Experimental Metabolic Syndrome Model Associated with Mechanical and Structural Degenerative Changes of the Aortic Valve

The purpose of this study was to test the hypothesis that an experimental high fat (HF) animal with metabolic syndrome results in structural degeneration of the aortic valve. Domestic pigs were divided (n = 12) and administered either a normal or HF diet. After 16-weeks, the HF diet group had increased weight (p ≤ 0.05), total cholesterol (p ≤ 0.05), and systolic and diastolic pressure (p ≤ 0.05). The aortic valve extracellular matrix showed loss of elastin fibers and increased collagen deposition in the HF diet group. Collagen was quantified with ELISA, which showed an increased concentration of collagen types 1 and 3 (p ≤ 0.05). In the HF diet group, the initial stages of microcalcification were observed. Uniaxial mechanical testing of aortic cusps revealed that the HF diet group expressed a decrease in ultimate tensile strength and elastic modulus compared to the control diet group (p ≤ 0.05). Western blot and immunohistochemistry indicated the presence of proteins: lipoprotein-associated phospholipase A2, osteopontin, and osteocalcin with an increased expression in the HF diet group. The current study demonstrates that experimental metabolic syndrome induced by a 16-week HF diet was associated with a statistically significant alteration to the physical architecture of the aortic valve.

Scientific REPORTS | (2018) 8:17835 | DOI: 10.1038/s41598-018-36388-y (LPC) 3 . Recently, studies have investigated the potential role of oxLDL and Lp-PLA 2 in aortic valve (AV) degeneration 3,5,14,15 . The final process involves valvular endothelial cells (VECs) that communicate with valvular interstitial cells (VICs) to initiate extracellular remodeling. Unlike atherosclerotic disease, there are no known preventive measures for the progression of AV calcification and degeneration where the current treatment is aortic valve replacement (AVR) 5 . The current study was designed to test the hypothesis that a clinically relevant experimental animal model given a HF diet will lead to the physical and structural changes of the aortic valve.

Methods
Ethical Statement. American Yorkshire domestic pigs were housed together at the Mayo Clinic Institutional Hills Farm and treated according to the Guide for the Care and Use of Laboratory Animals (National Institute of Health, USA). Standard of care was approved by Mayo Clinic's Institutional Animal Care and Use Committee (Protocol # A65014) throughout the study.
Animal Care, Sample Preparation, and Ex vivo Tissue Analysis. Pigs (n = 12) were nursed for 28 days and weened into a normal diet for 4 weeks before being transported to the Mayo Clinic facilities. After the initial 7 weeks, 6 pigs were randomly selected and given a HF diet while the remaining 6 pigs continued a normal feeding regimen for 16 weeks 10,12,16,17 . Animals in the control group (n = 6; female) were fed a normal diet consisting of a standard swine feed containing 14.5% protein and 3% fat with 3.3 Kcal/g of feed (Purina Animal Nutrition LLC, Shoreview MN). Animals in the treatment HF group (n = 6; female) were fed a high-fat and high-fructose feed containing 17% protein and 20% fat with 4.1 Kcal/g of feed (TestDiet, St. Louis MO). Additionally, all 12 pigs were implanted with a DSI telemetry system L11 implant (Data Sciences International, New Brighton, MN). Telemetry was surgically implanted 6-weeks into the normal and HF diet treatment and recorded temperature, heart rate, and blood pressure for 10 weeks. At approximately 23-24 weeks of age, blood samples were collected, pigs were euthanized with 100 mg/kg IV pentobarbital, and the aortic valve (including the root and cusps) was excised.
Radiologic Imaging: Microscopic Computerized Tomography (CT) Scan. One aortic cusp from each group (n = 4) was evaluated using a custom made micro CT scanner (North Star Imaging Company, Rogers, MN) to determine the degree of aortic cusp microcalcification observed in the HF versus the normal diet groups. The scanner has a fixed micro-focus X-ray source (Hamamatsu Photonics K.K, Hamamatsu City, Japan) and a flat panel X-ray detector (Varian Medical Systems, Palo Alto, CA). The scanner includes flexible geometry with spatial resolution ranging from 5-127 um at different focus modes (small, medium, large) and power levels ranging from 40 kVp-150 kVp 12,17,18 . Histology and Immunohistochemistry. One aortic cusp from each group (n = 10) was removed, sectioned, and paraffin-embedded. Antibodies were used for immunohistochemical (IHC) staining to express calcification, subendothelial lipid accumulation, and presence of interstitial-like cells in the explanted tissue. from each aortic valve was stored in cool PBS for no more than 30 minutes before processing. The ultimate tensile strength and elastic modulus were measured to determine the mechanical properties of aortic cusps from each group. Cusps were cut into rectangular 12 mm (height) and 4 mm (width) shapes. Collagen fibers were aligned circumferentially and digital calipers were used to measure the thickness at the center. Three thickness measurements were taken, and the average was used for data analysis 6,21 . Two rectangular paper window frames (20 mm × 9 mm) with a rectangular window (12 mm × 5 mm) at the center was used to hold and prevent unwanted damage while mounted into the Instron 5965 tensile tester (Instron Corporation, Norwood MA) 20 .
Testing was performed at crosshead displacement speed of 0.1667 mm/s per ASTM standards (ASTM F2150-13) 22 . The ultimate tensile strength and elastic modulus were calculated from the resulting stress-strain graph using load versus displacement data from the Bluehill 3 program (Instron Corporation, Norwood MA).

