RESUMO
CO2, the primary gaseous product of respiration, is a major physiologic gas, the biology of which is poorly understood. Elevated CO2 is a feature of the microenvironment in multiple inflammatory diseases that suppresses immune cell activity. However, little is known about the CO2-sensing mechanisms and downstream pathways involved. We found that elevated CO2 correlates with reduced monocyte and macrophage migration in patients undergoing gastrointestinal surgery and that elevated CO2 reduces migration in vitro. Mechanistically, CO2 reduces autocrine inflammatory gene expression, thereby inhibiting macrophage activation in a manner dependent on decreased intracellular pH. Pharmacologic or genetic inhibition of carbonic anhydrases (CAs) uncouples a CO2-elicited intracellular pH response and attenuates CO2 sensitivity in immune cells. Conversely, CRISPR-driven upregulation of the isoenzyme CA2 confers CO2 sensitivity in nonimmune cells. Of interest, we found that patients with chronic lung diseases associated with elevated systemic CO2 (hypercapnia) display a greater risk of developing anastomotic leakage following gastrointestinal surgery, indicating impaired wound healing. Furthermore, low intraoperative pH levels in these patients correlate with reduced intestinal macrophage infiltration. In conclusion, CO2 is an immunomodulatory gas sensed by immune cells through a CA2-coupled change in intracellular pH.
Assuntos
Dióxido de Carbono , Anidrase Carbônica II , Dióxido de Carbono/metabolismo , Anidrase Carbônica II/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Hipercapnia/enzimologia , Hipercapnia/metabolismo , IsoenzimasRESUMO
BACKGROUND: Patients with borderline resectable colorectal liver metastases (CRLM) frequently receive neoadjuvant chemotherapy (NC) to reduce tumour burden, thus making surgical resection feasible. Even though NC can induce severe liver injury, most studies investigating tissue-based prognostic markers focus on tumour tissue. Here, we assessed the prognostic significance of pyruvate-dehydrogenase-kinase isoenzyme 4 (PDK4) within liver tissue of patients undergoing surgical resection due to CRLM. METHODS: Transcript levels of hypoxia-adaptive genes (such as PDK isoenzymes) were assessed in the tissue of healthy liver, corresponding CRLM, healthy colon mucosa and corresponding tumour. Uni- and multivariate analyses were performed. Responses to chemotherapy upon up- or down-regulation of PDK4 were studied in vitro. RESULTS: PDK4 expression within healthy liver tissue was associated with increased overall survival and liver function following surgical resection of CRLM. This association was enhanced in patients with NC. PDK4 expression in CRLM tissue did not correlate with overall survival. Up-regulation of PDK4 increased the resistance of hepatocytes and colon cancer cells against chemotherapy-induced toxicity, whereas knockdown of PDK4 enhanced chemotherapy-associated cell damage. CONCLUSION: Our findings suggest that up-regulated PDK4 expression reduces hepatic chemotherapy-induced oxidative stress and is associated with improved postoperative liver function in patients undergoing multimodal treatment and resection of CRLM.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Colorretais/terapia , Hepatócitos/efeitos dos fármacos , Neoplasias Hepáticas/terapia , Piruvato Desidrogenase Quinase de Transferência de Acetil/genética , Idoso , Animais , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Terapia Combinada , Regulação para Baixo , Feminino , Fenofibrato/farmacologia , Fluoruracila/farmacologia , Técnicas de Introdução de Genes , Técnicas de Silenciamento de Genes , Células Hep G2 , Hepatectomia , Hepatócitos/metabolismo , Humanos , Técnicas In Vitro , Fígado/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundário , Masculino , Metastasectomia , Camundongos , Pessoa de Meia-Idade , Terapia Neoadjuvante , Oxaliplatina/farmacologia , Prognóstico , Piruvato Desidrogenase Quinase de Transferência de Acetil/metabolismo , RNA Mensageiro/metabolismo , Taxa de Sobrevida , Regulação para CimaRESUMO
