Clinica Chimica Acta 448 (2015) 238–246

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Invited critical review

MMPs and ADAMTSs in intervertebral disc degeneration Wen-Jun Wang a,⁎, Xiao-Hua Yu b, Cheng Wang a, Wei Yang a, Wen-Si He a, Shu-Jun Zhang a, Yi-Guo Yan a, Jian Zhang c a b c

Department of Spine Surgery, the First Affiliated Hospital, University of South China, Hengyang, Hunan 421001, China Life Science Research Center, University of South China, Hengyang, Hunan 421001, China Department of Hand and Micro-surgery, the First Affiliated Hospital, University of South China, Hengyang, Hunan 421001, China

a r t i c l e

i n f o

Article history: Received 24 April 2015 Received in revised form 22 June 2015 Accepted 23 June 2015 Available online 8 July 2015 Keywords: IDD MMPs ADAMTSs Col II Aggrecan

a b s t r a c t Intervertebral disc degeneration (IDD) is the most common diagnosis in patients with low back pain, a leading cause of musculoskeletal disability worldwide. The major components of extracellular matrix (ECM) within the discs are type II collagen (Col II) and aggrecan. Excessive destruction of ECM, especially loss of Col II and aggrecan, plays a critical role in promoting the occurrence and development of IDD. Matrix metalloproteinases (MMPs) and a disintegrin and metalloprotease with thrombospondin motifs (ADAMTSs) are primary enzymes that degrade collagens and aggrecan. There is a large and growing body of evidence that many members of MMPs and ADAMTSs are highly expressed in degenerative IVD tissue and cells, and are closely involved in ECM breakdown and the process of disc degeneration. In contrast, targeting these enzymes has shown promise for promoting ECM repair and mitigating disc regeneration. In the current review, after a brief description regarding the biology of MMPs and ADAMTSs, we mainly focus on their expression profiles, roles and therapeutic potential in IDD. A greater understanding of the catabolic pathways involved in IDD will help to develop potential prophylactic or regenerative biological treatment for degenerative disc disease in the future. © 2015 Elsevier B.V. All rights reserved.

Contents 1. 2. 3. 4. 5.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structure and function of IVDs . . . . . . . . . . . . . . . . . . . Etiology of IDD . . . . . . . . . . . . . . . . . . . . . . . . . . Matrix synthesis and degradation during disc degeneration . . . . . . Biology of MMPs and ADAMTs . . . . . . . . . . . . . . . . . . . 5.1. MMPs . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. ADAMTSs . . . . . . . . . . . . . . . . . . . . . . . . . Expression profiles of MMPs and ADAMTSs in degenerative IVD tissue 6.1. Disc MMP expression . . . . . . . . . . . . . . . . . . . . 6.2. Disc ADAMTS expression . . . . . . . . . . . . . . . . . . . Roles of MMPs and ADAMTSs in IDD . . . . . . . . . . . . . . . . . 7.1. MMPs and IDD . . . . . . . . . . . . . . . . . . . . . . . 7.2. ADAMTSs and IDD . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: IVD, intervertebral disc; IDD, intervertebral disc degeneration; ECM, extracellular matrix; MMPs, matrix metalloproteinases; ADAMTSs, a disintegrin and metalloprotease with thrombospondin motifs; NP, nucleus pulposus; AF, annulus fibrosus; Col I, type I collagen; Col II, type II collagen; IL-1, interleukin-1; MAPK, mitogen-activated protein kinase; MRI, magnetic resonance imaging; TIMP, tissue inhibitor of metalloproteinase; NGF, nerve growth factor; BMP, bone morphogenetic protein; TGF-β1, transforming growth factor-β1; SNPs, single nucleotide polymorphisms, miRNAs, microRNAs; LPS, lipopolysaccharide; TNF-α, tumor necrosis factor-α; NF-κB, nuclear factor κB; NO, nitric oxide; SDC4, syndecan-4; RSV, resveratrol; LfcinB, bovine lactoferricin; HO-1, heme oxygenase-1; MSCs, mesenchymal stem cells. ⁎ Corresponding author at: Department of Spine Surgery, the First Affiliated Hospital, University of South China, Hengyang, Hunan 421001, China. E-mail address: [email protected] (W.-J. Wang).

http://dx.doi.org/10.1016/j.cca.2015.06.023 0009-8981/© 2015 Elsevier B.V. All rights reserved.

W.-J. Wang et al. / Clinica Chimica Acta 448 (2015) 238–246

8. Therapeutic potential of targeting MMPs and ADAMTSs in IDD 9. Conclusions and future directions . . . . . . . . . . . . . Disclosure . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Low back pain is a chronic, expensive, common medical problem in the world. It is the most common cause of disability in people younger than 45 years of age. Over 80% of adults will experience low back pain during their lifetimes [1]. At present, low back pain has become the second most frequent cause for visits to the hospital. There is growing evidence that the majority of low back pain is associated with intervertebral disc (IVD) degeneration (IDD) [2]. IDD constitutes the pathological foundation of most musculoskeletal disorders of the spine, including spinal stenosis, instability, disc herniation, radiculopathy and myelopathy. Despite the pathogenesis of IDD has not been completely understood, the predominant changes to IDD are characterized by in active cell number reduction, extracellular matrix (ECM) degradation, altered phenotype of normal disc cells, and presence of inflammation [3,4]. Current treatment for IDD includes conservative management (bed rest, nonsteroidal anti-inflammatory drugs and physical therapy) and surgical procedures (laminectomy, corpectomy and fusion). If conservative treatment fails, then surgical fusion is commonly considered. All of these therapeutic methods are limited to treat the symptoms but do nothing to slow down or reverse the course of IDD. Thus, a greater understanding of IDD pathology is urgent to optimize treatment strategies and develop novel anti-IDD drugs. The major ECM components within the discs are collagens and proteoglycans. In healthy discs, the rates of synthesis and breakdown of ECM are in equilibrium because of intricate regulation by growth factors and catabolic cytokines. When catabolism of ECM prevails over its anabolism, IDD often occurs. It is well established that loss of collagens and proteoglycans play a critical role in the development of disc degeneration [5]. Matrix metalloproteinases (MMPs) and a disintegrin and metalloprotease with thrombospondin motifs (ADAMTSs) are primary enzymes that cleave collagens and proteoglycans. It has been already reported that many members of MMPs and ADAMTSs are highly expressed in degenerative IVD tissue and cells, and these enzymes are deeply implicated in ECM breakdown and IDD progression [6]. Moreover, inactivation or knockdown of MMPs and ADAMTSs has shown enormous potential in promoting ECM repair and retarding disc regeneration [7]. In this review, we summarize the expression patterns and roles of MMPs and ADAMTSs in IDD, and describe recent progress regarding their inhibition as a promising biological therapeutic approach for disc degeneration.

