Eur Spine J DOI 10.1007/s00586-017-4981-2

ORIGINAL ARTICLE

Myofibroblast in the ligamentum flavum hypertrophic activity Junseok W. Hur1,2 • Taegeun Bae2,4 • Sunghyeok Ye2,4 • Joo-Hyun Kim1 Sunhye Lee3 • Kyoungmi Kim2 • Seung-Hwan Lee2 • Jin-Soo Kim2,5 • Jang-Bo Lee1 • Tai-Hyoung Cho1 • Jung-Yul Park1 • Junho K. Hur6,7

•

Received: 3 September 2016 / Revised: 23 January 2017 / Accepted: 25 January 2017 Ó Springer-Verlag Berlin Heidelberg 2017

Abstract Purpose Majority of the previous studies compared lumbar spinal stenosis (LSS) and lumbar disc herniation (LDH) patients for analyses of LFH. However, the separation of normal/hypertrophied LF has often been ambiguous and the severity of hypertrophic activity differed. Here, we present a novel analysis scheme for LFH in which myofibroblast is proposed as a major etiological factor for LFH study. Methods Seventy-one LF patient tissue samples were used for this study. Initially, mRNA levels of the samples were assessed by qRT-PCR: angiopoietin-like protein-2 (ANGPTL2), transforming growth factor-beta1 (TGF-b1), vascular endothelial growth factor (VEGF), interleukin-6, collagen-1, 3, 4, 5, and 11, and elastin. Myofibroblasts were detected by immune stain using a-smooth muscle actin (aSMA) as a marker. To study the myofibroblast in TGF-b pathway, LF tissues were analyzed for protein

levels of aSMA/TGF-b1 by Western blot. In addition, from LF cells cultured with exogenous TGF-b1 conditioned medium, expression of aSMA/collagen-1 was assessed and the cell morphology was identified. Results The comparative analysis of mRNA expression levels (LSS vs LDH) failed to show significant differences in TGF-b1 (p = 0.08); however, we found a significant positive correlation among ANGPTL2, VEGF, TGF-b1, and collagen-1 and 3, which represent common trends in hypertrophic activity (p \ 0.05). We detected myofibroblast in the patient samples by aSMA staining, and the protein levels of aSMA were positively correlated with TGF-b1. In LF cell culture, exogenous TGF-b1 upregulated aSMA/collagen-1 mRNA levels and facilitated transdifferentiation to myofibroblast. Conclusions We conclude that the transition of fibroblast to myofibroblasts via TGF-b pathway is a key linker between inflammation and fibrosis in LFH mechanism.

Electronic supplementary material The online version of this article (doi:10.1007/s00586-017-4981-2) contains supplementary material, which is available to authorized users.

Keywords Ligamentum flavum  Hypertrophy  Myofibroblasts  Alpha-smooth muscle actin  Transforming growth factor beta1

& Junho K. Hur [email protected]

3

Department of Radiology, College of Medicine, Korea University, Seoul, Korea

Jang-Bo Lee [email protected]

4

Basic Science, IBS School, Korea University of Science and Technology, Seoul, Korea

Tai-Hyoung Cho [email protected]

5

Department of Chemistry, Seoul National University, Seoul, Korea

Jung-Yul Park [email protected]

6

Stem Cell Institute, College of Medicine, Korea University, Seoul, Korea

7

Korea University Anam Hospital, 73, Inchon-ro, Seongbukgu, Seoul 02841, Republic of Korea

1

Department of Neurosurgery, College of Medicine, Korea University, Seoul, Korea

2

Center for Genome Engineering, Institute for Basic Science, Seoul, Republic of Korea

