AKT pathway and promotes apoptosis in osteoarthritic osteoblasts

Biomedicine & Pharmacotherapy 118 (2019) 109357

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Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha

Ricolinostat (ACY-1215) inhibits VEGF expression via PI3K/AKT pathway and promotes apoptosis in osteoarthritic osteoblasts

T

Lei Lia,1, Fuen Liua,1, Wei Huanga, Jun Wanga, Yunpeng Wana, Meng Lib, Yiqun Pangc, ⁎ Zongsheng Yina, a

Department of Orthopaedics, The First Affiliated Hospital of Anhui Medical University, #269 Jixi Road, Hefei, Anhui, China Department of Orthopaedics, The First Affiliated Hospital of University of Science and Technology of China, #17 Lujiang Raod, Hefei, Anhui, China c Department of Radiology, The First Affiliated Hospital of University of Science and Technology of China, #17 Lujiang Raod, Hefei, Anhui, China b

ARTICLE INFO

ABSTRACT

Keywords: Osteoarthritis HDAC6 ACY-1215 PI3K/AKT Osteoblasts

Osteoarthritis (OA) is involved in these pathophysiological changes of articular cartilage, subchondral bone and synovium. As a selective HDAC6 inhibitor, Ricolinostat (ACY-1215) has demonstrated chondroprotective effects in OA. However, its efficacy remains unclear in subchondral bone. In this study, we found that the mRNA and protein levels of HDAC6 were elevated in human OA osteoblasts in vitro. PI3K/AKT signaling pathway was suppressed with downregulation of VEGF expression in osteoblasts after ACY-1215 treatment. ACY-1215 promoted apoptosis of OA osteoblast in a concentration-dependent manner, and the expression of apoptosis-related proteins was also changed by activating caspase pathway. Moreover, western blotting showed decreased expression of MMP9 and MMP13 in IL-1β-induced chondrocytes after co-culture with ACY-1215-stimulated osteoblasts. These data of immunohistochemistry and micro-CT from OA model mice also demonstrated the weak staining of MMPs in cartilage and prevention of aberrant subchondral bone formation after ACY-1215 injection. Therefore, high expression of HDAC6 in osteoblasts also contributed to the OA progression, and our study provided a new evidence that HDAC6 inhibitor may be a potential therapeutic drug for OA.

1. Introduction Osteoarthritis is the most common degenerative disease of joint, which affects hundreds of millions of elderly people in the worldwide [1]. OA pathophysiology is characterized by low-grade inflammatory infiltration, subchondral bone sclerosis, angiogenesis and osteophyte formation [2–4]. However, current therapeutic approaches that are used to attenuate the OA progression or restore the function of the damaged knee or hip joints have not been successful [5,6]. As a functional unit, subchondral bone and articular cartilage interact with each other during the progression of OA [7]. Due to excessive mechanical load, subchondral bone undergoes continuous adaptation beyond the extent of self-healing. This leads to uncoupled bone resorption with bone remodeling in OA [8]. Furthermore, a number of reports have shown that angiogenesis in subchondral bone contributes to OA development, and previous studies have also confirmed the occurrence of crosstalk between subchondral bone and

articular cartilage, which has been ascribed to biochemical connections of various signaling molecules through microfractures, vascular channels and blood vessels [9–11]. At the last-stage of OA, hypertrophic chondrocytes upregulate some genes expression, which promotes angiogenesis and more serious vascular invasion into cartilage [12,13]. However, abnormal angiogenesis signaling allows the transport of inflammatory factors, metalloproteinases (MMPs), and growth factors from subchondral bone to cartilage surface, which shape the deteriorated microenvironment surrounding chondrocytes [14]. Although the mechanism of angiogenesis remains unclear, the characteristic pathological changes of subchondral bone have provided another possibility to treat the OA. The histone deacetylase 6 (HDAC6) is a member of class IIb HDAC family and act as critical role for regulating endothelial cell migration. In addition, the special enzyme is involved in multiple pathophysiological processes of cell apoptosis and differentiation, tumor migration and proliferation by histone deacetylation [15–17]. Therefore, selective