2. Structure and function of IVDs The IVD is an important component of the spinal column and forms a shock absorber between each vertebra, allowing bending, flexion and torsion of the spine. Normal IVD is a complicated structure that contains three morphologically distinct regions: nucleus pulposus (NP), annulus fibrosus (AF) and cartilaginous end plates. The cartilaginous end plates are localized to the cranial and caudal aspects of each disc and contain the peripheral vasculature that nourishes the disc [8]. As a thick, dense structure, AF is divided into the outer and inner annuli. The outer annulus is made up of organized, collagenous concentric lamellae, which is primarily composed of fibroblast-like cells with elongated nuclei. The outer AF contains large amounts of type I collagen (Col I) together with collagen types III, V and VI. However, there is also a relatively low proteoglycan and water content within the outer annulus.

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243 244 244 244 244

In contrast to the outer annulus, the inner annulus is more fibrocartilaginous and is composed of both Col I and type II collagen (Col II), with a higher proteoglycan amount. Moreover, cells become more rounded and assume a chondrocyte-like phenotype. Taken together, collagen content in the AF comprises approximately 60% of dry weight, while proteoglycans account for approximately 25%. The biomechanical role of AF is to provide optimal tensile strength for containing NP [9]. NP locates in the central region of the IVD and is surrounded by the AF. The cell population in the NP varies with age. Cells in the NP at birth are predominantly composed of notochordal cells. However, with growth, these cells are converted to chondrocyte-like rounded cells, similar to those found in the inner annulus of AF [10]. As a gelatinous matrix, NP is rich in Col II and proteoglycans, and also includes small amounts of collagen types VI, IX and XI [11]. Among proteoglycans, aggrecan is the most common type and makes up as much as 50% of NP dry weight, which plays a critical role in absorbing water and contributes to the diffusion of nutrients from the periphery through maintenance of an osmotic gradient [12]. The hydrophilic nature of NP is responsible for a high swelling pressure. In a healthy IVD, the swelling pressure of NP and the tensile strength of AF keep balance and determine the intervertebral height, but allowing for the transformation of NP axial compression into AF hoop stresses [13]. A summary of structural and component differences between AF and NP is presented in Table 1.

3. Etiology of IDD Although the exact pathophysiology of IDD has not been completely understood, the environmental and genetic factors are thought to be the main contributors to IDD. Occupational exposures such as vibration [14], mechanical influences including heavy lifting and weight [15], lifestyle factors such as lack of exercise [16], and the long use of nonJapanese cars [17] are known to promote IVD degeneration. Injuries associated with lifting or trauma [18,19] and tobacco use [20,21] have been also reported to be involved in the pathology of IDD. However, there is accumulating evidence that these environmental factors may explain only a small portion of IDD, and heredity is predominant and probably accounts for more than 70% of an individual's risk for degenerative disc disease [22]. Recently, Videman et al. have confirmed a close association of 25 structural, degradative, and inflammatory candidate genes with lumbar disc desiccation, bulging, and height narrowing, revealing disc degeneration as a polygenetic condition [23]. Another research has demonstrated the existence of familial predisposition to IDD with generally high heritabilities that range from 34% to 61% in different Table 1 Structural and component differences between AF and NP. Characteristics

AF

NP

Location Cell shape and type Primary collagen type Collagen content Proteoglycan content Water content Biomechanical action Primary form of degradation

Surrounding of the NP Elongated, fibroblast-like CI High, dry weight (60%) Low, dry weight (25%) Low Tensile force to contain NP Destruction of structural integrity

Central region of the IVD Rounded, chondrocyte-like CII Low, dry weight (20%) High, dry weight (65%) High Resists axial compression Decreased proteoglycan and water contents