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Introduction A study on the global burden of disease announced that the global disability-adjusted life year rank of low back pain had jumped from 11th (1990) up to 6th (2010) among 291 diseases between 20 years [1]. Lumbar spinal stenosis (LSS) is one of the major causes of low back pain and shows high occurrences especially in elderly, which suggests that the prevalence will increase as the lifespan widens [2]. Ligamentum flavum hypertrophy (LFH) is a major contributor in lumbar spinal stenosis (LSS), but its pathophysiology is not completely known. The growth factors/cytokines related to inflammation, angiogenesis, and fibrosis, such as angiopoietin-like protein 2 (ANGPTL2), transforming growth factor beta 1 (TGF-b1), and vascular endothelial growth factor (VEGF) were claimed as important factors for LFH pathology [3–7]. Multiple histologic studies also found that collagen/elastin ratios were high in hypertrophied LF, suggesting that the accumulation of collagen (fibrosis) is one of late steps of the pathology [5, 8, 9]. Most previous patient tissue studies compared LSS and non-LSS groups, where lumbar disc herniation (LDH) patients were regarded as nonLSS group for practical reasons. The thickness of LF generally differed between LSS and LDH groups and, therefore, was regarded as a major variable that strongly correlates with the expression levels of pathologic growth factors/cytokines [10, 11]. However, in some studies, such strong correlation between the LFH factors with LF thickness was not observed [3, 12]. The ambiguity suggested that the current standard dichotomic patient sample grouping based on LF thickness may not fully reflect the intricate pathology of LFH. In this study, we anticipated that the mechanism of LFH is dynamic and complex. We hypothesized that all the progression steps are blended, and the expression levels of growth factors/cytokines that contribute to hypertrophy change similarly during the progression of ‘‘hypertrophic activity’’. Accordingly, we reasoned that thickened LF is already ‘hypertrophied’, and is not ‘hypertrophying’. Therefore, hypertrophic activity would show higher correlation with collagen synthesis level (one of late stage product of fibrosis) than with the thickness of LF. Finally, we sought to detect myofibroblasts that may explain how LF tissue manifests high hypertrophic activity.

Methods Patients This study was conducted after approval from the Institutional Review Board of our institute. We collected the

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specimen (LF) prospectively from the patients over 20 years who underwent posterior approach lumbar surgery for LSS or LDH treatment at our hospital. Informed consent was obtained from all patients. The exclusion criteria were spondylolisthesis, revision surgery, infection, trauma, combined medical systemic disease (heart failure, renal failure, etc), and presence of a tumor or inflammatory systemic disease (rheumatoid arthritis or any kind of inflammatory arthritis). Initially, 48 samples were provided from 37 patients (11 male and 16 female) who were diagnosed for LSS (29 samples from 20 patients) or LDH (19 samples from 17 patients) at level L3/4, 4/5, or 5/S1. These samples were used for Real-Time Quantitative Reverse Transcription polymerase chain reaction (qRTPCR). Additional 20 samples were provided from 12 patients (7 male and 5 female) who were diagnosed LDH (3 samples from 3 patients) or LSS (17 samples from 9 patients) at level L3/4, 4/5, or 5/S1. These samples were used for immunohistochemistry (IHC; 20 samples) and quantitative analyses of protein levels (8 samples). A sample was obtained from male patient diagnosed LSS and used for cell culture and immunocytochemistry (ICC) to detect myofibroblast. Finally, two samples from two patients diagnosed for LDH were used for exogenous TGFb1 conditioned medium (CM) assay. Measurement of ligamentum flavum thickness Midpoint LF thickness was measured from axial T1weighted magnetic resonance image at the facet joint level of the operative lesion [13]. Measurements were made by one radiologist and one neurosurgeon blinded to the clinical and experimental information and were then averaged. Real-time quantitative reverse transcription polymerase chain reaction for candidate gene analysis The LF tissues were minced and crushed with MagNA Lyser Instrument (Roche, Basel, Switzerland). Total RNA was extracted using TRIzol reagent (Invitrozen, Life Technologies, Carlsbad, CA, US), and the RNA was reverse transcribed to cDNA with Transcriptor First Strand cDNA Synthesis Kit (Roche, Basel, Switzerland). Realtime PCR was performed with LightCyclerÒ 480II and LightCyclerÒ 480 SYBR Green I Master (Roche, Basel, Switzerland). The relative expression levels of the target mRNAs (ANGPTL2, TGF-b1, VEGF, IL6, and collagen-1, 3, 4, 5, and 11) were internally standardized with GAPDH expression, and then compared between samples. Target mRNA primer sequences are provided as supplementary Table 1.

