Cysteine Proteases in Atherosclerosis A cc ep te d A rt ic le This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagin[.]
Accepted Article Cysteine Proteases in Atherosclerosis Tommy Weiss Sadan1, Israel Gotsman2 and Galia Blum*1 The Institute for Drug Research, School of Pharmacy, Faculty of Medicine, Hebrew University, Jerusalem, Israel Heart Institute, Hadassah University Hospital, Jerusalem, Israel *Corresponding author Galia Blum, Ph.D Institute for Drug Research, The School of Pharmacy The Faculty of Medicine, The Hebrew University of Jerusalem Jerusalem, Israel, 9112001 Phones: 972-2-675-8682 Fax: 972-2-675-8230 Email: galiabl@ekmd.huji.ac.il Article type : Review Article Key words: Cathepsins, atherosclerosis, vascular inflammation, autophagy, cholesterol, metabolism and activity based probes This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record Please cite this article as doi: 10.1111/febs.14043 This article is protected by copyright All rights reserved Abbreviations: Accepted Article ABCA1 and ABCG1, ATP Binding Cassettes A1 and G1; agLDL, aggregated LDL; Apo-AI, Apolipoprotein-Al; ApoE, apolipoprotein E; CC, cholesterol crystals; Cys, cysteine; CysC, cystatine C; EC, endothelial cells; ECM, extracellular matrix; GAG, glycosaminoglycans; HDAC6, histone deacetylase 6; HDL, High Density Lipoprotein; His, histidine; Hyp, hydroxyproline; LDLR, LDL receptor; Lox-1, Lectin like oxidized LDL receptor-1; NLRP, nucleotide-binding oligomerization domainlike receptor protein; oxLDL, oxidized LDL; PPAR- γ peroxisome proliferator activator gamma; Pro, proline; RCT, reverse cholesterol transport; SMC, smooth muscle cells; SR-A, Scavenger Receptor class A; SREBP, Sterol Regulatory Element Binding Protein; TFEB, transcription factor EB; TLR, Toll like receptors Abstract: Atherosclerosis predisposes patients to cardiovascular diseases, such as myocardial infarction and stroke Instigation of vascular injury is triggered by retention of lipids and inflammatory cells in the vascular endothelium Whereas these vascular lesions develop in young adults and are mostly considered harmless, over time persistent inflammatory and remodeling processes will ultimately damage the arterial wall and cause a thrombotic event due to exposure of tissue factors into the lumen Evidence from human tissues and pre-clinical animal models have clearly established the role of cathepsin cysteine proteases in the development and progression of vascular lesions Hence, understanding the function of cathepsins in atherosclerosis is important for developing novel therapeutic strategies and advanced point of care diagnostics In this review we will describe the roles of cysteine cathepsins in different cellular process that become dysfunctional in atherosclerosis such as lipid metabolism, inflammation, apoptosis and how they contribute to arterial remodeling and atherogenesis Finally, we will explore new horizons in protease molecular imaging, which may potentially become a surrogate marker to identify future cardiovascular events This article is protected by copyright All rights reserved Introduction: Accepted Article The global burden of atherosclerosis remains challenging in face of high prevalence of obesity and its related metabolic disorders such as hyperlipidemia, diabetes mellitus and hypertension Atherosclerosis is a chronic inflammatory disease and the most frequent cause of ischemic heart disease, stroke and premature death [1] This slow and progressive disease involves mostly the aorta, coronary and carotid arteries favoring the curvatures and branching points where blood flow is mostly turbulent [2] Early atherosclerotic lesions develop as fatty streaks with lipidengorged macrophages (a.k.a foam cells) deposition under the arterial wall Over time, these fatty streaks enlarge and form the lipid core of the atheroma, which composed of foam cells, macrophages, T-cells and a large fibrous cap of smooth muscle cells (SMC) and extracellular matrix (ECM) [3] Whereas most of these vascular lesions remain harmless and show no clinical symptoms, some of them may ultimately progress into advanced lesions These lesions are characterized by a large necrotic core of apoptotic cells, cholesterol crystals, components of innate and adaptive immune system and tissue remodeling proteases that collectively weakening plaque stability and render them prone to rupture [4] Consequently, this stimulates a molecular cascade of platelet aggregation, thrombosis and vascular occlusion, which suddenly strikes as myocardial infarction or stroke [5] Studies of culprit lesions causing myocardial infarction underscore the role of proteases in the pathogenesis of cardiovascular diseases, as they are frequently characterized by thin fibrous cap and high activity of matrix degrading