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Inhibitory effect of low‑intensity pulsed ultrasound on the fibrosis of the infrapatellar fat pad through the regulation of HIF‑1 α in a carrageenan‑induced knee osteoarthritis rat model

. Fibrotic changes in the infrapatellar fat pad (IFP) are involved in the pathogenesis of knee osteoarthritis (KOA). HIF‑1 α is a transcription factor that is activated during hypoxia and is suggested to play a role in fibrosis in various organs. However, its participation in the fibrotic changes in IFP remains unclear. Therefore, we investigated the role of HIF‑1 α in IFP fibrosis using a carrageenan‑induced KOA rat model and evaluated the potential of low‑intensity pulsed ultrasound (LIPUS) as a novel treatment for KOA. A rat model was prepared by intra‑articular injection of 0.5% carrageenan (50 µl) using 8‑week‑old male Wistar rats. Fibrosis of the IFP was evaluated histologically by hematoxylin and eosin and Sirius Red staining at 1 and 2 weeks after intra‑articular injection. The mRNA expression levels of HIF‑1 α and fibrosis‑related molecules, CTGF and VEGF, were analyzed using reverse transcription‑quantitative PCR, and the DNA binding activity of HIF‑1 α was assessed using a binding assay. In addition, the effect of irradiation with LIPUS on the fibrosis of IFP was verified. Histological studies demonstrated a significant increase in the fibrosis of IFP 1 and 2 weeks after intra‑articular injection of carrageenan, accompanied by overexpression of CTGF and VEGF, which was followed by upregulation of transcriptional activation of HIF‑1 α . Moreover, intervention with LIPUS for 2 weeks after injection of carrageenan attenuated fibrosis of IFP, accompanied by a significant reduction in the transcriptional activation of HIF‑1 α and decreased the gene expression levels of HIF‑1 α , CTGF, and VEGF. The present study demonstrated that activation of HIF‑1 α promoted fibrosis of IFP in carrageenan‑induced arthritis in rats and that intervention with LIPUS decreased the activity of HIF‑1 α and inhibited fibrosis. These results suggest that LIPUS cyto‑ kines and IL‑6 expression in the IFP and subcutaneous adipose


Introduction
Knee osteoarthritis (KOA) is a chronic disease characterized by degenerative changes in the articular cartilage, structural changes in the subchondral bone, and secondary synovitis. Patients with KOA have a limited range of motion, experience joint pain, and exhibit gait disturbances, and serious KOA interferes with daily life (1). Although radiographic findings do not necessarily correlate with the symptoms of KOA (2), and a previous report suggested that degenerative and structural changes in synovitis and bone marrow lesions are important factors that cause joint pain and impair the quality of life (3). In a clinical study using MRI, Hill et al (4) reported that synovitis in KOA correlates with pain severity, and more recently, it was reported that in addition to the synovial tissue, fibrotic changes in the infrapatellar fat pad (IFP) are also involved in the pathogenesis and mechanism of joint pain in KOA (5).
The IFP is the fatty tissue that fills the space in the knee joint surrounded by the patellar tendon, proximal tibia, and femoral condyle at the inferior border of the patella (6). The IFP is in contact with the superior edge of the patella, the medial patellar retinaculum, and the lateral patellar retinaculum, and plays an important role in the smooth movement of knee joints (7). Furthermore, the IFP is histologically a collagen-rich fibrous adipose tissue sequential to synovial tissue and is innervated by branches of the posterior tibial nerve, which are distributed by nerve fibers with free nerve endings (8). It also acts as a buffer against joint loading by adjusting the contact pressure on the knee joints during joint movement (9). Furthermore, the IFP is known to secrete several growth factors and cytokines and is reported to affect the metabolism of articular cartilage and synovial tissue. Distel et al (10) demonstrated that the expression of proinflammatory cytokines and IL-6 expression in the IFP and subcutaneous adipose tissue were elevated in obese patients, suggesting that cytokine secretion from the IFP may contribute to the degeneration of articular cartilage. It has also been reported that lymphocyte infiltration, angiogenesis (11), and an increasing number of cells expressing monocyte chemoattractant protein-1 (MCP-1) and IL-6 were observed in the IFP of KOA patients. Thus, the IFP is an important tissue in the progression of KOA from the perspective of induction of inflammation in knee joints. Moreover, the IFP is reported to be involved in the fibrosis of synovial tissues (12), and changes in the mechanical properties of IFP by fibrosis reduce the buffering capacity against joint loading (13,14). Therefore, fibrosis of the IFP is a major cause of functional impairment and pain in KOA patients. However, the mechanism underlying IFP fibrosis remains unclear.
