Influence of obesity on remodeling of lung tissue and organization of extracellular matrix after blunt thorax trauma

Abstract

Background: Previously, it has been shown that obesity is a risk factor for recovery, regeneration, and tissue repair after blunt trauma and can affect the rate of muscle recovery and collagen deposition after trauma. To date, lung tissue regeneration and extracellular matrix regulation in obese mice after injury has not been investigated in detail yet.

Methods: This study uses an established blunt thorax trauma model to analyze morphological changes and alterations on gene and protein level in lean or obese (diet-induced obesity for 16 ± 1 week) male C57BL/6 J mice at various time-points after trauma induction (1 h, 6 h, 24 h, 72 h and 192 h).

Results: Morphological analysis after injury showed lung parenchyma damage at early time-points in both lean and obese mice. At later time-points a better regenerative capacity of lean mice was observed, since obese animals still exhibited alveoli collapse, wall thickness as well as remaining filled alveoli structures. Although lean mice showed significantly increased collagen and fibronectin gene levels, analysis of collagen deposition showed no difference based on colorimetric quantification of collagen and visual assessment of Sirius red staining. When investigating the organization of the ECM on gene level, a decreased response of obese mice after trauma regarding extracellular matrix composition and organization was detectable. Differences in the lung tissue between the diets regarding early responding MMPs (MMP8/9) and late responding MMPs (MMP2) could be observed on gene and protein level. Obese mice show differences in regulation of extracellular matrix components compared to normal weight mice, which results in a decreased total MMP activity in obese animals during the whole regeneration phase. Starting at 6 h post traumatic injury, lean mice show a 50% increase in total MMP activity compared to control animals, while MMP activity in obese mice drops to 50%.

Conclusions: In conclusion, abnormal regulation of the levels of extracellular matrix genes in the lung may contribute to an aberrant regeneration after trauma induction with a delay of repair and pathological changes of the lung tissue in obese mice.

Keywords: Collagen; Extracellular matrix (ECM); High fat diet; MMP activity; Obesity; TIMP production; Thorax trauma.

Fig. 1

Body weight distribution of normal weight and obese mice. Values are given as mean ± standard error of the mean (SEM). Statistical analysis was done by two-way ANOVA, where **** indicates p ≤ 0.0001

Fig. 2

Regeneration process of the lung after blunt thorax trauma (BTT). Hematoxylin-eosin (HE) staining of lung tissue sections of male lean and obese C57BL/6 J mice after induction of blunt thorax trauma (n = 6). Figure shows lung tissue sections from control animals as well as lung sections from1 h, 6 h, 24 h, 72 h and 192 h post trauma. Arrow = lung tissue damage, arrowhead = lung tissue with intra-alveoli debris, thickened alveoli wall and alveoli collapse. Pictures were taken with an Olympus IX81microscope using Xcellence v.1.2. Scale = 50 μm at 40x magnification or 200 μm at 10x magnification. Abbreviations: HFD, high fat diet; ND, normal diet

Fig. 3

Gene expression status of lung tissue regarding ECM build-up post-BTT. Expression levels of collagen 1, collagen 3 and fibronectin in lung tissue were determined by qPCR using Gapdh as a housekeeping gene. Col1a, Col3a and Fn1 show the gene expression differences when comparing all the time-points after lung injury to the respective controls. Statistical significance was determined using two-way ANOVA followed by an uncorrected Fisher’s LSD test (α = 0.05). * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001. Indicators of significance directly above the bars of the diagram indicate a statistical difference to the control, while stars above the connector line show differences between the two diets at that specific time point

Fig. 4

ECM remodeling in lung tissue after BTT. A) ECM regulation and collagen amount in lung tissue post-BTT in ND and HFD mice. Sirius Red staining of lung tissue sections. Arrowhead = fibrosis, arrow = damaged lung tissue with blood cell infiltration. Pictures were taken with an Olympus IX81 using Xcellence v.1.2. Scale = 50 μm at 40x magnification or 200 μm at 10x magnification. B) Quantification of collagen in the lung using a chemical collagen quantification assay kit. Results are presented as mean ± SEM. C) Schematic presentation of Sirius Red stained whole lung tissue sections. Abbreviations: HFD, high fat diet; ND, normal diet

Fig. 5

Gene expression status of Mmps and Timps in lung tissue post-BTT. Expression levels of Mmp2, Mmp8, Mmp9, Timp2 and Timp1 in lung tissue were determined by qPCR using Gapdh as a housekeeping gene. Mmp2, Mmp8, Mmp9, Timp1and Timp2 show the gene expression differences by comparing all the time-points after lung injury to their respective controls. Statistical significance was determined using two-way ANOVA followed by an uncorrected Fisher’s LSD test (α = 0.05). * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001. Indicators of significance directly above the bars of the diagram indicate a statistical difference to the control, while stars above the connector line show differences between the two diets at that specific time point

Fig. 6

Activity of pro−/MMP9 and pro−/MMP2 in lung tissue of lean and obese mice after BTT. Activity of Pro-MMP9, MMP9, Pro-MMP2 and MMP2 was evaluated using gelatin zymography. Lung tissue from control animals as well as 1 h, 6 h, 24 h, 72 h and 192 h post trauma were used for this analysis. Statistical significance was determined using two-way ANOVA followed by an uncorrected Fisher’s LSD test (α = 0.05). * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001. Indicators of significance directly above the bars of the diagram indicate a statistical difference to the control, while stars above the connector line show differences between the two diets at that specific time point

Fig. 7

TIMP1/2 IHC staining of lung from lean and obese mice after BTT. A) TIMP1 and B) TIMP2 staining in lean and obese mice from control animals as well as 1 h, 6 h, 24 h, 72 h and 192 h post trauma. The area of positive staining was measured and is depicted as values next to the respective depiction of the staining for TIMP1 (C) and TIMP2 (D). The data is presented as mean ± SEM. Statistical significance was determined using two-way ANOVA followed by an uncorrected Fisher’s LSD test (α = 0.05) ** indicates p < 0.01

Fig. 8

Analysis of total MMP activity in lung tissue of lean and obese animals after BTT. The total MMP activity was measured in the presence of naturally occurring inhibitors in lung tissue from control animals as well as 1 h, 6 h, 24 h, 72 h and 192 h post trauma. Statistical significance was determined using two-way ANOVA followed by an uncorrected Fisher’s LSD test (α = 0.05). * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001. Indicators of significance directly above the bars of the diagram indicate a statistical difference to the control, while stars above the connector line show differences between the two diets at that specific time point