proteases such as the matrix metalloproteinases and cysteine cathepsins (hereafter referred as cathepsins) [6] This suggests that protease activity has a large impact on vascular pathology and hence, understanding the molecular mechanisms driven by protease function will provide new avenues for potential intervention points Cysteine Cathepsin Protease Super Family: Structure, Function and Regulation: Given that proteolytic activity has substantial impact on the transition from stable to pathologic atherosclerotic lesion, it is widely appreciated that cathepsins are pivotal in this process [7] Cathepsins belong to a broad family of the papain like proteases and the human genome encodes 11 proteins, namely cathepsins B, C, F, H, K, L, O, S, V/L2, W and X/Z Most cathepsins are endopeptidases (cathepsins F, K, L, S, V), cathepsins B and H also exhibit exopeptidase activity, while cathepsins C and X/Z and are strict exopeptidases [8, 9] Crystallographic structure and pairwise sequence alignment revealed highly conserved protein fold for most cathepsins with two major domains referred to as Left (L) and Right (R), Figure 1, that contribute the catalytic dyad cysteine and histidine [10, 11] These structural domains form the This article is protected by copyright All rights reserved active site cleft with histidine (His) emerging from the R domain and reactive cysteine (Cys) from L domain that drives a nucleophilic attack by an activated thiol towards the substrate’s amide Accepted Article carbon [12] Cathepsins are widely expressed in a variety of cells and tissues with an exception for cathepsins S and W, which are predominantly expressed in components of the immune system [13, 14] and http://www.proteinatlas.org/ This broad expression pattern in conjunction with their breadth of substrates and redundancy, as discovered by proteomics studies [15, 16] and analysis of substrate libraries [17], has led to conception that cathepsins are physiologically involved in protein turnover and degradation However, nowadays this view is refuted by evidences that cathepsins drive specific cellular functions such as cell cycle progression [18] or apoptosis [19] and that mice lacking the constitutively expressed cathepsin L display marked phenotypes in different tissues such as hair loss [20] or cardiomyopathy [21] Cathepsin expression is regulated by the transcription factors SP1, ETS1 and the lysosomal biogenesis regulator, transcription factor EB (TFEB) [22] Since proteolysis is a rapid and irreversible process, members of the C1 papain family including cathepsins are generated as inactive zymogens and post translationally modified by mannose 6-phosphate in the ER and Golgi to facilitate their delivery and isolation in the lysosomes [23, 24] Lysosomal cathepsin activity is further regulated by their prodomain region that folds on the catalytic site and prevents interaction with their substrates [10] Activation of cathepsin proteases is a complex and multistep process that involves the removal of the prodomain region by another protease or can be achieved by autocatalytic mechanism in acidic environments [9, 25] Cathepsins display optimal activity in acidic environments, with exception of cathepsin S that retains most of its activity even at slightly alkaline pH [26] Nevertheless, despite their natural preference for reducing environments such as the endo-lysosomal vesicles, cathepsins have also been found to act outside the lysosome In fact, cathepsins were found to participate in ECM remodeling [27], inflammation [28], cancer malignancy [29, 30] and atherosclerosis as will be discussed further in this review Extracellular cathepsin activity is enhanced in the presence of negatively charged polysaccharides, collectively termed glycosaminoglycans (GAG) [31] GAGs were found to stabilize cathepsin B, L and S and support their activity even at neutral pH [8] Furthermore, in case of cathepsin K, molecular interactions with GAG are essential for its collagenolytic function [31] Unregulated extracellular cathepsin activity underlie the long term sequela of inflammatory conditions such as atherosclerosis, therefore, their activity is counter balanced by the presence of endogenous inhibitors such as cystatins [32] Cystatins are small protein inhibitors 10-13 kDa that are present inside the cells (cystatin A and B) or extracellular in case of cystatin C [33, 34] Cystatins are potent inhibitors of cathepsins and their physiological importance is demonstrated by their broad immunomodulatory properties [35] and that low cystatin C (CysC) levels are associated with inflammatory conditions such as atherosclerosis and abdominal aortic aneurysms [36] Animal models This article is protected by copyright All rights reserved -/- Accepted Article further substantiate