Laser capture microdissection reveals dose-response of gene expression in situ consequent to asbestos exposure

Our plain-English summary of Laser capture microdissection reveals dose-response of gene expression in situ consequent to asbestos exposure, by Q. Yin, A.R. Brody, D.E. Sullivan (International Journal of Experimental Pathology, 2007).

The study

• Authors: Q. Yin, A.R. Brody, D.E. Sullivan
• Journal: International Journal of Experimental Pathology
• Published: 2007;88(6):415-25
• Cited by: 10 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

The genes that mediate fibroproliferative lung disease remain to be defined. Prior studies from our laboratory showed by in situ hybridization and immunohistochemistry that the genes coding for tumour necrosis factor alpha, transforming growth factor beta, the platelet-derived growth factor A and B isoforms, and alpha-1 pro-collagen are expressed in fibroproliferative lesions that develop quickly after asbestos inhalation. These five genes, along with matrix metalloproteinase 9, a collagenase found to be increased in several lung diseases, are known to control matrix production and cell proliferation in humans and animals. Here we show by laser capture microdissection that (i) The six genes are expressed at significantly higher levels in the asbestos-exposed mice when comparing the same anatomic regions 'captured' in unexposed mice. (ii) The bronchiolar-alveolar duct (BAD) junctions, where the greatest number of fibres initially deposit, were always significantly higher than the other anatomic regions for each gene. The first alveolar duct bifurcation (ADB) generally was higher than the second ADB, the ADBs were always significantly higher than the airway walls and pleura, and the airway walls and pleura were generally higher than the unexposed tissues. (iii) Animals exposed for 3 days always exhibited significantly higher levels of gene expression at the BAD junctions and ADBs than animals exposed for 2 days. To our knowledge, this is the first demonstration of a dose-response to a toxic particle in situ, and this response appears to be dependent on the number of fibres that deposits at the individual anatomic site.

In plain English

This was an animal inhalation experiment. Male C57BL/6 mice were exposed to an aerosol of chrysotile asbestos (about 10 mg/m3 respirable mass) or to room air for 5 hours a day for 2 or 3 days and were killed 48 hours after the last exposure. Frozen lung sections were cut with a laser (laser capture microdissection) to collect specific sites, the bronchiolar-alveolar duct (BAD) junctions, first and second alveolar duct bifurcations (ADBs), airway walls and visceral pleura, and expression of six genes (TNFa, TGFb, PDGFA, PDGFB, MMP9 and alpha-1 pro-collagen) was measured by real-time RT-PCR. The authors reported that expression at the BAD junctions and ADBs was significantly higher in exposed mice than at the same sites in unexposed mice, and that BAD junctions were always highest. With all six genes combined, 3-day exposure gave higher expression than 2-day exposure at the BAD junctions (P less than 0.0001), the first ADBs (P less than 0.005) and the second ADBs (P less than 0.0001), though for individual genes the 2-day versus 3-day difference was not always significant. Pleural and airway wall findings varied by gene. The authors described this as an in situ dose-response.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Animal experiment: mice exposed by inhalation to chrysotile asbestos, with laser capture microdissection of lung sites and real-time RT-PCR
• Who was studied: Specific-pathogen-free 6-week-old male C57BL/6 mice weighing 25-30 g, from Jackson Laboratories
• Exposure: Inhaled aerosol of chrysotile asbestos, approximately 10 mg/m3 respirable mass, 5 hours per day for 2 or 3 days; controls breathed room air
• Main results:
  • In the 2- and 3-day exposed mice, expression of all six genes at the BAD junctions and ADBs was significantly higher than at the same sites in unexposed animals
  • For each gene, the BAD junctions showed significantly higher expression than the other anatomic regions
  • The first ADB was generally higher than the second ADB, and the ADBs were always significantly higher than the airway walls and pleura
  • With all six genes combined at the BAD junctions, values from mice exposed 3 days were significantly higher than from mice exposed 2 days (P < 0.0001)
  • With all six genes combined, 3-day exposed mice had significantly higher expression than 2-day exposed mice at the first ADBs (P < 0.005) and the second ADBs (P < 0.0001)
  • Considered gene by gene, the 2-day versus 3-day difference was not always significant: TGFb at the BAD junction was significantly higher at 3 days (P < 0.01), while TNFa at the BAD junctions only approached significance
  • Pleural and airway wall expression was not consistently raised above unexposed tissue and varied by gene; for TGFb1 in pleura the comparison reached P = 0.06, while for PDGFB in pleura it was P < 0.0004
  • Parenchymal tissue remote from developing lesions in exposed mice showed no increase over normal gene expression levels (data not shown)
  • One-way ANOVA applied to the data for each gene individually showed a significant difference (P < 0.0001) in every case
  • A post-test for linear trend across the six anatomic sites was significant (P < 0.0001) for every gene
• The authors' conclusion: The authors concluded that, to their knowledge, this was the first demonstration of an in situ dose-response to an inhaled toxic agent, the site intercepting the most fibres showing the highest gene expression
• Limitations the authors state:
  • The authors stated they could not tell whether the higher gene expression came from more cells in the lesions or from greater up-regulation within specific cell populations
  • The authors noted that for individual genes the 2-day versus 3-day difference was not always statistically significant
  • The authors looked ahead to absolute quantitation of gene copy numbers in future work

Topics

The National Library of Medicine indexes this study under subjects our site covers: Asbestosis.

Medical terms in this study, explained in our Lexicon: Dose-response relationship, Asbestos Exposure, Immunohistochemistry, Necrosis, Platelet-Derived Growth Factor (PDGF), Collagen, Pleura.

Related studies

• Analysis of asbestos-induced gene expression changes in bronchiolar epithelial cells using laser capture microdissection and quantitative reverse transcriptase-polymerase chain reaction — C.B. Manning, B.T. Mossman, et al. (2006)
• Reduced tumor necrosis factor-alpha and transforming growth factor-beta1 expression in the lungs of inbred mice that fail to develop fibroproliferative lesions consequent to asbestos exposure — D.M. Brass, G.W. Hoyle, et al. (1999)
• Expression of genes coding for growth factors in experimental pneumoconiosis — G.F. Morris, J.Y. Liu, et al. (1996)
• Characterization of pulmonary responses in mice to asbestos/asbestiform fibers using gene expression profiles — N. Yanamala, E.R. Kisin, et al. (2018)
• Primary lung fibroblasts from the 129 mouse strain exhibit reduced growth factor responsiveness in vitro — D.M. Brass, S.Y. Tsai, et al. (2001)
• Mineral dusts directly induce epithelial and interstitial fibrogenic mediators and matrix components in the airway wall — J. Dai, B. Gilks, et al. (1998)

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