Dataset Information

Silencing Heat Shock Protein 47 (HSP47) in Fibrogenic Precision-Cut Lung Slices: A Surprising Lack of Effects on Fibrogenesis?

ABSTRACT: Idiopathic pulmonary fibrosis (IPF) is a chronic disease that is characterized by the excessive deposition of scar tissue in the lungs. As currently available treatments are unable to restore lung function in patients, there is an urgent medical need for more effective drugs. Developing such drugs, however, is challenging because IPF has a complex pathogenesis. Emerging evidence indicates that heat shock protein 47 (HSP47), which is encoded by the gene Serpinh1, may be a suitable therapeutic target as it is required for collagen synthesis. Pharmacological inhibition or knockdown of HSP47 could therefore be a promising approach to treat fibrosis. The objective of this study was to assess the therapeutic potential of Serpinh1-targeting small interfering RNA (siRNA) in fibrogenic precision-cut lung slices prepared from murine tissue. To enhance fibrogenesis, slices were cultured for up to 144 h with transforming growth factor ?1. Self-deliverable siRNA was used to knockdown mRNA and protein expression, without affecting the viability and morphology of slices. After silencing HSP47, only the secretion of fibronectin was reduced while other aspects of fibrogenesis remained unaffected (e.g., myofibroblast differentiation as well as collagen secretion and deposition). These observations are surprising as others have shown that Serpinh1-targeting siRNA suppressed collagen deposition in animals. Further studies are therefore warranted to elucidate downstream effects on fibrosis upon silencing HSP47.

SUBMITTER: Ruigrok MJR

PROVIDER: S-EPMC7917123 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Silencing Heat Shock Protein 47 (HSP47) in Fibrogenic Precision-Cut Lung Slices: A Surprising Lack of Effects on Fibrogenesis?

Ruigrok Mitchel J R MJR El Amasi Khaled E M KEM Leeming Diana J DJ Sand Jannie M B JMB Frijlink Henderik W HW Hinrichs Wouter L J WLJ Olinga Peter P

Frontiers in medicine 20210215

Idiopathic pulmonary fibrosis (IPF) is a chronic disease that is characterized by the excessive deposition of scar tissue in the lungs. As currently available treatments are unable to restore lung function in patients, there is an urgent medical need for more effective drugs. Developing such drugs, however, is challenging because IPF has a complex pathogenesis. Emerging evidence indicates that heat shock protein 47 (HSP47), which is encoded by the gene <i>Serpinh1</i>, may be a suitable therapeu ...[more]

PMID: 33659262

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Project description:BackgroundRespiratory diseases represent a global health burden. Because research on therapeutic strategies of airway diseases is essential, the technique of precision-cut lung slices (PCLS) has been developed and widely studied. PCLS are an alternative ex vivo model and have the potential to replace and reduce in vivo animal models. So far, the majority of studies was conducted with short-term cultivated PCLS (≤ 72 h). As there is large interest in research of chronic diseases and chronic toxicity, feasibility of cultivating human PCLS long-term over 2 weeks and recently over 4 weeks was investigated by another research group with successful results. Our aim was to establish a model of long-term cultivated rat PCLS over a period of 29 days.MethodsRat PCLS were cultured for 29 days and analysed regarding viability, histopathology, reactivity and gene expression at different time points during cultivation.ResultsCultivation of rat PCLS over a 29-day time period was successful with sustained viability. Furthermore, the ability of bronchoconstriction was maintained between 13 and 25 days, depending on the mediator. However, reduced relaxation, altered sensitivity and increased respiratory tone were observed. Regarding transcription, alteration in gene expression pattern of the investigated target genes was ascertained during long-term cultivation with mixed results. Furthermore, the preparation of PCLS seems to influence messenger ribonucleic acid (mRNA) expression of most target genes. Moreover, the addition of fetal bovine serum (FBS) to the culture medium did not improve viability of PCLS. In contrast to medium without FBS, FBS seems to affect measurements and resulted in marked cellular changes of metaplastic and/or regenerative origin.ConclusionsOverall, a model of long-term cultivated rat PCLS which stays viable for 29 days and reactive for at least 13 days could be established. Before long-term cultivated PCLS can be used for in-depth study of chronic diseases and chronic toxicity, further investigations have to be made.

Project description:BackgroundAsthma exacerbations evoke emergency room visits, progressive loss of lung function and increased mortality. Environmental and industrial toxicants exacerbate asthma, although the underlying mechanisms are unknown. We assessed whether 3 distinct toxicants, salicylic acid (SA), toluene diisocyanate (TDI), and 1-chloro-2,4-dinitrobenzene (DNCB) induced airway hyperresponsiveness (AHR) through modulating excitation-contraction coupling in human airway smooth muscle (HASM) cells. The toxicants include a non-sensitizing irritant (SA), respiratory sensitizer (TDI) and dermal sensitizer (DNCB), respectively. We hypothesized that these toxicants induce AHR by modulating excitation-contraction (EC) coupling in airway smooth muscle (ASM) cells.MethodsCarbachol-induced bronchoconstriction was measured in precision-cut human lung slices (hPCLS) following exposure to SA, TDI, DNCB or vehicle. Culture supernatants of hPCLS were screened for mediator release. In HASM cells treated with the toxicants, surrogate readouts of EC coupling were measured by phosphorylated myosin light chain (pMLC) and agonist-induced Ca2+ mobilization ([Ca2+]i). In addition, Nrf-2-dependent antioxidant response was determined by NAD(P) H quinone oxidoreductase 1 (NQO1) expression in HASM cells.ResultsIn hPCLS, SA, but not TDI or DNCB, potentiated carbachol-induced bronchoconstriction. The toxicants had little effect on release of inflammatory mediators, including IL-6, IL-8 and eotaxin from hPCLS. In HASM cells, TDI amplified carbachol-induced MLC phosphorylation. The toxicants also had little effect on agonist-induced [Ca2+]i. CONCLUSION: SA, a non-sensitizing irritant, amplifies agonist-induced bronchoconstriction in hPCLS via mechanisms independent of inflammation and Ca2+ homeostasis in HASM cells. The sensitizers TDI and DNCB, had little effect on bronchoconstriction or inflammatory mediator release in hPCLS.ImplicationsOur findings suggest that non-sensitizing irritant salicylic acid may evoke AHR and exacerbate symptoms in susceptible individuals or in those with underlying lung disease.

Dataset Information

Silencing Heat Shock Protein 47 (HSP47) in Fibrogenic Precision-Cut Lung Slices: A Surprising Lack of Effects on Fibrogenesis?

Publications

Silencing Heat Shock Protein 47 (HSP47) in Fibrogenic Precision-Cut Lung Slices: A Surprising Lack of Effects on Fibrogenesis?

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