Eur Spine J Table 1 Comparison between LSS and LDH groups LSS (n = 29)

LDH (n = 19)

Sex (m:f)

18:11

10:9

Agea

68.62 ± 1.98

51.00 ± 4.11

5.11 ± 0.37

3.89 ± 0.26

0.01*

LF thickness (mm)a a,b

p value 0.53 \0.01*

transferred to 0.2 lm nitrocellulose membrane. The membrane was then blocked for 1 h with 5% blocking solution at RT, and then was incubated with primary antibodies at 4°C overnight: anti-TGF-b1 (Santa Cruz, Santa Cruz, CA, USA), anti-aSMA (Sigma-AldrichÒ, St. Louis, MO, US), and antiGAPDH (Santa Cruz). The membranes were then stained with secondary antibodies: anti-mouse IgG and anti-rabbit IgG (Jackson ImmunoResearch labs, West Grove, PA, USA). Signal was detected by adding SuperSignal West Pico Chemiluminescent Substrate and SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific) to the membrane and exposing to X-ray film.

3.03 ± 0.49

1.00 ± 0.24

\0.01*

TGF-b1a,b

1.37 ± 0.14

1.00 ± 0.13

0.08

VEGFa,b

1.88 ± 0.28

1.00 ± 0.20

0.01*

IL6a

a,b

0.65 ± 0.28

1.00 ± 0.27

0.31

Collagen-1a,b

3.25 ± 0.71

1.00 ± 0.30

0.01*

Collagen-3a,b

3.25 ± 0.56

1.00 ± 0.26

\0.01*

Collagen-4a,b

0.94 ± 0.11

1.00 ± 0.20

0.78

Collagen-5a,b Collagen-11a,b

2.25 ± 0.49 3.15 ± 0.75

1.00 ± 0.34 1.00 ± 0.33

0.07 0.01*

LF cells isolation and culture

Elastina,b

1.24 ± 0.21

1.00 ± 0.16

0.42

LF tissue was harvested and washed in Phosphate Buffered Saline (PBS; Welgene, Gyeongsan-si, Gyeongsangbuk-do, Korea), minced, and incubated for 1 h at 37 °C with 0.2% collagenase type I (Gibco, Life Technologies, Grand Island, NY, US) in Dulbecco’s Modified Eagle Medium (DMEM; Welgene, Gyeongsan-si, Gyeongsangbuk-do, Korea) containing 1% penicillin–streptomycin (Welgene). The suspension was filtered with 100 lm mesh cell strainer (Falcon BD, Franklin Lakes, NJ, US) and centrifuged at 300 rcf for 5 min. Pellet was resuspended and seeded into six-well plate (CostarÒ, Corning, NY, USA) with DMEM containing 10% fetal bovine serum (FBS; Welgene) and 1% penicillin–streptomycin. Subsequent experiment was conducted using cells from the second passage.

ANGPTL2

a

The values are given as mean ± SEM

b

Relative mRNA expression level standardized after GAPDH

* p \ 0.05

Immunohistochemistry for myofibroblast LF tissues were fixed in 4% paraformaldehyde (PFA) for 48 h, freezed with OCT in -80 °C deep freezer, and then dissected in 10 lm thickness. 5% normal goat serum (Vector Laboratories, Burlingame, CA, US) was used as a blocking agent. The samples were then treated at 4°C overnight with mouse anti-human a-Smooth Muscle Actin (aSMA; SigmaAldrichÒ, St. Louis, MO, USA) as a primary antibody [14]. Next, the samples were stained with goat anti-mouse Alexa FluorÒ 488 conjugate (Invitrogen, Rockford, IL, USA) for 1 h at room temperature (RT) as secondary antibody, and nuclei were counterstained with 40 ,6-diamidino-2-phenylindole (DAPI) for 15 min at room temperature. The samples were then mounted and examined using a confocal microscope (LSM710, Carl Zeiss Microscopy GmbH, Germany). Images were collected using 405 and 488 nm laser lines for excitation and BP420-480 and BP505-550 emission filters for DAPI and Alexa488 fluorophores, respectively. Western blotting for TGF-b1 and aSMA LF tissues were minced and lysed in RIPA buffer (Biosesang, Seongnam-si, Gyeonggi-do, Korea) using TaKaRa BioMasher Standard (TaKaRa, Kusatsu, Shiga, Japan) followed by 2 cycle of sonication with BioruptorÒ Plus sonication device (Diagenode, Denville, NJ, USA). After centrifuge in 13,000 rpm, 15 min at 4°C, supernatant was collected and protein concentration was measured by the Bradford method using PierceTM BCA Protein Assay Kit (Thermo Scientific, Pierce Biotechnology, Rockford, IL, USA). Total protein 20 lg per each samples were separated by SDS-PAGE and

