Hyaluronic acid

Dulbecco’s modified Eagle’s medium (DMEM), F12, Trypsin-EDTA, phosphate-buffered saline (PBS), FBS, and amphotericin B were purchased from Gibco (Rockville, MD). Dispase II was purchased from Roche Diagnostics Corporation (Indianapolis, IN). Enzyme-linked immunosorbent assay (ELISA) kit for TGF-β1 was purchased from RayBiotech, Inc. (Norcross, GA). ELISA kit for human EGF was obtained from eBioscience (San Diego, CA). Hyaluronic acid (HA) concentrations and PDGF-AB were measured using ELISA kits from R&D Systems (Minneapolis, MN). ELISA kit for fibronectin was purchased from Assaypro (Missouri, USA). ELISA kit for PDGF-BB was obtained from Peprotech (New Jersey, USA). All other reagents and chemicals were from Sigma-Aldrich (St. Louis, MO).

Blood was collected from the hearts of mice at the determined end points and centrifuged at 3000 rpm to isolate the plasma by a microvolume high-speed cooling centrifuge MX-201 (TOMY, Tokyo, Japan) and stored at −20 °C. The dorsal skin, livers, and kidneys were excised and homogenized with lysis buffer (Kurabo, Osaka, Japan), and then, the tissue lysate was centrifuged at 10,000 rpm. Plasma concentrations of interleukin (IL)-6, tumor necrosis factor (TNF)-α, creatinine, hyaluronic acid, endostatine, glutamate oxaloacetate transaminase (GOT), glutamic acid pyruvate transaminase (GPT), matrix metalloproteinase (MMP)-1, histamine, and angiopoitin 1 and 2 were measured using appropriate ELISA kits according to manufacturers’ instructions (IL-6, Enzo Life Sciences, Farmingdale, NY, USA; TNF-α, hyaluronic acid, and angiopoietin 2, R&D Systems, Minneapolis, MN, USA; MMP-1, MyBioSource, San Diego, CA, USA; histamine, Bertin Pharm, Montigny-le-Bretonneux, France; creatinine, Cayman, Ann Arbor, MI, USA; Endostatine, BioMedica, Vienna, Austria; angiopoietin 1, EIAAB Science, Wuhan, China; GOT and GPT, Wako, Osaka, Japan; ROS, Cell Biolabs Inc., San Diego, CA, USA). Optical density was measured using a microplate reader (Molecular Devices, Sunnyvale, CA, USA).

Fluvastatin sodium (PHR1620) was obtained from Sigma Aldrich (St Louis, MO, USA). Hyaluronic acid (Molecular Weight: 15-40 kDa) (GLR001) was obtained from R&D Systems (Minneapolis, MN, USA). Primary antibodies against YAP (#14074, 1:100 for immunofluorescence or 1:1000 for Western blotting; Cell Signaling Technology, Danvers, MA, USA), p-YAP (#13008, 1:1000 for Western blotting; Cell Signaling Technology), TAZ (560235, 1:500 for Western blotting; BD Pharmingen, Franklin Lakes, NJ, USA), RHAMM (CD168) (ab170527, 1:250 for Western blotting; Abcam, Cambridge,UK), α-tubulin (T5168, 1:4000 for Western blotting; Sigma Aldrich), and FLAG M2 (F3165, 1:1000 for Western blotting; Sigma Aldrich), and DAPI solution (D523, 1:1000 for nuclear staining, Dojindo Laboratories, Kumamoto, Japan) were used in accordance with the manufacturer’s instructions. Anti-Rabbit IgG, HRP-Linked Whole Ab Donkey (NA934), and Anti-Mouse IgG, HRP-Linked Whole Ab Sheep (NA931) were obtained from GE Healthcare (Little Chalfont, Buckinghamshire, UK).

We extracted blood samples from the heart of the test mice on the final day of experiments. The plasma levels of MMP-1, MMP-2, MMP-9, histamine, hyaluronidase, hyaluronic acid, IL-6, TNF-α, and AGEs were determined using commercial ELISA kits (MMP-1: MyBioSource, San Diego, CA, USA; MMP-2, MMP-9, hyaluronic acid, and TNF-α: R&D Systems, Minneapolis, MN, USA; histamine: Bertin Pharm., Montigny-le-Bretonneux, France; hyaluronidase: AB Clonal Inc., Tokyo, Japan; IL-6: Proteintech, Rosemont, IL, USA; and AGEs: OxiSelect AGE connection ELISA kit, Cell Biolabs Inc., San Diego, CA, USA), according to the instructions of the respective manufacturers.