Experimental Cell Research Mechanical strain attenuates cytokine-induced ADAMTS9 expression via transient receptor potential vanilloid type 1

The synovial ﬂ uids of patients with osteoarthritis (OA) contain elevated levels of in ﬂ ammatory cytokines, which induce the expression of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) and of the matrix metalloproteinase (MMP) in chondrocytes. Mechanical strain has varying e ﬀ ects on organisms depending on the strength, cycle, and duration of the stressor; however, it is unclear under in ﬂ ammatory stimulation how mechanical strain act on. Here, we show that mechanical strain attenuates in ﬂ ammatory cytokine-induced ex- pression of matrix-degrading enzymes. Cyclic tensile strain (CTS), as a mechanical stressor, attenuated interleukin (IL)-1 β and tumor necrosis factor (TNF)- α -induced mRNA expression of ADAMTS4 , ADAMTS9 , and MMP- 13 in normal chondrocytes (NHAC-kn) and in a chondrocytic cell line (OUMS-27). This e ﬀ ect was abolished by treating cells with mechano-gated channel inhibitors, such as gadolinium, transient receptor potential (TRP) family inhibitor, ruthenium red, and with pharmacological and small interfering RNA-mediated TRPV1 inhibition. Furthermore, nuclear factor κ B (NF- κ B) translocation from the cytoplasm to the nucleus resulting from cytokine stimulation was also abolished by CTS. These ﬁ ndings suggest that mechanosensors such as the TRPV protein are potential therapeutic targets in treating OA.


Introduction
Osteoarthritis (OA) is the most common chronic disorder affecting the joints and constitutes a major burden on public health. It is believed that the articular cartilage-which consists of chondrocytes and extracellular matrix (ECM) molecules including aggrecan, collagen, and hyaluronan (HA) [1,2]-in patients with OA is destroyed by the aberrant up-regulation of matrix-degrading proteinases; however, it has been demonstrated that the synovial fluid contains elevated levels of inflammatory cytokines [3] that induce expression and activity of matrix metalloproteinase (MMP), a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) [4][5][6][7]. ADAMTS4, ADAMTS5, and ADAMTS9 cleave aggrecan, which holds water via its charged chondroitin sulfate and keratan sulfate chains; thereby conferring physical strength and stress resistance to cartilage [8]. In cartilage affected by OA, aggrecan and collagen are degraded [5]. We recently reported that inflammatory cytokines induce the expression of ADAMTS9 to a greater extent than that of ADAMTS4 and of ADAMTS5 in chondrocytes [6], suggesting that ADAMTS9 plays an important role in developing OA. We also found that HA treatment decreased ADAMTS9 levels in human and rat chondrocytes, it protected articular cartilage in a rat model with OA by down-regulating aggrecanase [6].
Mechanical strain can induce the expression of ECM proteins including collagen and aggrecan; however, excessive amounts of mechanical stress can actually stimulate proteases in chondrocytes instead [9][10][11]. Various molecules, including integrins, function as sensors that transduce mechanical stress [12]. Integrins are transmembrane glycoproteins composed of α and β subunits, each with an extracellular domain that directly binds ECM proteins such as collagen, fibronectin, and vitronectin [13], as well as with a cytoplasmic domain that interacts with the cytoskeleton via focal adhesion molecules. Transient receptor potential (TRP) channels were first identified in Drosophila as sixtransmembrane proteins that function in phototransduction [14]. The TRP family is subdivided into seven subfamilies: TRPC, TRPV, TRPM, TRPP, TRPML, TRPA, and TRPN; these channels are expressed in a variety of tissues and cell types and are mostly permeable to calcium, some also presumably to act as mechanosensors [15,16].
Currently, it is unknown whether mechanical strain can suppress or attenuate inflammatory cytokine-induced cellular responses. To address these questions, the present study investigated changes in gene expression in chondrocytes subjected to uniaxial cyclic stretching. Our results show that mechanical strain attenuates inflammatory cytokineinduced expression of matrix-degrading enzymes. Moreover, we determined that calcium channels function as mechanosensors and that mechanical strain inhibits the release of cytokine-induced calcium in chondrocytes; furthermore, it also blocks translocation of nuclear NF-κB from the cytoplasm to the nucleus induced by cytokine stimulation. These findings can provide a molecular basis for developing novel strategies for regenerating cartilage and treating OA.