Immunocyotochemistry for myofibroblast Cultured cells were fixed with 4% PFA for 15 min in a 35 mm Glass Bottom Culture Dish (MakTek, Ashland, MA, USA) subsequently blocked with 5% normal goat serum for 1 h at RT and incubated with anti-aSMA antibody for overnight at 4°C. Goat anti-mouse Alexa FluorÒ 488 conjugate was stained for 1 h at RT, and nuclei were counterstained with DAPI for 10 min at RT, then mounted. Sample was examined using a light microscope (DMI4000 B, Leica, Germany). Images were collected using 405 and 543 nm light by exciting DAPI and Alexa488 fluorochromes, respectively. Exogenous TGF-b1 conditioned medium assay The LF tissues were isolated and cultured as described above and sub-cultured in 24-well plate at a density of 1 9 105 cells/ well at passage 3. After 48 h, the culture medium was replaced with DMEM without FBS/antibiotics for both control and experimental groups. Subsequently, experimental group was treated with exogenous recombinant human TGF-b1 (Peprotech, Rocky Hill, NJ, USA) at the final concentration of 0.5 lg/ ml. After 24 h incubation, cells were harvested for qRT-PCR

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Eur Spine J Table 2 Pearson’s correlation among growth factors/cytokines and collagens LF thickness

ANGPTL2

TGF-b1

VEGF

Collagen1

Collagen3

LF thickness

–

0.078 (0.60)

0.059 (0.69)

0.41 (\0.01)*

0.23 (0.12)

0.21 (0.16)

ANGPTL2

–

–

0.62 (\0.01)*

0.49 (\0.01)*

0.47 (\0.01)*

0.60 (\0.01)*

TGF-b1

–

–

–

0.38 (\0.01)*

0.53 (\0.01)*

0.50 (\0.01)*

VEGF

–

–

–

–

0.33 (0.02)*

0.37 (0.01)*

Collagen1

–

–

–

–

–

0.90 (\0.01)*

Collagen3

–

–

–

–

–

–

Data shown as rho (p value) * p \ 0.05 20

15

r=0.5333 p<0.01

15

Collagen-3

Collagen-1

Fig. 1 Pearson’s analyses of mRNA expression levels indicate that TGF-b1 is positively correlated with collagen-1 and collagen-3

10 5 0

r=0.4998 p<0.01

10

5

0 0

1

2

TGFb1

3

4

0

1

2

3

4

TGFb1

and ICC analyses. The aSMA and collagen-1 mRNA levels were assessed and standardized with GAPDH. For cell morphology assessment, cells were stained with aSMA antibody. Experiments were conducted in duplicates.

between LSS and LDH groups (p \ 0.05). However, there was no significant difference for sex, TGF-b1, IL6, collagen-4, and collagen-5 between the two groups. Details are shown in Table 1.

Statistical analysis

Positive correlation between ANGPTL2, TGF-b1, VEGF, collagen-1, and collagen-3

Student’s t test was used for comparison between LSS and LDH groups. Pearson correlation analysis was performed among qRT-PCR measurements of growth factors/cytokines and clinical/radiographic parameters. Data are shown as the mean ± SEM, and a P value less than 0.05 was used to determine statistical significance. The IBM SPSS Statistics version 23.0 software (IBM, New York, NY, US) was used for all analyses.