the role of CysC in atherogenesis as mice grafted with bone marrow from Cys +/+ developed larger atheromas compared to CysC cells [37] In conclusion, cathepsin activity is highly regulated process and it is becoming clear that unbalanced cathepsin activity is a major contributor to vascular injury Cysteine Cathepsin Expression in Atherosclerosis Human tissue analyses show that increased cathepsin expression and activity are implicated in a variety of inflammatory conditions including rheumatoid arthritis, abdominal aortic aneurysms and atherosclerosis [38, 39] In atherosclerosis, cathepsins are over expressed in almost every cell type comprising the plaque tissue, especially in macrophages, endothelial cells (EC) and smooth muscle cells (SMC) [40] For example, in human vascular lesions, expression of cathepsins K and S was significantly higher in SMCs and macrophages compared to normal tissues [41] Furthermore, cathepsin K expression in vascular endothelium positively correlated with the number of elastin breaks in advanced human atheromas [42], implying their contribution to vascular lesion complications Likewise, cathepsin L showed similar expression pattern in advanced human vascular lesions that colocalized with macrophages, SMCs and ECs [40] In vitro studies further elaborated on these findings by showing that inflammatory mediators such as IFN-γ, IL-1β and TNF- can elevate cathepsin expression and potentiate their catabolic activity of vascular constituents [40, 41] Furthermore, a recent paper by Stellos et al uncovered a novel mechanism through which IFN-γ and TNF- regulate cathepsin S expression in human vascular ECs, involving two independent cohorts Their findings show that inflammatory stimulation invokes changes in cathepsin S mRNA metabolism and stabilization, which ultimately enhances its translation in atherosclerotic lesions [43] Despite of convincing evidence indicating atypical expression of cathepsins in human vascular lesions, much of our understanding regarding cathepsin activity and function in atherosclerosis came from preclinical models such as ApoE deficient mice (ApoE -/-) and LDL receptor deficient mice (LDLR-/-) In ApoE-/- mice fed on high fat diet, cathepsin expression was higher in vascular lesions compared to controls [44] Furthermore, deficiency of cathepsin L or cathepsin S in the mice models attenuated atherosclerotic lesion burden [45, 46], strengthening the deduction that cathepsins have a pathogenic role in atherosclerosis Since these animal models closely resemble the remodeling sequence in humans, information gained from these animal models might be further translated to clinical practice This article is protected by copyright All rights reserved Cysteine Cathepsins Connect Lysosomal Function, Lipid Accepted Article Metabolism and Vascular Inflammation Lysosomal Cathepsins and Lipid Metabolism Since their early discovery, more than 70 years ago by Christian de Duve, the lysosomes were simply perceived as the cellular “waste bags” for managing bulk catabolic reactions in eukaryotic cells [47] Indeed, the lysosomes provide excellent environment for macromolecular degradation such as lipids, proteins and branched sugars due to their highly denaturing conditions and their diversity of hydrolytic enzymes Ever since their discovery, lysosomes have been widely appreciated for their myriad functions such as nutrient sensing, coordination of immune response and maintenance of cellular homeostasis by elimination of dangerous substances or damaged organelles [48-50] The lysosomes are insulated micro-domains composed of spherical phospholipid bilayer and present in virtually all cell types As a part of the endocytic network, the lysosomes communicate with the extracellular environment through endocytosis / phagocytosis in which extracellular matter is delivered by an endocytic vesicle budding from the cytoplasmic membrane [51] In addition, endogenous cargo is conveyed for degradation in the lysosomes in a process known as autophagy [52, 53] In the context of atherosclerosis, lysosomal trafficking mechanisms are considerably important as they go awry during the course of the disease and thus contribute to its pathogenesis One of the most significant hallmarks in early atheromas is the retention of lipid-laden macrophages in the arterial wall Normally, intracellular cholesterol levels are kept under very tight balance This is achieved when cholesterol is recognized by its cell surface receptor, low density lipoprotein receptor (LDL-R) and internalized into the lysosomes to liberate free cholesterol [54] Elevated cholesterol levels are sensed by the endoplasmic reticulum, which immediately turns-off the expression of LDL-R, driven by the transcription factors Sterol Regulatory Element Binding Protein (SREBP) [55, 