Abbreviations: OA, osteoarthritis; HDAC6, histone deacetylase 6; MMP, metalloproteinase; PI3K, phosphoinositide 3-kinase; AKT, protein kinase B; VEGF, vascular endothelial growth factor ⁎ Corresponding author. E-mail address: [email protected] (Z. Yin) 1 Co-first authors. https://doi.org/10.1016/j.biopha.2019.109357 Received 3 July 2019; Received in revised form 9 August 2019; Accepted 14 August 2019 0753-3322/ © 2019 Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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HDAC6 inhibitors presented unique application in the treatment of cancer and degenerative diseases. In fact, we have found the anti-inflammatory and chondroprotective effects of ACY-1215 in human OA chondrocytes and IL-1β-stimulated C28/I2 cells in a previous study [18]. But little information is available so far regarding the effect of HDAC6 in bone and a report only indicated that it may be involve in angiogenesis and the interaction with runt-related transcription factor 2 (RUNX2) [19,20]. In this study, the expression levels of HDAC6 in OA osteoblasts and its contribution to pathological changes in subchondral bone were determined. Furthermore, the apoptosis of OA osteoblasts was evaluated in response to different concentrations of ACY-1215. In addition, we investigated whether ACY-1215 suppressed VEGF expression in osteoblasts by inhibiting PI3K/AKT signaling pathway in vitro. Moreover, the destabilization model medial meniscus (DMM) in C57/BL mice was conducted to induce osteoarthritis, and intraperitoneal injection with ACY-1215 was used to observe its effect for OA.

culture flasks at a density of 1 × 105 cells/ml, and cultured at 37 °C incubator with a volume fraction of 5% CO2.

2. Materials and methods

2.4. Histochemistry, immunohistochemistry and cell immunofluorescence staining

2.3. Mice Twelve-week-old male C57BL/6 mice were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Mice were fed in a standard food diet without specific pathogens on a 12 h light/dark cycle. Mice were anesthetized and the surgery of destabilization of the medial meniscus were performed on the left knees to induce surgical osteoarthritis, while only a joint capsule incision was performed on mice of sham group. Mice without any treatment were used as the control group. The 25 mg/kg ACY-1215 was injected into OA mice every two days from 3 days after surgery. The same amounts of saline were given to the sham group and other OA mice (vehicle group) (n = 3–6 per group). All mice were killed at 8 weeks after injection for micro-CT scanning and immunohistochemical staining.

2.1. Reagents

The subchondral bone samples of mice were fixed in formalin, decalcified with 10% ethylene diaminete traacetid acid (EDTA) and then embedded in paraffin using standard procedures. Tissue slides were treated with hematoxylin-eosin (HE) and Safranin-O and fast green staining based on the instruction. Slides were dehydrated three times in xylene for 5 min each time and subsequently rehydrated in 100%, 95% and 80% ethanol. Slides were soaked in sodium citrate solution buffer at 92 °C for 10 min, and 0.3% H2O2 was added in tissue slides to eliminate endogenous peroxidase. In the next step, slides were incubated with MMP13 (1:200 dilution; E-AB-70107, Elabscience) and MMP9 (1:100 dilution; 10375-2-AP, proteintech) at 4 °C overnight, followed by staining with goat anti-rabbit second antibody conjugated with HRP (E-AB-1043, Elabscience, Wuhan, China). A light microscope was employed to visualize immunostained slides, and results were captured by image J software. According to the instructions of apoptosis detection kit (Roche, Basel, Switzerland), the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was performed in mice and images were obtained under a fluorescence microscope BX51 (Olympus, Tokyo, Japan). Additionally, for the immunofluorescent detection, OA osteoblasts in 6-well plates were fixed in 4% paraformaldehyde and were blocked using QuickBlock™ immunostained blocking solution (Beyotime, Shanghai, China) for 15 min, and then were incubated with anti-HDAC6 antibody (1:1000 dilution, ab133493, Abcam), followed by secondary antibody conjugated with Cy3 at 1:50 dilution (E-AB-1010, Elabscience) for 1 h to perform detection. The percentage of positive cells were obtained by image J software.

