Induction of Vascular Smooth Muscle Cell Growth by Selective Activation of the Proteinase Activated Receptor-2 (PAR-2)

doi:10.1006/bbrc.1997.7847

Biochemical and Biophysical Research Communications

Volume 241, Issue 3, 29 December 1997, Pages 762-764

https://doi.org/10.1006/bbrc.1997.7847 Get rights and content

Abstract

The proteinase-activated receptor (PAR-2) which belongs to the family of proteolytically cleaved receptors is activated by trypsin and by a synthetic peptide (SLIGKV) derived from the new amino terminus. Here, we have studied the mitogenic effect of trypsin and of SLIGKV on human aortic smooth muscle cells (SMC). Like trypsin, SLIGKV was a potent mitogen for SMC and exhibited the same activity as that of SFLLRN, a peptide mimicking the new amino terminus created by cleavage of the thrombin receptor. SLIGKV stimulated the proliferation of growth-arrested SMCs with a half-maximum mitogenic response at 80 nM. Under the same experimental conditions, the retro analogue or SLIGKV (VKGILS) did not show any mitogenic activity. Two specific inhibitors of the enzymatic activity of trypsin, α1-antitrypsin and aprotinin, specifically inhibited trypsin-induced SMC growth (IC₅₀= 0.87 ± 0.09 and 0.74 ± 0.11 μM, respectively) but remain without effect on the mitogenic effect of the agonist peptide. The mitogenic effect of trypsin and SLIGKV was due to the release of platelet derived growth factor as demonstrated by the inhibitory activity of a neutralizing monoclonal anti-PGDF-BB antibody. These results demonstrate that the mitogenic effect of trypsin for SMCs is intimately linked to its esterolytic activity and mediated by the activation of PAR-2.

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Cited by (68)