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spine locations [24]. Segregation analysis further shows that the mode of inheritance is complex with multiple factors and multiple genes involved in intergenerational transmission [24]. 4. Matrix synthesis and degradation during disc degeneration In healthy discs, the rates of synthesis and breakdown of the ECM are in equilibrium. When ECM breakdown prevails over its synthesis, IDD usually occurs. In the early stage of degeneration, collagen synthesis, in general, is increased. Especially, a clear increase in Col II content is seen within the NP, possibly signifying an attempted repair mechanism [7]. With more advanced degeneration, Col II content markedly decreases, resulting in a reduced ratio of Col II to Col I. Moreover, the distribution of collagens is altered, with more Col II appearing in the outer annulus. Col I forms stronger collagen fibrils within the inner annulus and NP. Type X collagen appears in degenerate discs and is related to chondrocyte clusters and cleft formation, indicating abnormal cellular activity [25]. Proteoglycan amount also changes, with a progressive loss of aggrecan within the NP [26]. Degradation of the disc ECM components produces a number of severe consequences. For example, decreased levels of aggrecan and collagens lead to many characteristic features of disc degeneration, including disorganization of the AF, dehydration and fibrosis of the NP, and calcification of the cartilaginous end plates [27]. Furthermore, loss of NP proteoglycan matrix coupled with an increased content of nonaggregating proteoglycan breakdown products attenuates the hygroscopic properties of ECM, resulting in reduced water content, swelling pressure and ability to withstand load. Dehydration of the NP also inhibits the availability of nutrients and growth factors to the disc cells, leading to further impairment of IVD function [28]. 5. Biology of MMPs and ADAMTs 5.1. MMPs MMPs, also called matrixins, are a very large family of calciumdependent, zinc-containing endopeptidases. There are 24 MMP genes identified in humans to date, among which MMP-23 gene includes two genetic duplications. Thus, humans have 23 MMPs. MMPs are traditionally divided into six categories depending on their substrate specificity, protein structure and subcellular localization: collagenases, gelatinases, stromelysins, matrilysins (minimal domain), membrane-type MMPs, and unclassified types [29]. Collagenases, including MMP-1, MMP8, MMP-13 and MMP-18, predominantly cleave fibrillar collagens. Gelatinases (MMP-2 and MMP-9) degrade the denatured collagens, gelatins, and laminin. Stromelysins are composed of MMP-3, MMP10, and MMP-11, which can proteolyze proteoglycans, gelatins and collagens. Matrilysins (MMP-7 and MMP-26) primarily act on aggrecan. MMP14, MMP-17, MMP-24 and MMP-25 are membrane-type MMPs that are localized to plasma membranes and have cytoplasmic domains. These MMPs play an important role in transduction of signaling pathways and activation of other MMPs [30]. The rest of the MMPs (MMP-12, MMP19, MMP-20, MMP-21, MMP-23, MMP-27, and MMP-28) belong to unclassified types, which are also involved in degradation of ECM components. However, their substrate specificity is not well defined. Notably, MMPs are usually secreted in inactive forms and require to be activated by the regulatory activator proteins. Although the main function of MMPs has been considered to be the degradation and removal of ECM molecules from the tissues, there is accumulating evidence that many non-ECM molecules are also potential substrates of MMPs [31]. 5.2. ADAMTSs ADAMTSs are a newly discovered type of metalloproteinase family, and contain 19 members. They are multi-domain proteins synthesized as pre-pro-enzymes containing from the N- to C-terminal end: a peptide

signal, a pro-domain, a zinc binding metalloproteinase domain, a disintegrin-like domain, a thrombospondin domain, a cysteine-rich domain, a spacer domain and finally a variable number of thrombospondin motifs in the C-terminal end [32,33]. The pre-pro-ADAMTSs are then cleaved by furin or furin-like proteases via removal of the prodomain for conversion to activated forms. Once activated, ADAMTSs can bind to ECM components via their thrombospondin motifs, leading to degradation of ECM components through the metalloproteinase domain [34]. ADAMTSs are classified into four groups based on their structure and function: hyalectanase (ADAMTS-1, ADAMTS-4, ADAMTS5, ADAMTS-8, ADAMTS-9, ADAMTS-15 and ADAMTS-20), von Willebrand factor (ADAMTS-13), pro-collagen N-peptidase (ADAMTS-2, ADAMTS-3 and ADAMTS-14), and a fourth group with unknown function (ADAMTS-6, ADAMTS-7, ADAMTS-10, ADAMTS-12, ADAMTS-16, ADAMTS-17, ADAMTS-18 and ADAMTS-19).

6. Expression profiles of MMPs and ADAMTSs in degenerative IVD tissue 6.1. Disc MMP expression MMP expression in normal IVD tissue is low and even devoid. In infant and preadolescent IVDs, MMP-1, MMP-2, MMP-3 and MMP-9 cannot be detected using immunohistochemistry; however, MMP-1 and MMP-3 appear in adult IVDs [35]. Le Maitre et al. have demonstrated that MMP-3 and MMP-13 expression is absent but a minimal expression of MMP-1 exists in non-degenerative discs [36]. In porcine discs, the level of MMP-1 mRNA increased with aging [37]. In contrast, the greatest expression of MMP-19 is observed in young IVDs, and little expression is seen in older IVDs [38]. It is likely that the low expression of these MMPs in non-degenerative IVDs are involved in normal tissue repair and remodeling. Nevertheless, such an effect remains to be further investigated. Recently, increased expression of different MMPs has been reported in degenerative IVD tissue (Table 2). Xu et al. analyzed the relationship between degenerative IVD grade and MMP-1 expression in disc specimens from patients who had undergone operation for lumbar disc herniation. They found that with the increase in degenerative IVD grade, the expression of MMP-1 is gradually upregulated [39]. Microarray analysis of genes demonstrated that MMP-2 mRNA expression is upregulated in human degenerative IVD tissue when compared with normal IVD tissue [40]. In a rabbit IDD model that was established by an annular stab with a 16-gauge needle to the L2–L3, L3–L4, and L4–L5 discs, MMP3 gene expression in NP tissue markedly increases at 3 weeks, decreases to a low level from 6 to 12 weeks, and then has a second, late peak at 24 weeks, revealing a double peak characteristic [41]. Wei et al. also reported that the levels of MMP-3 mRNA are significantly increased at 15 months in degenerative IVD tissue induced by bleomycin in the rhesus monkey [42]. Interleukin 1 (IL-1) is potentially important in the pathogenesis of IDD, because it can promote production of matrix degradation enzymes and inhibit matrix synthesis. Mice lacking the natural inhibitor of IL-1 receptor develop spinal abnormalities that resemble characteristic features associated with human disc degeneration, and display a marked increase in disc MMP-3 and MMP-7 expression [43]. MMP-8 and MMP-10 mRNA levels are found to be consistently and substantially upregulated in samples with histological evidence for disc degeneration [44,45]. Immunocytochemical localization analysis revealed that MMP-12 is abundantly present in both the annulus and nucleus portions of human degenerative discs [46]. With the progression of IVD degeneration, MMP-13 expression is significantly increased in canine NP tissue [47]. In the post-surgery disc specimens obtained from lumbar disc herniation patients, the expression of MMP-1, MMP-2 and MMP-14 is upregulated and shows a positive correlation with the severity of disc degeneration [48]. Together, these observations suggest that highly expressed MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-10,

