“Matrix metalloproteinases and their inhibitors in pulmonary hypertension” – A Review

This past week, I delved into the arena of Matrix Metalloproteinases and their role in disease and Pulmonary Hypertension. One of the first papers that I read with regards to this is by S.S. Pullamsetti et al. entitled “Matrix metalloproteinases and their inhibitors in pulmonary hypertension”. My review and summary of this paper is below.

Abbreviations:

ECs = Endothelial Cells
SMCs = Smooth Muscle Cells
ECM = Extracellular Matrix
PAH = Pulmonary Arterial Hypertension
MMPs = Matrix Metalloproteinases
TIMPs = Tissue Inhibitors of Matrix Metalloproteinases

In PAH, complex remodeling is observed in the pulmonary vasculature that involves all components of the blood vessel. In addition to this complex vascular remodeling, there is enhanced synthesis and degradation of ECM components, imbalanced turnover, as well as accumulation of ECM components. It is believed that these processes within the ECM (imbalanced turnover, etc.) may contribute to the pathogenic vascular remodeling seen in PAH.

Overview of Blood Vessel and ECM Physiology

The blood vessel in the pulmonary artery is comprised of the following distinct layers: ECs (innermost layer of blood vessel, or intima), SMCs (middle layer of blood vessel, or media), adventitia (the outermost layer of the blood vessel), and the ECM (layer that is interspersed between all other layers).

The ECM of the vasculature is a complex matrix interspersed between all other layers of the blood vessel, and provides blood vessels with mechanical strength, elasticity, and compressibility. It also functions to maintain homeostasis within the vasculature since interactions and crosstalk between cells of the ECM, and between cells and the ECM components, tightly regulate cell migration, proliferation and differentiation.

The innermost layer of the blood vessels is comprised of EC’s, which line the pulmonary arteries and rest on a basement membrane for support. They are tightly regulated and altogether form a semi-permeable membrane. The EC’s are responsible for synthesizing vasoactive compounds, as well as components of the basal membrane on which it lies.

The middle layer, or media, contains several layers of SMCs in a concentric fashion, together with fenestrated elastic lamina. In muscular arteries, diffusion can occur between EC and SMCs due to the fenestrated layer of elastin separating the intima and media beneath the ECs.

The SMC’s in the pulmonary arteries can exist in multiple states: quiescent, synthetic, or intermediate. Normally, SMCs are quiescent and exhibit a differentiated contractile phenotype, regulating vascular tone in response to vasoactive compounds, maintaining contractility, and possessing low rates of proliferation and synthesis. However, the synthetic phenotype, which is usually observed in PH, exhibits increased proliferation, cell migration, synthesis (synthesizing and secreting multiple ECM components such as elastin, fibronectin and collagens), and ECM turnover.

It has been found that certain ECM components can play a role in determining the SMC phenotype. For example, “elastin, one of the major constituents in the ECM of the medial layer, is a potent autocrine regulator of vascular SMC activity. Besides rendering resilience and elasticity to the arteries, elastin is critical for stabilisation of the arterial structure by inducing a quiescent contractile state. Several in vitro studies indicate an inverse correlation between elastin expression and SMC proliferation.”

The adventitia is the thick outermost layer of the vasculature, of which fibroblasts are the most prominent cell. “Fibroblasts also regulate synthesis and secretion of adventitial ECM components such as collagen types I and III, which constitute the chief ECM content of the adventitia. Besides rendering structural integrity to the vasculature, adventitia has resident immunomodulatory and progenitor cell populations that contribute to the growth and repair processes of the vessel wall.”

Proteases of the ECM, and their inhibitors

ECM turnover and homeostasis is controlled by the balance between the proteolytic enzymes matrix metalloproteinases (MMPs) and serine elastases, as well as their respective inhibitors. There are a vast number of MMPs and they are grouped together and categorized into six groups according to their respective substrates as follows: “1) interstitial collagenases (MMP-1, MMP-8, MMP-13 and MMP-18); 2) type IV collagenases or gelatinases (MMP-2 and MMP-9); 3) stromelysins (MMP-3, MMP-10, MMP-11 and MMP-19); 4) matrilysins (MMP-7 and MMP-26); 5) transmembrane MMPs (membrane type (MT)-MMPs: MMP-14, MMP-15, MMP-16, MMP-17, MMP-24 and MMP-25); and 6) other MMPs (MMP-12, MMP-20, MMP-21, MMP-22, MMP-23, MMP-27 and MMP-28).”