Cells and cell culture
Normal human articular chondrocytes from knee cells (NHAC-kn) were purchased from Lonza (Walkersville, MD, USA) and cultured at 37°C in chondrocyte basal medium (Lonza) containing chondrocyte growth medium, fetal bovine serum (FBS), transforming growth factor β, R 3 insulin-like growth factor, transferrin, insulin, gentamicin, and amphotericin-B (CDM Bullet Kit; Lonza) as previously described [6]. The medium was changed every 3 days, only cells at passages 3-6 were used for all experiments.
OUMS-27 chondrosarcoma cells were prepared as previously described [6,17,18]. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS and penicillin/streptomycin at 37°C in a humidified atmosphere with 5% CO 2 . Cells were passaged to cell ratio of 1:2 or 1:4 using trypsin with ethylenediaminetetraacetic acid every 7-10 days, with medium replacement every 3 days. Only cells at passages 7-12 were used for experiments; 2.5 × 10 5 cells were seeded in 6-well plates for 2 days, and the medium was replaced with serum-free medium for 24 h before cytokine stimulation, after which the cells were cultured in the presence of IL-1β and TNF-α (both at 10 ng/mL).

Mechanical stimulation
Cells (1.5 × 10 5 ) were seeded in a stretch chamber coated with 0.1 mg/mL collagen and cultured for 2 days, then transferred to serumfree DMEM for 24 h. Cells were exposed to cycles of uniaxial stretching in the presence of IL-1β and TNF-α (both at 10 ng/mL) using a ShellPa mechanical stretch system (Menicon Life Science, Aichi, Japan) (n = 6 each), which allowed for uniform stretching of the entire silicone membrane.

Real-time quantitative reverse transcription PCR (qRT)-PCR
Following cytokine stimulation, cells were washed twice with phosphate-buffered saline (PBS) and total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions, and reverse transcribed into cDNA as previously described [19][20][21]. Briefly, genomic DNA was removed by treatment with 5 U DNase I (Roche Diagnostics, Lewes, UK) at 37°C for 15 min, followed by enzyme inactivation at 65°C for 10 min; 2 μg total RNA were reverse transcribed with random primers (Toyobo, Osaka, Japan). qRT-PCR was carried out on a StepOnePlus system (Applied Biosystems, Foster City, CA, USA) as previously reported [22][23][24], with slight modifications. Briefly, each reaction mixture contained 5 μl TaqMan Fast Advanced Master mix, 0.5 μl TaqMan Gene Expression assay for the target genes (ADAMTS4, ADAMTS9, MMP-13, TRPV1, TRPV2, and TRPV4) and the endogenous control (glyceraldehyde 3phosphate dehydrogenase; GAPDH), and 4 μl cDNA. Cycling conditions were as follows: 95°C for 20 s; and 40 cycles at 95°C for 1 s and 60°C for 20 s. All samples were analyzed in triplicate. TaqMan  as well as the TaqMan Fast Advanced Master Mix were purchased from Applied Biosystems. GAPDH was used to normalize the levels of target RNAs with the comparative Ct (ΔΔCT) method as previously described [25][26][27]. Values obtained from the untreated cells served as the control.

Evaluation of NF-κB translocation
Cells (1.5 × 10 5 ) were seeded in a stretch chamber coated with 0.1 mg/mL collagen and cultured for 2 days before being transferred to serum-free DMEM for 24 h. After treatment with IL-1β and TNF-α (both at 10 ng/mL), with or without 0.5-Hz cycles of uniaxial stretching (n = 6 each), the cells were treated with cold methanol for 30 min, followed by cold acetone treatment for 10 min for fixation with permeabilization. After washing the cells in PBS, samples were blocked with 3% bovine serum albumin/PBS for 2 h, washed in PBS, and incubated overnight at 4°C with anti-NF-κB p65 antibody (Santa Cruz Biotechnology, Dallas, TX, USA; 1:500, sc-372). After PBS washes, cells were incubated for 1 h at 20°C with Alexa 488-conjugated anti-rabbit secondary antibody (Invitrogen), then washed in PBS. The nuclei were stained with Hoechst 33,258 (1:5000) and the samples were mounted with coverslips and stored in the dark at 4°C. Images were obtained with a microscope (BZ-X700; KEYENCE).

