Pulmonary Arterial Hypertension is a complex disease associated with a poor prognosis. Many therapeutical strategies have been introduced to PAH patients. All approved therapies targeting vasodilation are effective by improving hemodynamics, exercise capacity and give small improvements in survival in PAH patients (78), however, they failed to reverse the disease. Therefore the focus of the treatment in recent years has changed from vasodilators to anti-proliferative agents and new signaling pathways are needed to be evaluated for future therapies. In our studies we focused on the Wnt signaling pathway, which was recently implicated to many cancerous diseases. In addition it has been shown that this pathway is crucial for cardiovascular diseases. In our study we observed downregulation of well-known canonical Wnt signaling ligands in experimental PAH in rats. GSK3beta and beta-Catenin, which are downstream targets of canonical Wnt signaling, are upregulated in lungs as well as in PASMCs from MCT-induced PAH rats compared to controls on protein level. Further, stimulation of MCT-PASMCs with both PDGF-BB and Wnt3a induced inactivation of GSK3beta, whereas only Wnt3a regulated beta-Catenin accumulation in PASMCs. Constitutive activation of GSK3beta by amino acid substitution S9A (9th serine replaced to alanine) or Y216D (tyrosine replaced to aspartic acid) inhibited serum-induced PASMC proliferation influencing ERK phosphorylation. PDGF-BB-induced proliferation was inhibited by Y216D mutant, which seems to be implicated in PDGF signaling in experimental PAH. The fact that PDGF-induced MCT-PASMC proliferation could be significantly decreased by GSK3beta amino acid residues modulation, collectively indicate the importance of GSK3beta in the pathogenesis of PAH. Significant upregulation of GSK3beta in lung explants of patients with iPAH support a concept that this protein plays a role in development of PAH. Profound changes in pulmonary vasculature in PAH are followed by heart hypertrophy and heart failure. Wnt signaling was previously reported to play crucial role in cardiovascular maintenance and one of our candidate genes was sFRP-1, an extracellular Wnt signaling modulator. We found that sFRP-1 is abundantly expressed in normal mice hearts at 3, 6 and 12 months of age and predominantly localized in cardiomyocytes and endothelial cells suggesting that it may play a significant role in heart function maintenance. To evaluate the role of sFRP-1 in adult hearts we used sFRP-1 KO mice up to 12 months of age. No significant phenotype was observed in these mice up to 6 months of age. At one year of age, sFRP-1 KO mice exhibited significant increase in heart size with age-dependent increase in heart weight to body weight ratio. Additionally to this phenotype, an increase in left ventricular dimensions, posterior wall thickness, and a decrease in fractional shortening occurred in sFRP-1 KO mice at 1 year of age. Increased formation of fibrotic lesions in myocardium of 1-year-old mice collectively suggest that loss of sFRP-1 leads to cardiac hypertrophy and finally cardiomyopathy. Determination of wide mRNA expression profile showed that parts of Wnt signaling in heart of 1-year-old mice are significantly upregulated mainly the Frizzled 2 pathway and pro-hypertrophic, pro-fibrotic Wnt dependent genes like Fgf4, WIF1 and already well described Wisp1. Increased ß-catenin expression and its accumulation in intercalated disks suggested a decrease in Wnt/ß-catenin transcriptional activity that was confirmed by significant down regulation of cyclin D and cMyc. Suppression of canonical Wnt signaling is possible mechanism of downregulation of connexin43 and loss of heart function both described in human idiopathic and dilated cardiomyopathies. In conclusion, we describe that sFRP-1 is a critical factor in maintaining normal cardiovascular function and loss of this gene induces cardiac remodeling which progresses to dilated cardiomyopathy. The mechanism is a supression of Wnt mediated canonical signaling pathway followed by decrease in connexin 43 expression, protein that is very important in heart function and its contraction ability. Yet more studies needs to be done to elucidate and propose final role of Wnt signaling in mainaining cardiovascular system function.
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