Liver fibrosis, eventually progressing to cirrhosis necessitating liver transplantation, poses a significant clinical problem. Oxygen shortage (hypoxia) and hypoxia-inducible transcription factors (HIFs) have been acknowledged as important drivers of liver fibrosis. The significance of oxygen-sensing HIF prolyl-hydroxylase (PHD) enzymes in this context has, however, remained elusive. In this study, we demonstrate that loss of PHD1 (PHD1-/-) attenuates the development of liver fibrosis in mice subjected to chronic bile duct injury, induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine. This effect was accompanied with reduced recruitment of inflammatory leukocytes and attenuated occurrence of profibrotic myofibroblasts in PHD1-/- livers. Further analyses focused on the significance of PHD1 in the activation of hepatic stellate cells (HSCs), which represent the driving force in liver fibrosis. Primary HSCs isolated from PHD1-/- mice displayed significantly attenuated myofibroblast differentiation and profibrogenic properties compared with HSCs isolated from wild-type mice. Consistently, the expression of various profibrogenic and promitogenic factors was reduced in PHD1-/- HSCs, without alterations in HIF-1α protein levels. Of importance, PHD1 protein was expressed in HSCs within human livers, and PHD1 transcript expression was significantly increased with disease severity in hepatic tissue from patients with liver fibrosis. Collectively, these findings indicate that PHD1 deficiency protects against liver fibrosis and that these effects are partly due to attenuated activation of HSCs. PHD1 may represent a therapeutic target to alleviate liver fibrosis.
Assuntos
Ductos Biliares/patologia , Fibrose/patologia , Células Estreladas do Fígado/patologia , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Cirrose Hepática/patologia , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Índice de Gravidade de Doença , Animais , Ductos Biliares/metabolismo , Células Cultivadas , Fibrose/metabolismo , Células Estreladas do Fígado/metabolismo , Humanos , Cirrose Hepática/metabolismo , Camundongos , Camundongos KnockoutRESUMO
Rifampicin and rifabutin can activate the pregnane X receptor (PXR, NR1I2), thereby inducing pharmacokinetically important genes/proteins and reducing exposure to co-administered drugs. Because induction effects vary considerably between these antibiotics, differences could be due to unequal rifamycin-induced activation or tissue expression of the three major NR1I2 splice variants, PXR.1 (NM_003889), PXR.2 (NM_022002), and PXR.3 (NM_033013). Consequently, PXR activation (PXR reporter gene assays) and mRNA expression levels of total NR1I2, PXR.1, PXR.2, and PXR.3 were investigated by polymerase chain reaction in colon and liver samples from eleven surgical patients, in LS180 cells, and primary human hepatocytes. Compared to the colon, total NR1I2 mRNA expression was higher in the liver. Both tissues showed similar expression levels of PXR.1 and PXR.3, respectively. PXR.2 was not quantifiable in the colon samples. Rifampicin and rifabutin similarly enhanced PXR.1 and PXR.2 activity when transfected into LS180 cells, while PXR.3 could not be activated. In LS180 cells, rifampicin (10 µM) reduced total NR1I2 and PXR.3 expression 2-fold after 24 h, while rifabutin (10 µM) increased total NR1I2, PXR.1, PXR.2, and PXR.3 mRNA by approx. 50% after 96-h exposure. In primary human hepatocytes, rifampicin (10 µM) suppressed total NR1I2, PXR.1, and PXR.3 after 48-h exposure, and rifabutin (10 µM) had no significant impact on total NR1I2 or any of the splice variants studied. In conclusion, both antibiotics activated the studied PXR splice variants similarly but modified their expression differently. While rifampicin can suppress mRNA of PXR forms, rifabutin rather increases their expression levels.
