RESUMO
Inflammatory bowel disease such as chronic colitis promotes colorectal cancer, which is a common cause of cancer mortality worldwide. Hypoxia is a characteristic of inflammation as well as of solid tumors and enforces a gene expression response controlled by hypoxia-inducible factors (HIFs). Once established, solid tumors are immunosuppressive to escape their abatement through immune cells. Although HIF activity is known to 1) promote cancer development and 2) drive tumor immune suppression through the secretion of adenosine, both prolyl hydroxylases and an asparaginyl hydroxylase termed factor-inhibiting HIF (FIH) negatively regulate HIF. Thus, FIH may act as a tumor suppressor in colorectal cancer development. In this study, we examined the role of colon epithelial FIH in a mouse model of colitis-induced colorectal cancer. We recapitulated colitis-associated colorectal cancer development in mice using the azoxymethane/dextran sodium sulfate model in Vil1-Cre/FIH+f/+f and wild-type siblings. Colon samples were analyzed regarding RNA and protein expression and histology. Vil1-Cre/FIH+f/+f mice showed a less severe colitis progress compared with FIH+f/+f animals and a lower number of infiltrating macrophages in the inflamed tissue. RNA sequencing analyses of colon tissue revealed a lower expression of genes associated with the immune response in Vil1-Cre/FIH+f/+f mice. However, tumor occurrence did not significantly differ between Vil1-Cre/FIH+f/+f and wild-type mice. Thus, FIH knockout in colon epithelial cells did not modulate colorectal cancer development but reduced the inflammatory response in chronic colitis.
Assuntos
Neoplasias Associadas a Colite/patologia , Colite/patologia , Neoplasias Colorretais/patologia , Mucosa Intestinal/patologia , Oxigenases de Função Mista/metabolismo , Adenosina/metabolismo , Animais , Azoximetano/toxicidade , Hipóxia Celular/fisiologia , Colite/induzido quimicamente , Colite/genética , Neoplasias Associadas a Colite/genética , Colo/patologia , Neoplasias Colorretais/genética , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Células Epiteliais/patologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxigenases de Função Mista/genética , Prolil Hidroxilases/metabolismo , Transdução de Sinais/fisiologia , Evasão Tumoral/imunologia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
This work aimed at the development of a stable albumin-perfluorocarbon (o/w) emulsion as an artificial oxygen carrier suitable for clinical application. So far, albumin-perfluorocarbon-(o/w) emulsions have been successfully applied in preclinical trials. Cross-linking a variety of different physical and chemical methods for the characterization of an albumin-perfluorocarbon (PFC)-(o/w) emulsion was necessary to gain a deep understanding of its specific emulsification processes during high-pressure homogenization. High-pressure homogenization is simple but incorporates complex physical reactions, with many factors influencing the formation of PFC droplets and their coating. This work describes and interprets the impact of albumin concentration, homogenization pressure, and repeated microfluidizer passages on PFC-droplet formation; its influence on storage stability; and the overcoming of obstacles in preparing stable nanoemulsions. The applied methods comprise dynamic light scattering, static light scattering, cryo- and non-cryo-scanning and transmission electron microscopies, nuclear magnetic resonance spectroscopy, light microscopy, amperometric oxygen measurements, and biochemical methods. The use of this wide range of methods provided a sufficiently comprehensive picture of this polydisperse emulsion. Optimization of PFC-droplet formation by means of temperature and pressure gradients results in an emulsion with improved storage stability (tested up to 5 months) that possibly qualifies for clinical applications. Adaptations in the manufacturing process strikingly changed the physical properties of the emulsion but did not affect its oxygen capacity.
Assuntos
Fluorocarbonos , Albuminas , Emulsões/química , Fluorocarbonos/química , Oxigênio , Tamanho da PartículaRESUMO
Developing biocompatible, synthetic oxygen carriers is a consistently challenging task that researchers have been pursuing for decades. Perfluorocarbons (PFC) are fascinating compounds with a huge capacity to dissolve gases, where the respiratory gases are of special interest for current investigations. Although largely chemically and biologically inert, pure PFCs are not suitable for injection into the vascular system. Extensive research created stable PFC nano-emulsions that avoid (i) fast clearance from the blood and (ii) long organ retention time, which leads to undesired transient side effects. PFC-based oxygen carriers (PFOCs) show a variety of application fields, which are worthwhile to investigate. To understand the difficulties that challenge researchers in creating formulations for clinical applications, this review provides the physical background of PFCs' properties and then illuminates the reasons for instabilities of PFC emulsions. By linking the unique properties of PFCs and PFOCs to physiology, it elaborates on the response, processing and dysregulation, which the body experiences through intravascular PFOCs. Thereby the reader will receive a scientific and easily comprehensible overview why PFOCs are precious tools for so many diverse application areas from cancer therapeutics to blood substitutes up to organ preservation and diving disease.