56] In atherosclerosis, circulating LDL-cholesterol tend to aggregate (Ag-LDL) in focal regions of the arteries and are prone to chemical modification, especially in the form of oxidation (oxLDL) [57, 58] Modified cholesterol particles are recognized by the innate immune system through class of pattern recognition receptors (PRR) such as CD36, Scavenger Receptor class A (SR-A) and Lectin like oxidized LDL receptor-1 (Lox-1) Unlike LDL-R, cholesterol uptake through this alternative pathway is not subjected to negative feedback and thus, contribute to cholesterol build up especially in macrophages and triggers the formation of foam cells [59-61] Intriguingly, cathepsins were implicated in atherogenic lipoprotein modification in several studies For example, Ưưrni et al showed that LDL cleavage by cathepsin F resulted in aggregated and fused lipoprotein particles and this activity was potentiated (up to folds) by the presence of proteoglycans [62] This article is protected by copyright All rights reserved Accepted Article Furthermore, comparative gene expression analysis of human monocytes suggested that cathepsin H is a bona fide lipoprotein modifying enzymes In vitro analysis further confirmed this hypothesis as incubation of LDL particles with recombinant cathepsin H / Cholesterylesterase resulted in foam cell formation and folds increase in macrophages intracellular lipid content [63] Together these studies provide valuable insight into the contribution of cathepsins to the onset of early atheroma Lipoproteins are taken up by LDLR or by alternative routes and delivered to the lysosomes for degradation by acid hydrolyses [64] Linke et al emphasized the role of cathepsins in lipoprotein metabolism by showing that cathepsins B, F, K, L and V were able to degrade ApoB-100 under acidic conditions (e.g pH 5.5) [65] Since cholesterol is highly reactive substance and mediates numerous biological functions, ingested cholesterol is stored primarily in lipid droplets as cholesterol-ester conjugates [66] [67] To avoid the cytotoxic effects caused by hypercholesterolemia, macrophages evolved alternative routes to eliminate excess cholesterol, a process known as reverse cholesterol transport (RCT) [68] RCT involves the hydrolysis of lipid droplets and mobilization of cholesterol to members of the ATP Binding Cassettes (ABCA1 and ABCG1) that transfer cholesterol to its extracellular acceptors, Apolipoprotein-Al (Apo-AI) and High Density Lipoprotein (HDL) [69], Figure 2a The pivotal role for lysosomal degradation to RCT has been recently addressed by Ouimet et al that showed that delivery of cholesterol in macrophage foam cells to Apo-AI and HDL is mediated by autophagy Furthermore, perturbations to this transporting mechanism caused by small molecule inhibitors or genetic engineering, resulted in decreased cholesterol transport to nascent Aop-AI or HDL [70] The significance of cathepsins to cholesterol trafficking and excretion can be inferred from both genetic studies and in vitro models For example, expression of cathepsins B, F, H, and L was lower in monocytes from patients with low HDL levels a major risk factor for cardiovascular disease, compared to normal age matched subjects [71] Furthermore, Becker et al uncovered that cathepsin L is engaged in a protein network that is important for cholesterol homeostasis and is disturbed in lipid-loaded macrophages [72] While expression studies can only imply on a possible relationship between cathepsin function and RCT, studies using cell culture models consolidated this hypothesis Several independent studies provide compelling evidence that oxLDL particles attenuate cathepsin function, leading to lysosomal cholesterol retention and impaired cholesterol efflux to HDL particles [73-76], see Figure 2b In that sense, Oneil and colleagues suggested a direct mechanism using cathepsin B as a model, by which oxidized lipid metabolites attenuate cathepsin activity by forming protein-lipid adducts on reactive cysteine and histidine residues [77, 78] In addition, cathepsins can hamper cholesterol secretion by digesting nascent HDL particles For example, cathepsins F and S were shown to cleave pre-βHDL and therefore, impair cholesterol efflux from macrophage [79] More recently Dinnes et al elaborated on these observations by showing that cathepsin B, secreted from human monocytes derived macrophages, impairs cholesterol efflux to HDL by cleaving its core protein Apo-AI Mass spectrometry analysis uncovered serine 228 as the cleavage site on Apo-AI and functional studies using truncated protein confirmed its impaired capacity to stimulate RCT [80], Figure 2b This article is protected by copyright All rights reserved In conclusion, we provide evidence that cathepsins are important in cholesterol homeostasis