Results Clinical, radiographic, and quantitative mRNA data analyses

ANGPTL2, TGF-b1, and VEGF showed positive correlation among each other (p \ 0.01). They were also positively correlated with collagen-1 and collagen-3, respectively (p \ 0.05). In addition, ANGPTL2 and TGFb1 showed positive correlation with collagen-5 and collagen-11, respectively (p \ 0.05). VEGF was the only growth factor that manifested positive correlation with age (r = 0.33, p \ 0.02) and LF thickness (p \ 0.01). IL6 had no significant correlation with any of the other growth factors/cytokines or collagens (1, 3, 5, and 11). Representative data are summarized in Table 2 and Fig. 1. Immunohistochemistry analysis (aSMA stain for myofibroblast)

LSS group shows higher mRNA expression levels of ANGPTL2, VEGF, collagen-1, 3, and 11 compared to LDH group

Myofibroblasts exist in LF tissues and the proportion of myofibroblasts to fibroblasts is variable

Age, LF thickness, ANGPTL2, VEGF, collagen-1, collagen-3, and collagen-11 showed significant differences

Four samples showed almost all LF cells positive for aSMA. Twelve samples were partially positive for aSMA.

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Fig. 2 Representative results of tissue samples stained with aSMA are shown for myofibroblasts. Four tissues showed concurrent stain of DAPI (blue) and aSMA (green) (a). Twelve tissues were partially positive with aSMA (b), and four tissues had background signal for aSMA (c)

In other four samples, aSMA staining was absent in most of the LF cells. Representative images are shown in Fig. 2.

expression of aSMA and TGF-b1, whereas the aSMA (-) group showed low expressions for both proteins (Fig. 3).

Western blotting analysis (aSMA and TGF-b1)

Immunocytochemistry analysis (aSMA stain for myofibroblast)

aSMA and TGF-b1 protein expression levels showed positive correlation

LF cells include myofibroblasts

Four aSMA strongly positive tissues and four weakly positive (or negative) tissues by IHC were selected for protein analysis by Western blot. The aSMA (?) group showed high

Tissues with LF cells that were partially positive for aSMA represents mixed population of fibroblasts and myofibroblasts (Fig. 4).

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Fig. 3 Western blot analysis shows positive correlation between aSMA and TGF-b1. Four tissue samples with strong positive signal for aSMA by IHC (left) also showed high intensity bands for TGF-b1,

compared with four samples with weak aSMA signal (right). GAPDH showed similar expression levels for both groups

Fig. 4 Immunocytochemistry for cultured LF cells from LSS patient. DAPI (blue) indicates all LF cells. A subset of cells were aSMA positive, indicating that fibroblasts and myofibroblasts are mixed in the population

Exogenous TGF-b1 conditioned medium assay TGF-b1 upregulated aSMA and collagen-1 mRNA synthesis and facilitated the trans-differentiation of fibroblast to myofibroblast LF cells cultured with exogenous TGF-b1 showed higher level of aSMA and collagen-1 mRNA levels compared

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to control. Sample 1 showed 3.31 ± 1.09 fold higher for aSMA and 5.78 ± 0.98 fold higher for collagen-1. Sample 2 showed increase of 3.87 ± 0.41 fold for aSMA and 4.37 ± 0.33 fold for collagen-1 (Fig. 5). ICC showed that cells cultured with exogenous TGF-b1 CM expressed higher level of aSMA compared to control group (Fig. 6).

Eur Spine J

Relative expression

(A)

alpha-SMA

5

Sample 1 Sample 2

4 3 2 1

Relative expression

(B)

TG Fb1

C

on tr

ol

0

Collagen-1

8

Sample 1 Sample 2

6 4 2

TG Fb1

C

on

tr

ol

0

Fig. 5 Relative mRNA expression levels are shown. The aSMA (a) and collagen-1 (b) are overexpressed for both samples 1 and 2 in exogenous TGF-b1 CM group (0.5 lg/ml)

Discussion Many spine researchers studied the pathologic mechanism of LFH for decades. The following cascade was suggested as a mechanism of LFH: mechanical stress, inflammation, angiogenesis, and fibrosis occurring in consecutive order [3, 4]. Previous studies revealed that numerous growth factors/cytokines are important factors for LFH pathology, and TGF-b1 has been widely considered as a key factor at the inflammation step, and was shown to be highly expressed at LSS group compared to LDH group [6, 10, 11]. Surprisingly, we found no significant difference of TGF-b1 mRNA levels between LSS and LDH groups. Consistent with our observation, a previous study reported negative correlation between TGF-b1 and LF thickness [12]. This discrepancy might be explained by a model, where LDH patients’ LFs underwent various degrees pathologic changes prior to development of hypertrophy. In addition, even the LSS patients’ samples could have various levels of severity. This model suggests that LF thickness may not fully represent the complexity in patient group splitting or expectations of the pathologic severity. We use our lumbar spine extensively throughout our lives during which micro injuries, the very initial step of