BAPTA-AM treatment
Cells (2.5 × 10 5 ) were seeded in a 6-well plate and cultured for 2 days, then transferred to serum-free DMEM for 24 h. The cells were treated IL-1β and TNF-α (both at 10 ng/mL) for 6 h with or without a 1 h pretreatment with 30 μM BAPTA-AM. ADAMTS9 transcript levels were evaluated relative to levels in unstimulated cells by qRT-PCR.

Statistical analysis
Data are expressed as the mean ± SD. Differences among groups were evaluated by analysis of variance followed by Bonferroni's test. P values < 0.05 were considered statistically significant.

Mechanical strain attenuates inflammatory cytokine-induced expression of ADAMTS and MMP in OUMS-27 cells
First, we examined whether mechanical strain attenuates expression of ADAMTS4, ADAMTS9, and MMP13 induced by IL-1β and TNF-α in OUMS-27 cells. Application of 5% CTS at a frequency of 0.5 Hz resulted in the downregulation of ADAMTS4, ADAMTS9, and MMP13 transcript levels in OUMS-27 cells stimulated with cytokines ( Fig. 1).

Mechanical strain attenuates inflammatory cytokine-induced expression of ADAMTS9 in human chondrocytes
We examined whether mechanical strain attenuates expression of ADAMTS9 mRNA induced by IL-1β and TNF-α in chondrocytes from non-OA patients. As expected, treatment of NHAC-kn cells with 5% tensile strain at 0.5-Hz frequency reduced cytokine-induced expression ADAMTS9 mRNA in these cells (Fig. 2).

Mechanical strain conditions
We examined the effects of different mechanical strain conditions on expression ADAMTS9 mRNA induced by cytokines in OUMS-27 cells. The levels of ADAMTS9 mRNA in cells treated with IL-1β and TNF-α decreased as the frequency increased (0.16, 0.33, and 0.5 Hz at 5% tensile strain; Fig. 3A) and tensile strain (2.0%, 3.0%, and 5.0% at 0.5-Hz frequency; Fig. 3B).

Pretreatment with integrin binding motif peptide does not influence response to mechanical strain
Previous studies have shown that integrin functions as a mechanosensor [10]. Integrin binds to the ECM and associates with cytoskeletal actin filaments via linker proteins to physically connect intra-and extracellular structures. We found here that pretreatment with the integrin binding motif sequence GRGDS and its analog GRDSP did not alter expression of ADAMTS9 mRNA in OUMS-27 cells (Fig. 4), indicating that integrin binding is unlikely involved in the cellular response to mechanical strain.

Mechanosensor inhibitors abrogate the impact of mechanical strain
Pretreatment with gadolinium (a broad-spectrum mechanosensor inhibitor) reversed the negative effects of mechanical strain on cytokine-induced ADAMTS9 upregulation (Fig. 5A), whereas gadolinium on its own had no effect on the expression of ADAMTS9 mRNA. Similar effects were observed with ruthenium red (TRPV family inhibitor) (Fig. 5B) and capsazepine (TRPV1 inhibitor) pretreatment. On the other hand, tranilast (TRPV2 inhibitor) and HC-067047 (TRPV4 inhibitor) pretreatment did not downregulate strain-induced expression of ADAMTS9 mRNA in cytokine-treated cells (Fig. 5B).