Assuntos
Substitutos Sanguíneos/uso terapêutico , Fluorocarbonos/uso terapêutico , Oxigênio/sangue , Animais , Substitutos Sanguíneos/efeitos adversos , Substitutos Sanguíneos/química , Composição de Medicamentos , Emulsões , Fluorocarbonos/efeitos adversos , Fluorocarbonos/química , HumanosRESUMO
Background/Objectives: Hereditary hemorrhagic telangiectasia (HHT) is a rare disorder characterized by dilated blood vessels. Different immunological changes have been described in these patients. In this study, the predisposition of patients with HHT to infections and allergic diseases was assessed. Methods: Patients with HHT completed an online survey in English or German. Their data were compared to non-affected partners or friends. Results: A total of 430 out of 588 respondents with HHT answered our questions about infections and allergies. Patients with HHT suffered significantly more often from various types of allergies than their partners, especially type I allergies (n = 226/276, 82%), and had a higher risk for sinusitis, urinary tract infections, pulmonary infections, and abscesses. A total of 38% of the patients took antibiotics prior to dental or surgical procedures (n = 57/152), and, in 10% of these patients, pulmonary arteriovenous malformations (PAVMs) were not detected. On the other hand, 51% of patients with PAVM did not report a prophylactic antibiotic intake (n = 40/79). The patients who needed iron supplementations suffered more often from sepsis (OR: 9.00, 95%CI: 0.92-88.16). Conclusions: Compared to their non-affected controls, patients with HHT showed an increased risk for infections in different organs and allergic diseases. There is a need for campaigns raising greater awareness recommending prophylactic antibiotic intake in patients with PAVM.
RESUMO
The ability of cells to communicate with their surrounding is a prerequisite for essential processes such as proliferation, apoptosis, migration, and differentiation. To this purpose, primary cilia serve as antennae-like structures on the surface of most mammalian cell types. Cilia allow signaling via hedgehog, Wnt or TGF-beta pathways. Their length, in part controlled by the activity of intraflagellar transport (IFT), is a parameter for adequate function of primary cilia. Here we show, in murine neuronal cells, that intraflagellar transport protein 88 homolog (IFT88) directly interacts with the hypoxia-inducible factor-2α (HIF-2α), hitherto known as an oxygen-regulated transcription factor. Furthermore, HIF-2α accumulates in the ciliary axoneme and promotes ciliary elongation under hypoxia. Loss of HIF-2α affected ciliary signaling in neuronal cells by decreasing transcription of Mek1/2 and Erk1/2. Targets of the MEK/ERK signaling pathway, such as Fos and Jun, were significantly decreased. Our results suggest that HIF-2α influences ciliary signaling by interacting with IFT88 under hypoxic conditions. This implies an unexpected and far more extensive function of HIF-2α than described before.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Cílios , Camundongos , Animais , Cílios/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Proteínas de Transporte/metabolismo , Hipóxia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Mamíferos/metabolismoRESUMO
The interplay between hypoxia-inducible factors (HIFs) and transforming growth factor beta (TGF-ß) is critical for both inflammation and angiogenesis. In hereditary hemorrhagic telangiectasia (HHT), we have previously observed that impairment of the TGF-ß pathway is associated with downregulation of HIF-1α. HIF-1α accumulation is mandatory in situations of altered energy demand, such as during infection or hypoxia, by adjusting cell metabolism. Leukocytes undergo a HIF-1α-dependent switch from aerobic mitochondrial respiration to anaerobic glycolysis (glycolytic switch) after stimulation and during differentiation. We postulate that the decreased HIF-1α accumulation in HHT leads to a clinically observed immunodeficiency in these patients. Examination of HIF-1α and its target genes in freshly isolated peripheral blood mononuclear cells (PBMCs) from HHT patients revealed decreased gene expression and protein levels of HIF-1α and HIF-1α-regulated glycolytic enzymes. Treatment of these cells with the HIF-prolyl hydroxylase inhibitor, Roxadustat, rescued their ability to accumulate HIF-1α protein. Functional analysis of metabolic flux using a Seahorse FX extracellular flux analyzer showed that the extracellular acidification rate (indicator of glycolytic turnover) after Roxadustat treatment was comparable to non-HHT controls, while oxygen consumption (indicator of mitochondrial respiration) was slightly reduced. HIF stabilization may be a potential therapeutic target in HHT patients suffering from infections.
RESUMO
Patients with hereditary haemorrhagic telangiectasia (HHT), also known as Rendu-Osler-Weber syndrome, suffer from the consequences of abnormal vessel structures. These structures can lead to haemorrhages or shunt effects in liver, lungs and brain. This inherited and rare disease is characterized by mutations affecting the transforming growth factor-ß (TGF-ß)/Bone Morphogenetic Protein (BMP) pathway that results in arteriovenous malformations and studies indicate an impaired immune response. The mechanism underlying this altered immune response in HHT patients is still unknown. TGF-ß interacts with hypoxia inducible factors (HIF), which both orchestrate inflammatory and angiogenic processes. Therefore, we analysed the expression of HIF and related genes in whole blood samples from HHT patients. We could show significantly decreased expression of HIF-1α on the mRNA and protein level. However, commonly known upstream regulators of HIF-1α in inflammatory responses were not affected, whereas HIF-1α target genes were significantly downregulated. There was no correlation between HIF1A or HIF2A gene expression and the severity of HHT detected. Our results represent a rare case of HIF-1α downregulation in a human disease, which underlines the relevance of HIFs in HHT. The study indicates an interaction of the known mutation in HHT and the dysregulation of HIF-1α in HHT patients, which might contribute to the clinical phenotype.
