Thrombin induces degradation of murine intervertebral discs via angiogenesis and M1-like polarization of macrophages

doi:10.21203/rs.3.rs-4912446/v1

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Thrombin induces degradation of murine intervertebral discs via angiogenesis and M1-like polarization of macrophages

https://doi.org/10.21203/rs.3.rs-4912446/v1

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With the marked aging of the global population, the prevalence of musculoskeletal disorders owing to low back pain has escalated, with an associated increase in the number of individuals requiring nursing care. To help identify novel therapies for treating disc degeneration, we examined the roles of thrombin and macrophages in intervertebral disc degeneration, a significant cause of low back pain. Experiments using a needle-puncture mouse model of disc degeneration confirmed that this process led to the production of thrombin and MCP-1. Thrombin altered macrophage markers, increasing the population of M1 markers and decreasing that of M2 markers. Therefore, increased thrombin and MCP-1 production may induce disc degeneration by inducing M1-type polarization of migrated macrophages. Disk-tissue M1-macrophage levels were elevated 3 weeks after puncture. Administering SCH79797, a thrombin receptor (PAR1) antagonist, suppressed thrombin-induced disc degeneration and inhibited macrophage migration, M1 polarization, VEGF production, and angiogenesis. These findings suggest the suppression of thrombin function in intervertebral disc inflammation as a novel and promising approach for treating disc degeneration.

Biological sciences/Molecular biology

Biological sciences/Molecular biology/Cell division/Cytokinesis

Disc degeneration

low back pain

thrombin

macrophage polarization

angiogenesis

Low back pain, which is common in the older population and linked to frailty and sarcopenia, hinders movement and well-being. This global health issue affected 619 million people in 2020 and is projected to increase to 843 million by 2050, owing to population growth and aging^{[1, 2]}. Low back pain reduces the quality of life, loss prevents people from working and imposes a significant economic burden ^[3–6]. “Non-specific” low back pain refers to pain not explained by other diagnoses, such as underlying disease, pathology, or tissue damage ^[7]. Risk factors include low physical activity, smoking, obesity, and high physical stress at work ^[8].

Normal intervertebral discs lack blood vessels and consist primarily of a cartilage matrix. Inflammation and sensory nerve intrusion owing to disc degeneration cause low back pain ^[9–13]. Vascular invasion of the fibrotic ring has been observed in degenerated discs, but the inflammatory process in disc degeneration remains unclear ^[14–16].

We previously employed magnetic resonance imaging (MRI) and reported spontaneous regression of herniated discs, with contrast-enhanced MRI revealing more cases of regression ^{[17, 18]}. Examination of surgically excised herniated discs revealed inflammatory cell infiltration, primarily by macrophages. Stimulation of mouse intervertebral discs with inflammatory cytokines increased vascular endothelial growth factor (VEGF) production and promoted angiogenesis in the discs ^[19].

Thrombin (coagulation factor IIa), produced from prothrombin by factor Xa, plays a central role in blood coagulation and is closely associated with inflammatory and immune responses ^{[20, 21]}. In cultured murine intervertebral discs, thrombin administration induces matrix metalloproteinase 3 (MMP3) production and disc degeneration ^[22]. In human primary nucleus pulposus culture, thrombin induces epidermal growth factor receptor expression and disc degeneration ^[23].

Of the two macrophage subtypes, M1 macrophages are induced during tissue damage and inflammation, whereas M2 macrophages act in tissue repair ^{[24, 25]}. In intervertebral disc degeneration, M1 macrophages initially dominate, switching to M2 dominance over time ^[26], with differences in polarization depending on the degree of degeneration ^[27].

However, the roles of M1 and M2 macrophage infiltration and polarization in intervertebral disc inflammation remain unclear. To address this, we examined the mechanisms whereby thrombin and macrophages induce inflammation. Clarifying these mechanisms may help establish novel therapies for intervertebral disc degeneration.