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241

Table 2 Expression profiles of MMPs and ADAMTSs in degenerative IVD tissue. Enzymes

Expression

Experimental models

References

MMPs MMP-1 MMP-2 MMP-3 MMP-7 MMP-8, MMP-10 MMP-12 MMP-13 MMP-14

Upregulation Upregulation Upregulation Upregulation Upregulation Upregulation Upregulation Upregulation

Human degenerative IVD tissue Human degenerative IVD tissue Rabbit degenerative NP tissue, monkey and mice degenerative IVD tissue Mice degenerative IVD tissue Human degenerative IVD tissue Human degenerative IVD tissue Canine degenerative NP tissue Human degenerative IVD tissue

[39,48] [40,48] [41–43] [43] [44,45] [46] [47] [48]

ADAMTSs ADAMTS-1 ADAMTS-3 ADAMTS-4 ADAMTS-5 ADAMTS-7, ADAMTS-12 ADAMTS-10 ADAMTS-15

Upregulation Downregulation Upregulation, no change Upregulation Upregulation Downregulation Upregulation

Human degenerative IVD tissue Human degenerative IVD tissue Human and rat degenerative IVD tissue Human degenerative IVD tissue Human and rat degenerative IVD tissue Human degenerative IVD tissue Human degenerative IVD tissue

[49] [57] [49,51,52,55,56] [49,51,52,54–56] [50,53] [57] [49]

MMP-12, MMP-13 and MMP-14 may play an important role in promoting the progression of IDD.

6.2. Disc ADAMTS expression Several studies have identified the presence of ADAMTS-1, ADAMTS4, ADAMTS-5, ADAMTS-9 and ADAMTS-15 mRNA and protein in human normal IVD tissue [36,49]. Similar to MMPs, these aggrecanases may contribute to normal physiologic function of IVDs as well. In contrast, the expression of ADAMTS-1, ADAMTS-4, ADAMTS-5, ADAMTS-7, ADAMTS-12 and ADAMTS-15 is significantly increased in human degenerated IVD tissue compared with non-degenerated tissue [49,50]. In a rat tail disc degeneration model induced by static compression at 1.3 MPa, a significant increase in disc ADAMTS-4, ADAMTS-5, ADAMTS7 and ADAMTS-12 mRNA levels is seen [51–53]. ADAMTS-5 levels in human herniated IVD tissue are also higher than those in normal IVD tissue [54]. However, Patel et al. compared ADAMTS-4 and ADAMTS-5 protein expression in human IVDs at early and advanced stages of IDD. They found that the content of ADAMTS-4 is higher in degenerated tissue at advanced stage than early stage, whereas the content of ADAMTS-5 does not differ between the two stages [55]. In human degenerative cartilaginous end plate tissue, a marked upregulation of ADAMTS-5, but not ADAMTS-4, is observed [56]. Additionally, Gruber et al. reported that the expression of ADAMTS-3 and ADAMTS-10 is significantly decreased in human degenerative discs [57]. Thus, unlike MMPs, ADAMTS expression patterns in IDD are still unclear and controversial. ADAMTS-4 and ADAMTS-5 expression may be dependent on degenerative stages and locations. Table 2 summarizes ADAMTS expression in degenerative IVD tissue. With continued efforts, this list is expected to grow.

7.1. MMPs and IDD MMPs are the most important enzymes that hydrolyze collagens, the main components of ECM. Recently, there has been a large body of evidence supporting the involvement of MMPs in IDD pathogenesis. A higher expression of MMP-1 and MMP-3 is seen in patients of recurrent lumbar disc herniation than in primary herniation [58]. The proteolytic fragments of ECM are known to aggravate IDD [59,60]. Quero et al. observed that treatment of human degenerative IVD cells with hyaluronic acid (one component of ECM) fragments (HAFs) markedly increases the expression of MMP-1, MMP-3 and MMP-13 through activation of the mitogen-activated protein kinase (MAPK) signaling pathway [61]. This suggests that HAFs-mediated IDD is associated with collagen breakdown driven by MMPs. In IVD tissue specimens obtained from patients who had undergone operation for lumbar disc herniation, the expression levels of MMP-3 show significant positive correlations with magnetic resonance imaging (MRI) grading and IVD histopathological

7. Roles of MMPs and ADAMTSs in IDD The initiation and development of IDD is a multifactorial and complex process, and the involved mechanisms are not fully elucidated. The disruption of ECM is a major hallmark of disc degeneration. Given the key role of MMPs and ADAMTSs in ECM degradation, it is not surprising that these enzymes are involved in the pathology of IDD (Fig. 1). The effects of MMPs and ADAMTSs on IDD progression will be discussed in detail below. Simultaneously, agents that affect the course of IDD by regulating the expression of MMPs and ADAMTSs are presented in Table 3.

Fig. 1. A schematic presentation of MMPs and ADAMTSs in IDD. The normal IVDs maintain a dynamic balance between ECM catabolism and anabolism. Under pathological stimulation, the expression of MMPs and ADAMTSs is significantly upregulated. These increased matrix degradation enzymes then promote the hydrolation of disc ECM components, especially Col II and aggrecan. Consequently, catabolism of ECM outweighs its anabolism, leading to the occurrence and development of IDD.

