Cigarette smoke exposure inhibits extracellular MMP-2 (gelatinase A) activity in human lung fibroblasts

BACKGROUND: Exposure to cigarette smoke is considered a major risk factor for the development of lung diseases, since its causative role has been assessed in the induction and maintenance of an inflamed state in the airways. Lung fibroblasts can contribute to these processes, due to their ability to produce proinflammatory chemotactic molecules and extracellular matrix remodelling proteinases. Among proteolytic enzymes, gelatinases A and B have been studied for their role in tissue breakdown and mobilisation of matrix-derived signalling molecules. Multiple reports linked gelatinase deregulation and overexpression to the development of inflammatory chronic lung diseases such as COPD. METHODS: In this study we aimed to determine variations in the gelatinolytic pattern of human lung fibroblasts (HFL-1 cell line) exposed to cigarette smoke extract (CSE). Gelatinolytic activity levels were determined by using gelatin zymography for the in-gel detection of the enzymes (proenzyme and activated forms), and the subsequent semi-quantitative densitometric evaluation of lytic bands. Expression of gelatinases was evaluated also by RT-PCR, zymography of the cell lysates and by western blotting. RESULTS: CSE exposure at the doses used (1–10%) did not exert any significant cytotoxic effects on fibroblasts. Zymographic analysis showed that CSE exposure resulted in a linear decrease of the activity of gelatinase A. Control experiments allowed excluding a direct inhibitory effect of CSE on gelatinases. Zymography of cell lysates confirmed the expression of MMP-2 in all conditions. Semi-quantitative evaluation of mRNA expression allowed assessing a reduced transcription of the enzyme, as well as an increase in the expression of TIMP-2. Statistical analyses showed that the decrease of MMP-2 activity in conditioned media reached the statistical significance (p = 0.0031 for 24 h and p = 0.0012 for 48 h), while correlation analysis showed that this result was independent from CSE cytotoxicity (p = 0.7833 for both exposures). CONCLUSION: Present work describes for the first time that, apart well characterized proinflammatory responses, human lung fibroblasts may react to CSE with a significant reduction of extracellular MMP-2 lytic activity. Therefore, fibroblasts may actively participate to the alteration of the proteolysis/antiproteolysis balance, which reflects the defective repair of the extracellular matrix. Such event should provide a further contribution to the maintenance of the inflamed state in the lungs

Amplified and selective assay of collagens by enzymatic and fluorescent reactions

Sensitive and selective assay of collagen is of substantial importance to the diagnostic study of health- and aging-related failures. In this paper, we describe a highly specific and sensitive method for the assay of whole collagens in biological samples using a novel fluorogenic reagent, 3,4- dihydroxyphenylacetic acid (3,4-DHPAA). The 3,4-DHPAA reagent can selectively detectN-terminal Gly-containing peptides (NGPs) in the presence of sodium borate and NaIO4. Under conditions optimized, this assay format for collagen, termed 3,4-DHPAA assay method showed a good linear relationship between the amplified FL signals and the collagen concentrations from 0.18 to 12 μg/ml. Therefore the sensitive determination of intracellular collagens in cheek tissue and HeLa cells was individually possible without any separation protocol. The dual recognitions of the collagens in the samples could be performed by the enzymatic digestion and the FL reaction. The proposed assay method enables the determination facile, specific, sensitive and quantitative for biogenic collagens

Virtual Reconstruction and Prey Size Preference in the Mid Cenozoic Thylacinid, <i>Nimbacinus dicksoni</i> (Thylacinidae, Marsupialia)

<div><p>Thylacinidae is an extinct family of Australian and New Guinean marsupial carnivores, comprizing 12 known species, the oldest of which are late Oligocene (∼24 Ma) in age. Except for the recently extinct thylacine (<i>Thylacinus cynocephalus</i>), most are known from fragmentary craniodental material only, limiting the scope of biomechanical and ecological studies. However, a particularly well-preserved skull of the fossil species <i>Nimbacinus dicksoni</i>, has been recovered from middle Miocene (∼16-11.6 Ma) deposits in the Riversleigh World Heritage Area, northwestern Queensland. Here, we ask whether <i>N. dicksoni</i> was more similar to its recently extinct relative or to several large living marsupials in a key aspect of feeding ecology, i.e., was <i>N. dicksoni</i> a relatively small or large prey specialist. To address this question we have digitally reconstructed its skull and applied three-dimensional Finite Element Analysis to compare its mechanical performance with that of three extant marsupial carnivores and <i>T. cynocephalus</i>. Under loadings adjusted for differences in size that simulated forces generated by both jaw closing musculature and struggling prey, we found that stress distributions and magnitudes in the skull of <i>N. dicksoni</i> were more similar to those of the living spotted-tailed quoll (<i>Dasyurus maculatus</i>) than to its recently extinct relative. Considering the Finite Element Analysis results and dental morphology, we predict that <i>N. dicksoni</i> likely occupied a broadly similar ecological niche to that of <i>D. maculatus</i>, and was likely capable of hunting vertebrate prey that may have exceeded its own body mass.</p></div