Thrombin and monocyte chemotactic protein-1 (MCP-1) induction in ex vivo and in vivo disc degeneration models

To elucidate the mechanisms of disc inflammation, we compared the ex vivo model with the in vivo puncture model. Murine intervertebral discs (mIVD) were cultured in an organ culture system in the presence or absence of 100 nM thrombin for 72 h. Under unstimulated conditions, Safranin-O dye specifically binds to acidic proteoglycans present in the cartilage matrix, forming a reddish-orange complex around the nucleus pulposus and cartilage endplate cells. In the presence of thrombin, mIVD degradation was confirmed by the loss of Safranin-O staining after 72 h of culture (Fig. 1a). Thrombin stimulation increased monocyte chemotactic protein-1 (MCP-1) expression, as confirmed using quantitative real-time PCR (p = 0.043) and ELISA (p < 0.001) [Fig. 1b].

In vivo, in the puncture model, punctured mIVDs exhibited reduced Safranin-O staining, with morphological degeneration over time (Fig. 1c). Immunostaining revealed that thrombin and MCP-1 expression was abundant in punctured mIVDs, especially in the nucleus pulposus and cartilage endplate (Fig. 1d, e). Disc degeneration and thrombin and MCP-1 production were therefore observed both ex vivo and in vivo.

Thrombin promotes M1 macrophage polarization

Next, we examined the effects of thrombin on MCP-1-induced macrophages. PCR revealed that thrombin stimulation of mouse bone marrow macrophages increased the levels of CD86, a common M1 marker, and reduced those of CD163, an M2 marker (Fig. 2a). Western blotting revealed that thrombin stimulation increased the expression of both M1 markers, CD86 and iNOS, and reduced that of M2 markers, CD163 and arginase (Fig. 2b; Supplementary Fig. S1). These results indicate that thrombin induced M1-type polarization.

In vivo induction of M1 macrophages in the puncture model

We confirmed macrophage polarization in vivo by examining mIVDs in the puncture model. At 3 weeks after treatment, mIVDs were collected for immunostaining and purified, and RNA and protein levels were quantified. Based on immunostaining, the expression of Iba1, a macrophage marker, was higher in the punctured discs than in the control discs. Staining for the M1 marker iNOS was stronger in the model mIVDs than in the control, whereas staining for the M2 marker arginase was negligible in the model mIVDs (Fig. 3a; Supplementary Fig. S2). The model exhibited significantly higher CD86 expression (p = 0.0040) and lower CD163 expression (p = 0.0013) than those of the control, based on western blotting (Fig. 3b, c). These findings suggest that macrophages infiltrating the puncture model in vivo were predominantly M1 macrophages.

PAR1 antagonist SCH79797 suppresses thrombin-induced mIVD degeneration

We hypothesized that, in the puncture model, thrombin induces M1-type macrophages, causing disc degeneration. We therefore examined whether this degeneration could be suppressed by inhibiting proteinase-activated receptor 1 (PAR1), the major receptor for thrombin. In the model, safranin staining of chondrocytes in the nucleus pulposus and endplates declined significantly over time, whereas this did not occur in the healthy control (Fig. 4a; Supplementary Fig. S3). A disc degeneration score was generated ^[28]. At 3 weeks after puncture, the group treated with SCH79797, a PAR1 antagonist, showed significantly less disc degeneration than that of the vehicle group (Fig. 4b; Supplementary Fig. S3).

PAR1 antagonist inhibits thrombin production and macrophage M1 polarization

Thrombin production and macrophage polarization were examined to elucidate the mechanisms by which the PAR1 antagonist suppresses disc degeneration. At 3 weeks after puncture, the mIVDs were removed and immunostained. Thrombin expression was higher in punctured discs than in the controls but was inhibited by the PAR1 antagonist. Levels of thrombin and that of CD86 and iNOS (M1 markers) were significantly elevated in punctured discs and were suppressed by the PAR1 antagonist. In contrast, levels of CD163 and arginase (M2 markers) were slightly lower in the punctured discs than in the healthy control and were not significantly altered by the PAR1 antagonist (Fig. 5).