TRPV silencing abolishes the effects of mechanical strain
To identify which TRPV family molecules mediate the effects of mechanical strain, we knocked down the expression of TRPV1, TRPV2, and TRPV4. Each of the siRNAs-but the not the scrambled control siRNAs-reduced the level of its target mRNA (Fig. 6B). Pretreatment with siRNAs (V1-1 and V1-2) against TRPV1 abolished the impact of mechanical strain in OUMS-27 cells (Fig. 6A); however, pretreatment with siRNAs against TRPV2 (V2-1, V2-3, and V2-3) and TRPV4 (V4-1, V4-2, and V4-3) did not alter the effects of mechanical strain (Fig. 6A). None of the siRNAs or the scrambled control RNAs used in this experiment affected cell viability or expression of ADAMTS9 mRNA (data Fig. 2. CTS attenuates cytokine-induced expression of ADAMTS9 mRNA in NHAC-kn cells. NHAC-kn cells were subjected to 5% tensile strain at a frequency of 0.5 Hz and treated with 10 ng/mL IL-1β and TNF-α for 6 h. The number ADAMTS9 transcripts was measured relative to the levels of mRNA found in unstimulated the control cells by qRT-PCR. Values represent mean ± SD (n = 6 per group). ## P < 0.01 vs. control; **P < 0.01 vs. cytokine-treated group.  Integrin binding motif peptide does not inhibit the effects of mechanical strain. Cells were pretreated with the integrin-binding peptide GRGDS (5 μg/ mL) or its analog GRADSP (5 μg/mL) for 1 h and then cultured in the presence of IL-1β and TNF-α (both at 10 ng/mL) with cyclic tensile strain (5%, 0.5 Hz) for 6 h. The number of ADAMTS9 transcripts was measured relative to the levels of mRNA found in unstimulated the control cells by qRT-PCR. Values represent mean ± SD (n = 6 per group). ## P < 0.01 vs. control; **P < 0.01 vs. cytokine-treated group.

Effects of inflammatory cytokines on intracellular calcium concentration in OUMS-27 cells
We examined whether inflammatory cytokines can influence intracellular calcium concentrations in OUMS-27 cells using Fluo-4 AM (Fig. 7). Treatment with IL-1β and TNF-α (both at 10 ng/mL) increased intracellular calcium content after 10 and 30 min of cytokine stimulation; however, after 60 min, the concentration declined.

BAPTA-AM suppresses expression of ADAMTS9 in OUMS-27 cells
We examined whether NF-κB inhibition by BAPTA-AM alters the expression of ADAMTS9 mRNA and found that BAPTA-AM pretreatment decreased cytokine-induced expression of ADAMTS9 after 6 h (Fig. 8).

Mechanical strain blocks cytokine-induced NF-κB nuclear translocation in OUMS-27 cells
We explored the intracellular mechanisms of mechanical strain by analyzing nuclear translocation of NF-κB-a key transcription factor involved in signal transduction of inflammatory cytokines-by immunofluorescence analysis. Rapid nuclear translocation of NF-κB was observed within 10 min of IL-1β and TNF-α treatment (Fig. 9); on the other hand, NF-κB was retained in the cytoplasm under mechanical strain for 6 h with IL-1β and TNF-α treatment (data not shown). In contrast, cells treated simultaneously with mechanical strain and cytokines exhibited strong suppression of IL-1β-and TNF-α-induced NF-κB nuclear translocation (Fig. 9).

Mechanical strain inhibits the cytokine-induced calcium increase in OUMS-27 cells
We investigated intracellular calcium kinetics by immunofluorescence analysis. Rapid calcium upregulation was observed within 10 min of adding IL-1β and TNF-α (Fig. 10). In contrast, cells subjected to CTS with simultaneous cytokine treatment showed strong suppression of the IL-1β-and TNF-α-induced increase in calcium content (Fig. 10). CST alone caused a slight elevation in intracellular calcium.