DMEM and DMEM/F12 mediums were purchased from HyClone (South Logan, UT, USA) and trypsin, cell lysis solution (RIPA), penicillin-streptomycin were supplied by Beyotime (Shanghai, China). Fetal bovine serum (FBS) was purchased from CLARK (CLARK Bioscience, Australia). Type I and II collagenases were purchased from Sigma (Sigma-Aldrich, St. Louis, USA). TRIzol reagent was obtained from Invitrogen (Carlsbad, CA, USA). ACY-1215 and LY294002 were bought from MCE China (Shanghai, China). Anti-HDAC6 antibody was purchased from Abcam (Cambridge, UK). Anti-cleaved caspase-3, Bax and Bcl-2 antibodies were obtained from Cell Signaling Technology (CST, Boston, USA). Anti-VEGF, AKT, phosphorylated AKT and MMP13 antibodies were bought from Elabscience Biotechnology Co.Ltd (Wuhan, China). Anti-MMP9 antibody was purchased from proteintech (WUHAN SANYING, China). ELISA kit was bought from Dakewe company (Dakewe Biotech, Shenzhen, China). Reagents for RNA reverse transcription and quantitative polymerase chain reaction (PCR) system were procured from TaKaRa (TaKaRa Bio, Japan). 2.2. Human OA osteoblast and chondrocyte extraction and culture Knee tibial plateaus from sixteen patients (12 females and 4 males) with mean age 70.3 years ( ± 7.9) undergoing total knee joint replacement and 5 healthy knee specimens (2 male and 3 females) with mean age 66.8 years ( ± 8.2) from amputees were enrolled in this study. All participants had no history of hormone uses and some diseases that affect bone metabolism. The informed consents were obtained from all participants. Clinical criteria of the American College of Rheumatology were applied to confirm the diagnosis of OA [21]. This study design was approved by the Ethical Committees of the first affiliated hospital of Anhui Medical University and was performed based on the Helsinki Declaration. All bone samples of OA were defined as KL grade 4 according Kellgren-Lawrence (K–L) grade [22]. Sclerotic zones (medial tibial plateau) in subchondral bone were cut into small pieces of approximately 2 mm2 after removing the trabecular bone and cartilage. The 1 mg/ml type I collagenase mixed in DMEM/F12 medium without FBS was used to digest these bone pieces at 37 °C for 20, 20 and 240 min (min) sequentially, and then was cultured in flasks containing 20% FBS. Additionally, based on previous report [23], articular cartilage overlying tibial plateaus were cut into small chips and digested with 5 g/l trypsin in 37 °C. After 30 min, the 0.5 mg/ml type II collagenase was added and digested in 37 °C for 12–16 hours (h). The digested cells were centrifuged at 1000 rpm /min for 5 min and the supernatant was aspirated into the medium containing 10% FBS. Cells were seeded in

2.5. CCK-8 detection, western blotting and Enzyme-linked immunosorbent assay The viability of osteoblasts was detected by cell counting kit-8 assay (CCK-8, Beyotime) after the treatment of LY294002 and ACY-1215. Briefly, 10 μl of CCK-8 solution was added to cell culture medium of 96well plate and incubated for 2 h. Absorbance at 450 nm was measured by a microplate reader (Thermo Scientific, USA). Total protein of osteoblasts was extracted using the mixture of RIPA buffer and PMSF (100:1) and protein concentration was measured using BCA assay kit. Osteoblasts lysate was centrifuged at 12000×g for 3 min, and 15 μg protein from each sample was added to polyacrylamide gels and separated by 10% SDS-PAGE according to standard electrophoresis conditions. Proteins were transferred to 0.45 μm polyvinylidene fluoride (PVDF) membranes (Millipore, USA) and blocked with TBST-5% skim milk for 2 h at room temperature. The membranes were incubated in primary antibodies of anti-HDAC6 at 1:20000 dilution (ab133493, 2

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Fig. 1. The increased expression of HDAC6 in human OA osteoblasts. (A) The mRNA level of HDAC6 in normal and OA osteoblasts. (B) The HDAC6 protein expression levels based on western blotting data in normal and OA osteoblasts. (C) Red fluorescent labeling of the HDAC6 and quantitative analysis of positive cells in per visual field are shown among two NC and OA group (Original magnification: 200×). Scale bar, 100 μm. The data are presented as the mean ± SD. **p < 0.01 and ***p < 0.001 compared to the normal control group.

