The Role of ADAMTS-4 in Atherosclerosis and Vessel Wall Abnormalities

Introduction

The extracellular matrix (ECM) is a dynamic three-dimensional scaffold composed of fibrillar macromolecules that provide structure for tissue cells to reside in. Specific proteolytic enzymes of the ECM mediate its continuous and controlled remodeling, a feature that is of vital importance throughout the life of all multicellular organisms [1]. The physiology of some major ECM proteins is regulated by metzincin proteases, including the disintegrin metalloproteinases with thrombospondin motifs (ADAMTS) [2]. This family belongs to the metzincin protease superfamily that has one conserved Met residue in the metalloproteinase domain (MPD) and a catalytic zinc ion in the active site [3^, 4]. The ADAMTS family includes 19 secreted metalloproteinases and seven ADAMTS-like proteins that have no catalytic activity [5^, 6]. They are largely evolutionarily conserved, reflecting their ubiquitous importance in embryogenesis and tissue homeostasis. Unsurprisingly, ADAMTS family members have recently been implicated in the pathophysiology of a growing number of vascular diseases, such as coronary artery disease (CAD) and coagulation disorders [5]. This review focuses on ADAMTS-4 and its implications in vascular pathology. ADAMTS-4 (also known as aggrecanase-1) is expressed by an array of tissues, most prominently in endocrine organs, lungs, and brain, but also in the cardiovascular system [7^, 8]. Notably, the majority of ADAMTS-4 substrates are principal proteoglycans expressed physiologically in smooth muscle cells (SMCs) of blood vessels and the developing heart. A good example is versican, a large ECM proteoglycan that is targeted by ADAMTS-4, which is an important reason why this metzincin protease was originally implicated in the pathophysiology of vascular diseases with an atherosclerotic background [9]. Atherosclerosis is a major underlying cause of cardiovascular and cerebrovascular disease and plays a crucial role in aortic aneurysms and dissections; therefore, the necessity to scrutinize the role of ADAMTS-4 in plaque formation and progression is apparent [5^, 10^, 11]. Therefore, this review highlights the current state of knowledge on the specific functions and action mechanisms of ADAMTS-4 in the complex pathophysiology of major vascular diseases.

Molecular Structure and Expression of ADAMTS-4

The architecture of ADAMTS-4 is rather simple in comparison to other ADAMTS family members: it consists of a proteinase domain and ancillary domains that is rather simple in comparison to other ADAMTS family members. This is a basic template that is then, didactically speaking, “upgraded” by protein and glycan structures in other ADAMTS molecules. Changing structure reflects various steps the protein passes as it traverses discrete cellular compartments on its way towards secretion and activation in the extracellular space. The ADAMTS-4 sequence starts with a signal peptide that targets the protein for secretion, followed by a prodomain whose cleavage activates the coming latent MPD (Fig. 1). The activity of MPD is likely strongly influenced by the neighboring disintegrin-like domain. This domain interacts with the substrates of ADAMTS-4 through a rather peculiar active site that can have an open or closed conformation, depending on whether a Ca²⁺ ion is bound to the domain [12]. This rather uncommon metzincin feature might serve the function of binding to accessory proteins, substrates, or even to ADAMTS-domains. The structure ends with an ancillary domain that likely also modulates ADAMTS-4 activity through ECM binding, regulation of proteinase activity, and substrate specificity fine-tuning [13]. This domain consists of a thrombospondin type I motif (TSR), a cysteine-rich domain, and a spacer domain, and it varies in other ADAMTS family members in the number of TSR and other specialized domains.

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Although the expression regulation of ADAMTS-4 is not fully understood, it is tightly controlled on multiple levels, including transcription, translation and through physiologic enhancers or inhibitors. Several enhancers like interleukin 1 (IL-1), tumor necrosis factor, transforming growth factor beta (TGF-β), IL-17, fibronectin, retinoic acid, and neprilysin have been identified. Conversely, alpha-2-macroglobulin and tissue inhibitor of metalloproteinases-3 (TIMP-3) are known to be its physiologic inhibitors [14^, 15]. Additionally, TIMP-3 inhibition appears to be dependent upon ADAMTS-4 TSR and SR interaction with its aggrecan glycosaminoglycan chains [2^, 16].