MMPs are initially inactive (they are zymogens), and become active through proteolytic cleavage. After activation, cleavage can still occur, either from the MMP cleaving itself, or via exogeneous means, each of which results in smaller forms of MMP.

Once active, MMPs can degrade many components of the ECM and in doing so mediate a wide range of physiological processes including embryogenesis, angiogenesis, cellular reproduction, bone remodeling and tissue repair. They are also implicated in a variety pathological settings, such as PAH.

MMPs are controlled by transcription, translation, and posttranslational mechanisms, as well as by the activity of their endogenous inhibitors. There are two primary endogenous inhibitors of MMPs: 1) the tissue inhibitors of MMPs (TIMPs), and 2) Alpha 2-macroglobulin.

The TIMP family tightly regulates MMPs via reversible inhibition, and has four members, TIMP-1 through TIMP-4, which form complexes with MMPs in a 1:1 stoichiometric ratio through binding of the TIMP to the MMP active site. Of all the TIMPs, TIMP-3 is unique “due to its direct binding to ECM proteins, and thus can provide a means for stabilising MMP-TIMP complexes within the interstitial space. TIMP-3 has also been demonstrated to inhibit ADAM (a disintegrin and metalloproteinase), notably ADAM-17.”

Αlpha2-macroglobulin is an abundant plasma protein and is the other major endogenous inhibitor of MMP activity, specifically in the plasma. Alpha2-macroglobulin binds to MMP complexes, which are then recognized by macrophages/monocytes and removed via scavenger receptor-mediated endocytosis. Thus, inhibition of MMPs via alpha2-macroglobulin is an irreversible process.

Another enzyme present in the ECM that is similar to MMP is ADAM (A Disintegrin And Metalloproteinase). There are two forms of this enzyme: membrane bound (ADAMs) and secreted (ADAMTSs). “The domain structure of the ADAMs consists of a pro-peptide domain, a metalloprotease domain, a disintegrin domain, a cysteine- rich domain, an endothelial growth factor (EGF)-like domain, a transmembrane domain, and a cytoplasmic tail. Like most proteases, the ADAMs are initially synthesised as enzymatically inactive precursor proteins… [Upon activation, the] metalloprotease domain hydrolyses protein substrates such as cytokines and growth factors, as well as their respective receptors. This process is termed ectodomain shedding and ADAM is often termed “sheddases”. The disintegrin domain binds integrin receptors such as α9β1 and, therefore, mediates cell–cell and cell–matrix interactions.”

The different domains within the ADAM and ADAMTS enzymes implicate that these enzymes can participate in proliferation, migration, and angiogenesis processes. For example, the “EGF receptor ligands (amphiregulin and heparin-binding EGF) released/shed by ADAM-17 enhance cell proliferation of cancer cells and induce angiogenesis. In accordance, altered expression of specific ADAMs has been implicated in different diseases; their best documented role is in cancer formation and progression. However, to date, no data are available about the roles of those proteins in the pathogenesis of PH.”

Serine proteases are the last form of proteolytic enzymes within the ECM that are involved in degrading the ECM. Serine proteases are enzymes that cleave peptide bonds in proteins. “Among several serine elastases, neutrophil elastases secreted by neutrophils and… endogenous vascular elastase (EVE) have been strongly implicated in vascular diseases. EVE is produced by vascular SMCs and degrades elastin and several other ECM proteins. Regulatory control of serine elastase activity is accomplished by the concomitant production of endogenous inhibitors like α1-antitrypsin, α2-macroglobulin and elafin, with the most relevant inhibitor in vasculature being elafin.”

Expression and Regulation of MMPs

The major sources of MMPs in the pulmonary vasculature during physiological and pathological states are the endothelium, SMCs, and fibroblasts and immune cells that infiltrate the vessel wall. In both physiological and pathological states, biological, mechanical, haemodynamic and/or neurohormonal stimuli can modulate MMP expression and activity.

During either angiogenesis or wound-healing, the endothelium can be a major source of MMP expression and activity. Basal MMP expression is typically low in ECs, but MMP expression in ECs can be activated by angiogenic factors, pro-inflammatory molecules, as well as organic compounds such as plant-derived phorbol esters. VEGF, and angiogenic compound, was found to increase MMP-2 and reduce TIMP-1 and TIMP-2 in ECs. In addition to inducing MMP-2, the downregulation of TIMP by VEGF allows for activation of other pre-existing collagenases within the ECM. This most likely contributes to the invasive migration of endothelial cells into the basement membrane and interstitial matrix.

