Inhibition of chymase-dependent production of IL-1β by smooth muscle cells in the fibrous caps of human atherosclerotic plaques: A reasonable approach to prevent cap rupture?

Traditionally, the chymotrypsin-like neutral serine protease chymase is considered to be synthesized, stored, and secreted by mast cells present in various human tissues [[1]Andersson M.K. Enoksson M. Gallwitz M. Hellman L. The extended substrate specificity of the human mast cell chymase reveals a serine protease with well-defined substrate recognition profile.Int. Immunol. 2009 Jan; 21 (Epub 2008 Dec 10. PMID: 19073880): 95-104https://doi.org/10.1093/intimm/dxn128Crossref PubMed Scopus (45) Google Scholar]. Regarding the cardiovascular system, immunohistochemical studies on both human coronary and carotid arteries have revealed the presence of chymase-containing mast cells in the subendothelial intimal tissue [[2]Kovanen P.T. Mast cells as potential accelerators of human atherosclerosis-from early to late lesions.Int. J. Mol. Sci. 2019 Sep 11; 20 (PMID: 31514285; PMCID: PMC6770933): 4479https://doi.org/10.3390/ijms20184479Crossref PubMed Scopus (43) Google Scholar,[3]Jeziorska M. McCollum C. Woolley D.E. Mast cell distribution, activation, and phenotype in atherosclerotic lesions of human carotid arteries.J. Pathol. 1997 May; 182 (1<115::AID-PATH806>3.0.CO;2-9. Erratum in: J Pathol 1997 Oct;183(2):248. PMID: 9227350): 115-122https://doi.org/10.1002/(SICI)1096-9896(199705)182Crossref PubMed Scopus (0) Google Scholar]. Once atherosclerosis develops, mast cells accumulate in the rupture-prone areas of coronary atheromas where they become activated to expel their chymase- and tryptase-containing secretory granules into the surrounding interstitial space [[4]Bot I. Shi G.P. Kovanen P.T. Mast cells as effectors in atherosclerosis.Arterioscler. Thromb. Vasc. Biol. 2015 Feb; 35 (Epub 2014 Aug 7. PMID: 25104798; PMCID: PMC4304944): 265-271https://doi.org/10.1161/ATVBAHA.114.303570Crossref PubMed Scopus (109) Google Scholar]. In this issue of Atherosclerosis, Morales-Maldonado and coworkers [[5]Morales-Maldonado A. Humphry M. Figg N. Clarke M.C. Human vascular smooth muscle cells utilise chymase for the atypical cleavage and activation of interleukin-1β.Atherosclerosis. 2024; 390117308https://doi.org/10.1016/j.atherosclerosis.2023.117308Abstract Full Text Full Text PDF Scopus (0) Google Scholar] report that vascular smooth muscle cells (VSMCs) in the fibrous caps of human carotid atherosclerotic plaques express chymase and that primary VSMCs cultured from tissue explants obtained from patients undergoing aortic valve replacement surgery contain enzymatically active chymase. The authors also demonstrate that the intracellularly located active chymase possesses the ability to generate mature bioactive IL-1β by proteolytically cleaving its inactive precursor pro-IL-1β, which was synthesized by cultured VSMCs upon their stimulation with either LPS or IL-1α. Of note, in the present work, like in previous work from the same laboratory [[6]Clarke M.C. Talib S. Figg N.L. Bennett M.R. Vascular smooth muscle cell apoptosis induces interleukin-1-directed inflammation: effects of hyperlipidemia-mediated inhibition of phagocytosis.Circ. Res. 2010 Feb 5; 106 (Epub 2009 Nov 19. PMID: 19926874): 363-372https://doi.org/10.1161/CIRCRESAHA.109.208389Crossref PubMed Scopus (177) Google Scholar], the viable arterial VSMCs retained the generated mature IL-1β molecules without secreting them into the culture medium. For the release of IL-1β, cultured VSMCs have to undergo apoptosis and ensuing secondary necrosis under conditions of failed apoptotic cell clearance, such as hyperlipidemia [[6]Clarke M.C. Talib S. Figg N.L. Bennett M.R. Vascular smooth muscle cell apoptosis induces interleukin-1-directed inflammation: effects of hyperlipidemia-mediated inhibition of phagocytosis.Circ. Res. 2010 Feb 5; 106 (Epub 2009 Nov 19. PMID: 19926874): 363-372https://doi.org/10.1161/CIRCRESAHA.109.208389Crossref PubMed Scopus (177) Google Scholar]. Previous studies in our laboratory have shown that chymase released from activated mast cells causes apoptosis of co-cultured SMCs by degrading the fibronectin component of their pericellular matrix and thereby blocking the outside-in survival signaling necessary for