Assuntos
Malformações Arteriovenosas , Subunidade alfa do Fator 1 Induzível por Hipóxia , Telangiectasia Hemorrágica Hereditária , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Mutação , Telangiectasia Hemorrágica Hereditária/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
Artificial blood for clinical use is not yet available therefore, we previously developed artificial oxygen carriers (capsules) and showed their functionality in vitro and biocompatibility in vivo. Herein, we assessed the functionality of the capsules in vivo in a normovolemic hemodilution rat-model. We stepwise exchanged the blood of male Wistar-rats with medium either in the presence of capsules (treatment) or in their absence (control). We investigated tissue hypoxia thoroughly through online biomonitoring, determination of enzyme activity and pancreatic hormones in plasma, histochemical and immunohistochemical staining of small intestine, heart, liver and spleen as well as in situ hybridization of kidneys. After hemodilution, treated animals show higher arterial blood pressure and have a stable body temperature. Additionally, they show a more stable pH, a higher oxygen partial pressure (pO2), and a lower carbon dioxide partial pressure (pCO2). Interestingly, blood-glucose-levels drop severely in treated animals, presumably due to glucose consumption. Creatine kinase values in these animals are increased and isoenzyme analysis indicates the spleen as origin. Moreover, the small intestine of treated animals show reduced hypoxic injury compared to controls and the kidneys have reduced expression of the hypoxia-inducible erythropoietin mRNA. In conclusion, our capsules can prevent hypoxic tissue damage. The results provide a proof of concept for capsules as adequate erythrocyte substitute.
Assuntos
Albuminas/metabolismo , Substitutos Sanguíneos , Fluorocarbonos , Hemodiluição , Hipóxia/metabolismo , Oxigênio/metabolismo , Albuminas/química , Animais , Biomarcadores , Índices de Eritrócitos , Fluorocarbonos/química , Fluorocarbonos/metabolismo , Expressão Gênica , Hormônios , Humanos , Concentração de Íons de Hidrogênio , Imuno-Histoquímica , Especificidade de ÓrgãosRESUMO
The aim of this study was to prove whether albumin-derived perfluorocarbon-based nanoparticles (capsules) can operate as a novel artificial oxygen carrier in a rat Langendorff-heart perfusion model. Hearts perfused with capsules showed increased left ventricular pressure and rate pressure product compared to hearts perfused with pure Krebs-Henseleit (KH)-buffer. The capsules prevented the myocardium from functional fail when in their absence a noxious ischemia was observed. Capsules did not change rheological properties of KH-buffer and could repeatedly reload with oxygen. This albumin-derived perfluorocarbon-based artificial oxygen carrier preserved the function of rat hearts due to the transport of oxygen in a satisfactory manner. Because of these positive results, the functionality of the applied capsules should be verified in living animals.
Assuntos
Albuminas/química , Substitutos Sanguíneos/química , Fluorocarbonos/química , Fluorocarbonos/farmacologia , Coração/efeitos dos fármacos , Animais , Substitutos Sanguíneos/farmacologia , Relação Dose-Resposta a Droga , Feminino , Coração/fisiologia , Hemodinâmica/efeitos dos fármacos , Oxigênio/metabolismo , Perfusão , Ratos , Ratos WistarRESUMO
Until today, artificial oxygen carriers have not been reached satisfactory quality for routine clinical treatments. To bridge this gap, we designed albumin-derived perfluorocarbon-based nanoparticles as novel artificial oxygen carriers and evaluated their physico-chemical and pharmacological performance. Our albumin-derived perfluorocarbon-based nanoparticles (capsules), composed of an albumin shell and a perfluorodecalin core, were synthesized using ultrasonics. Their subsequent analysis by physico-chemical methods such as scanning electron-, laser scanning- and dark field microscopy as well as dynamic light scattering revealed spherically-shaped, nano-sized particles, that were colloidally stable when dispersed in 5% human serum albumin solution. Furthermore, they provided a remarkable maximum oxygen capacity, determined with a respirometer, reflecting a higher oxygen transport capacity than the competitor Perftoran®. Intravenous administration to healthy rats was well tolerated. Undesirable effects on either mean arterial blood pressure, hepatic microcirculation (determined by in vivo microscopy) or any deposit of capsules in organs, except the spleen, were not observed. Some minor, dose-dependent effects on tissue damage (release of cellular enzymes, alterations of spleen's micro-architecture) were detected. As our promising albumin-derived perfluorocarbon-based nanoparticles fulfilled decisive physico-chemical demands of an artificial oxygen carrier while lacking severe side-effects after in vivo administration they should be advanced to functionally focused in vivo testing conditions.