The path toward synthesizing a fully functional ADAMTS-4 molecule starts by intracellular cleavage of the prodomain, after which the protein is transported to the ECM and further activated by discrete proteases. In contrast to other ADAMTS family members which are processed by furin in the extracellular space (e.g., pro-ADAMTS-5 and -9), ADAMTS-4 is initially processed within the cell [7]. Latent ADAMTS-4 is cleaved in the trans-Golgi network at multiple N-terminal sites by furin proprotein convertase (Fig. 1) [3]. This cleavage releases the signal peptide and prodomain, yielding three forms (68, 53, and 40 kDa) that are secreted into the extracellular space [17]. Once outside the cell, ADAMTS-4 binds to the ECM, where the processing continues at the C-terminal part, mediated by matrix metalloproteinase (MMP)-17 (MT4-MMP) or autocatalytically [18^-21] (Fig. 1). The cleavage of the C-terminal spacer domain is a physiologic event that seems to release ADAMTS-4 from the ECM. It also activates the proteolytic function and likely increases its substrate range, thus regulating the processing of ECM proteoglycans [22]. Once the protein gets trafficked to the ECM, its biologic functions (as with other aggrecanases) are dependent upon the interplay between the specificity of its exosites that bind potential substrates, and the high specificity of the actual protease cleavage site. One of the main targets of ADAMTS-4 is the proteoglycan aggrecan (accordingly its initial label was aggrecanase-1); however, its family member, ADAMTS-5, was shown to be 20 times more active in degrading aggrecan [23]. Additionally, ADAMTS-4 cleaves other members of the hyalectan family including versican 1, -2, and brevican [17^, 24], hence the protein was also named as “hyalectanase” [22]. Other substrates of ADAMTS-4 include a known MMP inhibitor alpha-2-macroglobulin and ECM modulators like decorin and fibromodulin and also cartilage oligomeric matrix protein and transferrin [14^, 25^, 26]. Obika et al. [27] showed that ADAMTS-4 might also play a role in the degradation of biglycan, an ECM proteoglycan.

The Role of ADAMTS-4 in Atherosclerosis and Resulting Vascular Diseases

Atherosclerosis is the result of fatty and/or fibrous material accumulation in the inner wall of arteries [10]. Plaques mostly consist of lipids, cholesterol, inflammatory cells, and apoptosis debris; they reduce blood flow and increase the risk of blood clot formation and embolization. A hallmark of progressive lesion formation is inflammation that favors cell adhesion to the vascular endothelium through specific glycoproteins. The lesions initiate on damaged endothelial cells which raise their expression of adhesive molecules, decrease secretion of nitric oxide, and other compounds. The net result is enhanced adhesion of pro-inflammatory low-density lipoproteins (LDLs), monocytes/macrophages, and thrombocytes on vessel walls (Fig. 2). The LDL is oxidized which in turn drives macrophage phagocytosis and pro-inflammatory cytokine secretion, and over time lipid-laden macrophages transform to foamy cells that accumulate to clumps (Fig. 2). Inflammatory cytokines secreted by the immune cells foster the migration of SMCs which contribute to LDL uptake and migrate to the forming plaque. Chronic inflammation triggers fibrosis which leads to the accumulation of dense, irregular connective tissue that hardens blood vessels [2^, 9^, 10^, 13^, 28^-30].