and that aberrant cathepsin activity contributes to atheromatous plaque formation through different Accepted Article mechanisms Dysfunctional Lysosomes Promotes Vascular Inflammation via Cathepsins The inflammatory basis of atherosclerosis is well established [81, 82] Recent studies linking lysosomal dysfunction to sterile inflammation in hyperlipidemic conditions illuminate the contribution of cathepsins to atherosclerosis progression As macrophages tend to collect cholesterol, their lysosomes become overwhelmed by lipid accumulation and they eventually become dysfunctional [75, 83] This has shown to be a consequence of cholesterol crystal (CC) formation that results from accumulation of oxLDL, consequently triggering an inflammatory response mediated by nucleotide-binding oligomerization domain-like receptor protein (NLRP) inflammasomes and involves cathepsins B and L [84] Two papers from Kathryn J Moore’s laboratory demonstrated that oxLDL is ingested via CD36 dimmers with Toll Like Receptors (TLR) signaling pathway to provide both priming and processing steps required for IL-1β activation in macrophages [85, 86] In vitro studies demonstrated that macrophages lacking cathepsins B or L secrete less IL-1β in response to CC stimulation, and in vivo, these mice displayed decreased neutrophil recruitment in response to CC challenge [84] Mechanistic studies suggested that CC disrupted lysosomal membrane integrity and caused the release of lysosomal hydrolases into the cytosol, which provided an alternative pathway for IL-1β processing [84] In support of this mechanism, macrophages incubated with silica or aluminum mediated lysosomal disruption, suggesting that cathepsin B is released into the cytosol and spearhead IL-1β processing in the absence of caspase-1 [87], Figure 2b These data, in addition to the recent discovery that cathepsins regulate the transcription and activation of NLRP3 and IL-1β [88], suggest the mechanisms through which cathepsins promote the inflammatory response under hyperlipidemic conditions Cathepsins Meet Autophagy to Manage Inflammation and Cellular Damage In order to handle the cytotoxic effects of excessive inflammation, cells activate an evolutionary conserved degradation mechanism known as autophagy [89] Autophagy is responsible for bulk degradation of damaged proteins/protein aggregates and dysfunctional organelles Several lines of evidence suggest that dysfunctional autophagy underlies the development of advanced atherosclerosis lesions in part by enhancing cellular inflammation [90] Cathepsins are highly activated during autophagy for example the loss of cathepsin L in mouse embryonic fibroblasts impaired lysosomal function and increased cellular oxidative stress [91] Similar observations were This article is protected by copyright All rights reserved obtained in macrophages when cathepsin S activity was diminished; in this case, dysfunctional autophagy was associated with increased cardiac inflammation [92] In contrast, reverting Accepted Article lysosomal function through TFEB knock-in approach in macrophages, effectively improved autophagy, cholesterol clearance and decreased IL-1β secretion [93] Furthermore, using a recombinant protein therapy approach, Sun et al were able to enhance lysosomal function, as evident by the levels of mature cathepsins B and L, reduce vascular inflammation and atherosclerosis burden in mice [94] Overall, lysosomal cathepsin activity seems to play important role in the autophagy-lysosomal pathway and is likely to protect against lipotoxicity induced vascular inflammation Cysteine Cathepsins in Cell Death and Macrophage Clearance Large necrotic area composed of dead cells and cellular debris is a typical hallmark of advanced atheroma and is the culprit of symptomatic plaques ('unstable plaques') The development of necrotic regions in atherosclerosis can happen due to primary necrotic cell death or imbalance between apoptosis and clearance of dead cells alluded to as "secondary necrosis" [95] Necrosis is a special case of cell demise in which intracellular content is released as cells lose their membrane integrity Atherosclerosis-related bioactive molecules such as monosodium ureate, CC and ATP are sensed by neighboring cells and elicit an inflammatory immune response driven by pattern recognition receptors and inflammasomes [96] The involvement of cathepsins in myeloid necrotic cell death has been documented and shown to involve lysosomal membrane permeabilization and cysteine cathepsins B, C and S [97, 98] Impairment of lysosomal integrity and release of cathepsins to the cytosol is a common feature in atherosclerosis as described by Duewell et al [84] and is likely to contribute to necrotic core formation; however, this mechanism requires further investigation Apoptosis