RESUMEN
Fibrosis results from aberrant wound healing and is characterized by an accumulation of extracellular matrix, impairing the function of an affected organ. Increased deposition of extracellular matrix proteins, disruption of matrix degradation, but also abnormal post-translational modifications alter the biochemical composition and biophysical properties of the tissue microenvironment - the stroma. Macrophages are known to play an important role in wound healing and tissue repair, but the direct influence of fibrotic stroma on macrophage behaviour is still an under-investigated element in the pathogenesis of fibrosis. In this review, the current knowledge on interactions between macrophages and (fibrotic) stroma will be discussed from biochemical, biophysical, and cellular perspectives. Furthermore, we provide future perspectives with regard to how macrophage-stroma interactions can be examined further to ultimately facilitate more specific targeting of these interactions in the treatment of fibrosis. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.
Asunto(s)
Matriz Extracelular/fisiología , Fibrosis/fisiopatología , Macrófagos/metabolismo , Cicatrización de Heridas/fisiología , Animales , HumanosRESUMEN
Our knowledge of complex pathological mechanisms underlying organ fibrosis is predominantly derived from animal studies. However, relevance of animal models for human disease is limited; therefore, an ex vivo model of human precision-cut tissue slices (PCTS) might become an indispensable tool in fibrosis research and drug development by bridging the animal-human translational gap. This study, presented as two parts, provides comprehensive characterization of the dynamic transcriptional changes in PCTS during culture by RNA sequencing. Part I investigates the differences in culture-induced responses in murine and human PCTS derived from healthy liver, kidney and gut. Part II delineates the molecular processes in cultured human PCTS generated from diseased liver, kidney and ileum. We demonstrated that culture was associated with extensive transcriptional changes and impacted PCTS in a universal way across the organs and two species by triggering an inflammatory response and fibrosis-related extracellular matrix (ECM) remodelling. All PCTS shared mRNA upregulation of IL-11 and ECM-degrading enzymes MMP3 and MMP10. Slice preparation and culturing activated numerous pathways across all PCTS, especially those involved in inflammation (IL-6, IL-8 and HMGB1 signalling) and tissue remodelling (osteoarthritis pathway and integrin signalling). Despite the converging effects of culture, PCTS display species-, organ- and pathology-specific differences in the regulation of genes and canonical pathways. The underlying pathology in human diseased PCTS endures and influences biological processes like cytokine release. Our study reinforces the use of PCTS as an ex vivo fibrosis model and supports future studies towards its validation as a preclinical tool for drug development.
Asunto(s)
Técnicas de Cultivo de Órganos/métodos , Transcriptoma/genética , Animales , Análisis por Conglomerados , Fibrosis/patología , Perfilación de la Expresión Génica/estadística & datos numéricos , Regulación de la Expresión Génica , Humanos , Masculino , Redes y Vías Metabólicas , Ratones Endogámicos C57BL , Análisis de Componente Principal , Análisis de Secuencia de ARNRESUMEN
Inhibitors of the proprotein convertase furin might serve as broad-spectrum antiviral therapeutics. High cellular potency and antiviral activity against acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been reported for (3,5-dichlorophenyl)pyridine-derived furin inhibitors. Here we characterized the binding mechanism of this inhibitor class using structural, biophysical, and biochemical methods. We established a MALDI-TOF-MS-based furin activity assay, determined IC50 values, and solved X-ray structures of (3,5-dichlorophenyl)pyridine-derived compounds in complex with furin. The inhibitors induced a substantial conformational rearrangement of the active-site cleft by exposing a central buried tryptophan residue. These changes formed an extended hydrophobic surface patch where the 3,5-dichlorophenyl moiety of the inhibitors was inserted into a newly formed binding pocket. Consistent with these structural rearrangements, we observed slow off-rate binding kinetics and strong structural stabilization in surface plasmon resonance and differential scanning fluorimetry experiments, respectively. The discovered furin conformation offers new opportunities for structure-based drug discovery.
