Protease Activated Receptors: A Pathway to Boosting Mesenchymal Stromal Cell Therapeutic Efficacy in Acute Respiratory Distress Syndrome?

Acute Respiratory Distress Syndrome is the most common cause of respiratory failure among critically ill patients, and its importance has been heightened during the COVID-19 pandemic. Even with the best supportive care, the mortality rate in the most severe cases is 40-50%, and the only pharmacological agent shown to be of possible benefit has been steroids. Mesenchymal stromal cells (MSCs) have been tested in several pre-clinical models of lung injury and been found to have significant therapeutic benefit related to: (a) potent immunomodulation; (b) secretion of epithelial and endothelial growth factors; and (c) augmentation of host defense to infection. Initial translational efforts have shown signs of promise, but the results have not yielded the anticipated outcomes. One potential reason is the relatively low survival of MSCs in inflammatory conditions as shown in several studies. Therefore, strategies to boost the survival of MSCs are needed to enhance their therapeutic effect. Protease-activated receptors (PARs) may represent one such possibility as they are G-protein coupled receptors expressed by MSCs and control several facets of cell behavior. This review summarizes some of the existing literature about PARs and MSCs and presents possible future areas of investigation in order to develop potential, PAR-modified MSCs with enhanced therapeutic efficiency.

Protease Activated Receptors and Arthritis.

The catabolic and destructive activity of serine proteases in arthritic joints is well known; however, these enzymes can also signal pain and inflammation in joints. For example, thrombin, trypsin, tryptase, and neutrophil elastase cleave the extracellular N-terminus of a family of G protein-coupled receptors and the remaining tethered ligand sequence then binds to the same receptor to initiate a series of molecular signalling processes. These protease activated receptors (PARs) pervade multiple tissues and cells throughout joints where they have the potential to regulate joint homeostasis. Overall, joint PARs contribute to pain, inflammation, and structural integrity by altering vascular reactivity, nociceptor sensitivity, and tissue remodelling. This review highlights the therapeutic potential of targeting PARs to alleviate the pain and destructive nature of elevated proteases in various arthritic conditions.

Roles of Coagulation Proteases and PARs (Protease-Activated Receptors) in Mouse Models of Inflammatory Diseases.

Activation of the blood coagulation cascade leads to fibrin deposition and platelet activation that are required for hemostasis. However, aberrant activation of coagulation can lead to thrombosis. Thrombi can cause tissue ischemia, and fibrin degradation products and activated platelets can enhance inflammation. In addition, coagulation proteases activate cells by cleavage of PARs (protease-activated receptors), including PAR1 and PAR2. Direct oral anticoagulants have recently been developed to specifically inhibit the coagulation proteases FXa (factor Xa) and thrombin. Administration of these inhibitors to wild-type mice can be used to determine the roles of FXa and thrombin in different inflammatory diseases. These results can be compared with the phenotypes of mice with deficiencies of either Par1 (F2r) or Par2 (F2rl1). However, inhibition of coagulation proteases will have effects beyond reducing PAR signaling, and a deficiency of PARs will abolish signaling from all proteases that activate these receptors. We will summarize studies that examine the roles of coagulation proteases, particularly FXa and thrombin, and PARs in different mouse models of inflammatory disease. Targeting FXa and thrombin or PARs may reduce inflammatory diseases in humans.

The emerging role of coagulation proteases in kidney disease.

A role of coagulation proteases in kidney disease beyond their function in normal haemostasis and thrombosis has long been suspected, and studies performed in the past 15 years have provided novel insights into the mechanisms involved. The expression of protease-activated receptors (PARs) in renal cells provides a molecular link between coagulation proteases and renal cell function and revitalizes research evaluating the role of haemostasis regulators in renal disease. Renal cell-specific expression and activity of coagulation proteases, their regulators and their receptors are dynamically altered during disease processes. Furthermore, renal inflammation and tissue remodelling are not only associated, but are causally linked with altered coagulation activation and protease-dependent signalling. Intriguingly, coagulation proteases signal through more than one receptor or induce formation of receptor complexes in a cell-specific manner, emphasizing context specificity. Understanding these cell-specific signalosomes and their regulation in kidney disease is crucial to unravelling the pathophysiological relevance of coagulation regulators in renal disease. In addition, the clinical availability of small molecule targeted anticoagulants as well as the development of PAR antagonists increases the need for in-depth knowledge of the mechanisms through which coagulation proteases might regulate renal physiology.