Assuntos
Receptores de Esteroides , Rifampina , Humanos , Receptor de Pregnano X , Rifampina/farmacologia , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Rifabutina , Antibacterianos , RNA Mensageiro , Citocromo P-450 CYP3ARESUMO
AIM OF THE STUDY: Arthroscopy ranks among the frequently performed interventions in orthopedics. The aim of this study was to compare the palpation technique with the ultrasound technique for locating the dorsal glenohumeral joint space (JS) in shoulder joint punctures. MATERIAL AND METHODS: Participants inexperienced in ultrasound examinations were included. Palpatory and ultrasound finding of the joint space by the participants was performed according to current recommendations and was initially demonstrated by an instructional video. The ideal point (IP) was marked under ultrasound visualization by an experienced ultrasound examinator and shoulder-elbow surgeon. Furthermore, a corridor for a safe puncture was defined. The palpatorily determined point (pdP) was marked by the participants and evaluated by means of a coordinate system. The evaluation of the sonographically determined point (sdP) was performed similarly to that of the palpatory procedure. RESULTS: Fifty-four participants were included in the study, and the mean length of work experience was 6.3 years. On average, participants had performed 16.5 punctures of the shoulder joint and 6.8 arthroscopies of the shoulder joint. The mean experience in performing sonographic examinations of the shoulder was 27.6 examinations. A total of 100 shoulder joints were examined (54 left, 46 right shoulders). The mean deviation from the ideal point (IP) for the palpatory approach was 17.1 mm with a maximum deviation of 59.5 mm; for the sonographic technique, the mean deviation was 10.3 mm (max. 30.2 mm). Overall, 22% of pdPs were within the defined corridor, while 42% of sdPs were within the target corridor. The average difference between palpatory and sonographic approaches was 9.0 mm in favor of the sonographic technique (max. 46.5 mm). A significantly greater deviation (p < 0.001) from the IP was observed with the palpatory approach than with the sonographic approach. CONCLUSION: Based on the results, the authors recommend ultrasound imaging of the shoulder joint as well as ultrasound-assisted punctures, especially for inexperienced users. Furthermore, training in ultrasound-assisted interventions should be implemented in future training curricula.
RESUMO
OBJECTIVE: This study aimed to investigate whether the asphericity of the neck-head junction of the femur confirmed via ultrasound is associated with further pathology due to femoro-acetabular impingement (FAI). METHODOLOGY: After a clinical examination with positive FAI tests, an ultrasound examination of the hip was performed. In the case of asphericity, a quantitative ultrasound-assisted assessment of the hip was performed, followed by contrast-enhanced arthro-MRI with the question of cartilage or labral damage. RESULTS AND CONCLUSIONS: We included 51 patients with a mean age of 35.25. According to the examination algorithm, asphericity was present in all patients via ultrasonography. The average anterior alpha angle (AAA) determined in ultrasonography was 43.49°. The average AAA on the arthro-MRI was 44.19°. The mean anterior head neck offset (AHNO) in ultrasound was 5.27 mm, and in arthro-MRI, it was 5.36 mm. Arthro-MRI confirmed a bump in 47 patients and a talization disorder in 4 patients. In 49 patients, a labral lesion was found, with one being a re-rupture. Furthermore, in one patient, labral degeneration was identified. Cartilage damage to the hip joint was found in 25 patients. Two patients had neither labral nor cartilage damage in the arthro-MRI. In our study, sonographically confirmed asphericity of the head-neck junction was found in 49 cases, which was associated with further pathology and, according to the current doctrine, was attributable to the FAI and required surgical intervention. This study shows that the detection of a pathologic head and neck contour via ultrasound in combination with positive clinical signs, as present in FAI, is associated with chondrolabral lesions detected via arthro-MRI in 96.1% of cases.
RESUMO
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.
Assuntos
Metformina , Animais , Humanos , Hipoglicemiantes , Hipóxia/complicações , Metformina/farmacologia , Metformina/uso terapêutico , Camundongos , Transdução de Sinais , Aderências Teciduais/etiologia , Aderências Teciduais/prevenção & controleRESUMO
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.