Abcam), anti-VEGF at 1:1000 dilution (E-AB-64131, Elabscience), anticleaved caspase-3 at 1:1000 dilution (9661S, CST), anti-Bcl-XL at 1:1000 dilution (2764S, CST), anti-Bax at 1:1000 dilution (2774S, CST), anti-AKT at 1:1000 dilution (E-AB-30467, Elabscience), anti-phosphorylated AKT at 1:1000 dilution (E-AB-21187, Elabscience), antiMMP13 at 1:1000 dilution (E-AB-70346, Elabscience), anti-MMP9 at 1:1000 dilution (10375-2-AP, proteintech) and anti-GAPDH at 1:1000 dilution (GB11002, Servicebio, Wuhan, China) overnight at 4 °C. Then, membranes were incubated with goat anti-rabbit IgG antibody (E-AB1041, Elabscience) after rinse using TBST, and images of protein bands were visualized and captured by the Bio-Rad XRS + imaging system (BIO-RAD, USA). Relative intensities of protein bands were measured by image J software. Additionally, secretion levels of VEGF in the chondrocyte supernatant were detected by ELISA following the manufacturer’s instructions, and absorption was measured at 450 nm using microplate reader. The standard curve was obtained by gradient dilution of cytokines, and the sensitivity of assay was 1 pg/ml.

CT values of both target and reference genes. The following primer sequences were used to amplify the human genes of interest: HDAC6: Forward: 5′-GCATCCGAACTCATACTCCT GTG-3′, Reverse: 5′-GCGGTGTTTCTGTTGAGCATAG-3′; VEGF: Forward: 5′-AGGGCAGAATCATCACGAAGT-3′, Reverse: 5′-AGGGTCTC GATTGGATGGCA-3′; caspase-3: Forward: 5′-TGGAAGCGAATCAATGG ACTCT-3′, Reverse: 5′-TGAATGTTTCCCTGAGGTTTGC-3′; Bcl-XL: Forward: 5′-ATCGCCCTGTGGATGACTGA-3′, Reverse: 5′-GAGACAGCC AGGAGAAATCAAAC-3′; MMP9: Forward: 5′-GCACGACGTCTTCCAGT ACC-3′, Reverse: 5′-GGTTCAACTCACTCCGGGAA-3′; MMP13: Forward: 5′-ACTGAGAGGCTCCGAGAAATG-3′, Reverse: 5′-GAACCCCGCATCTT GGCTT-3′; and Actin: Forward: 5′-CACCCAGCACAATGAAGATCAA GAT-3′, Reverse: 5′−CCAGTTTTTAAATCCTGAGTCAAGC-3′.

2.6. Total RNA extraction and RT-qPCR

OA osteoblasts were treated with the dose of 0 μM, 5 μM, 10 μM, and 20 μM ACY-1215 respectively, cultured in DMEM/F12 medium without FBS, and seeded into the Transwell chambers (Costar, Cambridge, MA), which were inserted into the 6-well plates of DMEM medium containing 10% FBS and 10 ng/ml IL-1β-stimulated chondrocytes from the same OA patient for 48 h culture. The number of cells in each chamber and well was consistent by the cell counting plate. The level of VEGF secretion and MMP-9, MMP-13 expression of chondrocytes was measured.

