Keywords

MMPs Structure, Function and Regulation

Matrix metalloproteinases (MMPs) constitute a group of over 20 structurally-related proteins which include a Zn++ ion binding site that is essential for their proteolytic activities. These enzymes play important role in extracellular matrix (ECM) turnover in order to maintain a proper balance in its synthesis and degradation [1]. Thus, MMPs substrates are mainly collagens, but also many other ECM proteins, including fibronectin, laminin, tenascin, aggrecan and osteonectin [2].

MMPs can be classified in four groups accordingly to their domain organization, sequence similarities and their substrate specificity. The group of (i) gelatinases comprises MMP-2 and MMP-9 and is characterized by an additional domain, called collagen binding domain, which represents the preferential binding domain for fibrillar collagen I [3]. MMP-2 is generally expressed in endothelial cells, fibroblasts, keratinocytes and chondrocytes; while MMP-9 is present in alveolar macrophages, trophoblasts, osteoclasts and polymorphonuclear leukocytes [4, 5]. The group of (ii) matrilysins is composed of MMP-7 and MMP-26 which are able to process collagen IV but not collagen I [6], and are involved in degradation of ECM constituents in the uterus at postpartum and implantation of embryos [7]. The (iii) typical MMPs group comprises three subgroups, namely stromelysins (MMP-3,-10 and-11), which participate of ECM turnover but are not able to cleave fibrillar collagen I; Collagenases (MMP-1,-8 and -13), which are responsible for degrading several native collagen (type I-V, XI) and other MMPs such as MMP-12, MMP-19, MMP-20 and MMP-27 [4, 8]. The last group is referred as (iv) furin-activated MMPs and consists of membrane type MMPs (MT-1,-2,-3,-4,-5-MMPs), transmembrane MMPs type II and secreted MMPs [4]. With a different structural arrangement from that of MMPs, the disintegrin and metalloproteinase domain with thrombospondin-1 motifs (ADAMTS) is another family of metalloproteinases and consists of 19 members which specifically process proteoglycans, in particular aggrecan, and pro-collagen [9, 10].

The MMPs are synthesized as inactive zymogens with a pro-peptide domain that must be withdrawn by several proteases, such as plasmin, thrombin, chimase, and MT-MMPs for protease activation. Activated MMPs can be either cell-secreted or membrane-bound exhibiting catalytic activity at the cell surface, intracellularly and extracellularly [5]. Due to its high proteolytic potential, MMPs are subjected to tight control and restrictive regulatory mechanisms that maintain homeostasis at the intracellular and extracellular level. One of the regulation mechanisms of the MMPs activity is by their endogenous inhibitors, tissue inhibitors of MMPs (TIMPs), which comprise a family of four members (TIMP-1 to 4) [8]. TIMPs are expressed in a tissue specific pattern and can operate through direct MMPs inhibition or by controlling their activation [11].

MMPs and TIMPs are associated to several physiological processes such as embryogenesis, wound healing, proliferation, cell motility, remodeling, angiogenesis and key reproductive events [4, 8]. On the other hand, recent evidence has shown that pathologically increased MMP activity or imbalance between MMP and TIMP expression can exacerbate a variety of diseases [12]. Indeed, MMPs are correlated to the pathogenesis of abnormalities of the growth plate and wound healing, heart failure, arthritic synovial joints, cancer, periodontal disease, ischemic brain injury [13] and even tendinopathies [14]. This chapter aims to relate MMPs changes in the diabetes mellitus (DM) pathogenesis with special focus on the tendon tissue.

MMPs and Pathogenesis of Diabetic Complications

DM is a group of metabolic disorders considered one of the major health problems worldwide [15, 16]. Chronic hyperglycemia resulting from defects in the secretion and/or insulin action is the main feature of the disease and may lead to vascular damage in vessels, nerves and many internal organs including the eyes, kidneys and heart.

Currently, the development mechanism of DM and its complications is subject of intense research. It has been observed the involvement of the MMPs in the development and progression of various complications of DM [17] and proteases are speculated to have importance for diabetes pathogenesis [18]. Previous studies have demonstrated higher concentrations of MMPs in the serum from type 1 DM [19], type 2 DM patients [20] and in a cell culture model for type 2 DM [21]. By using cultures of endothelial cells, Death and colleagues [22] demonstrated that hyperglycemia enhanced activity and expression of MMP-1,-2, and macrophage-derived MMP-9; while MMP-3 and TIMP-1 had expression and protein levels decreased and unaffected, respectively. These results indicate augmented ECM degradation in hyperglycemic conditions and suggest that MMPs can be useful markers for risk and/or progression of diabetic complications such as diabetic nephropathy and microangiopathy [23].

High levels of blood glucose are also associated to dysfunction of several intracellular signal transduction cascades including generation of reactive oxygen species (ROS), modulation of protein kinases and accumulation of advanced glycation end products (AGEs) (see Chap. 18) [24]. The receptor for AGE (RAGE) represents the main binding site for AGEs on resident and/or inflammatory cells and this interaction is capable to stimulate the production, expression and activity of MMPs in specific cell lines [25]. RAGEs are mainly located on the surface of macrophages, where they bind to the ECM proteins.