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Table 3 Experimently verified regulators of disc MMP and ADAMTS expression and their effects on IDD. Agents

Expression of MMPs Mechanisms and ADAMTSs

Actions

HAFs

↑MMP-1, MMP-3, MMP-13 ↓MMP-13 ↑MMP-3, MMP-9 ↓MMP-3, MMP-13 ↓MMP-1, MMP-3, MMP-13 ↓MMP-1

↑MAPK

Promotion [61]

? lipocalin 2 ↓NF-κB ?

Protection Promotion Protection Protection

[62] [65,66] [67] [68]

?

Protection

[69]

↓MMP-2 ↓MMP-13 ↑MMP-1, MMP-13 ↑ADAMTS-4, ADAMTS-5 ↑ADAMTS-4, ADAMTS-5 ↑ADAMTS-4, ADAMTS-5 ↑ADAMTS-4, ADAMTS-5

? ? ? ↑p38

Protection Protection Promotion Promotion

[72] [83] [84] [87]

?

Promotion [91]

↑NF-κB, SDC4

Promotion [95,97]

↑NO, MAPK, NF-κB, SDC4

Promotion [54,96,97]

IGF1R NGF LMP-1 Salmon calcitonin BMP-2, TGF-β1 Ulinastatin miR-146a Col II peptide Leptin MyD88 TNF-α IL-1β

References

alterations [62]. Knockdown of insulin-like growth factor 1 receptor (IGF1R) can also elevate MMP-13 levels and subsequently reduce the amounts of Col II and aggrecan in the discs, leading to accelerated IVD degeneration in mice [63]. In another study, intradiscal injection of the Smad-7 overexpression lentivirus is found to aggravate the progression of disc degeneration in a MMP-13-dependent manner in rats [64]. Increased neurotrophin activity is one potential cause of IDD. Nerve growth factor (NGF) stimulation has been shown to upregulate MMP3 expression and increase the ratio of MMP-3 to tissue inhibitor of metalloproteinase (TIMP)-3 in rat and human IVD cells and tissue, which is abolished by Ro 08-2750, a specific inhibitor of NGF [65]. A later study by the same authors has demonstrated that NGF also augments the expression and activity of MMP-9 in rat AF cells by upregulating lipocalin 2 expression [66]. Therefore, NGF has potential effects on matrix turnover activity and influences the catabolic/anabolic balance of IVD cells in an adverse way that may potentiate IVD degeneration. On the other hand, overexpression of LIM mineralization protein-1 (LMP-1) has been reported to abrogate TNF-α-induced production of MMP-3 and MMP-13 through inhibition of nuclear factor κB (NF-κB) and then increase Col II and aggrecan contents in NP cells [67]. Postmenopausal osteoporosis is known to induce IDD. In an ovariectomized rat model of IDD, administration of salmon calcitonin, an antiresorptive medication can protect the lumbar discs from degeneration by inhibiting the expression of MMP-1, MMP-3 and MMP-13 [68]. Treatment of porcine degenerative AF cells with a combination of minimum doses of both bone morphogenetic protein (BMP)-2 and transforming growth factor-β1 (TGF-β1) also leads to a greater decrease in MMP-1 levels and a greater increase in aggrecan content than either cytokine alone, suggesting a synergistic effect of both cytokines for amplifying the production of ECM components [69]. In addition to MMP-1, MMP-3, MMP-9 and MMP-13, several MMPs are implicated in IDD. It has been demonstrated that MMP-2 activity is significantly increased in degenerative AF cells of rats, and AF cells appear to use MMP-2 in a very directed fashion for local matrix degradation and collagen remodeling [70]. Moreover, MMP-14 is essential for increased activity of MMP-2 during disc degradation [71]. In a degenerated rabbit NP cells induced by IL-1β in vitro, addition of ulinastatin, a trypsin inhibitor, effectively enhances Col II levels by downregulating MMP-2 expression, revealing a protective effect of ulinastatin on IDD [72]. MMP-7, an important member of matrilysins, is known to cleave the major matrix molecules found within the IVDs,

especially Col II and aggrecan. Immunohistochemical staining showed that a significant increase in the proportion of MMP-7 immunopositive cells is seen in the NP of human degenerative discs, and the expression of MMP-7 is positively related to the degree of disc degeneration [73]. Likewise, the expression and activity of MMP-8 are significantly increased in tissue samples from human disc degeneration, followed by an enhanced ratio of MMP-8 to TIMP-1 or TIMP-2, the tissue-specific, endogenous inhibitors of MMPs [44]. However, whether other members of MMP family such as MMP-4, MMP-5, MMP-6, MMP-11, MMP-15, MMP-16, MMP-17 and MMP-18 are involved in IDD remains unclear. Future studies are required to evaluate the roles of these MMPs in IDD. Given that genetics have a dominant role in IDD, the genetic variants in genes encoding proteins such as MMPs also affect disc degeneration. The rs1799750 2G allele in the promoter region of the MMP-1 gene is known to upregulate MMP-1 expression in vitro. A recent study by Jacobsen et al. found that the MMP-1 rs1799750 2G allele is not directly related to disc degeneration but contributes to low back pain, sciatica and disability after human lumbar disc herniation [74]. Dong and colleagues have demonstrated that the frequency of MMP-2 1306CC genotype is significantly increased in Chinese young adult patients with lumbar disc degeneration when compared with healthy controls, and this genotype is found to correlate with more severe grades of disc degeneration observed on MRI scan [75]. In contrast, the 735CT and TT genotypes of MMP-2 contribute to decreasing the risk of lumbar disc degeneration in the Chinese Han population [76]. It is suggested that the mutant allele 5A of MMP-3 is closely associated with the severity of lumbar disc degeneration in an Egyptian population [77]. Moreover, synergistic interactions exist between the mutant allele 5A of MMP-3 and occupational exposures such as whole-body vibration and bending/twisting in lumbar disc degeneration cases [78]. This suggests that subjects who carry the mutant allele 5A of MMP-3 are more susceptible to IDD when they are exposed to occupational risk factors. Rajasekaran et al. analyzed the association between single nucleotide polymorphisms (SNPs) of 35 candidate genes and three highly specific markers for degenerative disc disease in an Indian population, namely disc degeneration, end plate damage and annular tears. Among these SNPs, the rs2252070 SNP of MMP-13 shows a significant positive correlation with end plate damage [79]. In the young adult population in North China, the frequency of the MMP-9 1562T genotype in patients with lumbar disc degeneration is significantly higher than that in healthy controls, and subjects carrying this genotype have a higher risk to develop lumbar disc degeneration [80]. Collectively, these findings suggest that screening MMP gene polymorphisms may be important for early detection of susceptible individuals and disease prevention. microRNAs (miRNAs) are a class of endogenously expressed, small noncoding RNAs, which can inhibit gene expression by targeting mRNAs for translational repression and/or cleavage [81,82]. It has been reported that transfection of miR-146a mimics inhibits IL-1-induced MMP-13 expression and then increases Col II collagen content in NP cells isolated from IVD of bovine tails [83]. Conversely, when disc segments from miR-146a knockout mice were cultured ex vivo in the presence of IL-1 for 3 days, a more severe IDD is observed, with concomitant elevation of MMP-13 expression [83]. Thus, miR-146a appears to protect against IL-1-induced IVD degeneration. Stimulation of endogenous miR146a expression or exogenous delivery of miRNA-146a are viable therapeutic strategies that may relieve IDD and regain a normal homeostatic balance in ECM production and turnover. As mentioned previously, NP has the highest amount of Col II within IVD tissue. After cleavage of Col II by MMPs, numerous fragments are produced and accumulated in degenerative discs. It is worth noting that the Col II peptide (245–270), a fragment of Col II, also enhances transiently MMP-1 expression in both adult bovine AF and NP cells but upregulates MMP-13 expression only in NP cells [84], thereby providing a positive feedback loop between MMPs and Col II fragments to further degrade Col II. Thus, an enhanced removal of Col II degradation