their survival [[7]Leskinen M.J. Lindstedt K.A. Wang Y. Kovanen P.T. Mast cell chymase induces smooth muscle cell apoptosis by a mechanism involving fibronectin degradation and disruption of focal adhesions.Arterioscler. Thromb. Vasc. Biol. 2003 Feb 1; 23 (PMID: 12588765): 238-243https://doi.org/10.1161/01.atv.0000051405.68811.4dCrossref PubMed Google Scholar]. Thus, by triggering apoptosis, chymase-releasing activated mast cells in atherosclerotic lesions might allow IL-1β to be ultimately released from the adjacent VSMCs undergoing secondary necrosis (Fig. 1). Moreover, the extracellularly located granule heparin-bound chymase may possess the potential to activate any pro-IL-1β released from non-viable VSMCs into the extracellular fluid [[8]Afonina I.S. Müller C. Martin S.J. Beyaert R. Proteolytic processing of interleukin-1 family cytokines: variations on a common theme.Immunity. 2015 Jun 16; 42 (PMID: 26084020): 991-1004https://doi.org/10.1016/j.immuni.2015.06.003Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar]. Taken together, we may envision that in an atherosclerotic plaque of a hypercholesterolemic patient, multiple sites of action exist where the chymase enzyme could contribute to a locally increased concentration of IL-1β: firstly, by generating IL-1β within smooth muscle cells; secondly, after being released from mast cells it may trigger VSCM apoptosis with ensuing secondary necrosis and allow the generated IL-1β to be released from VSMCs; and thirdly, by converting any released pro-IL-1β into IL-1β. Such multisite local chymase-dependent generation of IL-1β could exert a strong proinflammatory effect on the surrounding viable VSMCs in the fibrous cap, in which it could induce the production and secretion of proinflammatory cytokines, as suggested by Clarke and coworkers [[6]Clarke M.C. Talib S. Figg N.L. Bennett M.R. Vascular smooth muscle cell apoptosis induces interleukin-1-directed inflammation: effects of hyperlipidemia-mediated inhibition of phagocytosis.Circ. Res. 2010 Feb 5; 106 (Epub 2009 Nov 19. PMID: 19926874): 363-372https://doi.org/10.1161/CIRCRESAHA.109.208389Crossref PubMed Scopus (177) Google Scholar]. Morales-Maldonado and colleagues [[5]Morales-Maldonado A. Humphry M. Figg N. Clarke M.C. Human vascular smooth muscle cells utilise chymase for the atypical cleavage and activation of interleukin-1β.Atherosclerosis. 2024; 390117308https://doi.org/10.1016/j.atherosclerosis.2023.117308Abstract Full Text Full Text PDF Scopus (0) Google Scholar] diligently speculate that inhibition of the non-canonical generation of IL-1β, i.e., by blocking the pro-IL-1β processing that does not utilize inflammasomes or caspases but chymase instead, such as it occurs in VSMCs in fibrous caps of atherosclerotic lesions, could potentially serve as a safe long-term strategy for the prevention of major acute cardiovascular events (MACEs). This concern is well taken when considering that MACEs are mainly caused by rupture of a fibrous cap covering an inflamed vulnerable atherosclerotic plaque and by the ensuing acute occlusive thrombosis [[9]Arbab-Zadeh A. Nakano M. Virmani R. Fuster V. Acute coronary events.Circulation. 2012 Mar 6; 125 (PMID: 22392862; PMCID: PMC3322378): 1147-1156https://doi.org/10.1161/CIRCULATIONAHA.111.047431Crossref PubMed Scopus (233) Google Scholar]. Notably, the fibrous cap is a dynamic structure consisting of VSMCs and a connective tissue matrix produced and maintained by VSMCs. When the cap becomes fragile, some areas gradually become thin and prone to rupture as a result of a localized net loss of VSMC numbers and a parallel dwindling of the extracellular matrix, facts that show the importance of preventing any loss of VSMCs beyond their normal turnover in the fibrous cap [[10]Alonso-Herranz L. Albarrán-Juárez J. Bentzon J.F. Mechanisms of fibrous cap formation in atherosclerosis.Front Cardiovasc Med. 2023 Aug 21; 10 (PMID: 37671141; PMCID: PMC10475556)1254114https://doi.org/10.3389/fcvm.2023.1254114Crossref PubMed Scopus (2) Google Scholar,[11]Lutgens E. de Muinck E.D. Kitslaar P.J. Tordoir J.H. Wellens H.J. Daemen M.J. Biphasic pattern of cell turnover characterizes the progression from fatty streaks to ruptured human atherosclerotic plaques.Cardiovasc. Res. 1999 Feb; 41 (PMID: 10341847): 