Approval of the first protease-activated receptor antagonist: Rationale, development, significance, and considerations of a novel anti-platelet agent
2015, Blood Reviews
Citation Excerpt :
PARs are expressed on the surface of numerous cell types. In the cardiovascular system this includes platelets [17–19], and also leukocytes [20], vascular endothelial and smooth muscle cells [21–23], cardiomyocytes [24,25], and cardiac fibroblasts [26]. Humans express four PARs, with PAR1, PAR3 and PAR4 being activated by thrombin [17–19], and PAR2 by trypsin, tryptase, coagulation factors VIIa and Xa, and membrane-bound serine proteases MTSP1 and TMPRSS2 [27–31].
Twenty-three years after the discovery of the first thrombin receptor, now known as protease-activated receptor 1 (PAR1), the first drug targeting this receptor is available for human use. The PAR1 inhibitor, vorapaxar (Zontivity, MSD), was recently approved by the FDA for use in the USA for the prevention of thrombotic cardiovascular events in patients with a history of myocardial infarction or peripheral artery disease. In this review, we detail the rationale, development, as well as the clinical significance and considerations of vorapaxar, the original PAR antagonist and the latest anti-platelet agent in the pharmaco-armoury against arterial thrombosis.
Functional role of protease activated receptors in vascular biology
2014, Vascular Pharmacology
Citation Excerpt :
ECs express PAR1, PAR2 and PAR4 [32]. VSMCs express PAR1 and PAR2, although their expression in normal arteries is low [33,34]. Human platelets express PAR1 and PAR4 [1].
Protease activated receptors (PARs) are a small family of G protein-coupled receptors (GPCR) mediating the cellular effects of some proteases of the coagulation system, such as thrombin, or other proteases, such as trypsin or metalloproteinase 1. As the prototype of PARs, PAR1 is a seven transmembrane GPCR that, upon cleavage by thrombin, unmasks a new amino-terminus able to bind intramolecularly to PAR1 itself thus inducing signaling. In the vascular system, thrombin and other proteases of the coagulation-fibrinolysis system, such as plasmin, factor VIIa and factor Xa, activated protein C, are considered physiologically relevant agonists, and PARs appear to largely account for the cellular effects of these enzymes. In the vasculature, PARs are expressed on platelets, endothelial cells (ECs) and vascular smooth muscle cells (VSMCs). In the vessel wall, under physiological conditions, PARs are mainly expressed in ECs and participate in the regulation of vascular tone, by inducing endothelium-dependent relaxation. PAR activation on ECs promotes conversion of these cells into a proinflammatory phenotype, causes increase of vascular permeability, and the exposure/secretion of proteins and cytokines mediating the local accumulation of platelets and leukocytes. These effects contribute to the vascular consequences of sepsis and of diseases such as acute lung injury and acute respiratory distress syndrome. In normal arteries PARs are to a much lesser amount expressed on VSMCs. However, in conditions associated with endothelial dysfunction, PARs mediate contraction, proliferation, migration, hypertrophy of VSMCs and their production of extracellular matrix, thereby contributing to the pathophysiology of atherosclerosis and hypertension. Inhibition of protease–PAR interaction might thus become a potential therapeutic target in various vascular diseases.
Physiology, pharmacology, and therapeutic potential of protease-activated receptors in vascular disease
2012, Pharmacology and Therapeutics
Citation Excerpt :
Finally, constitutive endothelial PAR1 activation, at least in mice, also plays important roles in vasculogenesis: half of PAR1-deficient mouse embryos fail to develop through mid-gestation due to a defect in blood vessel formation (Connolly et al., 1996) which can be rescued by replacing PAR1 expression in endothelial cells alone (Griffin et al., 2001). Cultured human vascular smooth muscle (Bono et al., 1997) and endothelial cells (Molino et al., 1997c) also express PAR2. In human vasculature in situ, PAR2 mRNA and protein has been detected in the smooth muscle and endothelium of both arteries and veins (D'Andrea et al., 1998; Molino et al., 1998).
It has been 20 years since the discovery of the prototypical protease-activated receptor (PAR). In the time since this landmark work, significant advances have been made in our understanding of this family of four G protein-coupled receptors. Initially discovered and characterized in an attempt to determine the mechanism by which thrombin activates platelets, PARs have since been found to be widely expressed throughout the body, respond to multiple proteases, and to be involved in a vast array of physiological processes. Yet despite their wide-ranging expression, the function of PARs has been most extensively studied in the vascular system. In particular, the importance of PAR1 for platelet activation during arterial thrombosis has been thoroughly investigated and has led to the development of a host of PAR1 antagonists—two of which are currently in Phase 3 trials as antiplatelet agents. Given the impending clinical use of the first PAR antagonists, it is timely to review the physiological roles of PARs in cells of the blood and blood vessels, the development and experimental use of PAR antagonists in these systems, and the potential clinical application of these agents as therapeutics for vascular diseases.
Bikunin (Urinary Trypsin Inhibitor): Structure, Biological Relevance, And Measurement
2007, Advances in Clinical Chemistry
Citation Excerpt :