Thrombin induces VEGF production and angiogenesis

Finally, VEGF production and angiogenesis, which are important factors in disc degeneration, were investigated in the ex vivo and in vivo puncture models. Ex vivo, mIVD cells from the control and thrombin-treated groups, with or without PAR1-antagonist treatment, were incubated in a disc culture system for 72 h to generate intervertebral disc RNA, and the supernatant was collected. qPCR analysis revealed that thrombin increased the levels of VEGF RNA in the mIVD, whereas inhibition of PAR1 suppressed this (Fig. 6a). Quantification of VEGF in the culture supernatant using ELISA generated similar results (Fig. 6b).

At 3 weeks after puncture treatment, the mIVDs were removed and immunostained. VEGF expression was higher in punctured discs than that in the healthy controls, but this increase was abrogated by PAR1-antagonist administration. Relative to the controls, the punctured discs exhibited stronger staining for VEGF and CD31, a platelet-endothelial cell-adhesion molecule, around the nucleus pulposus, whereas this was abrogated by the PAR1 antagonist (Supplementary Fig. S4).

Intervertebral disc degeneration involves multiple processes, including changes in gene expression, stress responses, cellular senescence, and blood-supply-related nutritional status. To further elucidate these processes, we examined the roles of thrombin and macrophage polarization in the pathogenesis of intervertebral disc degeneration. In vivo, we confirmed thrombin and MCP-1 production in the puncture model, consistent with our prior ex vivo findings ^{[22, 29]}. The puncture model exhibited a significantly higher proportion of M1-type macrophages and VEGF and CD31 production than those of the healthy control. Administration of SCH79797, a PAR1 antagonist, reduced disc degeneration by suppressing these processes. These results suggest that thrombin causes inflammatory M1-type macrophage polarization during disc degeneration and that PAR1 antagonists may be potential therapeutic candidates.

Needle puncture, a common method for inducing intervertebral disc degeneration, was used to examine both thrombin expression in the injured discs and the effects of thrombin on macrophages. These findings reveal that thrombin acts on the intervertebral disc, inducing the expression of MCP-1 (a chemotactic factor for macrophages), VEGF, and angiogenesis; this suggests that thrombin creates an environment supporting macrophage infiltration into the disc tissue. In the presence of thrombin, M1 macrophage marker expression was significantly elevated, and these markers were strongly expressed in the puncture model. M1 macrophages, which exert bactericidal effects by producing proinflammatory cytokines, are induced by IFN-γ, lipopolysaccharides LPS, or TNF-α, and exhibit overexpression of CD80, CD86, and iNOS ^[30]. In contrast, M2 macrophages, which play important roles in tissue repair, angiogenesis, and metabolism, are induced by anti-inflammatory factors such as IL-4, IL-10, IL-13, TGF-β, and glucocorticoids, and exhibit overexpression of Arg-1, CD163, and CD206 ^{[31, 32]}. Here, we examined the polarization of thrombin-polarizing naive macrophages to the M1 type; while such findings are valuable in many fields, these have been rarely reported in the context of thrombin mediated disc degeneration ^[33].

The puncture model exhibited the production of thrombin and MCP-1, disc degeneration, macrophage migration to the degenerated disc, and abundant M1 macrophage production. To clarify whether these events were inflammatory mechanisms caused by thrombin or were independent events, we tested the effects of SCH79797, a PAR1 antagonist, in the puncture model. SCH79797 administration abrogated the enhanced disc degeneration, M1 macrophage polarization, and the migration of both M1 and M2 types. This suggests that, following disc puncture, thrombin production causes inflammation, leading to macrophage migration and polarization, causing disc degeneration.