is a programmed cell death that enables safe removal of dying cells and prevents the loss of membrane integrity Apoptotic cells are taken up and digested by the immune system, especially by macrophages in a processed known as efferocytosis [99] Normally, efferocytosis is efficient and necessary for early stage plaque regression, however in advanced atheromas this mechanism is overwhelmed by accelerated cell death leading to dysfunctional efferocytosis [100] The engagement of cathepsins in apoptotic cell death was reported in the presence of hyperlipidemia and pro-inflammatory cytokines [101-104] Cathepsins mediated cell death involves their translocation to the cytosol where they cleave Bid and other members of the Bcl-2 family such as Bcl-x and triggers apoptotic cascade in parallel or independent of caspase activation [12] In human atheromas, cathepsins B and L were found in apoptotic phagocytic cells and in vitro studies further ascertained their redistribution into the cytoplasm and nucleus in the presence of toxic lipid substances [105, 106] Rapid and efficient efferocytosis is an important mechanism to temper excessive inflammatory response and prevent atherosclerosis [107, 108] Exclusion of apoptotic cells by efferocytosis is a This article is protected by copyright All rights reserved multistep process that requires recognition cell signaling cascades and efficient delivery and digestion of cargo by lysosomal proteases Impaired lysosomal function due to oxidized lipid Accepted Article metabolites has been shown to attenuate apoptotic cell clearance and promotes necrotic core formation in LDL-/- mice [109] Cathepsin L has also been found to be necessary for apoptotic cell clearance in a C elegans model [110], in agreement with its importance in lysosomal function [91] Taken together, this data suggests that cathepsins may play a role in efferocytosis; however, their precise molecular function in the setting of atherosclerosis requires further investigation Cysteine proteases in atherosclerosis arterial remodeling: Cathepsins Reshape Blood Vessels Arterial remodeling is a prominent clinical feature in atherosclerosis During the early phase of arterial plaque formation, lesions grow away from the lumen and remain clinically undetectable Later on these fatty streaks enlarge and start to intrude into the lumen and limit blood flow by narrowing the artery and give raise to clinical symptoms such as ischemia and unstable angina [111] These physical and structural changes of the blood vessel can happen due to excessive inflammation and proteolysis of the vascular ECM, especially collagen and elastin [112], Figure Collagen fibers (especially type I and II) have triple helical structure and are generally resistant to degradation by cathepsins An exception is cathepsin K, which is deemed to have physiologically relevant collagenase activity [113] This unique feature of cathepsin K among other cathepsins is largely attributed to GAG-cathepsin K complex (especially with chondrotin sulfate 4) and its dimerization with another cathepsin K molecule at the gap regions of packed collagen fibrils [113115] Another distinctive characteristic of cathepsin K is the ability to accommodate proline (Pro) or hydroxyproline (Hyp) at position S2, enabling cleavage of Gly-Pro-X or Gly-X-Hyp that are common motifs in collagen fibers [17, 112] This was confirmed by mutation analysis in which replacement of Tyr67 and Leu205 at S2 position of cathepsin K, completely abolished its collagenolytic activity without affecting its gelatinolytic activity [116] Elastin is another principal component of the ECM, which is severely damaged during vascular remodeling in atherosclerosis For example, human atheromas over-express cathepsins K and S and protein extracts from these samples enhanced elastin degradation compared to extracts from normal arteries In addition, stimulation of SMC with IL-1β or interferon- stimulated cathepsin S secretion and elastin degradation [41], this provides plausible mechanism for enhanced tissue remodeling in atherosclerosis Figure Moreover, in vitro studies identified human cathepsin V but not cathepsin L as the most potent elastolytic enzyme so far [117] Mechanistic studies This article is protected by copyright All rights reserved the patient's outcome, compared to standard assessment techniques [139] As such, noninvasive molecular imaging is highly appealing for this endeavor Molecular imaging is a rapidly growing Accepted Article field that is committed to translate novel biomarker discoveries into diagnostic tools In the case of atherosclerosis, novel methods for detection of protease activity, reflecting an active vascular remodeling process are continuously emerging In general, proteolytic substrates are recognized by the primary