Calcific aortic valve disease: investigating new pharmacological approaches and the role of diabetes on the progression of the disease

Background: Calcific aortic valve disease (CAVD) is the most frequent valvulopathy in the Western world. To date, no effective medical therapy has been approved to halt or delay the progression of CAVD and the only available treatment remains aortic valve replacement. Thus, the development of an effective pharmacological treatment for CAVD is a major unmet medical need. Diabetic individuals are at higher risk of developing CAVD; diabetes itself plays an active role on the progression of the disease. Although CAVD was traditionally considered an age-related degenerative process, the disease is currently thought to include active processes, including the differentiation of valve interstitial cells (VICs) towards an osteogenic phenotype. The pathophysiology of the disease needs further investigations to discover novel targets for pharmacological therapies. Aims: The first research project aimed to investigate some pathways and treatments capable of modulating the differentiation of VICs, including the nitric oxide (NO) precursor L-Arginine and the anti-inflammatory Formyl Peptide Receptor 2 (FPR2). We also selected valproic acid (VPA) for its broad spectrum of action as inhibitor of histone deacetylases (HDACs). In the second research project, we aimed to assess the molecular processes leading to diabetic CAVD. Methods: For the first project, a subclone of bovine VICs seeded in culture plates or type I collagen scaffolds was treated with lipopolysaccharide (LPS) for 13 days to acquire an osteogenic phenotype. These cells were also exposed to several compounds: three selective FPR2 agonists, VPA, L-Arginine and soluble guanylyl cyclase (sGC) modulators. ALP activity and calcium deposition were determined with colorimetric assays. Proteins and RNA were extracted for western blot and gene expression analyses. For the second project, we used two animal models of CAVD, which were fed a diabetogenic (HFD) or control diet (NC) for 6, 12 and 26 weeks. The aortic valves were collected for bulk RNA sequencing analyses. Results: The exposure to L-Arginine prevented inflammatory activation and osteogenic differentiation in isolated VICs, probably by activating NO/sGC/cGMP pathway and modifying the expression levels of key proteins involved in ECM remodeling and redox homeostasis. As regards to the receptor FPR2, it was overexpressed under pathological conditions. The treatment with selective agonists prevented inflammatory activation, pro-calcific differentiation of VICs, and mineralization of collagen scaffolds. Of note, the exposure of isolated VICs to VPA promoted their osteogenic differentiation and subsequent calcium deposition, reproducing the effects of LPS probably through HDACs inhibition. The transcriptomic analyses performed on aortic valve tissue from two animal models of CAVD detected an upregulation of inflammatory, immune, and osteogenic pathways and a downregulation of genes involved in cardiogenesis, glucose and lipid metabolism in HFD-fed mice compared to NC-fed mice. Conclusions: Overall, our in vitro findings in isolated VICs give new insights on the pathogenesis of CAVD and suggest novel therapeutic strategies, such as the activation of sGC (with NO donors), FPR2 (through anti-inflammatory agonists) or HDACs. As regards to our second project, diabetes seems to accelerate early progression of CAVD by promoting inflammatory response, immune infiltration, and osteogenic differentiation of VICs. These findings give new insights on the pathophysiology of diabetic CAVD and may lead to the discovery of novel therapeutic targets for patients with concomitant diabetes.Background: Calcific aortic valve disease (CAVD) is the most frequent valvulopathy in the Western world. To date, no effective medical therapy has been approved to halt or delay the progression of CAVD and the only available treatment remains aortic valve replacement. Thus, the development of an effective pharmacological treatment for CAVD is a major unmet medical need. Diabetic individuals are at higher risk of developing CAVD; diabetes itself plays an active role on the progression of the disease. Although CAVD was traditionally considered an age-related degenerative process, the disease is currently thought to include active processes, including the differentiation of valve interstitial cells (VICs) towards an osteogenic phenotype. The pathophysiology of the disease needs further investigations to discover novel targets for pharmacological therapies. Aims: The first research project aimed to investigate some pathways and treatments capable of modulating the differentiation of VICs, including the nitric oxide (NO) precursor L-Arginine and the anti-inflammatory Formyl Peptide Receptor 2 (FPR2). We also selected valproic acid (VPA) for its broad spectrum of action as inhibitor of histone deacetylases (HDACs). In the second research project, we aimed to assess the molecular processes leading to diabetic CAVD. Methods: For the first project, a subclone of bovine VICs seeded in culture plates or type I collagen scaffolds was treated with lipopolysaccharide (LPS) for 13 days to acquire an osteogenic phenotype. These cells were also exposed to several compounds: three selective FPR2 agonists, VPA, L-Arginine and soluble guanylyl cyclase (sGC) modulators. ALP activity and calcium deposition were determined with colorimetric assays. Proteins and RNA were extracted for western blot and gene expression analyses. For the second project, we used two animal models of CAVD, which were fed a diabetogenic (HFD) or control diet (NC) for 6, 12 and 26 weeks. The aortic valves were collected for bulk RNA sequencing analyses. Results: The exposure to L-Arginine prevented inflammatory activation and osteogenic differentiation in isolated VICs, probably by activating NO/sGC/cGMP pathway and modifying the expression levels of key proteins involved in ECM remodeling and redox homeostasis. As regards to the receptor FPR2, it was overexpressed under pathological conditions. The treatment with selective agonists prevented inflammatory activation, pro-calcific differentiation of VICs, and mineralization of collagen scaffolds. Of note, the exposure of isolated VICs to VPA promoted their osteogenic differentiation and subsequent calcium deposition, reproducing the effects of LPS probably through HDACs inhibition. The transcriptomic analyses performed on aortic valve tissue from two animal models of CAVD detected an upregulation of inflammatory, immune, and osteogenic pathways and a downregulation of genes involved in cardiogenesis, glucose and lipid metabolism in HFD-fed mice compared to NC-fed mice. Conclusions: Overall, our in vitro findings in isolated VICs give new insights on the pathogenesis of CAVD and suggest novel therapeutic strategies, such as the activation of sGC (with NO donors), FPR2 (through anti-inflammatory agonists) or HDACs. As regards to our second project, diabetes seems to accelerate early progression of CAVD by promoting inflammatory response, immune infiltration, and osteogenic differentiation of VICs. These findings give new insights on the pathophysiology of diabetic CAVD and may lead to the discovery of novel therapeutic targets for patients with concomitant diabetes