PAR1 participates centrally in thrombin-induced platelet aggregation. In a prior study, PAR1-knockout mice did not exhibit spontaneous bleeding; a PAR1-antagonist was established as an antiplatelet agent to reduce bleeding ^[34–36]. This PAR1-antagonist, approved by the FDA in 2014 as vorapaxar and in clinical use for 10 years, reduces the risk of heart attack, stroke, and death due to cardiovascular events ^[37]. It is administered orally, making it relatively safe and easy to take ^[38]. The incidence of disc degeneration, myocardial infarction, stroke, and cardiovascular death is very high in individuals older than 50 years of age ^[39]. Considering that the age range of clinical morbidity for these vorapaxar-indicated conditions is close to that of patients with lumbar disc degeneration, vorapaxar administration may effectively treat degenerative disc disease, possibly caused by chronic inflammation, in patients with back pain that has persisted for over 3 months without responding to standard analgesic medications.

This study had some limitations. First, when selecting animal models, it is important to choose those whose mechanisms of intervertebral disc degeneration are similar to those in humans; the models must also be able to generate consistent and reproducible outcomes and must be cost-effective and easy to maintain. However, the fact that mice are quadrupedal makes it difficult to subject their intervertebral discs to the same gravitational loading as those of bipedal humans. Therefore, a puncture model was used to artificially induce disc degeneration. Second, our findings suggest that MCP-1 induces macrophage production and that thrombin induces differentiation into the M1-type. Nonetheless, it is possible that the puncture injury directly induced M1 macrophage production. Third, while we found that thrombin and M1 macrophages were induced in mIVDs in the puncture model and that disc degeneration occurred, we did not examine whether the induced macrophages were directly responsible for disc degeneration.

Using an in vivo puncture model experimental system, we found that thrombin plays a vital role as a proinflammatory cytokine in causing intervertebral disc degeneration, consistent with our prior ex vivo model findings ^{[22, 29, 40, 41]}. The thrombin produced in mouse intervertebral discs may promote angiogenesis and migration of macrophages, which then polarize into the inflammatory M1 type. Finally, inhibition of the thrombin receptor PAR1 by a PAR1 antagonist abrogated macrophage polarization to the M1 type and suppressed intervertebral disc degeneration. Future research should focus on translating these findings into clinical trials to evaluate the efficacy and safety of PAR1 antagonists in humans. Additionally, further investigation is needed to elucidate the detailed molecular mechanisms underlying thrombin-induced inflammation and macrophage polarization in disc tissues. Finally, these findings may potentially help improve outcomes for patients with low back pain and enhance the quality of life for patients with degenerative disc diseases.

Animals

All methods were carried out in accordance with relevant guidelines and regulations. All methods are reported in accordance with ARRIVE guidelines (https://arriveguidelines.org), the 2020 AVMA guidelines (https://www.avma.org), and the Proper Conduct of Animal Experiments of the Science Council of Japan (https://www.scj.go.jp/en/animal). The protocols were approved by the Animal Care and Use Committee of the University of Yamanashi (No. A28_38). Homozygous WT C57BL/6J male mice (5–6 weeks old) were purchased from CLEA Japan, Inc. (Tokyo, Japan). The mice were housed at 22–24°C under a 12 h light/dark cycle and were fed standard mouse chow. Water was provided ad libitum. The mice were divided into a model group, where coccygeal intervertebral discs 5–6 and 6–7 were punctured with a 27-gauge needle under anesthesia (puncture group) and a control group without treatment. This method is less invasive and time-consuming than other methods and allows precise control of the extent and location of injury ^{[42, 43]}. All mice were euthanized for histological analysis 1, 2, or 3 weeks after treatment. Anesthesia was performed using the XGI-8 Veterinary Gas Anesthesia System (PerkinElmer, MA, USA). A vaporizer provided the appropriate concentration of isoflurane, and the concentration was introduced at 4–5% and maintained at approximately 2–3%. Euthanasia was performed using a non-prefilled chamber infused with CO₂ from a commercially available cylinder with a fixed pressure regulator and in-line restrictor, with gas flow controlled to 30–70% of the chamber volume per minute, conforming to the ARRIVE 2.0, and 2020 AVMA guidelines ^[44].

