Vascular smooth muscle cell phenotypic switching and plaque stability: a role for CHI3L1

This editorial refers to ‘Chitinase 3 like 1 (CHI3L1) is a regulator of smooth muscle cell physiology and atherosclerotic lesion stability’ by P. Tsantilas et al., pp. 2767–2780.

Introduction

Vascular smooth muscle cells (VSMCs) play important and varied roles in the development and progression of atherosclerosis. VSMCs form the medial layer of arteries where they maintain vessel tone via their contractile function. Under normal conditions, VSMCs remain quiescent, however, in atherosclerotic cardiovascular disease, these cells phenotypically switch to a ‘synthetic’ phenotype, characterized by down-regulation of the contractile machinery, secretion of extracellular matrix (ECM) proteins, proliferation, and migration. VSMCs were traditionally thought to play a dichotomous role in atherosclerosis; VSMC proliferation drives lesion growth whereas ECM production results in fibrous cap formation in mature plaque, which stabilizes lesions and prevents rupture.^1,2 Recently, however, lineage tracing and single-cell RNA-sequencing profiling have revealed additional VSMCs plasticity in disease and shown that VSMCs generate cells resembling foam cells, macrophages, mesenchymal stem cells, and osteochondrogenic cells.^3–5 The importance of these phenotypes in vivo, and their functional impact on plaque growth and stability, is yet to be resolved.

Novel findings

In an exciting new study by Tsantilas et al., CHI3L1 is identified as a novel regulator of VSMC phenotype (Figure 1)⁶. CHI3L1, or Chil1 in mouse, is a secreted glycoprotein expressed by several plaque-relevant cells including macrophages, chondrocytes, and VSMCs. CHI3L1 expression is up-regulated in a number of inflammatory conditions including asthma and arthritis and has been previously suggested as a biomarker of vulnerable plaque due to increased serum levels detected in patients with symptomatic carotid atherosclerosis.⁷

Figure 1.

CHI3L1 is a novel regulator of VSMC phenotype. CHI3L1 is detected at low levels in stable plaque, but high expression is detected in vulnerable plaques, correlating with areas of aSMA expression. Inhibition of CHI3L1 in cultured VSMCs induces downregulation of the contractile phenotype, and upregulation of CD68 and cell proliferation.

Initial investigation of human samples revealed that compared with stable lesions, CHI3L1 levels were elevated in plaques that were classed as vulnerable or had ruptured. The increased expression of CHI3L1 in unstable plaques could initially imply a negative role of CHI3L1 in atherosclerosis, yet within vulnerable plaque CHI3L1 predominantly co-localized with αSMA+ cells, which are generally regarded as stabilizing. This co-localization with VSMC contractile markers was not observed in stable plaques. Based on these observations, it is not straightforward to interpret how CHI3L1 affects plaque stability, or whether CHI3L1 expression is a cause or consequence of contractile gene expression in vulnerable lesions.

To assess the functional relationship between CHI3L1 and contractile gene expression, the authors performed loss-of-function studies. CHI3L1 knockdown in cultured human VSMCs resulted in increased proliferation, apoptosis, and down-regulation of ACTA2 combined with an increase in CD68, suggestive of VSMC transdifferentiation to a macrophage-like phenotype, effects generally regarded as deleterious.

In Apoe−/− mouse atherosclerosis models Chil1 knockout resulted in the formation of more advanced lesions with increased intimal thickening. Whilst results regarding rupture were inconclusive, fewer αSMA+ cells localized to the cap region of plaques in knockdown animals, suggestive of reduced stability.

The authors conclude that CHI3L1 expression stabilizes plaque and prevents maladaptive VSMC changes, and it is clear from the extensive experiments in this study that CHI3L1 knockdown exacerbates VSMC phenotypic switching and worsens disease outcome. These findings would suggest that augmenting CHI3L1 could be used to prevent phenotypic switching and stabilize plaque. In line with this idea, overexpression of CHI3L1 decreased VSMC proliferation, suggesting a muting of phenotypic switching, yet did not result in changes to ACTA2 or CD68 expression. Furthermore, the elevated expression of CHI3L1 in vulnerable and ruptured human plaque samples could imply that increased CHI3L1 expression alone is not sufficient to ‘rescue’ unstable plaque.

Wider outlook and mechanism of action

Taken together, these data may imply both dose- and context-specific effects of CHI3L1, suggesting that CHI3L1 could be necessary to prevent phenotypic switching, but whether its overexpression is sufficient to induce reversion to a contractile state remains to be demonstrated. In order to clarify the value of manipulating CHI3L1 levels as a therapeutic strategy, it is necessary to understand if the increased CHI3L1 expression seen in vulnerable plaque is directly responsible for the expression of αSMA. Furthermore, it will be important to investigate how induction of CHI3L1 impacts the severity and stability of established lesions.

Looking forward, understanding the mechanism by which knockdown of CHI3L1 regulates interconversion of contractile, αSMA+ VSMCs and CD68-expressing cells would reveal downstream pathways that would allow more specific targeting. Intriguingly, the study identified the tumour suppressor LATS2 as a potential downstream factor, finding a direct negative correlation between LATS2 and CHI3L1 levels. Importantly, LATS2 knockdown was able to suppress expression of macrophage markers, suggesting a potential therapeutic advantage that may warrant further investigation.

Fully understanding the role of CHI3L1 is complicated by its identity as a secreted protein, meaning its impact on the plaque niche must be considered. Firstly, while tissue culture experiments are insightful, these do not model the multiple cell types and inter-cell-communication underlying plaque development. Secondly, delineation of which cells may be responding to CHI3L1 expression and their mode of action is impeded by the lack of identification of cell surface binding partner for CHI3L1.

As the regulation and remodelling of the ECM is a key element in plaque stability, with profound implications for VSMC phenotypic switching, the influence of CHI3L1 on cell-ECM interactions should be considered. Vulnerable and ruptured plaques have been reported to contain less collagen and have elevated levels of proteases. This is of note as CHI3L1 is known to bind collagen I, and has a role in collagen fibril formation.⁸ Alongside providing structural support, the ECM acts in both mechanical and chemical signalling networks that influence VSMC behaviour. It may therefore be possible that CHI3L1’s role in atherosclerosis and VSMC phenotype is indirect—functioning via modification of the ECM landscape with which VSMCs interact.

Conclusions

In conclusion, the field is moving towards a greater understanding of the multifaceted roles VSMCs can play within plaques. Defining how distinct phenotypes are acquired, and how to steer VSMCs towards more favourable cell states may be key to strengthening vessels walls and preventing adverse outcomes in atherosclerosis. Furthermore, as highlighted by this study, it is becoming clear that we must consider the entire plaque environment, in particular communication between cells and the ECM, if we wish to fully understand VSMC regulation.

Conflict of interest: none declared.

Funding

The authors are funded by the British Heart Foundation (BHF) grants PG/19/6/34153 and CH/2000003.

 

The opinions expressed in this article are not necessarily those of the Editors of Cardiovascular Research or of the European Society of Cardiology.