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ADAMTS family members are recognized to play significant roles in the process of plaque formation [9^, 31]. Their importance lies partly in the control of expression levels of versican, one of the most abundant proteoglycans produced by SMCs [32]. Versican has numerous roles that promote vascular disease progression through cell proliferation, migration, adhesion, and ECM remodeling [33]. In stress conditions, like increased blood flow and shear stress, the damaged intimal SMCs overexpress versican and aggrecan molecules which are then cleaved by ADAMTS-4. This in turn promotes SMC apoptosis and worsens blood vessel atrophy, further driving ADAMTS-4 production, which was shown to be synthesized by endothelial cells and localized to endosomes on the cell surface [9^, 27^, 34^-36]. Moreover, the pro-inflammatory microenvironment present in the plaque drives the expression of ADAMTS-4 enzymes by macrophages and macrophage-like cells derived from vascular SMCs [9^, 10^, 28^, 37]. In brief, the pro-inflammatory plaque microenvironment supports a positive feedback loop where versican and aggrecan enhance plaque formation and corrode vessel walls (Fig. 2). Cleavage of these proteoglycans by ADAMTS-4 further destabilizes the atherosclerotic plaques leading to unfavorable patient outcomes [5^, 34^, 36^, 38].

Over time, the ECM degrades which can lead to breaks in the formed atherosclerotic plaques. This process can result in thrombosis with life-threatening complications, such as myocardial infarction or ischemic stroke. ADAMTS-1, -4, -5, and -7 also regulate the long-term stability and/or lipid deposition in the atherosclerotic plaque [5^, 39]. However, it is noteworthy that Lee et al. [39] indicated that ADAMTS-1 is more likely to be involved in plaque destabilization than -4 or -5. Furthermore, genome-wide association studies revealed functionally relevant single nucleotide polymorphisms in the ADAMTS-7 locus which were associated with CAD [5]. However, to the best of our knowledge, no such associations have been suggested for ADAMTS-4 in relation to CAD. Interestingly, in ADAMTS-4 knockout (KO) mice, a decrease in high fat diet-induced atherosclerosis and increased plaque stability was observed [40]. The impact of ADAMTS-4 on the development and progression of atherosclerosis is clearly present; however, its potential therapeutic use through direct targeting is questionable due to its diverse roles in ECM homeostasis and the seemingly overlapping functions of ADAMTS family members.

Conclusion

ADAMTS proteases are a highly conserved family of proteins that are expressed throughout different tissues and play important roles in differentiation and homeostasis. Their dysregulation is thus expectedly implicated in the pathogenesis and progression of numerous diseases. Here, we highlighted the emerging role ADAMTS-4 might play in vascular pathology and appreciated its importance in atherosclerosis and vessel wall abnormalities, as well as in diseases resulting from these two pathophysiologic processes. However, it is important not to oversimplify the role of ADAMTS-4 and not to consider it to be a sole culprit for the progression of these conditions: ADAMTS-4 is likely only a small piece of the complex puzzle that are the pathogeneses of atherosclerosis and vessel wall abnormalities. ADAMTS-4 exerts its effects in concert with a myriad of other molecules, probably in a time and tissue-context dependent matter. Furthermore, ADAMTS-4 is a highly conserved and pleiotropic molecule that exerts a plethora of functions, not limited to vasculature. For these reasons and due to the lack of such evidence, we believe that the possibility of therapeutic potential of ADAMTS-4 inhibition in the context of halting vascular disease progression at this point is somewhat premature, although the presented evidence indicates possible merits of this approach. For the near future, it appears to be more likely, that ADAMTS-4 might find its clinical purpose as a biomarker for vascular disease, perhaps as one of the indicators in a larger cytokine panel. However, further clinical and preclinical research is needed to clearly elucidate the role of this molecule and to expound the findings that were thus far made.

Conflict of Interest Statement

The authors do not have any conflicts of interest to disclose.

Funding Sources

This work was supported by the Scientific Center of Excellence for Reproductive and Regenerative Medicine (project “Reproductive and regenerative medicine – exploration of new platforms and potentials,”) Grant Agreement KK.01.1.1.01.0008 which is funded by the European Union through the European Regional Development Fund.

Author Contributions

L.G., R.N., S.H., G.S., J.B., and L.M. all made substantial contributions to the manuscript or conception and design of the work. L.G. contributed to conceptualization ideas, manuscript writing, and supervision; R.N. contributed to conceptualization ideas, manuscript writing, and design of figures; S.H., G.S., J.B. contributed to conceptualization ideas and manuscript writing; L.M. contributed to manuscript writing and design of figures. All authors read and approved the final manuscript.