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products from IVD may represent a novel and promising approach to reduce MMP synthesis and treat IDD. In contrast to these studies, other reports suggest that MMPs have no effects on IDD. MMP-28, also known as epilysin, is a recently discovered member of the MMP family. A recent study by Klawitter et al. showed that although MMP-28 expression is increased in individual cases with trauma or disc degeneration, there is no significant correlation between the grade of disease and MMP-28 expression [85]. Moreover, stimulation with LPS, IL-1β, tumor necrosis factor-α (TNF-α) or trichostatin A do not alter MMP-28 gene expression at any investigated time point or any concentration [85]. Thus, more investigations are needed to elucidate the role of MMP-28 in the disc degeneration. 7.2. ADAMTSs and IDD It is well known that loss of aggrecan is an early critical event in the degenerative cascade in IVD tissue. ADAMTS-4 (aggrecanase-1) and ADAMTS-5 (aggrecanase-2) are currently classified as the major aggrecanases due to their high efficiency in cleaving aggrecan [86]. The role of these two ADAMTSs in the pathogenesis of IDD has obtained great attention. In primary human NP cells, overexpression of leptin, a hormone with increased circulating levels in obesity that is a risk factor of IDD, markedly upregulates ADAMTS-4 and ADAMTS-5 expression and reduces aggrecan levels through induction of p38 phosphorylation, whereas knockdown of ADAMTS-4 and ADAMTS-5 partially reverses leptin-mediated degradation of aggrecan [87]. These findings provide a novel mechanistic insight into the molecular pathogenesis of obesity-associated IDD. Prolonged upright posture is reported to induce AF fissures, decrease disc height and ADAMTS-5 expression, and reduce Col II and aggrecan contents in IVDs of rat cervical spine, suggesting long-term upright stance as an environmental risk factor of IDD [88, 89]. Additionally, Wu et al. reported that the rs2252070 SNP of ADAMTS-5 contributes to predisposition of lumbar IVD degeneration in a Chinese Han population [90]. On the other hand, application of the inhibitor of MyD88, an adapter protein that links toll-like receptors and IL-1 receptors, markedly attenuates lipopolysaccharide (LPS)mediated induction of ADAMTS-4 and ADAMTS-5 and subsequent proteoglycan depletion in bovine NP cells [91]. In a rabbit model of IDD, injection of ADAMTS-5 small interference RNA (siRNA) induces the inhibition of ECM degradation in NP tissue, as shown by significantly improved MRI and histologic grades [92]. Overall, these data indicate that ADAMTS-4 and ADAMTS-5 play a key role in the development of IDD, and targeting these two enzymes may have considerable promise for repairing ECM and treating disc degeneration. Inflammation has been correlated with degenerative disc disease [93]. TNF-α and IL-1β, both major proinflammatory mediators, are thought to play a crucial role in the initiation and continuation of inflammatory response. Elevated levels of TNF-α and IL-1β are frequently detected in degenerative IVD tissue [7]. In an in vivo rat model of IDD induced by disc puncture, repeated disc injury promotes abundant production of TNF-α and IL-1β, leading to persistent inflammation and accelerated disc degeneration [94]. Of note, TNF-α and IL-1β also affect ECM metabolism within the discs by regulating ADAMTS expression. For example, treatment of human IVD cells with TNF-α dramatically induces ADAMTS-4 and ADAMTS-5 expression and subsequently stimulates the catabolism of aggrecan and Col II by promoting nuclear translocation of NF-κB, which can be blocked by BMP-7 [95]. IL-1βtreated rat NP cells display a significant increase in ADAMTS-4 and ADAMTS-5 levels and aggrecan degradation, which is attributed to activation of the MAPK and NF-κB pathways [96]. Exposure of rat NP cells to IL-1β is found to upregulate ADAMTS-5 expression in a nitric oxide (NO)-dependent manner [54]. Deficiency of the natural inhibitor of IL-1 receptor in mice leads to IDD and raises ADAMTS-4 expression in degenerative discs [43]. Also, syndecan-4 (SDC4) is involved in TNF-α and IL-1β-induced upregulation of ADAMTS-4 and ADAMTS-5 in the NP cells [97]. Thus, in addition to proinflammatory effects, TNF-α and