amino acids sequence adjacent to the cleavage site within target proteins and therefore, it is possible to target protease activity using molecules based on their recognition sequence [140] This approach in conjunction with a specific reporter such as a fluorophore or other contrasting agent is widely employed in the field of cardiovascular imaging to capture abnormal protease activity [141, 142] This is achieved through different classes of affinity based molecules such as substrate based probes and activity based probes (ABP), see [143, 144] for comprehensive discussion Briefly, substrate based probes are molecules that can change their spectral properties upon enzymatic cleavage, Figure For example, ProSense® (PerkinElmer) is a commercial pan cathepsin substrate based probe, composed of poly L-lysine embedded on methoxypolyethylene glycol scaffold [145] ProSense® is self-quenched due to high density of fluorophores and generates a fluorescent signal upon its cleavage, preferably by cathepsins This polymeric substrate has been widely employed for preclinical cardiovascular imaging [146-149] In these studies, fluorescent intensity correlated with vascular inflammation, macrophage content and cathepsin expression While ProSense®, represents an early initiative of substrate based probes and in some cases displayed slower kinetics compared to modern technologies [105], recent advent of lipidated cathepsin substrates looks promising with improved homing properties over other substrate probes [101] In the ABP approach, the molecules are designed to retain within the enzymes' active site by covalent modification of the protease reactive amino acid side chain, in the case of cysteine protease this is a reactive thiol group An improved ABPs class, quenched ABPs (qABP) harbor a quenching moiety, which makes them suitable for real time imaging applications such as in vitro live cell imaging and in vivo imaging [105, 105] Furthermore, the covalent binding of enzyme to the small molecule enables down-stream analysis, such as SDS-PAGE We recently applied this method on human carotid endarterectomy samples to show that cathepsin activity may be used as surrogate marker to distinguish symptomatic patients [138] In addition, labeling cathepsin activity with qABP has also been successfully implemented in pre-clinical animal models of atherosclerosis [105] Using fluorescence molecular tomography (FMT) we showed that qABP (GB137) home in on atherosclerotic plaque in mice, and confocal microscopy analysis of tissue sections delineated that fluorescent signal colocalizes with plaque associated macrophages [105] Reduced light penetrance is one of the major downsides of these fluorescent based technologies This article is protected by copyright All rights reserved Accepted Article thus, limiting their use to preclinical imaging, invasive clinical procedures and detection of superficial tissue (see [100] for comprehensive discussion) However, the fact that ABPs retain in tissue expressing high protease activity and can be decorated with tags that enable deeper tissue contrast provides a great advantage over substrate based probes and therefore, lay the ground for translation of molecular imaging into clinical practice [105] In that sense, Withana et al took a major step towards translating molecular imaging into clinical practice by developing a dual-modality ABP for PET/CT and optical analysis That study shows the novel molecular agent can accurately discriminate between inflamed and normal arteries in vivo, and ex vivo sections from these animals further supported their findings by showing that fluorescent signal colocalized with activated macrophages [105] The authors also demonstrated encouraging data using this imaging modality for idiopathic pulmonary fibrosis in human subjects [105], which further support their utility in the clinical arena With the advent of multimodal imaging [105], such novel contrasting agents can significantly improve the ability to identify rupture prone atherosclerotic lesions by gathering anatomical and molecular information Cathepsin Targeted Therapy for Atherosclerosis The ability to target potentially dangerous atherosclerotic lesions using ABP or substrates, has largely inspired the use small molecule inhibitors for preclinical assessment Two independent studies using cathepsin S inhibitors have successfully attenuated vascular lesion development in ApoE-/- mice Both studies report on decreased plaques size and macrophage content in mice treated with cathepsin S inhibitor [121, 155] Figueiredo et al also provide a compelling mechanism for decreased plaque macrophage content using a cathepsin S inhibitor, through the reduction of chemotactic molecule and growth differentiation factor 15 (GDF-15) [155] A different approach