Platelets mediate acetaminophen hepatotoxicity.

In this issue of Blood, Miyakawa et al show that platelets and protease-activated receptor (PAR)-4 contribute to acetaminophen (APAP)-induced liver damage. Using various strategies in a mouse model of APAP overdose, the authors demonstrate that platelets participate in the progression of liver damage, and that the direct thrombin inhibitor lepirudin and PAR-4 deficiency attenuate hepatotoxicity. These findings have the potential to help identify future therapeutic targets for APAP-induced hepatotoxicity.

The role of toll-like and protease-activated receptors in the expression of cytokines by gingival fibroblasts stimulated with the periodontal pathogen Porphyromonas gingivalis.

Porphyromonas gingivalis is a periodontitis-associated pathogen and interactions between the bacterium and gingival fibroblasts play an important role in development and progression of periodontitis, an inflammatory disease leading to degeneration of tooth-supporting structures. Gingival fibroblasts, which expresses protease activated receptors (PARs) as well as toll-like receptors (TLRs), produces inflammatory mediators upon bacterial challenges. In this study, we elucidated the importance of PAR1, PAR2, TLR2 and TLR4 for the expression and secretion of CXCL8, interleukin-6 (IL-6), transforming growth factor-ß1 (TGF-ß1) and secretory leukocyte inhibitor (SLPI). Human gingival fibroblasts were transfected with small-interfering RNA against the target genes, and then stimulated with P. gingivalis wild-type W50 and W50-derived double rgp mutant E8 and kgp mutant K1A. TLR2-silencing reduced P. gingivalis-induced CXCL8 and IL-6. IL-6 was also reduced after PAR1-silencing. No effects were observed for TGF-ß1. SLPI was suppressed by P. gingivalis and silencing of PAR1 as well as TLR2, gave additional suppression at the mRNA level. TLR4 was not involved in the regulation of the investigated mediators. CXCL8 and IL-6 are important for progression and development of periodontitis, leading to a chronic inflammation that may contribute to the tissue destruction that follows an exacerbated host response. Therefore, regulating the expression of TLR2 and subsequent release of CXCL8 and IL-6 in periodontitis could attenuate the tissue destruction seen in periodontitis.

Platelets and protease-activated receptor-4 contribute to acetaminophen-induced liver injury in mice.

Acetaminophen (APAP)-induced liver injury in humans is associated with robust coagulation cascade activation and thrombocytopenia. However, it is not known whether coagulation-driven platelet activation participates in APAP hepatotoxicity. Here, we found that APAP overdose in mice caused liver damage accompanied by significant thrombocytopenia and accumulation of platelets in the liver. These changes were attenuated by administration of the direct thrombin inhibitor lepirudin. Platelet depletion with an anti-CD41 antibody also significantly reduced APAP-mediated liver injury and thrombin generation, indicated by the concentration of thrombin-antithrombin (TAT) complexes in plasma. Compared with APAP-treated wild-type mice, biomarkers of hepatocellular and endothelial damage, plasma TAT concentration, and hepatic platelet accumulation were reduced in mice lacking protease-activated receptor (PAR)-4, which mediates thrombin signaling in mouse platelets. However, selective hematopoietic cell PAR-4 deficiency did not affect APAP-induced liver injury or plasma TAT levels. These results suggest that interconnections between coagulation and hepatic platelet accumulation promote APAP-induced liver injury, independent of platelet PAR-4 signaling. Moreover, the results highlight a potential contribution of nonhematopoietic cell PAR-4 signaling to APAP hepatotoxicity.

Proteases and their receptors as mediators of inflammation-associated colon cancer.