473-479https://doi.org/10.1016/s0008-6363(98)00311-3Crossref PubMed Google Scholar]. In the CANTOS trial systemic inhibition of IL-1β did reduce the incidence of MACEs, thereby proving the concept of IL-1β or its downstream effectors, such as IL-6, being major factors behind the events [[12]Ridker P.M. Everett B.M. Thuren T. MacFadyen J.G. Chang W.H. Ballantyne C. Fonseca F. Nicolau J. Koenig W. Anker S.D. Kastelein J.J.P. Cornel J.H. Pais P. Pella D. Genest J. Cifkova R. Lorenzatti A. Forster T. Kobalava Z. Vida-Simiti L. Flather M. Shimokawa H. Ogawa H. Dellborg M. Rossi P.R.F. Troquay R.P.T. Libby P. Glynn R.J. CANTOS Trial GroupAntiinflammatory therapy with canakinumab for atherosclerotic disease.N. Engl. J. Med. 2017 Sep 21; 377 (Epub 2017 Aug 27. PMID: 28845751): 1119-1131https://doi.org/10.1056/NEJMoa1707914Crossref PubMed Scopus (5704) Google Scholar,[13]Ridker P.M. Libby P. MacFadyen J.G. Thuren T. Ballantyne C. Fonseca F. Koenig W. Shimokawa H. Everett B.M. Glynn R.J. Modulation of the interleukin-6 signalling pathway and incidence rates of atherosclerotic events and all-cause mortality: analyses from the Canakinumab Anti-Inflammatory Thrombosis Outcomes Study (CANTOS).Eur. Heart J. 2018 Oct 7; 39 (PMID: 30165610): 3499-3507https://doi.org/10.1093/eurheartj/ehy310Crossref PubMed Scopus (341) Google Scholar]. Yet, in the CANTOS trial, the systemic inhibition of circulating IL-1β by the specific anti-IL-1β antibody canakinumab was also associated with an increase in fatal infections, so reflecting the significance of IL-1β not only in the generation of MACEs but also in the promotion of bacterial defense in the host. To circumvent the above problem, should we direct IL-1β inhibition precisely to the plaque-localized mechanisms behind the clinical scenarios of MACEs? Mechanistically, a local VSMC-protecting effect of the inhibition of IL-1β and/or its downstream actors can be envisioned. The majority of the generation of active IL-1β in the human body occurs in cells belonging to the innate immune system, macrophages, neutrophils, and even mast cells being essential for first-line control of common bacterial infections [[14]Boyalla V. Gallego-Colon E. Spartalis M. Immunity and inflammation in cardiovascular disorders.BMC Cardiovasc. Disord. 2023 Mar 23; 23 (PMID: 36959565; PMCID: PMC10035189): 148https://doi.org/10.1186/s12872-023-03185-zCrossref PubMed Scopus (3) Google Scholar,[15]Bonnekoh H. Scheffel J. Kambe N. Krause K. The role of mast cells in autoinflammation.Immunol. Rev. 2018 Mar; 282 (PMID: 29431217): 265-275https://doi.org/10.1111/imr.12633Crossref PubMed Scopus (32) Google Scholar]. In these inflammatory cells, sensing of infection or danger leads to activation of the NLRP3 inflammasome- and the caspase-1-dependent IL-1β-processing canonical pathway. Thus, inhibition of the majority of IL-1β in plasma, i.e., the fraction that has been generated via activation of the canonical pathway in the inflammatory cells, is one likely reason for the infectious complications during the relatively strong IL-1β inhibition in the CANTOS trial. Since the production of IL-1β in neutrophils and mast cells is not only caspase-dependent but also executed by other neutral proteases [[16]Guma M. Ronacher L. Liu-Bryan R. Takai S. Karin M. Corr M. Caspase 1-independent activation of interleukin-1βeta in neutrophil-predominant inflammation.Arthritis Rheum. 2009 Dec; 60 (PMID: 19950258; PMCID: PMC2847793): 3642-3650https://doi.org/10.1002/art.24959Crossref PubMed Scopus (252) Google Scholar], inhibition of the minor non-canonical generation of IL-1β executed by alternative proteolytic enzymes in these particular cell populations could potentially evade the above problem. Here, again, it may be appropriate to exercise caution, inasmuch as any inhibition of IL-1β production in these inflammatory cells may compromise their full capacity to fight against acute bacterial infections. On the other hand, inhibition of the solely chymase-dependent generation of IL-1β in VSMCs would be safe since these cells are not involved in host defense. Would systemic inhibition of the chymase enzyme be an efficient and safe strategy to combat MACEs? Systemic inhibition of chymase potentially affects all chymase-producing cells in the body, particularly mast cells, which are the major cell population for the synthesis and storage of chymase, and which participate in host defense [[17]Caughey G.H. Mast cell tryptases and chymases in inflammation and host defense.Immunol. Rev. 2007 Jun; 217 (PMID: 17498057; PMCID: PMC2275918): 141-154https://doi.org/10.1111/j.1600-065X.2007.00509.xCrossref PubMed Scopus (345) Google Scholar]. Accordingly, systemic chymase inhibition might compromise the protective role of mast cells in the settings of bacterial infection [[18]Johnzon C.F. Rönnberg E. Pejler G. The role of mast cells in bacterial infection.Am. J. Pathol. 