During pathological states, growth factors and cytokines can also modulate MMPs within the medial layer. “Exposure of cultured SMCs to pro-inflammatory molecules like interleukin (IL)-1α, tumour necrosis factor (TNF)-α, and oxidised low-density protein are shown to significantly increase the expression and activity of MT1-MMPs…”. In a separate study, it was found that “autocrine expression and activation of transforming growth factor (TGF)-β1 antagonises the platelet-derived growth factor (PDGF)-BB… induced upregulation of MMP-2 in SMCs during phenotypic modulation, suggesting TGF-β1 promotes contractile phenotype by modulating MMPs.” This is interesting since, while TGF-β1 is profibrotic and plays an active role in PH pathogenesis, it also plays a role in promoting differentiation of SMCs into quiescent phenotypes.

Within the adventitia, fibroblasts, which are typically quiescent, may become activated during growth/proliferation and can thus affect MMP activity. For example, during hypoxia, there is a significant increase in active MMP-2, TIMP-1 and TIMP-2 production in fibroblasts when compared to normoxia. MMP activity can also affect fibroblast activity: oxidative stress via reactive oxygen species (ROS) can increase MMP activity, and thus in turn modulate fibroblast proliferation and collagen synthesis.

Inflammatory cells are also a major source of MMPs and other proteases, as well as cytokines and growth factors within the vasculature. Major players typically are monocyte/macrophages, dendritic cells, neutrophils, mast cells, T-cells and B-cells, all of which are capable of infiltrating the vessel wall.

Exposure of human alveolar macrophages “to native or denatured collagen type I and III selectively stimulated the expression of interstitial collagenase and TIMPs, suggesting that ECM components can directly influence macrophage-mediated MMP secretion…”  Furthermore, immune cells can regulate MMPs via cytokines and other factors. For example, when “granulocyte-macrophage colony-stimulating factor is added in combination with either TNF-α or IL-1β-induced MMP-1 synthesis it synergistically enhanced MMP-9 and TIMP-1 expression. Whereas MMP-9 production is negatively regulated by IL-4, IL-10, IFN-β, IFN-γ and TGF-β in monocytes… Similarly, MMP-12 expression in human peripheral blood-derived macrophages is induced by several cytokines and growth factors including IL-1β, TNF-α, M-CSF, VEGF and PDGF-BB.”

Moreover, there are distinct patterns of MMP expression in circulating leukocytes and monocytes depending on their state of activation. These distinct patterns of MMP expression in monocytes can thus increase transmigratory capacity across certain barriers (e.g. the blood-brain barrier). In addition, T-cell extravasation into the ECM and perivascular tissue during inflammation is mediated by induction and localisation of MMP-2 on the surface of T-cells via interaction with vascular cell adhesion molecule-1 on ECs.

Neutrophils are another significant source of MMPs and other proteolytic enzymes. Neutrophils “participate in different inflammatory process by releasing enzymatically active neutrophil elastase, and other proteases including cathepsin G, proteinase 3, MMP-1, MMP-8, MMP-9 and MMP-12. In addition to the innate immune functions of neutrophils, several studies indicate the role of neutrophil-derived proteases in trans-endothelial neutrophil migration and in the initiation of the angiogenic switch. Specifically, neutrophil-derived MMP-9 is shown to play an important role in basal lamina type IV collagen degradation and in catalysing the angiogenic switch by facilitating MMP-9-dependent VEGF mobilisation and consequent pro-angiogenic signalling.”

Finally, mast cells are also players that can induce proteases in the ECM. While they are typically localized in the adventitia, they have also be found in the media of pulmonary arteries. Mast cells “release several factors including MMPs, neutral and serine proteinases, heparin, heparinase, tryptase, chymase, histamine, and angiogenic growth factors such as basic fibroblast growth factor (bFGF), VEGF and cytokines. Mast cell-derived neutral proteases like tryptase and chymase potentially mediate activation of latent MMPs in human carotid arteries and subsequently enhance angiogenic phenotypes and also degrade different components of BM.”

Physiological Functions of MMPs

MMPs have many functions in the vasculature. Aside from proteolysis, “MMPs play a crucial role in multiple physiological process like morphogenesis, angiogenesis, tissue remodelling and tissue repair, as well as modulating fundamental cellular processes like proliferation, migration, differentiation, apoptosis, permeability, host defence, release of ECM-bound chemotactic factors and chemokine processing.”