2.7. Three-dimensional (3D) co-culture model of human primary osteoblasts with chondrocytes

TRIzol lysate (Invitrogen, USA) was employed to extract total RNA from osteoblasts, and reverse transcription was performed using a PrimeScript™RT reagent kit (TaKaRa, Japan) based on the manufacture's protocol. cDNA was then amplified under the following conditions: 95 °C for 5 min; followed by 40 cycles of: 95 °C for 30 s, 60 °C for 30 s, 72 °C for 1 min; followed by 72 °C for 10 min, and the relative mRNA expression was calculated by the 2−ΔΔCT method based on the

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2.8. Flow cytometry

5 μM, 10 μM, and 20 μM did not show any marked toxic effect on osteoblasts. However, decreased cell viability was observed at a concentration of 50 μM (Fig. 2C). Furthermore, VEGF expression in OA osteoblasts was downregulated in a concentration-dependent manner of ACY-1215. As the key regulator of PI3K/AKT signaling pathway, phosphor-AKT was significantly decreased after ACY-1215 treatment. Additionally, indifference in AKT protein expression between the four concentrations further demonstrated the suppressed effect of HDAC6 inhibitor on the PI3K/AKT pathway (Fig. 2D). Meanwhile, the VEGF secretion level and positive cell number were also decreased after ACY1215 treatment in OA osteoblasts, and there were statistical differences among four concentrations (Fig. 2E).

According to manufacturer’s instruction, apoptosis of osteoblasts after ACY-1215 treatment was measured by FITC Annexin V apoptosis detection kit (BD Biosciences, USA) in CytoFLEX flow cytometry (Beckman Coulter, USA), and results were shown using CytExpert 2.1 software. 2.9. Micro-CT Left knee joints of mice that were free of adipose tissue and peripheral ligaments were anatomized and fixed in 98% ethanol for 24 h. The high-resolution in vivo X-ray microtomograph (Skyscan1176, Bruker, Germany) was used to visualize these specimens. The micro-CT scanner was set at a voltage of 58 kVp, a current of 431 μA, and a resolution of 9.0 μm per pixel. NRecon software was applied to image three-dimensional reconstruction in the sagittal compartment of the medial tibial plateau and relative parameters of the trabecular bone in different groups were analyzed. The parameters of BMD (bone mineral density) and BV/TV (bone volume/tissue volume) in 3D structure were measured to analyze subchondral bone changes.

3.4. ACY-1215 promoted apoptosis of abnormally proliferative osteoblasts in OA A gradual increase of the proportion of apoptotic osteoblasts treated with different doses of ACY-1215 in a concentration-dependent manner was observed by flow cytometry (Fig. 3A). Moreover, the expression levels of apoptosis-related proteins by western blotting and RT-qPCR showed that ACY-1215 contributed to osteoblasts apoptosis in OA. Upregulation of cleaved caspase-3 and Bax, and downregulation of BclXL at mRNA and protein levels were also found after ACY-1215 treatment (Fig. 3B, C).

2.10. Statistical analysis All experiments and measurements were repeated at least three times. All data were presented as the mean ± standard deviation (SD). Comparisons among different groups were performed by analysis of variance (ANOVA) SPSS V.23.0 (SPSS Inc., Chicago, USA) to evaluate differences. The threshold for statistical significance was set at P value of 0.05.

3.5. Co-culture model indicated the inhibition of HDAC6 in OA osteoblasts downregulated the expression of MMP9 and MMP13 in IL-1β-induced chondrocytes The above results have demonstrated that the HDAC6 may regulate VEGF secretion in OA osteoblast. Therefore, the levels of VEGF secretion and MMP9, MMP13 protein levels in chondrocyte were significantly downregulated after co-culture with different doses of ACY1215-stimulated osteoblasts (Fig. 4A, B). Taken together, inhibition of HDAC6 activity in osteoblasts not only prevented VEGF expression in osteoblasts but also ameliorated cartilage degradation indirectly during OA progression.

3. Results 3.1. Expression level of HDAC6 was elevated in human OA osteoblasts The RT-qPCR analysis and western blotting were firstly used to verify the expression levels of HDAC6 in healthy and osteoarthritic osteoblasts. As shown in Fig. 1A, B, the HDAC6 expression at mRNA and protein levels were higher in OA group compared to healthy group. Consistent strong cell immunofluorescence staining of HDAC6 in OA osteoblasts were represented in Fig. 1C. Therefore, the overproduction of HDAC6 in osteoblasts may contribute to OA development.