2016 Jan; 186 (Epub 2015 Oct 23. PMID: 26477818): 4-14https://doi.org/10.1016/j.ajpath.2015.06.024Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar]. Of more concern is the fact that, although systemic chymase inhibition is achievable by an oral drug, inhibition of the chymase-dependent production of IL-1β would block only a small fraction of its total body production, while the majority, i.e., the caspase-1–dependent canonical production in the inflammatory cells would be left uninhibited. Also, in advanced atherosclerotic plaques the majority of the professional inflammatory cells, notably macrophages, would be allowed to continue their IL-1β production and so negatively affect VSMCs present in their vicinity. Taken together, during systemic chymase-inhibition, VSCMs would be exposed to nearly pre-treatment levels of IL-1β derived either from the plasma or the inflammatory cells in the atherosclerotic plaques. Accordingly, systemic chymase inhibition achievable by any of the available oral drugs currently available, although considered to be safe [[19]Pejler G. Novel insight into the in vivo function of mast cell chymase: lessons from knockouts and inhibitors.J. Innate Immun. 2020; 12 (Epub 2020 Jun 4. PMID: 32498069; PMCID: PMC7506261): 357-372https://doi.org/10.1159/000506985Crossref PubMed Scopus (35) Google Scholar], would probably not be a reasonable choice when aiming at prevention of MACEs. However, the currently available option of using chymase inhibitors calls for future experimental studies. Finally, VSMCs are both sources and targets of IL-1β. If we allow chymase to remain active and so allow VSMCs to cleave pro-IL-1β into its biologically mature and active form, no acute danger is in sight, since the generated IL-1β would remain within the VSMC, where it appears not to cause any harm. Since the death of parent VSMCs is required for the release of bioactive IL-1β molecules, the best strategy to prevent fibrous cap from becoming vulnerable to rupture and causative of MACE would be to keep the matrix-producing cap VSMCs healthy and alive. A non-inflammatory fibrous cap environment lacking at least LPS and IL-1α, the two components capable of stimulating pro-IL-1β production in the cultured VSMCs [[5]Morales-Maldonado A. Humphry M. Figg N. Clarke M.C. Human vascular smooth muscle cells utilise chymase for the atypical cleavage and activation of interleukin-1β.Atherosclerosis. 2024; 390117308https://doi.org/10.1016/j.atherosclerosis.2023.117308Abstract Full Text Full Text PDF Scopus (0) Google Scholar], is worth pursuing. In contrast, inhibition of the chymase activity in the cap VSMCs is hardly part of any preventive strategy. The authors are to be congratulated and thanked for having sparked a topical discussion about novel strategies to safely control the inflammatory components that are driving atherogenesis and its acute complications. The author declares that he has no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Human vascular smooth muscle cells utilise chymase for the atypical cleavage and activation of Interleukin-1βAtherosclerosisVol. 390PreviewAtherosclerosis and other cardiovascular diseases (CVD) are well established to be both instigated and worsened by inflammation. Indeed, CANTOS formally proved that targeting the inflammatory cytokine IL-1β only could reduce both cardiovascular events and death. However, due to the central role of IL-1β in host defence, blockade increased fatal infections, suggesting targeting key immune mediators over the long natural history of CVD is unsuitable. Thus, discovering alternative mechanisms that generate vascular inflammation may identify more actionable targets. Full-Text PDF Open Access