**3. Discussion**

Oxidative stress and ROS contribute significantly to the pathogenesis of cardiovascular diseases [22], such as stroke and hypertension [23]. Elevated ROS could cause vascular damage via recruiting more leukocytes in blood reperfusion [24]. Moreover, oxidative stress can induce endothelial dysfunction and induce pro-fibrotic and pro-osteoblastic effects with subsequent calcification in the aortic valves, as shown in a mouse calcific aortic valve disease (CAVD) model [25].

Oxidative stress via ROS induces damage to DNA double strands, in addition to the rapid phosphorylation of H2AX by PI3K-related kinases with downstream Akt modulation [26]. Additionally, Akt phosphorylation induced the osteogenic early marker RUNX2 [17], which had the ability to increase osteogenic differentiation [27]. The progression of calcification in VIC cells also involved a significant increase in the osteogenic medium (OM) condition as well as the increased phosphorylation of the NF-κB, PI3K-Akt, TNF, and MAPK signaling pathways [19]. In this study, Fx treatment could alleviate the oxidative stress-induced ROS level (Figure 3) and decrease the progression calcification through Akt- and MAPK-related signaling pathways (Figure 4).

Aortic leaflets are composed of three main layers, named the fibrosa, spongiosa, and ventricularis. This composition builds up the valve function, which is mainly diastole and systole extension [28]. Considering that ECM is also the main component of valves, the structure and the arrangemen<sup>t</sup> of ECM play important roles in the valve function.

Increased inflammation and LDL oxidation induce the calcification of the valves [29]. On the other hand, oxidized high-density lipoprotein (ox-HDL) increases in CAVD patients' plasma to protect against the high oxidative stress induced by CAVD [30]. As ROS increase, chronic inflammation, with accompanying immunity components, increases, along with increased growth factors and proteolytic enzymes, such as MMP-9 and MMP-2 for ECM deposition and remodeling effects [31]. Therefore, the up-regulation of MMPs indicates valve remodeling and calcification [32].

Several studies have shown vascular diseases with ROS-related pathology in studies of animals fed with a high-cholesterol or high-fat diet to induce vascular calcification. There are different categories of antioxidant compound. Natural antioxidants, such as gallic acid [33], curcumin [34], and quercetin [35], or synthetic ROS scavengers such as N-acetylcysteine, pyrrolidine dithiocarbamate, and poly(1,8-octamethylene-citrate-cocysteine) with a reduction in ROS, PI3K/Akt or inflammation-related protein expression could reduce calcification and apoptosis through ROS scavenging [36].

Fx exerts antioxidant ability that has been shown to eliminate pregnancy-related hypertension [37], high glucose-induced diabetes retinopathy [38], and ox-LDL-induced endothelial damage [39] via ROS reduction. In our current in vitro study, Fx showed a potential protective effect against high oxidative stress-induced VIC damage through a reduction in apoptosis (Figure 2) and ROS and modulation of the phosphorylation of Akt and ERK to decrease the calcification and ECM accumulation (Figure 4).

The first-line diagnosis of heart valve disease in dogs includes the use of radiographs, ultrasound, and echocardiography [40]. Indicators of valve disease include an increase in VHS score or LA/AO. In echocardiography assessment, the veterinarian would use the Tei index to evaluate systolic and diastolic function. The E/e' ratio is used to assess the mitral valve inflow and analyze the mitral valve leakage and left ventricular diastolic dysfunction [41]. In our study, fucoxanthin treatments could reverse mitral valve and tricuspid valve leakage. Moreover, using in vivo experiments in dogs, we showed that the long-term supplementation of Fx could improve both compensatory cardiac hypertrophy and valve function (Figure 5).

#### **4. Materials and Methods**