In general, tissue fibrosis is observed as a physiological event with the deposition of extracellular matrix components such as collagen (15). Cirrhosis of the liver, renal disease, and pulmonary fibrosis are known to involve severe fibrosis, which is hypothesized to be a response to chronic inflammation (16,17). Hypoxia is also considered to be strongly involved in fibrosis in various adipose tissues in vivo (18). Studies on obese patients have demonstrated that an increase in the amount of adipose tissue results in hypoxic conditions in the tissue by delayed angiogenesis, which activates the transcription factor, HIF-1α, and causes fibrosis in adipose tissues (19). Thus, HIF-1α may also be a key molecule in the fibrosis of IFP in the pathogenesis of KOA. Regarding the relationship between hypoxia and KOA, it was reported that HIF-1α in articular cartilage and synovial fluid is activated and is correlated with the severity of KOA (20). In addition, Sotobayashi et al (21) demonstrated that the transcriptional activity of HIF-1α in the synovial tissue was increased in a contracture model of joint immobilization, resulting in synovial tissue fibrosis. These results strongly suggest that the increased expression and activation of HIF-1α are important factors that promote fibrosis in IFP in the pathogenesis of KOA.
In this study, we focused on HIF-1α and investigated the direct involvement of HIF-1α in the mechanism of fibrosis of IFP accompanied by the progression of KOA using an animal model. In addition, to examine a useful and practical management approach for KOA, the inhibitory effect of low-intensity pulsed ultrasound (LIPUS) on HIF-1α activation and fibrosis in IFP was evaluated, as it has been reported that LIPUS attenuates the fibrosis of synovial tissues followed by reduced activation of HIF-1α in a joint contracture model (23,24).

Materials and methods
KOA model. A KOA model was established and LIPUS intervention was performed on male Wistar rats weighing 300-400 g, obtained from Japan SLC. Animals were anesthetized with 1.5% isoflurane mixed with oxygen using inhalation anesthesia and were maintained on the same concentration of anesthetic throughout the entirety of the procedure. Animals were then subjected to 0.5% carrageenan injection into the bilateral knees (25). Animals were sacrificed by intraperitoneal administration of an overdose of 1-1.5 ml pentobarbitone (150-200 mg/kg), which amounted to 64.8 mg/ml pentobarbitone. Death was confirmed by checking for cardiac arrest, after which the animal was observed for ~5 min. After ensuring that there were no signs of recovery, tissues were harvested from the animals.
The unilateral knee joints of the rats were treated with LIPUS (BR Sonic-Pro, Ito Co., Ltd.). LIPUS was set at a frequency of 3 MHz and an output power of 120 mW/cm², based on a previous study, and was performed for 15 min a day, 4 times a week, every other day (23).
The samples were isolated and used for reverse transcription-quantitative (RT-q)PCR, ELISA, and histological analysis.
In addition, the IFP was harvested along with the synovial tissues after dissection at the inferior pole of the patella and flipped with the patellar tendon (26).
Ethics statement. All animal procedures were approved by the Ethics Committee for Animal Experiments of the Morinomiya University of Medical Sciences (approval no. 2019A001) and performed in accordance with our institutional guidelines. The animal procedures were also performed in compliance with the law (no. 105) and notification (no. 6) of the Japanese government and conducted in accordance with the guidelines of the National Research Council. All surgeries were performed under anesthesia, and all efforts were made to minimize suffering. Signs of significant distress in animals, such as joint infection, behavioral restriction due to excessive pain, and avoidance behavior were considered humane endpoints requiring immediate intervention. However, there were no cases requiring euthanasia due to observation of a humane endpoint and, therefore, animals were euthanized only at the end of the experimental period.
Histological and immunohistochemical studies. The excised joint was decalcified with Morse's solution as previously described (27) and fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) overnight at 4˚C. Then, the excised joint was processed for routine paraffin embedding. Tissue cross-sections (5 µm) were rehydrated and stained with immersion in hematoxylin and eosin (HE) solution for 3-5 min at room temperature (28).
Toluidine blue staining and collagen staining were also performed to evaluate the progression of osteoarthritis (OA) caused by carrageenan and for measuring the OASRI score (29). For toluidine blue staining, tissues were immersed in the Toluidine blue solution for 15 min at room temperature. For total collagen staining, sections were rehydrated and incubated using a Picrosirius Red Stain Kit for 2 h at room temperature according to the manufacturer's protocol (SR; Polysciences, Inc.), which stains collagen I and III. The stained area was measured using ImageJ (National Institutes of Health. Version 1.48) (21).