Asunto(s)
Antivirales , Furina , SARS-CoV-2 , Antivirales/química , Furina/química , Proproteína Convertasas , SARS-CoV-2/efectos de los fármacosRESUMEN
Fibrotic processes in the liver of non-alcoholic steatohepatitis (NASH) patients cause microcirculatory dysfunction in the organ which increases blood vessel resistance and causes portal hypertension. Assessing blood vessel function in the liver is challenging, necessitating the development of novel methods in normal and fibrotic tissue that allow for drug screening and translation toward pre-clinical settings. Cultures of precision cut liver slices (PCLS) from normal and fibrotic rat livers were used for blood vessel function analysis. Live recording of vessel diameter was used to assess the response to endothelin-1, serotonin and soluble guanylate cyclase (sGC) activation. A cascade of contraction and relaxation events in response to serotonin, endothelin-1, Ketanserin and sGC activity could be established using vessel diameter analysis of rat PCLS. Both the sGC activator BI 703704 and the sGC stimulator Riociguat prevented serotonin-induced contraction in PCLS from naive rats. By contrast, PCLS cultures from the rat CCl4 NASH model were only responsive to the sGC activator, thus establishing that the sGC enzyme is rendered non-responsive to nitric oxide under oxidative stress found in fibrotic livers. The role of the sGC pathway for vessel relaxation of fibrotic liver tissue was identified in our model. The obtained data shows that the inhibitory capacities on vessel contraction of sGC compounds can be translated to published preclinical data. Altogether, this novel ex vivo PCLS method allows for the differentiation of drug candidates and the translation of therapeutic approaches towards the clinical use.
Asunto(s)
Cirrosis Hepática/fisiopatología , Hígado/irrigación sanguínea , Enfermedad del Hígado Graso no Alcohólico/fisiopatología , Guanilil Ciclasa Soluble/fisiología , Vasoconstricción , Adenosina Trifosfato/metabolismo , Animales , Vasos Sanguíneos/efectos de los fármacos , Vasos Sanguíneos/fisiología , Tetracloruro de Carbono , Endotelina-1/farmacología , Ketanserina/farmacología , Hígado/metabolismo , Hígado/patología , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , Masculino , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , Estrés Oxidativo , Pirazoles/farmacología , Pirimidinas/farmacología , Ratas Sprague-Dawley , Ratas Wistar , Serotonina/farmacología , Transducción de Señal , Vasoconstricción/efectos de los fármacos , Vasodilatación/efectos de los fármacosRESUMEN
BACKGROUND: Animal models should display important characteristics of the human disease. Sheep have been considered particularly useful to study allergic airway responses to common natural antigens causing human asthma. A rationale of this study was to establish a model of ovine precision-cut lung slices (PCLS) for the in vitro measurement of airway responses in newborn and adult animals. We hypothesized that differences in airway reactivity in sheep are present at different ages. METHODS: Lambs were delivered spontaneously at term (147d) and adult sheep lived till 18 months. Viability of PCLS was confirmed by the MTT-test. To study airway provocations cumulative concentration-response curves were performed with different allergic response mediators and biogenic amines. In addition, electric field stimulation, passive sensitization with house dust mite (HDM) and mast cells staining were evaluated. RESULTS: PCLS from sheep were viable for at least three days. PCLS of newborn and adult sheep responded equally strong to methacholine and endothelin-1. The responses to serotonin, leukotriene D4 and U46619 differed with age. No airway contraction was evoked by histamine, except after cimetidine pretreatment. In response to EFS, airways in PCLS from adult and newborn sheep strongly contracted and these contractions were atropine sensitive. Passive sensitization with HDM evoked a weak early allergic response in PCLS from adult and newborn sheep, which notably was prolonged in airways from adult sheep. Only few mast cells were found in the lungs of non-sensitized sheep at both ages. CONCLUSION: PCLS from sheep lungs represent a useful tool to study pharmacological airway responses for at least three days. Sheep seem well suited to study mechanisms of cholinergic airway contraction. The notable differences between newborn and adult sheep demonstrate the importance of age in such studies.