Angiogenesis

A complex process involving multiple steps that are regulated both in space and time, physiological angiogenesis typically involves the interaction of “multiple growth factors that modulate the complex cell–cell and cell–matrix interactions through activation of different proteolytic systems in the vascular microenvironment. In response to an angiogenic stimulus, the endothelial-derived MMPs mediate proteolytic degradation of endothelial cell–cell and EC–matrix interactions and facilitate ECs to acquire a proliferative and migratory or invasive phenotype. In particular, angiogenic factor-induced secretion of MMP-2, MMP-9 and MT1-MMP in vascular ECs is critical for EC proliferation, migration and vascular ECM invasion associated with sprouting angiogenesis.” MMP-2 and MMP-9 are critical for sprouting angiogenesis because they degrade type IV collagen, which is a major component of the basement membrane of the intima.

Furthermore, the process of proteolysis itself “liberates ECM-sequestered pro-angiogenic factors, exposes cryptic pro-angiogenic integrin binding sites in the vascular ECM and also generates pro-angiogenic fragments, which collectively trigger the angiogenic switch.”

Pathological angiogenesis, on the other hand, is a dysregulated process. While their role isn’t entirely clear, it appears that MMPs are also major players in this process as evidenced by studies done on knockout mice: “MMP-2 deficient mice displayed reduced rates of tumour neovascularisation, total vascular area, number of vessels and tumour progression, as well as normal embryonic development of the vascular system. However, the combined deficiency of MMP-2/MMP-9 in the experimental model of tumour angiogenesis displayed an impaired angiogenic and invasive phenotype with strong reduction of gelatinolytic activity… Similarly, FGF-2-induced angiogenic response was lacking in the MT1-MMP deficient mice, suggesting that MT1-MMP might be important for initiation of angiogenesis. Furthermore, studies with MMP inhibitors revealed strong inhibition of angiogenic responses both in vitro and in vivo.”

Cell Proliferation & Migration

MMPs both directly and indirectly regulate proliferation, migration, and apoptosis of ECs and SMCs. For example, MMP-2 and MMP-9 act in synergy with MT1-MMPs to promote EC migration by proteolytically remodelling the BM, controlling the dissolution of ECM components, and releasing ECM derived chemotactic factors. It has been observed that when new vessels are being formed, at the leading edge of the developing vessel, there is EC proliferation, MT1-MMP-dependent activation of MMP-2, and MT1-MMP-dependent collagenolysis.

Arterial injury can induce medial hyperplasia that involves proliferation and migration of SMCs, as well as ECM remodelling. This process is regulated by various cytokines and growth factors, e.g. PDGF. In medial explants, it was found that migration of SMCs induced by PDGF-BB or -AB “was mediated by MMP-2, whereas the stimulatory effect of bFGF on medial SMC migration was mediated by both MMP-2 and MMP-9. Administration of potent MMP inhibitor (BB94) dose-dependently suppressed the PDGF-BB-induced migration of cultured SMCs and also suppressed the intimal thickening and medial SMC proliferation after arterial injury… Moreover, in situ and in vitro studies performed on pulmonary arteries and pulmonary artery smooth muscle cells (PASMCs) from patients showed increased MMP expression, suggesting a critical role for MMPs in different vascular remodelling processes that involves SMC proliferation, migration and intimal thickening.”

Cell Differentiation

As mentioned, SMCs can exist in multiple states. They can transition from a quiescent/contractile phenotype to a synthetic phenotype during either physiological or pathological conditions. Typically, the proliferative phenotype is migratory in nature and exhibits increased proteolytic activity and ECM turnover. This phenotype also undergoes a loss of cellular differentiation markers like myosin bundles and α-actin.

MMP-2 upregulation has been associated with the process of PDGF-BB induced SMC dedifferentiation from a quiescent to a synthetic phenotype. Although, as mentioned, TGF-β antagonizes this upregulation. It was also found that TGF-β upregulates TIMP-1 mRNA levels, further showing how TGF-β can promote a contractile SMC phenotype by altering MMP/TIMP ratios.

Moreover, both MMPs and serine elastases can produce elastin peptides within the ECM which stimulates the production of fibronectin. Fibronectin also influences the SMC phenotypic, switching it from a contractile to a synthetic state.

Interestingly, with regards to fibroblasts, “the homeostatic relationship between resident fibroblasts and the collagen matrix keeps them in a quiescent, undifferentiated state. However, normal fibroblasts subjected to hypoxia display hypoxia-induced phenotypic switching to myofibroblasts, through the MMP-2/TIMP mediated pathway. Specifically, PDGF, TGF-β1, tenascin-C (TN-C), fibronectin and ET-1 exhibit mitogenic activity and induce the myofibroblast phenotype both in vitro and in vivo, probably by regulating the MMP/TIMP balance.”