Chronic inflammation increases the risk of developing cancer. For example, patients with severe and prolonged inflammatory bowel disease, particularly ulcerative colitis, have a significantly higher risk of developing colorectal cancer. Serine proteases coordinating the coagulation cascade and immune cell proteases play important roles in regulating the inflammatory response through their actions on protease-activated receptors (PAR). PARs and their activating proteases have also been implicated in many cancers, including CRC. Importantly, the actions of proteases could be important for mediating the transition from chronic inflammation to cancer. PAR activation has been shown to have pro-tumourigenic effects including the production of matrix metalloproteinases that can promote tumour cell growth and metastasis, and transactivation of the epidermal growth factor receptor, which is a main target for cancer treatment. Additionally, PAR activation can also result in increased expression of cyclooxygenase (COX)-2, an important enzyme mediating inflammation, resolution, and cancer progression. In this review, we will highlight our current knowledge about the effects of proteases and their receptors on intestinal inflammation and cancer, and explore the potential role of PAR-induced COX-2 on colitis-associated cancer.

Protease-activated receptors and itch.

Protease-activated receptors (PARs) have been implicated in a variety of physiological functions, as well as somatosensation and particularly itch and pain. Considerable attention has focused on PARs following the finding they are upregulated in the skin of atopic dermatitis patients. The present review focuses on recent studies showing that PARs are critically involved in itch and sensitization of itch. PARs are expressed by diverse cell types including primary sensory neurons, keratinocytes, and immune cells and are activated by proteases that expose a tethered ligand. Endogenous proteases are also released from diverse cell types including keratinocytes and immune cells. Exogenous proteases released from certain plants and insects contacting the skin can also induce itch. Increased levels of proteases in the skin contribute to inflammation that is often accompanied by chronic itch which is not predominantly mediated by histamine. The neural pathway signaling itch induced by activation of PARs is distinct from that mediating histamine-induced itch. In addition, there is evidence that PARs play an important role in sensitization of itch signaling under conditions of chronic itch. These recent findings suggest that PARs and other molecules involved in the itch-signaling pathway are good targets to develop novel treatments for most types of chronic itch that are poorly treated with antihistamines.

The role of proteases in pain.

Proteinase-activated receptors (PARs) are a family of G protein-coupled receptor that are activated by extracellular cleavage of the receptor in the N-terminal domain. This slicing of the receptor exposes a tethered ligand which binds to a specific docking point on the receptor surface to initiate intracellular signalling. PARs are expressed by numerous tissues in the body, and they are involved in various physiological and pathological processes such as food digestion, tissue remodelling and blood coagulation. This chapter will summarise how serine proteinases activate PARs leading to the development of pain in several chronic pain conditions. The potential of PARs as a drug target for pain relief is also discussed.

Diabetic nephropathy and proximal tubular damage.

Diabetic nephropathy (DN) is a major cause of uremia in developed societies. Inflammation is emerging as an important mechanism for its pathogenesis and progression. Herein, we review 4 recently described cellular receptors that have been shown to mediate diabetic interstitial kidney disease. Peroxisome proliferator-activated receptor-γ attenuates STAT-1 activation and has shown promise in renoprotection. Its clinical utility is limited mainly by fluid retention through upregulation of sodium-hydrogen exchanger-3 and aquaporin-1 channels in the proximal tubule. The bradykinin receptor 2 of the kallikrein-kinin system has been shown to mediate diabetic kidney injury and its blockade conferred renoprotective effects in animal models of DN. The related protease-activated receptor, especially receptor 4, has recently been shown to participate in DN. Further studies are required to confirm its role. Finally, the toll-like receptor, especially TLR4 and TLR2, has been verified in multiple models to be a significant sensor of and reactor to hyperglycemia and other diabetic substrates that orchestrate interstitial inflammation in DN.

Endothelial barrier dysfunction in septic shock.

The endothelium provides an essential and selective membrane barrier that regulates the movement of water, solutes, gases, macromolecules and the cellular elements of the blood from the tissue compartment in health and disease. Its structure and continuous function is essential for life for all vertebrate organisms. Recent evidence indicates that the endothelial surface does not have a passive role in systemic inflammatory states such as septic shock. In fact, endothelial cells are in dynamic equilibrium with a myriad of inflammatory mediators and elements of the innate immune and coagulation systems to orchestrate the host response in sepsis. The barrier function of the endothelial surface is almost uniformly impaired in septic shock, and it is likely that this contributes to adverse outcomes. In this review, we will highlight recent advances in the understanding of the signalling events that regulate endothelial function and molecular events that induce endothelial dysfunction in sepsis. Endothelial barrier repair strategies as a treatment for sepsis include modulation of C5a, high-mobility group box 1 and VEGF receptor 2; stimulation of angiopoietin-1, sphingosine 1 phosphate receptor 1 and Slit; and a number of other innovative approaches.