Asunto(s)
Envejecimiento/inmunología , Pulmón/citología , Pulmón/fisiología , Ovinos , Alérgenos/farmacología , Animales , Animales Recién Nacidos , Aminas Biogénicas/farmacología , Broncoconstricción/efectos de los fármacos , Cimetidina/farmacología , Histamina/farmacología , Pulmón/efectos de los fármacos , Pulmón/inmunología , Supervivencia TisularRESUMEN
The peripheral airway innervation of the lower respiratory tract of mammals is not completely functionally characterized. Recently, we have shown in rats that precision-cut lung slices (PCLS) respond to electric field stimulation (EFS) and provide a useful model to study neural airway responses in distal airways. Since airway responses are known to exhibit considerable species differences, here we examined the neural responses of PCLS prepared from mice, rats, guinea pigs, sheep, marmosets and humans. Peripheral neurons were activated either by EFS or by capsaicin. Bronchoconstriction in response to identical EFS conditions varied between species in magnitude. Frequency response curves did reveal further species-dependent differences of nerve activation in PCLS. Atropine antagonized the EFS-induced bronchoconstriction in human, guinea pig, sheep, rat and marmoset PCLS, showing cholinergic responses. Capsaicin (10 µM) caused bronchoconstriction in human (4 from 7) and guinea pig lungs only, indicating excitatory non-adrenergic non-cholinergic responses (eNANC). However, this effect was notably smaller in human responder (30 ± 7.1%) than in guinea pig (79 ± 5.1%) PCLS. The transient receptor potential (TRP) channel blockers SKF96365 and ruthenium red antagonized airway contractions after exposure to EFS or capsaicin in guinea pigs. In conclusion, the different species show distinct patterns of nerve-mediated bronchoconstriction. In the most common experimental animals, i.e. in mice and rats, these responses differ considerably from those in humans. On the other hand, guinea pig and marmoset monkey mimic human responses well and may thus serve as clinically relevant models to study neural airway responses.
Asunto(s)
Broncoconstricción/efectos de los fármacos , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Animales , Bloqueadores de los Canales de Calcio/farmacología , Callithrix , Capsaicina/farmacología , Estimulación Eléctrica , Cobayas , Humanos , Imidazoles/farmacología , Técnicas In Vitro , Ratones , Ratas , Rojo de Rutenio/farmacología , OvinosRESUMEN
The precision-cut lung slice (PCLS) technique is widely used to examine airway responses in different species. We developed a method to study nerve-dependent bronchoconstriction by the application of electric field stimulation (EFS) to PCLS. PCLS prepared from Wistar rats were placed between two platinum electrodes to apply serial rectangular impulses (5-100 Hz), and bronchoconstriction was studied by videomicroscopy. The extent of airway contractions increased with higher frequencies. Stable repeated airway contractions were obtained at a frequency of 50 Hz, a width of 1 ms, and an output of 200 mA for 2.5 s each minute. Larger airways showed stronger responses. The EFS-triggered contractions were increased by the acetylcholine esterase inhibitor neostigmine (10 µM) and reversed by the muscarinic antagonist atropine (10 µM), whereas the thromboxane protanoid receptor antagonist SQ29548 (10 µM) had no effect. Magnesium ions (10 mM) antagonized airway contractions induced by EFS, but not by methacholine, indicating that nerve endings remain intact in PCLS. Our data further show that the electrically evoked airway contractions in PCLS are mediated by cholinergic nerves, independent of thromboxane and more prominent in larger airways. Taken together these findings show that nerve endings remain intact in PCLS, and they suggest that the present method is useful to study neurogenic responses in airways of different size.