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 1

Pharmacologic inhibition of HIF-prolyl hydroxylases improves healing of ischemic anastomoses.

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Pharmacologic inhibition of HIF-prolyl hydroxylases improves healing of ...
(A) Schematic drawing of blood vessel ligation to induce ischemia in colonic anastomoses. (B) Bursting pressure analysis of ischemic (ligation, red dots) and nonischemic colonic anastomoses (sham, black dots). (C) Schematic overview of pretreatment with PHI (DMOG, 100 μg/g BW) or vehicle control in vivo. (D) Left: Representative images of ischemic colonic anastomoses (dotted white lines) from vehicle- and DMOG-treated mice. Note indirect signs of anastomotic leakage, such as severe adhesion (§) and abscess (white arrowheads). Right: Quantification of indirect signs of anastomotic leakage (n = 8–10 animals per group; **P = 0.003 by χ2 test). (E) Bursting pressure analysis of ischemic colonic anastomoses from vehicle- and DMOG-treated mice (n = 8–10 animals per group; Student’s t test). (F) Left: Representative Masson-Trichrome-Goldner (MTG) stainings, revealing the thickness of collagen capsules (black dashed lines) close to sutures (§) in ischemic anastomoses from vehicle- and DMOG-treated mice (scale bar represents 100 μm). Right: Histomorphometric quantification of collagen capsule thickness in ischemic colonic anastomoses from vehicle- and DMOG-treated mice (n = 3–4 animals per group; Mann-Whitney U test). (G) Real-time PCR analysis of Acta2 mRNA expression within ischemic anastomoses from vehicle- and DMOG-treated mice (n = 3–4 biological replicates; Student’s t test). (H) Representative CD45 (upper panels) and F4/80 (lower panels) immunostainings of ischemic colonic anastomoses from vehicle- and DMOG-treated mice (n = 3–4 animals per group; scale bar represents 200 μm; § and # in F and H indicate positions of [extracted] sutures and gut lumen, respectively). (I) Antibody array, revealing the relative protein abundance of M1 macrophage–associated cytokines within whole tissue from vehicle- and DMOG-treated ischemic colonic anastomoses (n = 4 biological replicates; *nonadjusted P value reported, after correction for multiple comparison with Holm-Šidák method, P = 0.402).