Differential cellular effects of old and new oral anticoagulants: consequences to the genesis and progression of atherosclerosis.

The main purpose of anticoagulants is to diminish fibrin formation, thereby decreasing the risk of venous or arterial thrombosis. Vitamin K antagonist have been used for many decades in order to achieve reduced thrombotic risk, despite major drawbacks of this class of drugs such as cumbersome dossing and monitoring of anticoagulant status. To overcome these drawbacks of VKA, new classes of anticoagulants have been developed including oral anticoagulants for direct inhibition of either thrombin or factor Xa, which can be administrated in a fixed dose without monitoring. Coagulation factors can activate cellular protease-activated receptors, thereby inducing cellular processes as inflammation, apoptosis, migration, and fibrosis. Therefore, inhibition of coagulation proteases not only attenuates fibrin formation, but may also influence pathophysiological processes like vascular calcification and atherosclerosis. Animal models revealed that VKA therapy induced both intima and media calcification and accelerated plaque vulnerability, whereas specific and direct inhibition of thrombin or factor Xa attenuated atherosclerosis. In this review we provide an overview of old and new oral anticoagulants, as well discuss potential pleiotropic effects with regard to calcification and atherosclerosis. Although translation from animal model to clinical patients seems difficult at first sight, effort should be made to fully understand the clinical implications of long-term oral anticoagulant therapy on vascular side effects.

Cytoprotective-selective activated protein C therapy for ischaemic stroke.

Despite years of research and efforts to translate stroke research to clinical therapy, ischaemic stroke remains a major cause of death, disability, and diminished quality of life. Primary and secondary preventive measures combined with improved quality of care have made significant progress. However, no novel drug for ischaemic stroke therapy has been approved in the past decade. Numerous studies have shown beneficial effects of activated protein C (APC) in rodent stroke models. In addition to its natural anticoagulant functions, APC conveys multiple direct cytoprotective effects on many different cell types that involve multiple receptors including protease activated receptor (PAR) 1, PAR3, and the endothelial protein C receptor (EPCR). Application of molecular engineered APC variants with altered selectivity profiles to rodent stroke models demonstrated that the beneficial effects of APC primarily require its cytoprotective activities but not its anticoagulant activities. Extensive basic, preclinical, and clinical research provided a compelling rationale based on strong evidence for translation of APC therapy that has led to the clinical development of the cytoprotective-selective APC variant, 3K3A-APC, for ischaemic stroke. Recent identification of non-canonical PAR1 and PAR3 activation by APC that give rise to novel tethered-ligands capable of inducing biased cytoprotective signalling as opposed to the canonical signalling provides a mechanistic explanation for how APC-mediated PAR activation can selectively induce cytoprotective signalling pathways. Collectively, these paradigm-shifting discoveries provide detailed insights into the receptor targets and the molecular mechanisms for neuroprotection by cytoprotective-selective 3K3A-APC, which is currently a biologic drug in clinical trials for ischaemic stroke.

Tissue factor, protease activated receptors and pathologic heart remodelling.

Tissue factor is the primary initiator of coagulation cascade and plays an essential role in haemostasis and thrombosis. In addition, tissue factor and coagulation proteases contribute to many cellular responses via activation of protease activated receptors. The heart is an organ with high levels of constitutive tissue factor expression. This review focuses on the role of tissue factor, coagulation proteases and protease activated receptors in heart haemostasis and the pathological heart remodelling associated with myocardial infarction, viral myocarditis and hypertension.

Protease-activated receptor signalling by coagulation proteases in endothelial cells.

