) Representative immunolabeling of leukocytes (CD45, left upper panels) and macrophages (F4/80, left lower panels) in healthy and septic (LPS i.p.) anastomoses from WT, Phd1–/–, and Phd2+/– mice and histomorphometric quantification (right). (B and C) ELISA-based quantification of IL-6 in whole tissue lysates from healthy and septic (LPS i.p.) anastomoses harvested from WT and Phd2+/– mice (B) or in cell culture medium (CMM1) conditioned with LPS-treated M1-polarized primary WT or Phd2+/– BMDMs (C) (n = 3, pooled data from individual biological replicates; ANOVA with post hoc test in B and Student’s t test in C). (D) Representative immunolabeling of proinflammatory M1 macrophages (iNOS and CXCR2, left upper panels) and immunomodulatory M2 macrophages (Arginase 1, left lower panels) in healthy and septic (LPS i.p.) anastomoses from WT, Phd1–/–, and Phd2+/– mice and histomorphometric quantification (right) (in A and D: n = 6–7 animals per group; ANOVA with post hoc or Kruskal-Wallis [only CXRC2] test; scale bar represents 200 μm; § indicates positions of [extracted] sutures).