For immunohistochemical (IHC) staining, the anti-RM-4 antibody (cat. no. KT014, Medical Chemistry Pharmaceutical Inc.) was used to analyze the infiltration of macrophages in the IFP. IHC staining was performed using the high polymer HISTOFINE simple stain mouse MAX-PO (Nichirei Bioscience Inc.) method, as described previously (21). Briefly, 5 µm-thick sections were deparaffinized, rehydrated before blocking endogenous peroxidase activity with 3% hydrogen peroxide, and preincubated with 1.5% blocking reagent (Roche Applied Science) in TBS at room temperature for 1 h. Diluted primary antibodies (1:500) were then applied to the sections, and these sections were further incubated at room temperature for 1 h. Following this, the sections were rinsed twice with TBS for 5 min each and incubated with HISTOFINE simple stain mouse MAX-PO (rat) (Nichirei Bioscience Inc.) for 30 min at room temperature. Peroxidase activity was visualized by treatment with 0.05% diaminobenzidine containing 0.3% hydrogen peroxide. The sections were rinsed in water, dehydrated, cleared, and mounted.
RT-qPCR. Total RNA was extracted from the knee capsule, excluding the cartilage and meniscus. Excised tissues were homogenized in cold PBS and centrifuged at 20,000 x g for 15 min at 4˚C. Total RNA was extracted from the tissue samples using ISOGEN II (Nippon Gene) and resuspended in PBS, and its purity was assessed by spectrophotometry. Only samples with an A260/A280 ratio in the range of 1.8-2.0 were used. cDNA was synthesized using an iScript cDNA synthesis kit according to the manufacturer's protocol (Bio-Rad Laboratories, Inc.). Amplification reactions were performed using SsoFast EvaGreen Supermix (Bio-Rad Laboratories), with 100 µm of each primer and 1 µg cDNA in a final volume of 20 µl. Amplification reactions were performed in a MiniOpticon Real-Time PCR Detection System (Bio-Rad Laboratories, Inc.), with the following amplification protocol: Initial denaturation of 1 min at 95˚C; followed by 40 cycles of 1 sec at 95˚C and 5 sec at 61-65˚C, with each primer. The expression levels of hypoxanthine-guanine phosphoribosyltransferase (HRPT) as a housekeeping gene were used as the internal control, and the comparative Cq method (2 -ΔΔCq ) was used to quantify the gene expression levels (30). Oligonucleotides were synthesized using Gene Design Inc. (Ibaragi, Osaka, Japan). The primer sequences used were obtained from previous studies (21,22,27,31) and are listed in Table I. ELISA. Synovial tissue including IFP was removed from the knee joint. Proteins were extracted from the knee IFP. Extraction was performed using a Nuclear Extract Kit (Active Motif) according to the manufacturer's protocol. The binding activity of HIF-1α in the homogenized tissue was measured using an HIF-1α transcription factor assay kit, according to the manufacturer's instructions (cat. no. ab133104, Abcam).
Statistical analysis. All data are presented as the mean ± SEM. The distribution of the data was analyzed first and subsequently analyzed using a one-way ANOVA followed by a Tukey-Kramer post-hoc test or a Kruskal-Wallis followed by a Steel-Dwass post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

Results
Histological analysis of fibrosis in IFP. Histological analysis revealed that intra-articular administration of carrageenan resulted in significant progress in the pathogenesis of OA, OARSI score: Saline, 0.6±0.55; Car 1 week, 1.8±0.84 (P<0.05 vs. Saline); and Car 2 weeks, 2.4±0.55 (P<0.01 vs. Saline). HE, SR, and IHC staining for macrophages showed cellular infiltration and fibrotic tissue proliferation in the adipose tissue of IFP at 1 and 2 weeks after intra-articular injection of carrageenan, and cellular infiltration and fibrosis were higher after 2 weeks compared with after 1 week (Fig. 1A).
SR staining, which is specific for type I and III collagen, demonstrated that significant collagen fibril proliferation was observed after both 1 and 2 weeks (middle-left and middle-right panels in Fig. 1A). The area stained was significantly increased 1 and 2 weeks after treatment with carrageenan compared with that of the saline group. Furthermore, the stained area was greater after 2 weeks of treatment with carrageenan compared with after 1 week (Fig. 1B).