Endothelial cells express several types of integral membrane protein receptors, which upon interaction and activation by their specific ligands, initiate a signalling network that links extracellular cues in circulation to various biological processes within a plethora of cells in the vascular system. A small family of G-protein coupled receptors, termed protease-activated receptors (PAR1-4), can be specifically activated by coagulation proteases, thereby modulating a diverse array of cellular activities under various pathophysiological conditions. Thrombin and all vitamin K-dependent coagulation proteases, with the exception of factor IXa for which no PAR signalling has been attributed, can selectively activate cell surface PARs on the vasculature. Thrombin can activate PAR1, PAR3 and PAR4, but not PAR2 which can be specifically activated by factors VIIa and Xa. The mechanistic details of the specificity of PAR signalling by coagulation proteases are the subject of extensive investigation by many research groups worldwide. However, analysis of PAR signalling data in the literature has proved to be challenging since a single coagulation protease can elicit different signalling responses through activation of the same PAR receptor in endothelial cells. This article is focused on briefly reviewing the literature with respect to determinants of the specificity of PAR signalling by coagulation proteases with special emphasis on the mechanism of PAR1 signalling by thrombin and activated protein C in endothelial cells.

G protein-coupled receptors in rheumatology.

G protein-coupled receptors (GPCRs) are transmembrane receptor proteins that allow the transfer of signals across the cell membrane. In addition to their physiological role, GPCRs are involved in many pathophysiological processes including pathways relevant in rheumatoid arthritis (RA), osteoarthritis (OA) and psoriatic arthritis. Two-thirds of all currently available drugs target GPCRs directly or indirectly. However, the detailed mechanism of GPCR signalling is still unclear. Selective modification of GPCR-dependent signalling cascades to inhibit disease progression in rheumatic diseases is now being investigated. One approach is to use antibodies against ligands activating GPCRs. However, several GPCRs are known to be activated by only one ligand. In this case, targeting the receptor itself is a promising approach. So far, more information is available on GPCR action in RA as compared with OA, and even less information is available for other rheumatic diseases. Additional research on the role of GPCRs involved in the pathophysiology of rheumatic diseases is required to develop specific therapeutic approaches.

Endosomal signaling by protease-activated receptors.

Protease-activated receptors (PARs) are a family of G protein-coupled receptors (GPCRs) that are uniquely activated by proteolysis. There are four members of the PAR family including: PAR1, PAR2, PAR3, and PAR4. PARs are expressed primarily in the cells of the vasculature and elicit cellular responses to coagulant and anticoagulant proteases. PAR1 exemplifies the unusual proteolytic mechanism of receptor activation. Thrombin binds to and cleaves the N-terminal exodomain of PAR1, generating a new N-terminus that functions as a tethered ligand. The N-terminal tethered ligand domain of PAR1 binds intramolecularly to the receptor to trigger transmembrane signaling and cannot diffuse away. Similar to other GPCRs, activation of PARs promotes coupling to heterotrimeric G proteins at the plasma membrane. After activation, PARs are rapidly internalized to endosomes and then sorted to lysosomes and degraded. Internalization functions to uncouple PARs from heterotrimeric G proteins at the cell surface. However, recent studies indicate that activated internalized PARs signal from endosomes through the recruitment of ß-arrestins and potentially other pathways. Here, we provide an overview of methods and strategies used to examine endosomal signaling by PARs.

Proteinase-activated receptors in fibroproliferative lung disease.

The coagulation cascade plays a central role in the pathogenesis of fibroproliferative lung diseases such as the acute respiratory distress syndrome (ARDS) and idiopathic pulmonary fibrosis (IPF) through multifaceted effects on haemostasis, inflammation and tissue repair. However, targeting the coagulation cascade using traditional anticoagulant approaches has not resulted in improved outcomes for these patients. The cellular effects of the coagulation cascade are mediated via a family of four proteinase-activated receptors (PAR(1-4)). PARs are G protein-coupled receptors that have a unique method of activation involving proteolytic cleavage. They play key roles in mediating the interplay between coagulation and inflammation and tissue repair and fibrosis. Current evidence suggests a central role for PAR(1) and PAR(2) in influencing these responses, although data from animal models suggest that their contribution is highly dependent on both the nature of the insult and disease status. Nonetheless, these receptors may represent important targets in conditions associated with uncontrolled coagulation signalling responses including IPF, ARDS, asthma and chronic obstructive pulmonary disease.