3.6. Intraperitoneal injection of ACY-1215 attenuates cartilage degradation in vivo Based on the in vivo findings, we next examined the function of inhibition of HDAC6 activity on the knee joints in OA mice. Due to the lack of reports on the dose of intraperitoneal injection of ACY-1215 in OA mice, we referred to the related literatures about tumor model mice and experimental acute liver failure and regarded 25 mg/kg as the optimal dose [24,25]. The strong staining of HDAC6 were found in cartilage and subchondral bone at 8 weeks after surgery in mice (Fig. 5A). HE staining showed that the thickness of the calcified cartilage zone in OA mice with ACY-1215 injection was lower, and the cartilage degradation in ACY-1215 group was significantly alleviated compared to vehicle group by Safranin-O and fast green staining. These immunohistochemical results confirmed that the percentages of MMP9-, MMP13-positive cells in articular cartilage were also obviously decreased in ACY-1215 group (Fig. 5B). The increased TUNEL-positive cells in subchondral bone indicated the upregulated OA osteoblasts apoptosis (Fig. 5C). Additionally, 3D micro-CT images represented the aberrant subchondral bone formation in OA mice and suggested the pathological process of OA has entered the bone sclerosis stage dominated by osteoblasts at 8weeks after surgery. However, increased BMD and BV/TV proved that the process was also delayed after ACY-1215 treatment (Fig. 5D). OARSI scoring of OA severity was also decreased in

3.2. VEGF expression in OA osteoblasts was influenced by activation of PI3K/AKT pathway To explore the possible signaling pathway of VEGF expression, we examined the effect of different concentrations of 0 μM, 10 μM, 25 μM, 50 μM and 75 μM LY294002 (the inhibitor of PI3K) on osteoblasts. The cell viability was assessed using CCK-8 assay and showed that a concentration of75 μM was toxic for osteoblasts (Fig. 2A). Thus, the effect of LY294002 in OA osteoblasts was investigated at concentrations of 0–50 μM, and protein levels of phosphor-AKT and VEGF were downregulated with the increase of LY294002 concentration (Fig. 2B). However, there still was no effect on the protein level AKT. 3.3. ACY-1215 inhibited VEGF expression viaPI3K/AKT pathway in OA osteoblasts To investigated the role of HDAC6 inhibitor in OA osteoblasts, the cell viability was assessed in response to different concentrations of ACY-1215. The result of CCK-8 detection demonstrated that the doses of

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Fig. 2. The PI3K/AKT pathway was required in VEGF expression in OA osteoblasts, which was inhibited after ACY-1215 treatment. (A) OA osteoblasts were treated with different concentrations of LY294002 for 24 h and cell viability was detected. (B) Phosphor-AKT, AKT and VEGF protein levels after LY294002 treatment and relative quantification. (C) The detection of osteoblasts vitality in response to different concentration of ACY-1215. (D) Phosphor-AKT and AKT protein levels after different dose of ACY-1215 treatment and quantitative analysis. (E) The VEGF secretion level and VEGF-positive cells in per visual field gradually decreased after ACY-1215 treatment in a dose-dependent manner, and relative quantification is shown on the right. (original magnification: 200×). Scale bar, 100 μm. Values are presented as the means ± SD. *p < 0.05 and ***p < 0.001.

ACY-1215 treatment group (Fig. 5E).

Society for Clinical and Economic Aspects of Osteoporosis, Osteoarthritis and Musculoskeletal Diseases) recommend that CS serves as disease-modifying OA drug [29]. However, some reports also questioned the efficacy of CS. The reason is that only 12% of CS is absorbed into the body, and most of it is also degraded into low molecular weight disaccharide units, such as oligosaccharides [30]. Therefore, the effective concentration reaching the plasma membrane may be lower than the concentration used in vitro experiments. The higher quality clinical trials are necessary to verify the efficacy of CS in human. As multiple malignant tumors aggressive inducer, HDAC6 contributes to angiogenesis, inflammatory factor secretion, and occurrence of immune system disorders [31–33]. Meanwhile, previous study and our team have confirmed that OA progression partly attributed to the excessive activation of HDAC6 in chondrocytes, and inhibited the overexpression

