Decreased Laminin Expression by Human Lung Epithelial Cells and Fibroblasts Cultured in Acellular Lung Scaffolds from Aged Mice.

The lung changes functionally and structurally with aging. However, age-related effects on the extracellular matrix (ECM) and corresponding effects on lung cell behavior are not well understood. We hypothesized that ECM from aged animals would induce aging-related phenotypic changes in healthy inoculated cells. Decellularized whole organ scaffolds provide a powerful model for examining how ECM cues affect cell phenotype. The effects of age on ECM composition in both native and decellularized mouse lungs were assessed as was the effect of young vs old acellular ECM on human bronchial epithelial cells (hBECs) and lung fibroblasts (hLFs). Native aged (1 year) lungs demonstrated decreased expression of laminins α3 and α4, elastin and fibronectin, and elevated collagen, compared to young (3 week) lungs. Proteomic analyses of decellularized ECM demonstrated similar findings, and decellularized aged lung ECM contained less diversity in structural proteins compared to young ECM. When seeded in old ECM, hBECs and hLFs demonstrated lower gene expression of laminins α3 and α4, respectively, as compared to young ECM, paralleling the laminin deficiency of aged ECM. ECM changes appear to be important factors in potentiating aging-related phenotypes and may provide clues to mechanisms that allow for aging-related lung diseases

Decreased expression of laminin α3, tissue factor and N-cadherin in human bronchial epithelial cells incubated in old versus young lung matrix.

<p><b>A)</b> Gene expression (qRT-PCR) of hBECs after 1 week incubation in young and old decellularized B10.BR mouse lung matrix. N = 3/group; *p<0.05 for 1 yo vs 3 wo. Data are normalized against hBECs grown on tissue culture plastic. UD = undetected. <b>B)</b> Immunofluorescence staining shows decreased human Lama3 deposition (green) by hBECs infused in 1yo decellularized lung matrix compared to 3wo matrix. Non-infused, age-matched, decellularized lungs are shown as controls. DAPI nuclear staining is shown in blue. Distal alveolar regions are shown. Magnification 200X. Images are 1 representative of 3 lung matrices per condition.</p

Differentially expressed ECM proteins in young versus old decellularized murine lungs identified by iTRAQ.

<p>Differentially expressed ECM proteins in young versus old decellularized murine lungs identified by <u>iTRAQ</u>.</p

Differential expression of matrix-associated genes and proteins in young versus old mouse lungs.

<p><b>A</b>) Comparison of ECM component levels in native and decellularized lungs of different aged B10.BR mice. Statistical comparisons were done only between ages within a treatment group, not between treatment groups. *p<0.05 comparing ages indicated by bar. N = 6 for all groups. For hydroxyproline, **p<0.05 for 4 do vs. all other ages in treatment group; for elastin, **p<0.05 for 3 mo decell vs. other decell ages; for total laminin, **p<0.05 for 4 do decell vs. other decell ages, ***p<0.05 for 3 wo decell vs. other decell ages; for vitronectin, **p<0.05 for 4 do decell vs. other decell ages; for sulfated Glycosaminoglycans, **p<0.05 for 4 do or 3 mo non-decell vs. 3 wo or 1 yo non-decell, ***p<0.05 for 3 wo or 3 mo decell vs. 4 do and 1 yo decell. Abbreviations used: do = day old, wo = week old, mo = month old, yo = year old. <b>B)</b> Gene expression in native lungs from young and old B10.BR mice as assessed by qRT-PCR. N = 6/group; *p<0.05 old vs young. Values are normalized to a representative 3 wo mouse. <b>C)</b> Immunofluorescence images of mouse Lama3, Lama4, fibronectin and vitronectin staining of decellularized lungs from 3-week vs 1-year old B10.BR mice. Magnification 200X. Images are 1 representative of 3 decellularized lungs per group.</p

Human LFs deposit less laminin α4 in old lung matrix compared to young lung matrix.

<p>HLFs were incubated for 1 week in decellularized B10.BR mouse lungs. <b>A)</b> and <b>B)</b> Gene expression (qRT-PCR) of hLFs after 1 week incubation in young and old decellularized BALB/c mouse lung matrix. N = 3/group; *p<0.05 for 2 yo vs 4 wo. All values in <b>A)</b> and <b>B)</b> were normalized to cells grown on tissue culture plastic. UD = undetected. <b>C)</b> Immunofluorescence staining shows decreased human laminin α4 deposition (green) by hLFs infused in 1yo decellularized lung matrix compared to 3wo matrix. Non-infused, age-matched, decellularized lungs are shown as controls. DAPI nuclear staining is shown in blue. Distal alveolar regions are shown. Magnification 200X. Images are one representative of three lung matrices per condition.</p