Background and Aims: Ideal preclinical models of Pulmonary Hypertension (PH) mimic pulmonary vascular and right ventricular (RV) cardiac tissue remodelling processes and the accompanied re-organisation of the extracellular matrix. Within the latter process, a relevant re-occurrence of fetal variants of cell adhesion modulating proteins, e.g. fibronectin (Fn), can be observed. Since these molecules are virtually absent in healthy adult organs, they qualify as potential diagnostic markers or even therapeutic targets. To plan preclinical treatment studies in a dedicated animal model, the longitudinal re-expression dynamics of target molecules are of great interest. Thus, the aim of the current study was to perform longitudinal analyses of the re-expression and extracellular deposition dynamics of extra-domain A containing Fn (ED-A⁺ Fn) in a well-established preclinical model of PH.

Methods:

The rat model of Monocrotaline (MCT) induced PH was chosen to study the re-expression dynamics of ED-A⁺ Fn on mRNA as well as protein level in lung and cardiac tissue at different time points (day 7, 14, 21 and 28 after PH induction; 7 animals each) compared to sham-treated controls (n=3). All animals underwent echocardiographic evaluation of RV morphology and function (e.g., RV basal diameter, RV_basal or tricuspid annular plane systolic excursion, TAPSE), right heart catheterization to asses right ventricular systolic pressure (RVP_sys) and organ harvesting at each time-point. Lung and RV cardiac tissue samples were subjected to further analyses.

Results:

We could demonstrate a continuous significant increase of RVP_sys over time (p<0.001) reaching maximum values at day 28 (83.98 ± 22.29 mmHg) compared to controls (27.4 ± 6.6 mmHg, p=0.017).

These hemodynamic effects were accompanied by significant changes of surrogate parameters of increased right ventricular load. Thus, among others, RV_basal significantly increased within the experimental period (p=0.006), especially when comparing day 14 to 21 (p=0.035) or 28 (p=0.032). Correspondingly, TAPSE decreased over time (p=0.008). Histological lung tissue damage with respect to typical PH associated remodelling patterns significantly increased until day 21 (p<0.001) with no further changes at day 28 (p=n.s, compared to day 21). Evaluation of RV tissue revealed gradually increased inflammation (p=0.002) as well as fibrosis (p=0.002) with rising PH duration. Microscopic assessment of ED-A⁺ Fn tissue deposition displayed a spatial association to pulmonary vasculature and, to a lesser extent, lung parenchyma. In RV tissue, there was an abundant deposition in areas of interstitial inflammation and fibrosis. Quantitative analysis of ED-A⁺ Fn in lung and RV demonstrated significantly elevated expression levels in diseased compared to healthy animals with a continuous increase over time (p<0.001 for both, lung and RV).

Conclusion: In the current study, we could show for the first time that the re-expression of ED-A⁺ Fn, which has been evidenced to play a crucial role for pathogenesis of PH, shows a continuous increase over time in lung and cardiac tissue. Thus, the molecule reliably represents disease development and progression with clear association to certain PH specific tissue remodelling patterns. Against that background, this particular preclinical PH model is suitable to further study ED-A⁺ Fn targeting as promising disease-modifying concept to stop or even reverse detrimental cardiovascular remodelling.