4. Discussion Growing evidences indicate that the OA is not a pure articular cartilage disorder, and high vascularization of subchondral bone is considered to be an important factor in OA development [26,27]. Traditional non-steroidal anti-inflammatory drugs (NSAIDs) did not exhibit relieved effect on cartilage degradation of OA, but may cause severe gastrointestinal side effects after long-term use. Nowadays, a meta-analysis has concluded that chondroitin sulfate (CS) is effective in relieving pain and swelling caused by osteoarthritis, and the side effects of CS are significantly lower than that of NSAIDs [28]. Therefore, EULAR (European League Against Rheumatism) and ESCEO (European

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Fig. 3. (A) OA osteoblasts were treated with different concentrations of ACY-1215 for 48 h and stained using propidium iodide (PI) and fluorescein isothiocyanate (FITC). The results of cell apoptosis were analyzed by flow cytometer. (B–C) The expression of apoptosis-related proteins in osteoblasts treated with ACY-1215 at mRNA and preotein levels. The data are presented as the mean ± SD. *p < 0.05, **p < 0.01 and ***p < 0.001.

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Fig. 4. (A) The level of VEGF secretion in IL-1β-induced chondrocytes supernatant after co-culture with ACY-1215- stimulated OA osteoblasts. (B) The MMP9 and MMP13 protein levels and quantitative analysis in IL-1β-induced chondrocytes after co-culture with ACY-1215-stimulated OA osteoblats. The data are presented as the mean ± SD. *p < 0.05, **p < 0.01 and ***p < 0.001.

further demonstrated the reliability of these results. In fact, it has been suggested that the biological and physical crosstalk of cartilage-bone lead to entire knee joint disorder during the evolution of OA, and thus the intrinsic connection determines the importance of co-culture models of osteoblasts and chondrocytes in vitro. A study on the use of co-culture model of OA chondrocytes and osteoblasts has displayed the effect of reduced aggrecan loss and upregulated of MMP13 and MMP3 in osteoblasts and OA development [43]. Consistently, chondrocytes were stimulated with IL-1β to simulate the inflammatory environment of OA, and we found that MMP9 and MMP13 proteins expression in chondrocytes were reduced when HDAC6 was inhibited in OA osteoblasts using the transwell assay. Previous studies have also demonstrated that VEGF in synovial fluid promoted the expression of metalloproteinases in OA [44,45]. Hence, our results demonstrated, on the one hand, the intrinsic crosstalk between chondrocytes and osteoblasts each other, and on the other hand, more profoundly suggested the important role of ACY-1215 in inhibiting VEGF expression in osteoblasts. To fully explicate the effect of the HDAC6 inhibitor in vivo, we firstly examined the HDAC6 expression in cartilage and subchondral bone from OA mice at 8 weeks after DMM surgery. Unexpectedly, immunohistochemistry revealed that a strong staining of HDAC6 was found in knee joint of OA mice compared to the sham group and control group. Consistent with in vitro results, changes on reduced proteoglycans loss, MMP9 and MMP13 expression levels in articular cartilage after ACY-1215 treatment revealed that inhibition of HDAC6 could prevented cartilage degradation in vivo. Moreover, TUNEL assay showed that ACY-1215 can effectively increase the apoptosis of abnormally proliferating osteoblasts. To our knowledge, this is the first animal model study, which has investigated and shown the potential use of ACY-1215 in OA. Furthermore, the increased BMD and BV/TV, and lower thickness of the calcified cartilage zone after ACY-1215 injection implied that the amelioration of aberrant subchondral bone formation. Whether this mechanism is similar to the fibrotic process in some tissues may require further study [46]. In summary, we showed that HDAC6 acts as the harmful factor during OA. ACY-1215 involved in the inhibition of VEGF expression in

