Effect of Metformin on HIF-1α Signaling and Postoperative Adhesion Formation.

BACKGROUND: Peritoneal adhesion formation is common after abdominal surgery and results in severe complications. Tissue hypoxia is one of the main drivers of peritoneal adhesions. Thus, we determined the clinical role of hypoxia-inducible factor (HIF)-1 signaling in peritoneal adhesions and investigated whether the biguanide antidiabetic drug metformin shows HIF-inhibitory effects and could be repurposed to prevent adhesion formation. STUDY DESIGN: As part of the ReLap study (DRKS00013001), adhesive tissue from patients undergoing relaparotomy was harvested and graded using the adhesion grade score. HIF-1 signaling activity within tissue biopsies was determined and correlated with adhesion severity. The effect of metformin on HIF-1 activity was analyzed by quantification of HIF target gene expression and HIF-1 protein stabilization in human mesothelial cells and murine fibroblast under normoxia and hypoxia. Mice were treated with vehicle or metformin 3 days before and until 7 days after induction of peritoneal adhesions; alternatively, metformin treatment was discontinued 48 hours before induction of peritoneal adhesions. RESULTS: HIF-1 signaling activity correlated with adhesion severity in patient biopsies. Metformin significantly mitigated HIF-1 activity in vitro and in vivo. Oral treatment with metformin markedly prevented adhesion formation in mice even when the treatment was discontinued 48 hours before surgery. Although metformin treatment did not alter macrophage polarization, metformin reduced proinflammatory leucocyte infiltration and attenuated hypoxia-induced profibrogenic expression patterns and myofibroblast activation. CONCLUSIONS: Metformin mitigates adhesion formation by inhibiting HIF-1-dependent (myo)fibroblast activation, conferring an antiadhesive microenvironment after abdominal surgery. Repurposing the clinically approved drug metformin might be useful to prevent or treat postoperative adhesions.

Inhibition of HIF-prolyl hydroxylases improves healing of intestinal anastomoses.

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.

Pharmacological HIF-inhibition attenuates postoperative adhesion formation.

Peritoneal adhesions represent a common complication of abdominal surgery, and tissue hypoxia is a main determinant in adhesion formation. Reliable therapeutic options to reduce peritoneal adhesions are scarce. We investigated whether the formation of postsurgical adhesions can be affected by pharmacological interference with hypoxia-inducible factors (HIFs). Mice were treated with a small molecule HIF-inhibitor, YC-1 (3-[5'-Hydroxymethyl-2'-furyl]-1-benzyl-indazole), or vehicle three days before and seven days after induction of peritoneal adhesions or, alternatively, once during induction of peritoneal adhesions. Pretreatment or single intraperitoneal lavage with YC-1 significantly reduced postoperative adhesion formation without prompting systemic adverse effects. Expression analyses of cytokines in peritoneal tissue and fluid and in vitro assays applying macrophages and peritoneal fibroblasts indicated that this effect was cooperatively mediated by various putatively HIF-1α-dependent mechanisms, comprising attenuated pro-inflammatory activation of macrophages, impaired recruitment and activation of peritoneal fibroblasts, mitigated epithelial-mesenchymal-transition (EMT), as well as enhanced fibrinolysis and impaired angiogenesis. Thus, this study identifies prevention of postsurgical peritoneal adhesions as a novel and promising field for the application of HIF inhibitors in clinical practice.

Influence of neoadjuvant chemotherapy on resection of primary colorectal liver metastases: A propensity score analysis.

BACKGROUND AND OBJECTIVES: There is ongoing debate about whether patients planned for liver resection of colorectal liver metastases (CRLM) benefit from neoadjuvant chemotherapy (NC). Therefore, we performed a retrospective survival analysis of patients with and without NC prior to surgery. METHODS: Data prospectively collected from 468 consecutive patients were analyzed in a retrospective design. We performed a survival analysis and added propensity score matching (PSM). Univariate and multivariate analysis was performed to determine independent prognostic risk factors. RESULTS: NC was performed in 145/468 patients. NC did not have a significant influence on overall survival (OS) either before or after PSM. Patients receiving NC showed increased complication rates, especially concerning non-surgical complications after primary resection (P = 0.025) of CRLM. Multivariate analysis before and after PSM revealed that the Memorial Sloan Kettering Cancer Center (MSKCC) score and CEA values are strong predictors for OS in patients with CRLM. CONCLUSIONS: NC was not associated with increased OS in patients suffering from CRLM. Additionally, potentially harmful chemotherapy prior to surgery increases the risk of postoperative complications in these patients.

Neither Isolated Hepatic Arterial Clamping nor Hepatic Arterial Ligation Induce Ischemic Type Biliary Lesions in Rats.