Gene expression of fibrosis-related molecules and HIF-1α in the IFP. We quantitatively analyzed the mRNA expression levels of CTGF and VEGF, well-known major factors involved in the mechanisms of fibrosis (32)(33)(34), and found that their expression was significantly increased 1 week after intra-articular injection of carrageenan compared with the expression after saline injection and remained high after 2 weeks ( Fig. 2A and B). Moreover, the mRNA expression levels of HIF-1α, which regulates and induces the expression of CTGF and VEGF at the genetic level, were higher 1 and 2 weeks after intra-articular injection of carrageenan compared with that after saline injection (Fig. 2C). Furthermore, the transcriptional activity was significantly increased 2 weeks after the intra-articular injection of carrageenan compared with that after saline injection (saline, 0.558±0.028; carrageenan, 0.597±0.020; P<0.05).

Inhibitory effect of LIPUS on fibrosis in IFP.
We also examined the inhibitory effect of LIPUS on fibrosis in IFP. Histological analysis showed that the area stained by SR was significantly decreased in IFP treated with LIPUS for 2 weeks after intra-articular injection of carrageenan (Car + LIPUS group) compared with that of IFP without LIPUS after injection (Car group) (inside the square box of left panels in Fig. 3A and B), indicating that synovial fibrosis was attenuated by LIPUS intervention. The HIF-1α mRNA levels were significantly lower in the Car + LIPUS group in both 1 and 2-week time points compared with that in the Car group (Fig. 4A). The HIF-1α binding activity was lower in the Car + LIPUS group compared with the Car group 1 week after intra-articular injection of carrageenan (Car group, 0.531±0.012; Car + LIPUS group, 0.513±0.011; P<0.05). In addition, intervention with LIPUS resulted in the inhibition of the expression of CTGF and VEGF 1 and 2 weeks after injection of carrageenan ( Fig. 4B and C).

Discussion
The IFP in patients with KOA has been reported to show hypo-intensity on MRI scans, which is indicative of fibrosis (35,36), and fibrotic IFP has been hypothesized to be correlated with the symptoms of KOA and articular cartilage damage (36). Fibrosis of IFP has a major impact on the pathogenesis of KOA and controlling the fibrosis of IFP may be a novel avenue for the management of KOA, in addition to conventional drug-based and physical therapy.
Pathological fibrosis induced by inflammation is widely observed in various diseases such as liver cirrhosis, nephrosclerosis, and pulmonary fibrosis and is caused by the activation of myofibroblasts, which play a major role in tissue repair and induce the production and accumulation of extracellular matrix components such as collagen (16). TGF-β and various inflammatory cytokines secreted by innate immune cells such as infiltrated macrophages are considered  .05, ** P<0.01 vs. saline. n=5 per group. Data are presented as the mean ± SEM. CTGF, connective tissue growth factor; VEGF, vascular endothelial growth factor; Saline, a rat knee injected only with saline; Car 1 week, a rat knee injected 1 week after intra-articular injection of carrageenan; Car 2 week, a rat knee injected 2 weeks after intra-articular injection of carrageenan.
to activate myofibroblasts and promote the production of collagen (37). The same mechanisms occur in the IFP, which include inflammatory cell infiltration followed by fibrosis, in carrageenan-induced animal models of KOA, and in other OA-induced models using ACLT or monoiodoacetate (38)(39)(40)(41). Furthermore, it has been demonstrated that macrophages migrate into hypoxic tissues to secrete inflammatory cytokines (42), and HIF-1α, which is a transcription factor that is activated in response to hypoxic conditions, is intricately involved in macrophage migration and its functions (43). These findings suggest that a hypoxic environment that induces macrophage infiltration via activation of HIF-1α may contribute to fibrosis in IFP. In the present study, significant macrophage infiltration was observed in the IFP of the rat KOA model immediately after carrageenan administration, suggesting the presence of a hypoxic environment in the IFP (Fig. 1A). In addition, the results demonstrated that the expression and activity of HIF-1α increased after 1 week in the IFP.
Importantly, pathological fibrosis is primarily caused by fibrosis-associated growth factors such as CTGF and VEGF, whose expression is regulated by the transactivation of HIF-1α (32-34). Sotobayashi et al (21) demonstrated that HIF-1α activation induced by joint immobilization promoted the expression of CTGF and VEGF, resulting in the development of synovial tissue fibrosis that leads to joint contracture, followed by immobilization and disuse. In addition, they also provided important results indicating that inhibition of HIF-1α activity by decoy oligonucleotides attenuated fibrosis of synovial tissue through suppression of the expression of CTGF and VEGF. Yabe et al (44) showed that the expression of HIF-1 was increased in a joint contracture animal model. These results strongly suggest that the increased expression and activation of HIF-1α shown in the present study are involved in the mechanisms of fibrosis in IFP through upregulation of CTGF and VEGF. In fact, the present study demonstrated not only increased gene expression and activation of HIF-1α, but also induced expression of CTGF and VEGF, followed by accumulation of type I collagen. Therefore, it is hypothesized that HIF-1α is involved in the fibrosis of IFP in KOA and that regulation of HIF-1α may serve as a novel therapeutic strategy for the management of KOA.