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IL-1β can enhance ADAMTS-4 and ADAMTS-5 levels and lead to ECM degradation, which is another important mechanism of TNF-α and IL1β-associated IDD. Besides ADAMTS-4 and ADAMTS-5, other ADAMTSs are implicated in IDD. Recently, Zhang et al. examined ADAMTS-7 and ADAMTS-12 expression and Col II content in 64 degenerated lumbar end plate specimens from patients with degenerative disc disease categorized as type Modic I or II in MRI and 12 non-degenerative specimens (vertebra burst fracture patients without degenerative change in MRI) during surgical procedures. In comparison with non-degenerative specimens, ADAMTS7 and ADAMTS-12 expression is upregulated but Col II content is significantly decreased in degenerated lumbar end plates [50]. Among the etiological factors of IDD, mechanical stress plays an important role in the physiological and pathological processes of NP cells. It has been reported that compressive load is able to augment ADAMTS-1 levels in NP cells isolated from human degenerative discs, accompanied by a marked reduction of aggrecan amount [98]. Thus, ADAMTS-1-induced aggrecan degradation is likely to be, at least partially, responsible for mechanical stress-associated IDD. 8. Therapeutic potential of targeting MMPs and ADAMTSs in IDD Existing therapy options for IDD are restricted to treat the symptoms, and does not target its pathophysiology, highlighting the urgent need to develop biological therapeutic approaches. Since excessive ECM disruption is thought to the primary cause of IDD, delaying or stopping the characteristic ECM loss in disc degeneration has been suggested as a novel strategy which aims to restore matrix homeostasis by enhancing anabolism or reducing catabolism [99]. Considering the key role of MMPs and ADAMTSs in hydrolation of ECM components, these enzymes could be promising candidates for biologically-induced disc ECM repair, and their inhibition might provide an alternative to surgical intervention for early stage disc degeneration. Indeed, electroacupuncture intervention has been suggested to protect against disc degeneration by attenuating MMP-13 expression and enhancing Col II synthesis in a rabbit model of IDD [100]. Diabetes and obesity are highly correlated with IDD and low back pain. A recent experimental study has shown that streptozotocin-induced diabetic mice exhibit specific degenerative changes of NP morphology, with high expression of disc MMP-13 and ADAMTS-5 [101]. However, a combined treatment with pentosanpolysulfate (an anti-inflammatory drug) and pyridoxamine (an inhibitor of advanced glycosylation end product) alleviates NP degeneration due to decreased MMP-13 and ADAMTS-5 levels [101]. Resveratrol (RSV) is a natural compound found in various plants including grapes and red wines, and has been reported to protect against articular cartilage in rabbit models for arthritis [102]. Incubation of bovine NP cells with RSV for 21 days significantly blocks IL-1β-induced production of MMP-13 and ADAMTS-4 and thus results in increased proteoglycan synthesis, revealing its potential as a unique biologic treatment to retard the progression of IVD degeneration [103]. Activation of NF-κB, a major transcription factor, is known to induce the transcription of a number of genes including adhesion molecules, chemokines, MMPs and ADAMTSs both in vitro and in vivo [104]. It has been demonstrated that NF-κB activity is significantly increased in human degenerative NP cells, and shows a positive correlation with the degree of disc degeneration [105]. However, exposure of human NP cells to BAY11-7082, a specific inhibitor of NF-κB, significantly inhibits IL-1β-dependent upregulation of MMP-3, MMP-9, MMP-13, ADAMTS-4 and ADAMTS-5, and then enhances the contents of aggrecan and Col II, suggesting a potential therapeutic benefit of BAY11-7082 on IDD [106]. Given NF-κB as a potent positive regulator of most MMP and ADAMTS expression, inhibition of NF-κB may represent a novel avenue to promote ECM restoration and treat degenerative disc disease. Bovine lactoferricin (LfcinB) is a product obtained by acid-pepsin hydrolysis of the N-terminal region of lactoferrin from cow's milk, and has more potent biological actions than equimolar amounts of intact

