Background and Aims: Disease-associated cardiovascular tissue remodeling is accompanied by structural and functional reorganization of the extracellular matrix (ECM) entailing the re-occurrence of fetal variants of the cell-adhesion modulating proteins fibronectin (Fn) and tenascin-C (Tn-C). These variants are virtually absent in the healthy adult cardiovascular system, but are detectable during normal human aging. For the use of fetal Fn or Tn-C variants as biomarkers or therapeutic targets, the discrimination between disease- versus age-related re-expression is necessary. The main cellular source of these molecules is the activated cardiac fibroblast (myofibroblast). Thus, the aim of the current study was a detailed analysis of age-related changes in synthesis and extracellular deposition of Fn and Tn-C variants in primary cardiac fibroblasts derived from a well-defined non-pathologic mouse model of aging. 

Methods: Age-related growth behavior of healthy primary cardiac fibroblasts (cFb) isolated from C57BL/6 mice were studied. We compared three different groups of age: group 1 (3 weeks); group 2 (2 to 4 months) and group 3 (21 to 24 months). In all groups, cell growth rates and senescence-associated beta-galactosidase (beta-gal) were determined. Fetal variants of Fn (ED-A⁺ and ED-B⁺ Fn) and Tn-C (B⁺, C⁺ and D⁺ Tn-C) as well as alpha-smooth muscle actin (a-SMA) and collagen III alpha 1 (col3a1) were detected by real-time PCR and immunofluorescence staining.

Results: In cell culture, cFB reveal age-associated differences with respect to growth pattern with higher cell growth rates (+26.3%) after 3 days in culture observable in group 1 compared to group 2 (+6.2%) and 3 (+5.3%) along with an increased fraction of beta-gal positive cFB, in particular in group 3. 

Analysis of mRNA expression revealed an up-regulation ED-A⁺ Fn (8-fold), ED-B⁺ Fn (6-fold), a-SMA (3.6-fold) and col3a1 (4.8-fold) in group 1 and of ED-A⁺ Fn (3.7-fold) and ED-B⁺ Fn (2.5-fold) in group 3; each compared to group 2. For the three fetal Tn-C variants analyzed, no biological relevant changes in mRNA expression could be detected. The mRNA findings correlated with protein levels detected by immunofluorescence labeling. 

Conclusions: 

The current study showed an age-associated differential expression of fetal variants of Fn and Tn-C synthesized by activated cFB. While fetal Fn variants show relevant changes depending on age group, this could not be shown for Tn-C variants. Against the background of these preliminary findings, fetal Tn-C variants might better qualify as disease markers or therapeutic targets especially in elderly patients.