LFH mechanism, occur repetitively and constantly. This suggests that various steps of hypertrophy mechanism might co-exist asynchronously. As a result, our observation of patient specimens at given timepoints might actually be compound data: potentially an intrinsic characteristic of LF tissues. We empirically know that the acceleration of the LFH (hypertrophic activity) differs from person to person. Based on the observation, we could deduce that there might be a common tendency of signaling pathway albeit with different rates in individuals. The quantification results of the study showed a significant positive correlation among pathologic factors (ANGPTL2, TGF-b1, and VEGF). In addition, they correlated strongly with collagen-1 and 3, but weakly with LF thickness, which is consistent with the notion that collagen-1 and 3 might be important indicators for assessing the hypertrophic activity in LF tissues. The LFH is widely considered as a fibrotic disorder. Studies on connective tissue remodeling found that the differentiation of fibroblasts to myofibroblasts, which expresses aSMA, may affect the production of the contractile force, and secrete fibrotic factors [14–16]. As myofibroblasts emerge, collagen synthesis is enriched, leading to scaring of tissue instead of normal healing. Therefore, preventing or reversing fibroblast to myofibroblast trans-differentiation is considered as one of treatment goals in other connective tissue disease issues [17]. The TGF-b signaling pathway connected with myofibroblast has been widely studied but is not yet completely understood [18]. The TGF-b1 activates Smad2/3 complex via TGF-b1/2 receptors; then, this intrinsic transcriptional factor enhances the down-stream molecule expression, and eventually leads to trans-differentiation of fibroblast to myofibroblast [19]. In this study, we found that myofibroblasts exist in LF tissues and confirmed it with cell culture assay. We also observed that myofibroblast/fibroblast co-exists in single tissue and the proportions differ in different samples. Finally, we found that exogenous TGF-b1 can up-regulate mRNA synthesis of aSMA and collagen-1, and facilitate trans-differentiation of fibroblast to myofibroblast. We conclude that this study is the first report, in LFH field, that proposes myofibroblast as a major factor for future LFH studies and treatments. Limitations In the current study, we showed that TGF-b1 can up-regulate the expression of aSMA in LF. However, for the search of LFH prevention and treatment strategy, inhibition studies on TGF-b receptor pathway genes should be performed. Further investigation on how TGF-b1 and other signaling molecules regulate myofibroblast might also provide important clues for the future treatment of LFH.

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Fig. 6 Immunocytochemistry after treatment of exogenous TGF-b1 CM. Blue indicates DAPI, and green indicates aSMA. The exogenous TGFb1 CM group (a) shows higher expression of aSMA compared to the control group (b)

Conclusion We conclude that the state of LFH is an ensemble of heterogeneous progression state of fibrosis. Several growth factors and cytokines as TGF-b1, ANGPTL2, and VEGF related to LFH show positive correlation with collagen synthesis. We observed that aSMA expressing myofibroblasts are trans-differentiated from fibroblast-like LF cells and that the transition was facilitated by TGF-b1. We propose that myofibroblast is one of the key element in LFH mechanism.

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Acknowledgements The authors thank Bokkee Eun, Geukrae Jeong, Jaeyun Han, and Hyun Kim (Core-Laboratory for Convergent Translational Research, College of Medicine, Korea University) for the technical support. This research was supported by Grants from the Hanmi Pharm Co., Ltd. to Jung-Yul Park and Institute for Basic Science (IBS-R021-D1) to Jin-Soo Kim. Compliance with ethical standards Conflict of interest The manuscript submitted contains no information about medical device(s)/drug(s). No benefits in any form have been or will be received from a commercial party related directly or

Eur Spine J indirectly to the subject of this manuscript. The authors declare that they have no competing interests. Ethical standards This study was conducted after approval from the Institutional Review Board of Clinical Trial Center of Korea University Anam Hospital (Approval No. ED14278).

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