Statistical analysis.
Continuous variables are presented as mean ± SD or median (25th, 75th percentiles) depending on the normality of distribution. Given the low sample size (N < 25) and non-normal distribution of studied continuous variables, the Mann-Whitney U test was used to analyze HF diet and control diet differences in ultimate tensile strength and elastic modulus properties, histology and immunohistochemistry as percentage of positive signal area visualized under 20x magnification, and Western blot and ELISA as protein concentration visualized with film and plate reading. Statistical significance was defined as a 2-tailed P value ≤ 0.05. Statistical analyses were completed utilizing JMP ® Pro 10.0.0 (SAS Institute Inc, Cary, North Carolina).

Ex vivo Macroscopic Tissue Analysis.
After a 16-week period, there was no inflammation on the valve cusps from either group (n = 6 HF diet and n = 6 normal diet pigs). Vital signs, weight, blood pressure, and heart rate were routinely recorded via telemetry, which showed an increase in all areas for the HF diet group at sacrifice compared to 6 weeks into arrival. Blood draw obtained at sacrifice measured glucose and cholesterol levels and showed an increase in the HF diet group (Table 1). Additionally, gross calcific nodules were present on the cusps of the HF diet group (Fig. 1E).

Radiologic Imaging: Microscopic Computerized Tomography (CT) Results.
There was no evidence of microcalcification found in the control group; however, opaque areas signifying microcalcification were observed from the HF diet group in both 2D and 3D imaging (Fig. 1G). The mean percentage of total opaque surface area in HF diet is 10.7% ± 1.7 and control diet is 0% (p < 0.05).

Scanning and Transmission Electron Microscopy (SEM/TEM).
Collagen is aligned in the circumferential direction within the ECM. In the normal diet group, cusps imaged with SEM showed directional uniformity of collagen fibers while cusps from the HF diet were misaligned (Fig. 2D). The same cusps imaged with TEM showed the longitudinal view of collagen fibers in the normal diet group and an increase of the same collagen fibers in the HF diet group (Fig. 2F).

Analysis of Biomarkers and Quantification of Collagen and Elastin Content.
On H&E staining, Fig. 2 shows that the extracellular matrix (ECM) was uniformly arranged in both normal and HF diet groups; however, a small number of inflammatory cells were observed in the HF diet group.
Collagen and Elastin. Masson trichrome showed the collagen structure, muscle fiber, and nuclei in cusps from both groups (Fig. 3). There was increased collagen deposition within the fibrosa layer in the HF diet group compared to the control group ( Table 2). As expected, elastin fibers were present in cusps from the control group; however, there was loss of elastin in cusps from the HF diet group (Fig. 3D). Collagen quantification utilizing ELISA was performed and showed a statistically significant increase in the concentration of collagen types 1 and 3 in the HF diet compared to the normal diet group. In Fig. 3E, a statistical significance was observed for collagen type 1 (P < 0.05) and collagen type 3 (P < 0.05). Movat's pentachrome showed similar results in that there was an increase of collagen found within the fibrosa and spongiosa layers in the HF diet group, while only ground substances and mucin were observed in the normal diet group (Fig. 3E,F). Calcium. Von Kossa stains showed dark areas of microcalcification (Fig. 1H). On IHC analysis, there was an increased expression of OCN, OPN, and PAFAH in cusps from the HF diet group (Fig. 4). In addition, α-SMA was utilized to assess the cellularity of the cusps and showed myofibroblast-like cell infiltration in the HF diet group (Fig. 4H). Western blot protein analysis was performed with aortic cusp homogenates from each group. Both normal and HF diet cusps showed expression of OCN, OPN, and PAFAH; however, there was a statistically significant increase in the normalized band density to GAPDH of all biomarkers in the HF diet group (P < 0.05) (Fig. 5).