Interestingly, in this study, LIPUS was applied to decrease the expression of HIF-1α based on our previous study (23), and intervention with LIPUS inhibited the gene expression levels of fibrosis-related factors such as CTGF and VEGF, which were suppressed through attenuation of both gene expression and HIF-1α activation. These results are also supported by recent studies reporting similar findings regarding the effect of LIPUS on the activation of HIF-1α and its detailed mechanisms (23). miR-31-5p, which regulates the cytoskeleton, was identified as a mechanosensitive miRNA following LIPUS stimulation, and LIPUS prevented long-term hypoxia-induced myocardial fibrosis by regulating the HIF-1α/DNA methyltransferase 3α signaling pathway through the mechanosensitive protein TRAAK (45,46). These reports suggest that the unique stimulation of microscopic vibrations by LIPUS directly affects the regulation of transcriptional activation of HIF-1α via induction of mechanosensitive molecules. Moreover, LIPUS treatment successfully decreased the fibrotic lesions in the IFP in the rat model of KOA, suggesting that LIPUS is an effective device to regulate HIF-1α and attenuate the fibrotic process of IFP in KOA. In clinical settings, it has been reported that LIPUS treatment for KOA relieves joint pain, joint swelling, and a reduction in the limitation of joint range of motion (47), and our findings suggest that this phenomenon is mediated by the antifibrotic effects of LIPUS via the regulation of CTGF, VEGF, and HIF-1α in the IFP.
The present study aimed to clarify the involvement of HIF-1α in the development of fibrosis of the IFP in KOA. The IFP was harvested to investigate gene expression levels and the transcriptional activity of HIF-1α and related molecules.
Although the present study demonstrated that HIF-1α participated in the development of fibrosis in the IFP in KOA, there are several limitations. One of these is the technical limitation on the collection of IFP samples. The synovial tissue could not be completely removed from the IFP because of the continuous histological connection of the IFP with its surrounding tissues. Therefore, the gene expression and transcriptional activity may include some synovial tissue responses. Additionally, although the involvement of HIF-1α in the development of fibrosis of IFP was investigated, degeneration of the articular cartilage, another major factor in the pathogenesis of KOA, was not fully evaluated in this study. In addition, the effects of LIPUS on HIF-1α activation and degeneration of articular cartilage should be investigated in the future. Mechanical stress has also been suggested to be involved in the pathogenesis of KOA. However, given that carrageenan was used to induce synovitis in this study, the relationship between HIF-1α and fibrosis of IFP in OA models due to joint instability, such as destabilization of the medial meniscus or resection of the ligament, requires further investigation. Finally, the effect of LIPUS on fibrosis of IFP was evaluated only 2 weeks after LIPUS intervention; lack of data at 1 week is a limitation of the present study. Figure 4. Effect of LIPUS on the mRNA expression of HIF-1α, CTGF, and VEGF in the IFP of a rat knee. Relative mRNA expressions levels of (A) CTGF, (B) VEGF, (C) HIF-1α. Car 1 week, 2 weeks, a rat knee injected at 1 or 2 weeks after intra-articular carrageenan injection. * P<0.05, ** P<0.01 vs. saline; † P<0.05 vs. Car 1 week; # P<0.05 vs. Car 2 weeks. Data are presented as the mean ± SEM. LIPUS, low-intensity pulsed ultrasound; IFP, infrapatellar fat pad; CTGF, connective tissue growth factor; VEGF, vascular endothelial growth factor; Saline, a rat knee injected with saline only; Car 1 week, a rat knee injected 1 week after intra-articular injection of carrageenan; Car 2 week, a rat knee injected 2 weeks after intra-articular injection of carrageenan; Car + LIPUS 2 weeks, Car + LIPUS 1 week, a rat knee treated with LIPUS 1 week after injection of carrageenan; Car + LIPUS 2 weeks, Car + LIPUS 2 weeks, a rat knee treated with LIPUS 2 weeks after injection of carrageenan.
In conclusion, the present study using a rat animal model of KOA demonstrated that activation of HIF-1α, which is considered to be induced by hypoxic conditions in the IFP, promoted fibrosis of IFP, followed by upregulation of fibrosis-related molecules, CTGF and VEGF. Notably, intervention with LIPUS resulted in attenuation of fibrotic changes in the IFP through reduction of HIF-1α activity. Our findings reveal a novel therapeutic strategy for the treatment of KOA, focusing on HIF-1α.