Vascular Permeability and Cell Adhesion

The integrity and permeability of the EC barrier, as well as the quiescent EC phenotype, is usually sustained through cell-matrix and cell-cell interactions. However, pro-angiogenic factors such as VEGF and angiopoietins also regulate EC barrier, permeability, and phenotype: “VEGF promotes vascular permeability by uncoupling inter-endothelial junctions, inducing the formation of endothelial fenestrae and an increase in modification of caveolae. A recent study revealed that the hypoxia-induced vascular leakage in vivo and the associated rearrangement of tight junction protein occludin and its diminished expression is mediated by hypoxia-induced MMP-9 activation. However, VEGF inhibition attenuated vascular leakage, hypoxia-induced MMP-9 activation and dependent gelatinolytic activity. Also, MMP inhibition attenuated vascular hyperpermeability, and prevented gap formation and tight junction rearrangement.”

Integrins, cadherins, selectins and the immunoglobin family of endothelial cell adhesion molecules also play a role in normal cell–cell and cell–matrix interactions, as well as in pathophysiological events: “In particular, the αvβ3 and αvβ5 integrins are described to regulate endothelial cell adhesion and migration during angiogenesis and vasculogenesis. In addition, αvβ3 integrins interact with an array of ECM ligands such as vitronectin, fibronectin, collagen, laminin, von Willebrand factor, fibrinogen, osteopontin, thrombospondin and RGD-containing peptides, which are potential MMP substrates… Moreover, during endothelial sprouting, surface expression and MT1-MMP dependent activation of MMP-2 in ECs were found to be associated with integrin αvβ3 and TIMPs at basolateral focal contacts and mediate focal degradation of ECM.”

Cadherins are also known to play an important role in tissue cohesion and cell migration, as there is typically a correlation between tissue cohesion and E-cadherin expression. However, cells that exist in an environment with intermediary tissue cohesion but high MMP expression become migratory in nature. From MMP inhibition studies, it has been found “that the decrease in the expression of E-cadherin and increase in type IV collagenase activity (MMP-2 and MMP-9) would enhance detachment of tumour cells and facilitate invasion. In contrast, in ECs, an increase in VE-cadherin was observed in connection with the downregulated MMP production and pericellular proteolysis during stabilisation and maturation of the neovessel. This recent study confirms the mechanism of cell–cell contact dependent regulation of pericellular proteolysis in angiogenesis. This correlates with the finding that MMP inhibitor treatment enhanced colocalisation of cadherin/β-catenin at cell–cell contacts and promoted stabilisation of cadherin-mediated cell–cell adhesion in fibroblasts suggesting a possible feedback loop between MMP and cadherin/β-catenin systems.”

Mobilization of Growth Factors and Cytokine Processing

Aside from being a structural support for the vasculature, the ECM is a repository for a variety of compounds such as VEGF, bFGF, hepatocyte growth factor, insulin-like growth factor-1, TGF-β1 and connective tissue growth factor (CTGF). During digestion/proteolysis of the ECM, MMPs liberate these compounds: “VEGF is sequestered as an inactive form by the ECM components like CTGF, pleiotrophin, etc., resulting in reduced bioavailability. Several MMPs (MMP-1, -2, -7, -9, -16 and -19) mediate proteolytic cleavage of this inhibitory complex and increase the bioavailability of VEGF… Mice with targeted disruption of MMP-9 exhibited reduced hypertrophic cartilage vascularisation due to the lack of mobilisation of ECM-bound VEGF, confirming the essential role of MMP-9 in bioavailability of growth factors. In addition, in cultured SMCs, serine elastase (EVE) was shown to mediate degradation of elastin, which subsequently promotes liberation of ECM-bound bFGF.”

Proteolysis of ECM components by MMPs also release matrikines. Matrikines are fragmented matrix peptides that can regulate cell activities. For instance, proteolysis of the ECM components collagen and perlecan by MMPs generates anti-angiogenic matrikines (arrestin, canstatin, tumstatin, metastatin, endostatin, neostatin, vastatin, restin and endorepellin), whereas proteolysis of fibronectin, laminin, osteonectin and elastin liberates matrikines that promote cell proliferation, migration and angiogenesis.

MMPs also play a role in processing cytokines, such as TGF-β, which regulates cellular processes such as proliferation, migration, differentiation, cell survival and synthesis of vascular cells in the ECM. In the ECM, TGF-β isoforms are secreted and maintained in an inactive state by binding with elastic microfibrils within the ECM. Proteolytic enzymes such as MMP-9, MT1-MMP and plasmin degrade these microfibrils thus releasing the active form of TGF-β.