Additionally, the intracellular C‐terminus of PAR interacts with creatine kinase activating Rho kinase. Aprotinin has been shown to prevent activation of PAR with reduction of cell proliferation and signaling [72]. In contrast to traditional receptors that sense through binding, proteolytic activation is irreversible and the cleaved receptors are degraded in lysosomes.
Inflammatory processes, such as phagocytosis, coagulation, and vascular dilation, promote the release of serine proteases by neutrophils, macrophages, mast cells, lymphocytes, and the epithelial or endothelial cells. These proteases further facilitate the release of inflammatory cytokines and growth factors as well as take part in signal‐cell proliferation through protease‐activated receptors (PARs). Controlling the action of this cascade is necessary to prevent further damage to the normal tissues. One of the main anti‐inflammatory response mediators is bikunin (Bik) that is responsible for inhibiting the activity of many serine proteases such as trypsin, thrombin, chymotrypsin, kallikrein, plasmin, elastase, cathepsin, Factors IXa, Xa, XIa, and XIIa. During the acute‐phase response, Bik is released into plasma from proinhibitors primarily due to increased elastase activity. Bik is a glycoprotein, also referred to as urinary trypsin inhibitor, which in plasma inhibits the trypsin family of serine proteases by binding to either of the two Kunitz‐binding domains. Bik also accumulates in urine. In conditions such as infection, cancer, tissue injury during surgery, kidney disease, vascular disease, coagulation, and diabetes, the concentrations of Bik in plasma and urine are increased. Several trypsin inhibitory assays for urine and immunoassays for both blood and urine have been described for measuring Bik. In addition to presenting the synthesis, structure, and pathophysiology of Bik, we will summarize various diagnostic approaches for measuring Bik. Analysis of Bik may provide a rapid approach in assessing various conditions involving the inflammatory processes.
Expression of proteinase-activated receptors (PAR)-2 in articular chondrocytes is modulated by IL-1β, TNF-α and TGF-β
2006, Osteoarthritis and Cartilage
To investigate the modulation of expression of proteinase-activated receptor-2 (PAR-2) in articular chondrocytes by inflammatory cytokines.
Articular synovium and cartilage tissues were collected from eight patients with osteoarthritis (OA), and three patients without arthropathy (“normal”). Chondrocytes were stimulated with interleukin (IL)-1β, tumor necrosis factor (TNF)-α or transforming growth factor (TGF)-β1. The expression of PAR-2 was detected using reverse transcriptase-polymerase chain reaction (PCR), Western blotting and immunofluorescence. Quantitative PCR was performed to assess the expression levels of PAR-2 messenger RNA (mRNA).
The expression of PAR-2 mRNA was demonstrated in both OA and normal chondrocytes as well as in synovial fibroblasts. However, the level of PAR-2 in OA chondrocytes was much higher than in normal chondrocytes. Long-term culture revealed that PAR-2 mRNA expression was maintained up to three passages in OA but not in normal chondrocytes. IL-1β and TNF-α both upregulated PAR-2 expression in normal and OA chondrocytes. In contrast, TGF-β1 significantly decreased expression of PAR-2 in OA chondrocytes but increased PAR-2 in normal chondrocytes.
Overexpression of PAR-2 in OA chondrocytes is upregulated by proinflammatory cytokines IL-1β and TNF-α, and down-regulated by regulatory cytokine TGF-β1. PAR-2 may be involved in the pathogenesis of OA.
Inhibitors of mast cell tryptase beta as therapeutics for the treatment of asthma and inflammatory disorders
2005, Pulmonary Pharmacology and Therapeutics
A survey of the available biological data on tryptase inhibitors suggests that there is considerable interest in tryptase as a therapeutic target particularly for the treatment of allergic asthma and inflammatory disorders. This interest was driven primarily by data from studies carried out on the cellular and in vivo actions of this serine protease over the past decade, all of which have suggested a pro-inflammatory role for tryptase. Tryptase beta is the form of interest in allergic asthma and the data from numerous studies have shown that tryptase cannot only contribute to airway bronchoconstriction and hyperresponsiveness, but may have a key role in fibrosis and ECM turnover, hallmarks of the remodeling process. Hence, inhibitors of tryptase have the potential to make an impact on fibrosis and airway wall remodelling. However, few studies, if any, have been carried out to determine the effect of tryptase inhibitors on airway remodeling and this is an area that warrants further investigation with the appropriate models because the eventual positioning of tryptase inhibitors in asthma therapy will be strengthened by data supporting an impact on airway remodeling in addition to effects on bronchial hyperresponsiveness. This review has focused on tryptase inhibitors in the pipeline and it is clear that with a few exceptions, the majority of these compounds are targeted for inhaled delivery. Finally, judging by the interest from numerous pharmaceutical companies, it appears the stage is set for tryptase inhibitors to make their mark as drugs of the future for allergic asthma and the results from clinical trials is awaited with eager anticipation.

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Regular ArticleInduction of Vascular Smooth Muscle Cell Growth by Selective Activation of the Proteinase Activated Receptor-2 (PAR-2)

Abstract

FEBS Lett.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Blood Coag. Fibrinol.

Thromb. Haemost.

J. Clin. Invest.

Regular Article
Induction of Vascular Smooth Muscle Cell Growth by Selective Activation of the Proteinase Activated Receptor-2 (PAR-2)