BACKGROUND Ischemic type biliary lesions (ITBL) is a troublesome complication after liver transplantation. Little is known about its pathogenesis and there is particularly little data about morphological alterations. Prolonged warm and cold ischemia time and reduced hepatic arterial perfusion are risk factors leading to ITBL. There are only a few animal models described in literature. Therefore, we examined the effects of 3 h of hepatic artery ischemia-reperfusion (3 h I/R) and hepatic arterial ligation (HAL), both combined with ligation of the peribiliary plexus (PBP). MATERIAL AND METHODS 3 h I/R was performed by clamping the hepatic artery with microvascular clamps for 3 h. HAL was performed by ligation of the hepatic artery. Both procedures were combined with stenting of the common bile duct with double ligation of the PBP. A sham group without clamping served as control. Serum activities of aspartate transaminase (AST) and alanine transaminase (ALT), direct and total bilirubin (DB/TB), and lactate dehydrogenase (LDH) were measured. Bile flow was analyzed and histological examinations of leukocyte infiltration (CAE), cell proliferation (PCNA), apoptotic cells (HE), and bile ducts morphology (CK7) were performed. Western blots of the vascular endothelial growth factor (VEGF) and caspase 3 were made to investigate vascular growth expression and apoptotic cell death. RESULTS 3 h I/R and HAL were associated with a significant hepatocellular injury and inflammation, shown through increased AST and ALT, leukocyte infiltration, and apoptotic cell death. An increase of bile ducts and a reduction of arteries/bile duct ratio after 30 days was observed in the 3 h I/R group and HAL, but no ITBL-typical bile duct necrosis, intrahepatic strictures, or dilatations of bile ducts occurred. CONCLUSIONS Morphological alterations in a rat animal model of 3 h I/R and HAL could be demonstrated. However, a model of intrahepatic biliary lesions could not be established through hepatic arterial ligation or through 3-h hepatic arterial ischemia and reperfusion.

Preconditioning by cilostazol protects against cold hepatic ischemia-reperfusion injury.

BACKGROUND: In liver transplantation, prolonged cold storage and post-transplant reperfusion are associated with endothelial inflammation, organ dysfunction, and graft failure. We herein studied whether cilostazol, a phosphodiesterase-3-inhibitor, attenuates post-ischemic liver injury after prolonged cold storage. MATERIAL AND METHODS: Sprague-Dawley rats were assigned to 5 groups (n=6 each): sham animals (cold storage time (CST): 1 h), vehicle-treated (NaCl 0.9%) controls (CST: 24 h), and animals receiving 0.1, 1.0, or 10.0 mg/kg body weight (BW) cilostazol pre-treatment (CST: 24 h). After organ explantation, all livers were stored at 4°C in HTK solution, followed by 60 min of reperfusion with 37°C Krebs Henseleit buffer in a non-recirculating ex situ system. Bile flow was measured to evaluate liver function. To analyze inflammation and morphology, liver tissue samples were taken and histology, immunohistochemistry, and Western blotting were conducted. RESULTS: In vehicle-treated controls, prolonged cold storage and warm reperfusion induced inflammation, organ dysfunction, and cell death. This was indicated by an increase of hepatocellular vacuolization, endothelial ICAM-1 expression, and apoptotic cell death compared to sham animals. Cilostazol pre-treatment protected against cold hepatic ischemia-reperfusion injury by preventing hepatocellular disintegration, ICAM-1-associated endothelial inflammation, and apoptotic death. CONCLUSIONS: Inhibition of PDE3 reduces endothelial cell activation and hepatocellular injury in cold ischemia/reperfusion of the liver.

Influence of age on resection of colorectal liver metastases.

BACKGROUND AND OBJECTIVES: Only limited data exist investigating the outcome of patients older than 75 years after resection of colorectal liver metastases (CLM). Therefore, the present study aims to evaluate clinical and oncological outcome of elderly patients. METHODS: A cohort of 405 patients was divided into three age-groups: (1) <65 years; (2) 65-75 years; and (3) >75 years of age. Patients' data were prospectively collected and retrospectively analyzed. We performed survival analysis and added age-correction. Univariate and multivariate analysis was performed to determine independent prognostic risk factors. RESULTS: The 5-year survival rate of the >75 years age-group was distinctly decreased, compared to the other age-groups. After age-correction, the 5-year survival rates and the survival curves increased to the greatest extent in patients older than 75 years. The MSKCC score proved to be a sufficient independent prognostic factor in the total patient cohort, patients <65 years and patients 65-75 years. In patients older than 75 years, only localization of the primary tumors was a significant prognostic factor for overall survival. CONCLUSIONS: Patients' age is no reason to deny surgical treatment of CLM. Prognostic factors, such as MSKCC score, are not sufficient predictors of survival in patients older than 75 years.

Isoflurane does not protect from brain death-associated aggravation of cold hepatic ischemia-reperfusion injury.

BACKGROUND: Previous studies have shown that brain death aggravates cold ischemia-reperfusion injury in liver transplantation. Isoflurane, a volatile anesthetic, has been indicated to reduce warm hepatic ischemia-reperfusion injury. Herein, we studied in Sprague-Dawley rats whether isoflurane is capable of ameliorating brain death-associated aggravation of cold hepatic ischemia-reperfusion injury. MATERIAL AND METHODS: Brain-dead animals were treated for 30 min with isoflurane (MAC 1.5%; n=8). Animals without isoflurane treatment served as controls (n=8). Another 13 animals without induction of brain death served as sham controls. After a 4-h period portal venous blood perfusion, hepatic microcirculation and bile flow were determined. Livers were recovered and stored for 24 h in 4°C cold HTK solution, followed by reperfusion with 37°C Krebs-Henseleit-buffer for 60 min. Liver enzymes in the effluent and bile flow were analyzed. Hepatocellular morphology was determined by histology and immunohistochemistry. RESULTS: Brain death reduced portal venous blood perfusion and bile flow, induced heme oxygenase-1 (HO-1), and resulted in hepatocellular damage. Isoflurane treatment did not prevent the reduction of portal venous blood perfusion or bile flow or the induction of HO-1. Accordingly, isoflurane was not capable of reducing the hepatocellular injury. CONCLUSIONS: Isoflurane does not protect from brain death-associated aggravation of cold hepatic ischemia-reperfusion injury.