MMPs also influence inflammatory responses by mediating chemokines through alteration of their chemotactic properties: “MMP-2 mediated processing of CCL7/MCP-3 generated a cleaved MCP-3 that acts as a general chemokine antagonist and attenuates chemotaxis and the host inflammatory response in vivo.”

MMP Role in PAH Pathogenesis

MMP Expression in PAH

MMP/TIMP expression is altered/dysregulated in PAH:

• MT1-MMP is present in myofibroblasts and endothelial cells in onion-skin lesions and cellular plexiform lesions
• MT1-MMP and MMP-2 detected in myofibroblasts and endothelial cells in the cellular plexiform lesions
• Discontinuous type IV collagen and focal thinning was observed in onion-skin lesions and mature plexiform lesions
• MMP-2 expression and activity increased in PASMCs from IPAH patients
• MMP-3 is decreased and TIMP-1 is increased IPAH PASMCs, which favors ECM accumulation
• Circulating MMP-9 and TIMP-1 levels are increased in plasma of PH patients
• MMP-2, MMP-9, neutrophil collagenase (MMP-8), stromelysins (MMP-10, MMP-11), macrophage metalloelastase (MMP-12), MMP-20, PLAT (tPA) and SERPINB2 are upregulated in MCT-treated lungs.
• Transgenic expression of human MMP-9 in MCT-treated lungs resulted in extensive infiltration of macrophages. However, another study demonstrated that “macrophage-specific transgenic expression of human MMP-1 in a mouse model of MCT-induced PH resulted in attenuation of collagen deposition, SMC proliferation, infiltration of macrophages and consequently medial thickening.”
• MMP-2 expression is enhanced in both hypoxia- and MCT- induced PH in animal models
• Increased MMP-13 and collagenolytic activity was found in the pulmonary arteries of hypoxic rats.
• Increased serine elastase activity and elastin fragmentation was observed in pulmonary arteries of MCT-induced PAH rats.
• Preceding the development of MCT-induced PH, there is increased activity of endogenous vascular elastase (EVE)

However, despite the above, it appears that not all MMPs and proteolytic activity are “bad”. For example, there is a transient increase of MMP-1 and MMP-3 during normoxic recovery from hypoxia-induced PH. Even more interesting, there was a “significant increase in stromelysins and total proteolytic, collagenolytic and gelatinolytic activities was predominantly found in the media and adventitia of pulmonary arteries during normoxic recovery of hypertensive vessels. This observed increase in protease activity correlated with the rapid reduction in collagen and elastin content in pulmonary arteries, thus indicating a correlation between decreased vascular remodelling and increased MMP activity during early reversal of hypoxia-induced PH. In a similar study by Tozzi et al., increased activation of mast cell-derived interstitial collagenase was shown to mediate restoration of vascular architecture by facilitating collagen breakdown in remodelled pulmonary arteries during the early recovery phase from chronic hypoxia. Poiani et al. also reported accumulation of collagen and elastin in the main pulmonary arteries of rats during chronic hypoxia, while the normoxic recovery of hypertensive vessels were associated with decreased collagen and elastin content. Cumulatively, these experimental studies suggest an essential role of MMPs in reabsorption of vascular collagen during the de-remodelling process that occurs in the post-hypoxic recovery phase.”