Discussion
In this study, we examined the effects of mechanical strain on inflammatory cytokine-induced expression of ADAMTS9 mRNA and the underlying mechanisms in chondrocytes. We found that intracellular calcium and translocation of the NF-κB nuclear factor increased in the presence of cytokines, whereas mechanical strain reversed this effect via TRPV1, resulting in downregulation of ADAMTS9.
Cultured chondrocytes have been exposed to CTS stimulation at a wide range of strain magnitudes and durations with various systems [32][33][34]. Xu et al. (2000) reported that CTS acts as an antagonist of IL-1β actions in chondrocytes [32]. Although the mechanism has not yet been elucidated, they reported that CTS attenuates the induction of IL-1β-induced inflammatory responses. Interestingly, CTS had lower effect on inflammatory signals when inflammatory cytokines were not associated with it. Hayashi et al. (2015) reported that p21 plays a role in the expression of MMP13 mRNA in response to CTS [10]. CTS increased p21 expression and this effect was mediated by signal transducer and activator of transcription 3 (STAT3). Recently, Lohberger et al. (2019) reported that moderate tensile strain most effectively reduces ADAMTS5 and MMP13 expression levels in chondrocytes compared to strong tensile strain [33]. They also demonstrated that moderated tensile strain significantly decreases the expression of IL-6, a notable inflammatory marker. Their results suggested that different loading conditions cause different effects on the expression of inflammatory markers and MMPs/ADAMTSs in chondrocytes. Recently, Papadopoulou et al. (2019) reported that CTS immediately activates extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) in human periodontal ligament fibroblasts [34]. They also demonstrated that CTS induces c-fos activation. These accumulating evidences led to the hypothesis that CTS attenuates IL-1β-induced ADAMTS9 expression. Notably, the effect of CTS is altered depending on its conditions such as magnitudes, durations, and frequency. Our data suggests that CTS (0.16-0.50 Hz with 5% elongation or 0.5 Hz with 2.0%-5.0% elongation) reduces levels of ADAMTS9 mRNA in cytokinestimulated chondrocytes (Fig. 3B). The duration of cytokine exposure and mechanical strain treatment was an important factor; the degree of attenuation was reduced by a shorter duration of mechanical strain (data not shown). Based on these data, we considered that the CTS condition used in the present study demonstrated the antagonistic effects in IL-1β stimulated chondrocytes via TRPV.
Previous in vitro and in vivo studies revealed that there is a stronger expression of ADAMTS9 in chondrocytes than ADAMTS4 and ADAMTS5, indicating that it is more likely involved in articular cartilage degradation in OA. We have previously reported that nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent 1 (NFATc1) regulates expression of ADAMTS9 in chondrocytes via IL-1β, and that a treatment with the NFATc1-specific inhibitor reduces levels of ADAMTS9 [35], implying that expression of ADAMTS9 is regulated by different mechanisms. It is well known that NF-κB is widely used as transcription factor for matrix degradative molecules (ADAMTS4, 5,9,18,2,3,8,9,13) under inflammatory stimulation [36]. It appears that NF-κB regulation is important for OA therapy. Yang et al. (2019) reported that CTS alleviates the chondrocyte damage induced by IL-1β by activating AMP-activated protein kinase (AMPK) phosphorylation and suppressing nuclear translocation of NF-κB [37]. They proposed that moderate AMPK, activated by moderated CTS, reduces intracellular ROS production, which inhibits nuclear translocation of NF-κB. Their data were consistent with our results; therefore, a similar underlying mechanism is suggested. In order to analyze the molecular mechanism of OA, we measured the effects of mechanical strain on expression of ADAMTS9 using NHAC-kn and OUMS-27 cell lines, which exhibit particular chondrocyte properties such as expression of the chondrocyte-specific ECM genes type II, IX, and XI collagen and aggrecan; moreover, treatment with IL-1β and TNF-α induced expression of ADAMTS4 and ADAMTS9 after 6 h in both cell lines, and with similar kinetics [6,17]. Aggrecan cleavage by ADAMTS was also observed in OUMS-27 cells (data not shown), suggesting that these cells can serve as a model for investigating the effects of cytokines on expression of ADAMTS.