to eliminate inflammatory cells can be achieved through photodynamic therapy (PDT) PDT uses photosensitizer agent and a specific wavelength to produce focal burst of reactive oxygen species (ROS) and ultimately kill neighboring cells This approach holds great promise as it increased overall plaque stability and reduced inflammatory cell content in rabbits [151] On top of its therapeutic benefits, incorporating these photosensitizers to cathepsin targeted quenched ABP can generate a potent theranostic agent (PS-qABP) that becomes activated on demand This means that PS-qABP will produce a fluorescent signal / ROS when bound to an active protease as shown in cancer mice models at the site of inflammation [155] This feature should enable more accurate and effective treatment for potentially deleterious vascular lesions These benefits prompted the design of cathepsin B theranostic substrate molecule (L-SR15) using chlorin E6 embedded on poly lysine polymer [155] This study reported encouraging results when PDT light treatment diminished macrophage cell content without having effect on smooth muscle cells and without notable cytotoxic effects in ApoE -/- mice [155] One thing to consider with L-SR15 is the requirement for a complicated invasive procedure in order to apply PDT, which highlights the need for an improved design in pursuit of a non-invasive approach Thus, the high cathepsin expression and activity in atherosclerosis has led to the generation of targeted imaging and therapeutic entities that enable accurate tracking of This article is protected by copyright All rights reserved vulnerable plaques as well as treatment in animal models Accepted Article Conclusion and perspectives: Cathepsin activity is versatile and potentially affects almost every critical step in atherogenesis Over the past decade, a large body of data from humans and rodents emphasize the role of cathepsins in the etiology of vascular diseases Early in vitro studies demonstrated that cathepsins are capable of digesting the physical constituents of the vasculature such as collagen and elastin and therefore, much work in vivo has naturally focused on this aspect to investigate their impact on atherosclerosis Recently, due to significantly increase in risk of stroke, Merck's cathepsin K inhibitor, Odanacatib, was pulled out of clinical trials, despite the promising data with respect to bone mineral density [155] This dropout emphasizes the importance of cathepsin K function in the cardiovascular arena Nevertheless, the increase of stroke using a cathepsin K inhibitor is counterintuitive stressing the lack of comprehensive knowledge on the involvement of cathepsins in cardiovascular pathogenesis Cathepsins are chiefly lysosomal hydrolases and beside their significant role in shaping their microenvironment, they also participate in several important pathways "in house" One of them is autophagy, which regulates many important features of atherogenesis such as lipid metabolism, inflammation and clearance of damaged cellular components or apoptotic cells Cathepsins are functionally important to autophagy-lysosomal pathway, but comprehensive data regarding their specific functions under these conditions is currently lacking There is no doubt that future studies will inevitably expand our understanding on the role of cathepsins in atherosclerosis and will potentially help designing novel therapeutic strategies and avoid cytotoxic effects An interesting approach to study on cathepsin function has recently been introduced by Prudova A, et al In their study they used sophisticated mass spectrometry approach to explore the contribution cathepsins in the context of pancreatic cancer [561] We strongly believe that such methodologies are valuable and are likely to expand our future understanding of the physiological roles of cathepsins in atherosclerosis In the translational perspective, cathepsin activity provides a promising target for future theranostic agents in attempt to identify ruptureprone plaques Acknowledgements: This review was supported by the United States–Israel Binational Science Foundation (BSF) (2009010 and 2011480 to G.B.) 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TNF- regulate cathepsin S expression in human vascular ECs, involving two independent cohorts Their findings show that inflammatory stimulation invokes changes in cathepsin S mRNA metabolism... that cathepsins may play a role in efferocytosis; however, their precise molecular function in the setting of atherosclerosis requires further investigation Cysteine proteases in atherosclerosis. .. cathepsin activity to further clarify the interplay of cysteine cathepsins in the setting of atherosclerosis and suggest a pivotal role for cathepsins in ECM turnover and immune response Cathepsins