Consequences of MMP dysregulation in PAH

• SMC phenotype switching that is associated with SMC hyperplasia, hypertrophy and migration are regulated by the homeostasis and turnover of ECM components such as elastin, collagen, and fibronectin within the medial layer. An increased deposition of collagen and elastin responsible for the medial thickening during PAH pathogenesis, and this thickening is responsible for the stiffening of the pulmonary arteries observed in PAH.
• Fragmentation and gaps in the internal elastic lamina have been observed in lung tissue from PAH patients and in patients with PAH and neointimal lesions. Animal studies have also shown that elastin fragmentation precedes the development of changes in vascular morphology. The production of fibronectin within the ECM, which switches the phenotype of the SMC to a proliferative/migratory state, is stimulated by serine elastases and MMPs.
• When MMP and elastase activity increases, TN-C is increased which enhances PASMC proliferation via positive regulation. Furthermore, increased “expression of the TN-C is associated with progression of clinical and experimental PH. In support, treatment of organ cultures with either MMP-2 or an elastase inhibitor resulted in suppression of TN-C expression, and regression of medial hypertrophy associated with PASMC apoptosis. Subsequent studies in cultured PASMCs documented that TN-C amplifies the mitogenic response to FGF-2 and is a prerequisite for EGF-dependent SMC proliferation.”
• Crosstalk between MMPs/serine elastases, and growth factors and pro-hypertensive molecules has been observed. For example, MMP activity is affected by BMP: “Knockdown of BMPR1A in human PASMCs reduced MMP-2 and MMP-9 activity, attenuated serum-induced proliferation, and impaired PDGF-BB-directed migration. Furthermore, knockdown of MMP-2 or MMP-9 recapitulated these abnormalities…”.
• Serine elastases, through degrading the ECM, release bFGF, which may be amplified by MMPs.
• “MMPs also positively regulate EGF-dependent SMC proliferation by promoting TN-C induced clustering of αvβ3 integrin receptor. In addition, studies from genetic or pharmacological ablation of serotonin receptor 5-HT2BR suggests that 5-HT2BR-dependent increased elastase activity and MMP/TIMP imbalance, subsequently leading to latent growth factor release, including TGF-β. Vice versa, evidence suggests that TGF-β1 stimulates expression of pro-MMP-9 in IL-1β treated PASMCs. IL-1β, a major cytokine associated with PH, was shown to markedly increase MMP-2 and MMP-9 gelatinase activity.”

MMP Inhibitors in Experimental Models of PH

MMP inhibition studies have mostly been performed on the hypoxia- and MCT- experimental animal models of PH. Elastase and MMP inhibitor therapy has proven effective in the MCT-induced PH model by apparently reducing TN-C, suppressing proliferation of SMCs and by inducing apoptosis. Furthermore, “adenovirus-mediated overexpression of human TIMP-1 gene in the lungs of rats exposed to MCT reduced pulmonary vascular remodelling, right ventricular hypertrophy, gelatinase activity and muscularisation of peripheral pulmonary arteries, suggesting that balancing the MMP/TIMP ratio can reverse the disease.”

However, MMP inhibition studies yield mixed results when performed on the hypoxia-induced PH model. In hypoxia induced PH, there is increased synthesis and accumulation of collagen and elastin in the pulmonary arteries. Since MMPs can also degrade collagen, inhibiting their activity could result in increased collagen deposition. Furthermore, “using inhibitors for collagen synthesis (cis-4-hydroxy-L-proline) and crosslinking (β-aminopropionitrile) in the rats exposed to chronic hypoxia, demonstrated reduction of excess vascular collagen and attenuation of PH. However, in contrast, Vieillard-Baron et al. demonstrated that intratracheal instillation of the adenovirus-mediated overexpression of human TIMP-1 gene or administration of a broad-spectrum MMP blocker (doxycycline) in rats subjected to chronic hypoxia was associated with increased muscularisation and periadventitial collagen accumulation in distal arteries.”

Serine elastase inhibitors have also shown promise in reversing PH. Serine elastase inhibitors have been shown to reverse advanced PH in the MCT-induced PH rat model as well as attenuate PH in the hypoxia-induced animal model. “Furthermore, transgenic mice that overexpress the serine elastases inhibitor elafin when exposed to chronic hypoxia demonstrated reduced serine elastase and MMP activity compared to the non-transgenic mice. Importantly, elafin-transgenic mice displayed reduced right ventricular pressure, reduced muscularisation and preservation of the number of distal vessels as compared with control or non-transgenic mice.”

*Side note: In this talk by Marlene Rabinovitch, it is mentioned that 1) circulating elastase levels are significantly elevated in all forms of PAH, and 2) elafin levels, which are natural elastase inhibitors and are usually elevated with elevated levels of elastase to match activity/maintain homeostasis, are decreased in all forms of PAH.*

What’s interesting is that pharmacological agents responsible for regression/reversal of established PH appear to function by modulating the MMP/TIMPs ratio in the ECM as evidenced by the following:

• “…inhalation of a combined selective PDE3/4 inhibitor (tolafentrine) exhibited anti-proliferative, anti-migratory and anti-remodelling effects, and consequently reversed MCT-induced PH in rats… due to downregulation or even normalisation of the deregulated profile of several MMPs and adhesion molecules…”.
• “Lercanidipine, a vasoselective dihydropyridine calcium channel blocker, demonstrated beneficial effects in patients with PH by decreasing elevated circulating MMP-9 levels. This lercanidipine-induced effect was associated with a significant decrease in MMP-9 activity without affecting proMMP-2 activity and TIMP-1 concentration…”
• “Similarly, administration of a third-generation calcium channel blocker, amlodipine, immediately followed by MCT treatment suppressed the MCT-induced increase in MMP-2 activity, platelet activation, EC damage and SMC proliferation, and consequently inhibited the development of PH.”
• “Administration of the FDA-approved drug for PH, bosentan… also attenuated the MCT-induced upregulation of MMP-2, TIMP-1, endothelial NO synthase expression and MCT-induced PH.”
• “Amelioration of MCT-induced PH by fluoxetine (selective serotonin reuptake inhibitor) treatment was associated with suppression of MMP-2, MMP-9, TIMP-1 and TIMP-2 expression.”
• “…captopril (an angiotensin-converting enzyme inhibitor) and losartan (angiotensin II type 1 receptor antagonist) administration attenuated pulmonary vascular remodelling, probably associated with the regulation of the expressions of MMP-2, MMP-9 and TIMP-1, in pneumonectomy plus MCT injection-induced severe PAH.”

Overall, selective or complete MMP inhibition can either attenuate or enhance vascular remodelling and PH, depending on the circumstance and how the PH is initiated. As a result, using selective or complete MMP inhibition is not a currently effective strategy for treating PH.

MMP inhibitor therapy may be a viable treatment for PH once we understand more about MMPs, their functions, as well as their natural inhibitors. Increasing the selectivity of MMP inhibitors, as well as honing in on primary MMP culprits in PH is most likely the key in establishing MMP inhibitor therapy as an effective treatment for PH.

Furthermore, since there are multiple subgroups of PAH, it is most likely critical to obtain the MMP/TIMP profile of each subgroup.

KEY TAKEAWAYS/HIGHLIGHTS:

• Elastin is crucial for inducing a quiescent/differentiated SMC state
• VEGF downregulates TIMP levels and may allow activation of collagenases and contribute to the invasion of ECs into the basement membrane and interstitial matrix.
• TGF-β1 is profibrotic, but promotes contractile phenotype in SMCs by decreasing PDGF-BB induced increase in MMP-2. It also attenuates expression of MMP-9 in mast cells. However, TGF-β activates fibroblasts to become myofibroblasts.
• During inflammation, T-cells can “leak” into perivascular tissue due to expression of MMP-2 on surface of T-cell via interaction with VCAM-1 on ECs.
• Neutrophil-derived proteases (specifically MMP-9) are responsible for trans-endothelial neutrophil migration and initiating angiogenesis.
• During hypoxia the tight junction protein occludin is rearranged and its presence is diminished, all due to MMP-9 activation
• Pericellular proteolysis due to increased MMPs is associated with angiogenesis and dysregulated angiogenesis. VE-cadherin expression is associated with decreased MMPs and proteolysis, as well as with stabilization and maturation of blood vessels.  
• MMP-9 (and others) enhances the bioavailability of growth factors in the vasculature via degradation of ECM components.
• MMP-2 can attenuate inflammation via altering properties of chemokines
• Proteolysis of collagen and perlecan by MMPs generates anti-angiogenic matrikines (arrestin, canstatin, tumstatin, metastatin, endostatin, neostatin, vastatin, restin and endorepellin), whereas proteolysis of fibronectin, laminin, osteonectin and elastin liberates matrikines that promote cell proliferation, migration and angiogenesis.
• MMP-1 may be responsible for attenuation of collagen deposition, SMC proliferation, infiltration of macrophages and thickening of the media.
• Not all MMPs and proteolytic activity are “bad”. MMP-1 and MMP-3 increases during recovery from hypoxia induced PH. There is also increased proteolytic activity to reduce collagen and elastin content, as well as activation of mast cell-derived interstitial collagenase (to help facilitate collagen breakdown) in pulmonary arteries during the early recovery from chronic hypoxia.
• It appears that both excess proteolytic activity due to excess MMP activity, as well as excess collagen synthesis due to low MMP activity contribute to PH pathogenesis. Thus, this provides weight to the case that there must be a delicate balance of MMP/TIMP (proteases and their inhibitors) within the ECM.
• In PAH, there is a deregulated expression and activity profile of MMP/TIMP
• Overall, selective or complete MMP inhibition can either attenuate or enhance vascular remodelling and PH, depending on the circumstance. Thus, using selective or complete MMP inhibition is not a currently effective strategy for treating PH, and more studies are warranted. This may be due to the fact that there are particular MMP/TIMP profiles for both physiological and pathological states; certain MMPs may be “good” in one scenario but “bad” in another.
• Most clinical trials of MMP inhibitors fail to demonstrate efficacy since they induce severe side effects (e.g. musculoskeletal syndrome) most likely due to their lack of selectivity and poor oral bioavailability.