Control Diet High Fat Diet
Biomechanical Properties of Porcine Aortic Valves: Uniaxial Tensile Testing. As seen in Fig. 6, specimens were tested in the circumferential direction (n = 10). The stress-strain curves demonstrated a statistically significant decrease in elastic modulus in the HF diet group. There was also a statistically significant difference observed in the ultimate tensile strength when comparing the HF diet group to the control. The mean and standard deviation for cusp thickness was 0.56 mm ± 0.20 for the control group and 0.82 mm ± 0.30 for the HF diet group (p < 0.05).

Discussion
The study demonstrated that experimental metabolic syndrome (MetS) in pigs induced by a high fat (HF) diet for 16 weeks resulted in mechanical and structural aortic valve degeneration and calcification. These changes were characterized by a statistically significant decrease in biomechanical properties. The current study suggests that cardiovascular risk factors that are associated with atherosclerosis may have also contributed to the progression of aortic valve degeneration and calcification (Fig. 7). Pigs have become widely used in cardiovascular research for similarities to human anatomy and physiology 23 . Studies have utilized pigs with MetS to study the effects of HF diet on aortic calcification 10 . The aortic cusp is composed of a fibrosa, spongiosa, and ventricularis layer that are primarily composed of proteoglycans and the fibrous proteins collagen and elastin. Together, these components are responsible for the biomechanical support and structure of the aortic valve. Other fibrous proteins such as fibronectin and laminin within the extracellular framework have contributions to tissue remodeling. Fibronectin mediates cellular attachment and function, and it has been implicated with tenascin in the promotion of fibroblast migration during embryogenesis and wound healing [24][25][26] . Furthermore, components such as laminins are found in the basal lamina and regulate cellular differentiation, proliferation, and migration 27 . Although these components are integral in understanding endothelial injury and cellular differentiation, the emphasis of this manuscript was tailored specifically around the major fibrous proteins collagen and elastin. The mechanism by which a HF diet may contribute to aortic valve calcification and degeneration may be multifactorial. The decrease in tensile strength and elasticity is from an increase in collagen types 1 and 3 and loss of elastin in the HF diet group. Additionally, the initial stages of microcalcification were appreciated in the HF diet group with staining and imaging. The focus of the current study revolves around the observed architectural properties of the extracellular matrix (ECM) and the physical changes that resulted from a high fat diet.  In the same study, it was observed that patients diagnosed with severe calcific aortic stenosis had a combination of decreased total EPCs, but increased EPC-OCN percentage, which may have contributed to accelerated vascular and valvular calcification 32 . Chronic diseases, such as hypercholesterolemia, hypertension, and diabetes mellitus, have been linked to increased incidence of calcific aortic valve disease 33 . It was observed that in the HF diet group, an expression of osteoblast-derived proteins osteocalcin (OCN) and osteopontin (OPN) were concentrated along the aortic side of the valve cusp. These findings suggest that (1) proteins present in ectopic calcification of the coronary arteries are also present on the aortic cusp, (2) tissue calcification may be specific to the fibrosa and ventricularis layers, and (3) MetS facilitates osteoblast-derived protein mobilization (Fig. 7). Similar studies have shown OPN localization within the aortic fibrosa further promoting the theory of layer susceptibility [34][35][36] . The current study shows that hypertension was observed in the