Murine intervertebral disc organ culture

The coccygeal murine intervertebral disc (mIVD) organ-culture system was prepared as previously described ^[29]. Briefly, mIVD tissue specimens were obtained from the tail bones of mice using a dissecting microscope after the skin and soft tissues were removed. Whole mIVD tissue specimens were cultured at 37°C in 12-well plates in 1 mL of Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen/Gibco; Thermo Fisher Scientific, MA, USA) containing 0.1% FBS with or without thrombin (100 nM) obtained from Prolytix (Essex Junction, VT, USA), or with or without SCH79797 (a PAR1 antagonist; Santa Cruz Biotechnology, Inc.; TX, USA).

Tissue histological analysis

Histological analysis was performed as previously described ^[45]. The mIVDs were fixed in 4% paraformaldehyde for 3 days, defatted for 3 days, and decalcified with 10% EDTA for 7–21 days. Discs were paraffin-embedded, and consecutive 5 µm sections were stained with hematoxylin and eosin (H&E) or Safranin-O (Merck; Darmstadt, Germany) and fast green (Sigma-Aldrich, St Louis, MO). Immunohistochemical (IHC) staining was then performed for thrombin (sc271449, 1:100, Santa Cruz Biotechnology), MCP-1 (#2029, 1:100, Cell Signaling Technology, Danvers, MA, USA), Iba-1 (013-27691, 1:1000, Fujifilm, Tokyo, Japan), iNOS (MAB9502, 1:50, R&D Systems, Minneapolis, MN, USA), Arginase-1 (#93668, 1:100, Cell Signaling Technology), CD86 (NBP2-25208, 1:250, Novus Biologicals, Centennial, CO, USA), CD163 (ab182422, 1:500, Abcam, Cambridge, MA, USA), VEGF (sc152, 1:50, Santa Cruz Biotechnology, Inc.), CD31 (#77699, 1:100, Cell Signaling Technology), or control IgG, using the Liquid DAB + Substrate Chromogen System (Dako, Carpinteria, CA, USA) according to the manufacturer’s specifications, with counterstaining using hematoxylin.

Quantitative real-time reverse transcription PCR (qRT–PCR)

Quantitative RT–PCR was performed as previously described ^[46]. In brief, mIVD tissues (nine discs, ~ 10 µg in total) were collected, and total RNA was extracted using the RNeasy mini kit and a quality check analysis was performed using NanoDrop (Thermo Fisher Scientific), followed by treatment with DNase (Qiagen, Venlo, Netherlands), according to the manufacturer’s instructions. Complementary DNA was synthesized from 2 µg of total RNA using a Reverse Transcriptase System (Applied Biosystems, Foster City, CA, USA). Quantitative real-time PCR analysis of the cDNA from each mouse was performed using the StepOnePlus Real-Time PCR System (Applied Biosystems) according to the manufacturer’s instructions. The primers and probes for murine MCP-1 (Mm 00441242_m1), murine CD86 (Mm00444540_m1), murine CD163 (Mm00474091_m1), VEGF (Mm00437306_m1), and murine hypoxanthine phosphoribosyltransferase (Mm03024075_m1) were purchased from Applied Biosystems. Relative mRNA levels were quantified using the ΔCt method and normalized to those of HPRT ^[47].

ELISA

mIVDs were cultured in 12-well plates. The conditioned media were collected and prepared by microcentrifugation at 4,000 × g for 5 min, twice. Concentrations of MCP-1 (432704, BioLegend) and VEGF (DY493-05, R&D Systems) in the conditioned media were determined using a Quantikine Colorimetric Sandwich ELISA Kit, according to the manufacturer’s specifications. Absorbance was measured using a microplate reader SH-1100R (Lab; Corona Electric Co., Ltd., Ibaraki, Japan).