Chondrocytes are surrounded by ECM. Integrins are transmembrane receptors that facilitate cell-ECM adhesion by binding specific integrin subtypes to activate signal transduction [11]. There is evidence that integrins function as mechanosensors [38]. CTS increased expression of collagen (COL1A1 and COL3A1) via integrin αVβ3, which binds to the Arg-Gly-Asp (RGD) motif [11]. Integrins transduce mechanical signals in annulus fibrosus cells, whereas the RGD peptide suppresses phosphorylation of focal adhesion kinase induced by severe CTS (10%, Fig. 7. Cytokine treatment increases intracellular calcium levels. OUMS-27 cells were seeded in glass-bottomed dishes for 48 h. After culturing cells overnight serum starvation, the cells were treated for 1 h with 5 μM Fluo-4 AM to visualize intracellular Calcium (green), followed by IL-1β and TNF-α (both at 10 ng/mL) for 10, 30, and 60 min. Cells were fixed by placing them in cold methanol for 30 min and in cold acetone for 10 min. After washing, the nuclei were stained with Hoechst 33258 (blue). 1.0 Hz) [39]. Mechanical strain also induced hyperpolarization of the membrane in primary chondrocytes via α5β1 integrin, but could be reversed by RGD peptide [40]. On the other hand, we found that the RGD peptide did not attenuate cytokine-induced expression of ADAMTS9 in OUMS-27 cells, implying that this integrin does not participate in this mechanism. On the other hand, pretreatment with gadolinium, which inhibits several types of Ca + channels [41], abrogated the impact of mechanical strain on expression of ADAMTS9 mRNA, implying that calcium signaling is involved. We, therefore, focused on the function of calcium channels as possible mechanosensors using a variety of TRPV-specific inhibitors [42][43][44][45][46], and demonstrated that pretreatment with ruthenium red, a well-known TRPV family inhibitor [47], as well as capsazepine, a TRPV1 inhibitor, abolished the effects of mechanical stress. However, tranilast and HC-067047 (inhibitors of TRPV2 and TRPV4, respectively) did not have this effect. TRPV4 is highly expressed in both bone and cartilage [48,49]. Similarly, knocking down TRPV1 abrogated the effect of mechanical strain, whereas silencing other TRPV types (TRPV2 and TRPV4), on the other hand, did not. Based on these results, we conclude that TRPV1 mediates the effects of mechanical strain on expression of ADAMTS9. Intracellular calcium levels are tightly controlled in cells. In their resting state, cells maintain a 20,000-fold gradient of calcium concentration between the cytosol (100 nM) and the extracellular space (mM) [50]. The rapid increase in calcium in the cytoplasm is achieved through a calcium influx from the extracellular environment or through its release from intracellular calcium stores such as the endoplasmic reticulum (ER). The ER is also a major intracellular calcium reservoir [51]. In chondrocytes, both mechanisms are critical for regulating physical stimulus-induced responses [52]. Even a single cycle of mechanical stress was found to instantly increase cytoplasmic calcium concentrations in OUMS-27 cells (data not shown). Our results clearly demonstrated that CTS decreased intracellular levels of cytokine-induced calcium and blocked cytokine-induced translocation of NF-κB from the cytoplasm to the nucleus in OUMS-27 cells. NF-κB translocation needs several steps; intracellular calcium up-regulation, Calcium/ calmodulin-dependent protein kinase II (CaMKII) activation, IκB Fig. 9. Mechanical strain inhibits translocation of cytokine-induced NF-κB (p65) from cytoplasm to nucleus. OUMS-27 cells were seeded on a collagen-coated stretch chamber. After an overnight serum starvation, the cells were treated with IL-1β and TNF-α (both at 10 ng/mL) for 10 min with or without uniaxial strain (0.5-Hz cycles of stretch). Afterwards, the cells were fixed with cold methanol for 30 min followed by cold acetone for 10 min. After washing and blocking, NF-κB p65 (green) was detected by immunocytochemistry and nuclei were stained with Hoechst 33258 (blue). After an overnight starvation treatment, cells were treated with 5 μM Fluo-4 AM for 1 h to visualize intracellular Calcium (green), followed by IL-1β and TNF-α (both at 10 ng/mL) for 10 min with or without uniaxial strain (0.5-Hz cycles of stretch). The cells were fixed with cold methanol for 30 min, and cold acetone for 10 min. phosphorylation and release from complex, NF-κB delivery to the nuclear membrane via molecular motor and few more steps [53]. Therefore, we propose the following hypothetic model to explain our results: translocation of NF-κB requires high concentrations of calcium for CaMKII activation; cytokines stimulate the release of calcium from the ER but mechanical strain activates TRPV1 in the plasma membrane, resulting in the release of calcium into the culture medium, NF-κB inactivation, attenuation of cytokine induced ADAMTS9 expression.
In conclusion, mechanical strain attenuates cytokine-induced expression of ADAMTS and MMP in chondrocytes; the calcium channel and mechanosensor TRPV1 mediates the effect of mechanical strain on the expression of matrix-degrading enzymes. Our findings provide insight into the regulation of ADAMTS9 mRNA expression in chondrocytes and suggest that mechanosensors such as TRPV can serve as therapeutic targets for treating OA.

Conflicts of interest disclosure
The authors have no conflicts of interest to disclose in relation to this manuscript.