Role of Lipid Accumulation in Smooth Muscle Mineralization and Degeneration. Several studies
have suggested the role hyperlipidemia has in the mechanism of aortic valve degeneration 14,38 . Circulating lipids accumulate within the subendothelium and deposit on the aortic and ventricular sides of the valve. In Fig. 7, the enzyme lipoprotein-associated phospholipase A 2 (Lp-PLA 2 ) attaches to the substrate oxidized low density lipoproteins (oxLDL) to produce the proinflammatory lipid mediators free fatty acid (FFA) and lysophosphotidylcholine (LPC) [39][40][41] . In human clinical studies, Mahmut et al. observed varying expressions of Lp-PLA 2 with respect to the severity of aortic stenosis. Findings from these studies suggest that lipid metabolism of LPC and FFA may promote vascular smooth muscle cell mineralization and activation of the tissue degradation process (Fig. 7) 3,4,41 . In the current study, increased Lp-PLA 2 expression was similarly observed in specimens from the HF diet group. It is important to note that the HF diet group had a significantly increased lipid profile when compared to baseline. On histopathology and western blot quantification, an increased expression of Lp-PLA 2 , also referred to as platelet activating factor acetylhydrolase (PAFAH), was observed in the HF diet aortic cusps. Mahmut et al. showed that Lp-PLA 2 greatly contributes to valve mineralization by the production of LPC, and the plasma level of oxLDL is directly associated with stenotic aortic valve remodeling 3 . The current findings promote that the experimental MetS model is conducive to studying the effects of lipid metabolism on the aortic valve. It may be of further interest to investigate the role of Lp-PLA 2 inhibition to ameliorate the progression of tissue degradation 3,39 .   7) 42,43 . Although VIC phenotypes have been extensively studied, differentiation of these cells are not completely understood 44 . In the HF diet group, the myofibroblast-like phenotype was appreciated with alpha-smooth muscle actin (α-SMA) expression. There was a significant increase in collagen deposition and active remodeling, but loss of elastin that altered the aortic cusp mechanical   45 . Findings from mechanical testing further confirmed that when compared to the normal diet group, cusps from the HF diet group were less elastic due to loss of structural uniformity in the ECM. On average, cusps from the HF diet group were thicker compared to the normal diet group, which may be attributed to fibrotic change. Although calcification was present in the fibrosa and ventricularis layers, there was also an increase of collagen deposition in the fibrosa and spongiosa (Fig. 3). These findings show a possible connection between the mechanisms of collagen deposition alongside calcification specific to the fibrosa layer. Further investigation regarding this link should be evaluated in future studies. Continuous activation of VICs may lead  to increased valve cusp fibrosis and decreased valvular mechanical flexibility [47][48][49] . Despite different cell responsibilities, studies indicate that there might be an additional mechanistic step involving myofibroblast-like cells that leads to the initial stages of aortic calcification 43 .
The findings observed in this study have numerous implications in regards to VHD. For the first time, the effects of an experimental MetS pig model show that in addition to atherosclerosis, aortic calcification and degeneration may be related to a HF diet. The current study extends these observations and suggests a potential mechanism and therapeutic target for aortic valve degeneration in a high-risk population 5,36 . Other studies have investigated similar pathways and proposed therapeutic targets that would mitigate the effects of MetS and associated comorbidities 50,51 .

Limitations
In future studies, it is important to explore the effect of therapy and reversibility of factors associated with a HF diet. The duration of diet given in each group has been observed to be sufficient to study the effects of MetS; however, allowing the study to take place for a longer duration may induce more of the degenerative effects of HF diet paralleled with age. In addition to micro CT analysis, a high resolution non-contrast CT with calcium score may be beneficial to image the aortic root, providing better insight into the degree of aortic calcification present. Additionally, measuring the aortic cusp size and weight would help further assist in determining the physical properties of the aortic valves. Biomechanical forces exerted in valve cusps may be equal in both the circumferential and radial directions of the cusp. Variability of the cusps is based on the morphology and structural composition of the valve. The aortic valve cusps experience a complex bidirectional flexure. Since the circumferential direction is the major curvature in the cusps, uniaxial mechanical testing focused bending in that direction 6 .

Conclusions
In summary, the current study demonstrated that experimental metabolic syndrome is associated with mechanical and structural changes of the aortic valve leading to degeneration and early calcification. The pathological processes involved in the initial stages of aortic calcification are similar to the well-known pathological processes involved in atherosclerosis. Additionally, a HF diet was postulated to cause VIC differentiation leading to further expression of osteoblast-like factors and fibrosis of the aortic cusps. Clinical vigilance in improving these problems and understanding the mechanisms associated with VHD is essential to overall cardiovascular health. In the past decade, the main focus has been atherosclerotic risk factor assessment and modification. Currently, there are still no preventive medical approaches to early detection and attenuation of the progression of aortic valve calcification and degeneration. While surgical intervention remains the gold standard of therapy, findings from the current study suggest that an approach similar to atherosclerosis should be explored for aortic valve disease.