may be a novel target for osteoarthritic treatment [34,35]. However, some new findings in our study provided key insights into the efficacy of ACY-1215 and its underlying mechanisms in subchondral bone of OA. As the most important factor, VEGF plays a pivotal role in regulating angiogenesis. A report has shown that TGF-β signaling pathway, OPG/ RANKL system and mechanical overload were involved in angiogenesis in subchondral bone [36]. Ryu CH et al. have demonstrated that PI3K/ AKT signaling pathway exerted a crucial effect by promoting angiogenesis in human umbilical cord blood mesenchymal stem cells (hUCBSC) [37]. The activated PI3K and thereby induced the combination of second messenger of PIP3 with AKT containing pleckstrin homology (PH) domain, which mediates the process of vascular endothelial cell migration and proliferation [38,39]. We have confirmed that the PI3K/AKT signaling pathway participated in VEGF expression. ACY-1215 also effectively inhibited the VEGF secretion level, which may be attributed to the downregulated phosphor-AKT levels after ACY-1215 treatment in OA osteoblasts. Therefore, our results suggested that ACY-1215 could apparently inhibit VEGF expression by inducing inactivation of PI3K/AKT pathway in OA osteoblasts, thereby affecting angiogenesis in subchondral bone. In addition to these effects on the regulation of cell proliferation and vascular invasion, HDAC6 also plays an indispensable role in the cellular apoptosis. A recent study has revealed that ACY-1215 exhibits the promotion of cellular apoptosis in esophageal squamous cell carcinoma viaPI3K/AKT/mTOR pathway in a concentration-dependent manner [40]. Similarly, the selective HDAC6 inhibitor combined with ibrutinib against non-Hodgkin lymphoma and follicular lymphoma by inhibiting tumor cells viability and upregulating cellular apoptosis levels [41]. Coincidentally, abnormal changes in the biological phenotype of osteoblasts lead to subchondral bone sclerosis and show greater proliferative capacity compared to normal osteoblasts [42]. ACY-1215 also promotes osteoblast apoptosis in OA as indicated by, increased expression of cleaved caspase-3 and Bax protein and reduced Bcl-XL protein levels in our study proved the motivation of caspase pathway. The number of apoptotic osteoblasts were increased in a concentrationdependent manner after ACY-1215 treatment in OA osteoblasts, which

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Fig. 5. (continued)

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Fig. 5. Intraperitoneal injection of ACY-1215 reduced microvascular activity in the subchondral bone and diminished degradation of articular cartilage. (A) The HDAC6 expression of mice knee joints and quantitative analysis in the control group, sham group and OA group. Scale bars, left 100 μm (original magnification: 200×), right 50 μm (original magnification: 400×). (B) The MMP9, MMP13, and Safranin-O and fast green staining in articular cartilage after intraperitoneal injection of ACY-1215 in mice. HC: hyaline cartilage; CC: calcified cartilage; SBP: subchondral bone plate. Scale bars, left 100 μm (original magnification: 200×), right 50 μm(original magnification: 400×). (C) TUNEL assay showed the green fluorescence represents the apoptosis of cells in subchondral bone. Scale bars, 100 μm (original magnification: 200×). (D) 3D micro-CT images of the medial compartment in subchondral bone (sagittal view) of mice in the sham group, OA group, and ACY-1215 group. The parameter analysis of BMD and BV/TV in micro-CT was shown. Scale bar, 1 mm. (E) OARSI scoring of OA severity in sham group, vehicle group and ACY-1215 group. The data are presented as the mean ± SD. *p < 0.05, **p < 0.01 and ***p < 0.001.

Fig. 5. (continued)

OA osteoblasts viablocking PI3K/AKT pathway. The promotion of OA osteoblast apoptosis and prevent aberrant bone formation were also found in our study. Taken together, these findings provide some evidences that the protective effect of ACY-1215 for OA is comprehensive and involves more than one component of knee joint. Further studies will be needed to investigate whether ACY-1215 inhibits synovitis and osteophyte formation in OA and its biological mechanisms.

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Authors contributions Zongsheng Yin and Meng Li designed the experiments; Lei Li, Fuen Liu and Wei Huang performed this study. Lei Li, Jun Wang and Yunpeng Wan analyzed experimental results; Lei Li and Yiqun Pang wrote this manuscript. Funding This study was supported by the National Natural Science Foundation of China (No. 81672161). Declaration of Competing Interest All authors have no conflict of interest in this work. Acknowledgment We appreciate all patients' support for our study. References [1] J.W.J. Bijlsma, F. Berenbaum, F.P. Lafeber, Osteoarthritis: an update with relevance

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