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lactoferrin [107]. As a cell membrane-permeable molecule, it can interact electrostatically with negatively-charged matrix and cell surface glycosaminoglycans, heparin and chondroitin sulfate, contributing to ECM repair. In IL-1 and LPS-stimulated bovine or human NP cells, addition of LfcinB has been shown to reduce proteoglycan breakdown by antagonizing the production of MMP-1, MMP-3, MMP-13, ADAMTS-4 and ADAMTS-5 [108]. Simultaneously, the ability of LfcinB to restore proteoglycan accumulation is further confirmed in an en bloc intradiscal microinjection model followed by ex vivo organ culture using both mouse and rabbit IVD tissue [108]. Additionally, administration of Link-N, a naturally occurring peptide, strongly stimulates proteoglycan synthesis by decreasing the mRNA levels of ADAMTS-4 and ADAMTS5 in human degenerative IVD cells [109]. Furthermore, Link-N injection is reported to partially restore disc proteoglycan content in a rabbit model of IDD, which is attributed to decreased expression of ADAMTS4, ADAMTS-5 and MMP-3 [110]. These observations suggest that LfcinB and Link-N are potent inducers of pericellular matrix formation and function as promising candidates for biologically-induced disc regeneration. Autophagy is a homeostatic and evolutionarily conserved process that can degrade cellular organelles and proteins and maintain cellular biosynthesis, and it has been shown to exert protective effects on disc degeneration [111,112]. Jiang et al. have demonstrated that administration of glucosamine promotes autophagosome formation by suppressing the mammalian target of rapamycin (mTOR)/p70S6K signaling pathway, leading to decreased MMP-13 and ADAMTS-4 expression as well as increased Col II and aggrecan contents in IL-1β or hydrogen peroxide (H2O2)-treated human NP cells, whereas these effects are partially abolished in the presence of 3-methyladenine, an autophagy inhibitor [113]. Thus, induction of autophagy may be also a viable approach for ECM repair within the degenerative IVDs. Gene therapy involves the transfers of exogenous genetic material (DNA or RNA) into a target cell leading to gene expression and subsequent production of deficient or beneficial proteins. There is growing evidence that gene therapy has shown promise for slowing down IDD progression [8,114]. One important approach of gene therapy for IDD is to correct the imbalance between anabolism and catabolism within the disc ECM. It has been reported that administration of heme oxygenase-1 (HO-1) plasmid DNA carried by mixed polyplex micelles with thermo-responsive heterogeneous coronas can diminish MMP-3 activity and increase Col II and aggrecan contents in rat NP cells, indicating a potential protective effect of HO-1 on IDD [115]. Introduction of IL-1 receptor antagonist into human degenerate IVD explants using genetically engineered constructs leads to elimination of matrix degradation due to decreased expression of MMP-3, MMP-7 and MMP-13 [116]. TIMP-1 is a specific inhibitor of MMP-3. Gene delivery of TIMP1 has been shown to enhance proteoglycan synthesis in human degenerate IVD cells [117]. In a in vivo rabbit model of IDD, injection of adenoassociated virus serotype 2 (AAV2) vector carrying TIMP-1 gene into NP tissue remarkably enhances Col II content in the discs, followed up less MRI and histologic evidence of degeneration [118]. These data further confirm that an increased ECM production contributes to the delay of IDD progression. Thus, transgene of endogenous MMP and ADAMTS inhibitors may have important therapeutic application in the management of IDD. With continued efforts, gene therapy may prove to be an extremely powerful tool in the future treatment of disc degeneration. In agreement with gene therapy, several cell-based therapies to stimulate disc ECM repair have been proposed in recent years. Sun and colleagues have reported that adipose-derived stromal cells (ADSCs) protect human NP cells from compressive load-mediated degeneration partially by downregulating the expression of MMP-3, MMP-13, ADAMTS-1, and ADAMTS-5 [119]. In contrast to ADSCs, mesenchymal stem cells (MSCs) have many excellent properties such as long-term self-renewal, multi-differentiation potential and autologous transplantation, and thus have become one of the most attractive candidate cell types for IDD treatment. Injection of MSCs into NP has confirmed

them to migrate to inner AF for ECM production and disc regeneration, without evidence of systemic illness in the recipient rabbits [120]. In rabbits, synovial MSCs injected into the NP space is able to remain in the IVDs up to 24 weeks, and inhibit disc degeneration via a decreased expression of MMP-2, MMP-3 and MMP-13 as well as a subsequent increase in Col II content [121]. Moreover, MSC therapy for IDD has entered clinical trials. Recently, two patients with chronic discogenic low back pain were treated with human umbilical cord tissue-derived MSC (HUC-MSC) transplantation. After transplantation, the pain and function are effectively improved in both patients during a 2-year follow-up period, without obvious adverse safety concerns observed [122]. Overall, MSC transplantation may be an ideal approach for the treatment of IDD. However, additional, larger, and higher-quality studies are needed to assess the long-term safety and efficacy. 9. Conclusions and future directions It is clear that IDD primarily results from an imbalance between catabolism and anabolism of disc ECM components, especially Col II and aggrecan. As the primary mediators of Col II and aggrecan degradation, the majority of MMPs and ADAMTSs are highly expression in degenerative IVD tissue and cells, and play a crucial role in ECM breakdown and IDD progression. Despite the fact that inhibition of MMPs and ADAMTSs has shown efficacy and therapeutic potential in promoting ECM repair and preventing disc degeneration, many challenges are still ahead. Because MMPs and ADAMTSs exist throughout the body, suppression of MMPs and ADAMTSs must be exactly localized to the degenerative IVDs in a tissue-specific manner, such as intradiscal injection puncture. However, intradiscal injection puncture has been reported to aggravate disc degeneration via NP depressurization and/or AF damage [123,124]. To reduce these detrimental consequences, systemic delivery of therapeutic agents to the avascular IVDs may be more attractive, but this administrated method must overcome the barrier of reaching sufficient intradiscal concentrations to produce a protective effect. Therapeutic agents also face the challenges of limited nutrition, low acidity and altered mechanics in degenerative IVD environment. Given that ECM breakdown is only one of the mechanisms involved in IDD, the combination of MMP/ADAMTS inhibition and other anti-IDD drugs may be more effective than a single medication in the treatment of IDD. In addition, to our knowledge, the promising results deriving from suppression of matrix degradation enzymes are restricted primarily to in vitro studies, animal models and human degenerate IVD explants. There is still a lack of studies performed in patients with degenerative disc disease. Thus, further clinical trials and randomized controlled studies are urgently required for a better definition of the effectiveness and safety of MMP or ADAMTS inhibition for the management of IVD degeneration. In summary, addressing these questions will provide insightful knowledge about the roles of MMPs and ADAMTSs in IDD etiology, and enhance our chances to design matrix degradation enzyme-centered biological therapy so as to help further improve the prognosis of degenerative disc disease patients in the future. Disclosure The authors have declared no conflict of interest. Acknowledgment The authors gratefully acknowledge the financial support from the Natural Science Foundation in Hunan Province, China (2015JJ5003). References [1] M.W. Lively, Sports medicine approach to low back pain, South. Med. J. 95 (2002) 642–646. [2] T. Kadow, G. Sowa, N. Vo, et al., Molecular basis of intervertebral disc degeneration and herniations: what are the important translational questions? Clin. Orthop. Relat. Res. 473 (2014) 1903–1912.

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