It is recognized that AGEs are able to affect the production and structure of ECM proteins, mainly due to formation of collagen cross-links. Aside from altered mechanical properties of collagen, AGEs can modify the interaction of collagen with other molecules such as proteoglycans (PGs), MMPs and cell integrins [26]. This collagen modification by AGEs have been correlated to inhibited wound repair and exacerbated inflammation in a rotator cuff tendon-bone [27] and medial collateral ligament [28] healing models. If the mechanical effects of AGEs in the collagenous tissue is not well understood, it is known that AGE cross-links are involved in reduced remodeling capacity due to reduced sensitivity to collagenases [29, 30], possibly via alterations in collagen solubility and augmented collagen resistance to protease breakdown [26, 31].

In some tissues, matrix-glycation products and AGEs formation can attract and stimulate cells to release cytokines (e.g., IL-1 and IL-6), MMPs and growth factors (e.g., TNF-α) during wound repair, which in turn accelerates the inflammatory response [32]. It should be noted that certain growth factors (e.g., TGF-β and CTGF) seem to have a role in the pathogenesis of long-term diabetic complications since they have increased concentrations and are associated to ECM accumulation in diabetic nephropathy [17] and tenocyte death and scar formation in tendons [33]. In addition, elevated levels of inducible nitric oxide synthase (iNOS) and prostaglandin E2 (PGE2), disturbance of the insulin secretion/action and its key signalling proteins, genetic polymorphism of MMPs and participation of oxidative stress have all been attributed to play a role in the elevated MMP expression during hyperglycemia in diabetic patients and models [25, 34].

MMPs and Diabetic Tendon Disorders: Current Findings

A growing body of evidence has associated DM to changes in the tendon structure, including abnormalities in fiber structure and organization [35], increased tendon thickness, volume and disorganization obtained by image findings [36, 37], and a tendency of impairing biomechanical properties [38, 39]. Interestingly, these tendon alterations may represent features of the ECM, which is in a constant state of dynamic equilibrium between synthesis and degradation [40].

Besides the relevance of MMPs in the ECM turnover and their role in the tendon pathophysiology [41], data linking these proteases to the development and progression of diabetic tendon disorders is still scarce. In a recent review, Shi and colleagues [39] highlighted the participation of the MMPs in the tendon alterations based in two studies. The first work, from Ahmed and co-workers [42], aimed to investigate the mechanical properties and expressional changes of genes involved in the tendon healing process in a diabetic rodent model . The authors found strong expression of MMP-13 and MMP-3 in tenocytes both in diabetic and healthy tendons; whereas MMP-13 expression was upregulated and MMP-3 expression decreased in the tendon healing model . Moreover, Tsai and others [43] found upregulation of MMP-9 and MMP-13 and increased enzymatic activity of MMP-9 in a tendon cells culture treated with high glucose concentration. Overall, these results indicate that hyperglycemia might induce collagen degradation by increasing MMPs activity and lead to a low-quality tendon structure [39].

MMPs are also implicated in the development of diabetic fibrosis, a pathological finding characterized by ECM accumulation with or without changes in the ECM composition. Cardiac fibrosis, for example, is a major feature of diabetic cardiomyopathy and results in cardiac dysfunction [44]. Fibrosis is often a result of an imbalance between MMPs and TIMPs activity after tissue insults such as hyperglycemia, dyslipidemia and hypertension [17], but increased production of collagen and other ECM components may be also involved. To date, it is not clear if DM results in fibrosis in uninjuried or healing tendons. Previously, a streptozotocin (STZ)-induced diabetes study in Achilles tendons of rats demonstrated areas of higher density of collagen I and collagen fibers disorganization when compared to the controls [45]. It was suggested that increased vascularization associated with cell proliferation and possible migration caused hypercellularity and accounted for over-deposition of collagen in the diabetic group [45]. In fact, recruitment of inflammatory cells have been connected to dysregulation of tenocyte homeostasis followed by secretion of ECM proteins, which results in an increased turnover and remodeling of the tendon ECM [41, 46]. Moreover, MMPs are able to increase release of TGF-β1 (resident in the tendon’s matrix) which in turn may lead to an increased fibroblasts proliferation and collagen I deposition [47]. In this manner, a relationship between MMP/TIMP system and pro-fibrotic growth factors might contribute to fibrosis development due to ECM turnover dysregulation [17].

As previously mentioned, DM is known to produce a poor wound healing due to complications in the connective tissue metabolism, and higher expression of gelatinases in diabetes might be due to the prolonged inflammatory period [48]. In a recent work with STZ-induced diabetes in rats, Mohsenifar et al [49] showed impaired mechanical properties and altered inflammatory response in the diabetic group after Achilles tenotomy. The authors also found lower collagen content in the diabetic group at 15 days after surgery. Taken together, these results corroborate with other studies [42, 50] that point out for a delayed healing process in tendons of diabetic animals. It is noteworthy to mention that different MMPs operate in tendons, participating not only of the ECM degradation but also promoting micro-trauma healing and maintaining normal tendon function [41]. In this manner, besides augmented collagen turnover, other mechanism associated to the pathogenesis of tendinopathies may involve a “failed healing response” via decreased MMP-3 activity, for example [41]. Thus, although not fully elucidated, it is reasonable to highlight the participation of regulators of the ECM turnover (such as MMPs and others) in the tendon’s delayed healing response.

Conclusion

MMPs play a role in the mechanisms of several diabetic complications. Although little studied, evidence suggests that diabetes-induced MMP dysregulation may contribute to structural and biomechanical alterations and impair the healing process in tendons.