Murine macrophage preparation

Bone marrow-derived cells were collected from the femurs of 6-week-old C57BL/6J mice. After removing the soft tissue surrounding the femur, both bone ends were removed. A 23-gauge needle was inserted at one end, and PBS (Life Technologies Japan) was injected to wash out the bone marrow-derived cells. The cells were collected via centrifugation and placed in a 12-well plate. Cells were primary cultured in RPMI supplemented with 10% FBS at 37°C and 5% CO₂ for 3 days. The cells were then cultured in a growth medium comprising DMEM (4.5 g/L glucose; Invitrogen) containing 10% fetal calf serum, 100 U/mL penicillin, and 100 µg/mL streptomycin, in a humidified atmosphere at 37°C with 5% CO₂. The cells were routinely subjected to mycoplasma testing using MycoAlert® Mycoplasma Detection Kits (Lonza, Basel, Switzerland).

Western blotting

mIVD tissues (nine discs) were collected, and a protein assay was performed for quantitative analysis of the nucleus pulposus and annulus fibrosis (AF), using CelLytic MT cell lysis reagent (Sigma-Aldrich), according to the manufacturer’s instructions. Equal amounts of protein from each sample were analyzed, as previously described ^[48], using immunoblotting with primary antibodies against iNOS (MAB9502, 1:500, R&D Systems), CD86 (NBP2-25208, 1:250, Novus Biologicals), Arginase-1 (#93668, 1:1000, Cell Signaling Technology), CD163 (ab182422, 1:1000, Abcam), and β-actin (A2228, 1:1000, Sigma-Aldrich). The β-actin was used as for normalization of relative protein levels. After three washes, the membranes were treated with anti-mouse or anti-rabbit secondary antibodies conjugated with horseradish peroxidase (#7076, 1:2000, or #7074, 1:2000, Cell Signaling Technology) for 2 h. Images were captured using ChemiDoc Touch imager (Bio-Rad, Hercules, CA, USA) and quantified using ImageJ (National Institutes of Health, Bethesda, MA, USA).

Statistical analysis

Data are presented as the mean ± standard deviation (SD). Significance was determined using Student’s or Welch’s t-test after an F-test was performed for equality of variances, unless otherwise stated. The normality of data was assessed using the Shapiro–Wilk test, except when the raw data did not fit a normal distribution, when the Mann–Whitney U-test was used. Differences were considered significant at p < 0.05.

Competing interests

The authors declare no competing interests.

Funding

This research was funded by JSPS KAKENHI (19K09594, and 23K08584).

Author Contribution

Conceptualization: R.T., H.H., and T.A. Methodology: R.T., Y.T., and T.A. Data Analysis: R.T. and Y.T. Data Curation: R.T., T.A., T.F., Y.T., and T.O. Writing—Original Draft: R.T. and T.A. Writing—Review and Editing: R.T., T.A., and H.H. All authors take responsibility for the integrity of the data analysis, have reviewed the results, and have approved the final version of the manuscript.

Acknowledgement

The authors thank Mr. Tadashi Iwato, Ms. Chizuru Chino, and Ms. Mika Ubagai for their technical assistance, and Kahori Sano and Azusa Sakamoto for secretarial assistance. We would like to thank Editage (www.editage.com) for English language editing.

Data Availability

The datasets used and/or analyzed in the current study are available from the corresponding author upon reasonable request.

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No competing interests reported.

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Thrombin induces degradation of murine intervertebral discs via angiogenesis and M1-like polarization of macrophages

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Version 1

Abstract

Figures

Introduction

Results

Thrombin promotes M1 macrophage polarization

PAR1 antagonist SCH79797 suppresses thrombin-induced mIVD degeneration

PAR1 antagonist inhibits thrombin production and macrophage M1 polarization

Thrombin induces VEGF production and angiogenesis

Discussion

Materials and methods

Animals

Murine intervertebral disc organ culture

Tissue histological analysis

Quantitative real-time reverse transcription PCR (qRT–PCR)

ELISA

Murine macrophage preparation

Western blotting

Statistical analysis

Declarations

Competing interests

Funding

Author Contribution

Acknowledgement

Data Availability

References

Additional Declarations

Supplementary Files

Status:

Version 1