Abstract

As many as 70% of cells in atherosclerotic plaque are vascular smooth muscle cell (VSMC) in origin, and pathways and proteins which regulate VSMC migration, proliferation, and phenotype modulation represent novel targets for rational drug design to reduce atherosclerotic vascular disease. In this volume of Clinical Science, Karle et al. demonstrate that tumor suppressor, promyelocytic leukemia protein (PML) plays an important role in regulation of VSMC phenotype and response to inflammatory stimuli (Clin Sci (2021) 135(7), 887-905; DOI: 10.1042/CS20201399). This important work demonstrates that PML, previously unrecognized as a participant in development of atherosclerosis, may represent a novel target for anti-atherosclerotic therapeutic modalities.

Atherosclerosis

Despite advances in prevention and treatment, atherosclerotic vascular diseases are a cause of substantial morbidity, mortality, and economic burden in the Western and developing world. Atherosclerosis is a lipid-driven, chronic inflammatory process involving multiple cell types in various stages of activation, apoptosis, and death. The major effector cells in this process are macrophages, lymphocytes, endothelial cells, and vascular smooth muscle cells (VSMCs); the activation of these cells is initiated and sustained by a tightly regulated network of cytokines. Despite a focus on inflammatory cells, more recently, the role of VSMCs in atherogenesis has gained attention, and as many as 70% of cells in atherosclerotic plaque are VSMC in origin [1]. In the presence of inflammatory stimuli, normally quiescent VSMC assume a synthetic, de-differentiated phenotype, migrate from the media into the intima where they proliferate, and, in the case of advanced plaque, form a cellular ‘cap’ over the developing lipid core [2–4]. VSMCs are largely responsible for maintaining plaque stability, and are essential in maintaining in cap patency and the vulnerability of plaque to rupture, leading to thrombi formation and myocardial ischemia. With this background in mind, identification of pathways and proteins involved in functioning and communication of plaque cells and maintenance of VSMCs quiescence is key to development of modalities to combat vascular inflammatory diseases.

Promyelocytic leukemia protein

Interestingly, numerous similarities between atherosclerosis and cancer have been noted [5,6]. In terms of dysregulation of gene expression, cell proliferation, inflammatory response, and plaque VSMC clonality, some investigators have begun to focus their attention to proteins traditionally thought of as tumor promotors and tumor suppressor proteins as important players in atherogenesis. In this volume of Clinical Science, Karle et al. [7] investigate the expression and functional ramifications of promyelocytic leukemia protein (PML) in development of atherosclerosis. Fittingly named, PML is a protein first detected in acute promyelocytic leukemia initially and classified as a tumor suppressor protein [8,9]. PML is a component of nuclear structures termed PML-nuclear bodies (PML-NBs) which are associated with numerous fundamental cellular functions including inflammation, apoptosis, and cell division; many processes prominent in atherogenesis. PML is a reported potent mediator of NF-κB transcription and TNFα responses, all integral pro-inflammatory cellular events prominent in development of atherosclerosis [10,11]. Despite these intriguing similarities, there is a dearth of information linking PML expression in atherosclerotic plaque, and this study breaks new ground in testing the hypothesis that PML expression plays a previously unrecognized role in VSMC activation and atherosclerosis development.

As an initial approach, the authors determined that significantly more PML was found in lysates of atherosclerotic arteries compared with normal healthy arteries. Further, dissection of the arteries determined that PML was preferentially found in plaque-containing areas of those arteries compared with non-plaque sections. Extending this analysis, immunohistochemistry using cell-lineage specific antibody determined that VSMCs, and particularly VSMCs in the vulnerable shoulder regions of the plaque had the most robust PML immunoreactivity compared with other cell types in the plaque such as endothelial and mononuclear cells. VSMCs in plaque exist in an inflammatory milieu, and culture of human CaSMC with pro-inflammatory cytokines such as TNFα and IFNγ also increased abundance of PML, and formation of PML-NBs. This was in contrast with CaSMC exposed to TGFβ, which did not have any effect on PMBL-NB or PML abundance. This further strengthened a link between PML and the VSMC inflammatory response and suggested a functional role for PML in inflammatory vascular disease such as atherosclerosis.

VSMCs phenotype and PML expression

Smooth muscle cells demonstrate incredible plasticity and in fact are capable of two phenotypes, contractile or synthetic, as illustrated by the ‘response to injury’ hypothesis originally proposed by Raines and Ross [12]. In the healthy artery, quiescent medial VSMCs are fully differentiated and respond mainly to vasoconstricting or vasodilatory peptides. However, VSMCs respond to local inflammation by expressing numerous cytokines, matrix, and cell proliferation proteins and exhibit a de-differentiated phenotype and transcriptome. This process has been the subject of numerous studies and has identified a multitude of transcription factors and other proteins thought to regulate the VSMC phenotypic switch [3]. PML mutation or loss, and the subsequent dysregulation of these processes, has been implicated in a variety of cancers [13]. PML is a tumor suppressor and recognized regulator of cell differentiation, implying a similar role in regulation of VSMC de-differentiation and phenotypic switch atherogenesis [14]. Indeed, Karle et al. found that VSMCs in which PML was overexpressed by transfection proliferated more rapidly and demonstrated morphological and gene expression patterns associated with the de-differentiated VSMC phenotype and consistent with that found in atherosclerotic plaque. Conversely, siRNA knockdown of PML resulted in decreased VSMC proliferation, consistent with the differentiated, quiescent VSMC phenotype. Further, the de-differentiation induced by IFNγ was reduced when PML expression was reduced by siRNA. Together these observations confirm initial observations that increased expression of PML was observed in plaque VSMC more than uninvolved medial VSMC, and demonstrate a causative effect of PML abundance and the VSMC phenotypic state.

SUMOylation, PML, and atherosclerosis

PML-NB complexes are composed of PML associated with other proteins modified by the small ubiquitin-like modifier (SUMO). SUMOylation of PML itself is essential for the integrity of PML-NBs [15]. There is a large body of literature demonstrating SUMOlyation as an inflammatory regulatory process. For example, SUMOlyation of MAPK pathway proteins and other NF-κB pathway components such as IκBα can inhibit its ubiquitination and destruction resulting in down-regulation of the NF-κB pro-inflammatory pathway [16]. Its not surprising then that in contrast with PML there are a host of studies which investigate and implicate an important role for SUMOlyation in atherosclerosis (for review, [17]). The present study reported that similar to PML, abundance of SUMO-1 was significantly increased in atherosclerotic plaque compared with normal human coronary arteries. Further, the authors identified co-localization of PML with SUMO-1 in PML-NB in nuclei of plaque VSMC.

SUMO proteins are members of the ubiquitin-like family of proteins which covalently interact with other proteins in PML-NBs to modify their protein–protein interactions, subcellular nuclear localization, protein–DNA interactions and enzymatic activity [18]. SUMOylation is an important post-translational modification which regulates diverse cellular processes such as transcriptional regulation, apoptosis, response to inflammation and stress, and proliferation, all pro-atherogenic processes [18]. Karle et al. extended these data and tested the hypothesis that PML expression and SUMOylation participated in VSMC phenotype modulation and development of atherosclerosis, and number of interesting observations were noted. First, SUMO-1-dependent protein SUMOylation was significantly increased in hCaVSMC transfected with a PML expression vector. Secondly, if SUMOylation of PML were inhibited by transfection with a PML mutant lacking SUMOylation residues, proliferation and expression of VSMC de-differentiation markers did not increase as they did with transfection with the functional PML expression vector. This indicates that it is not PML overexpression itself that leads to increased VSMC proliferation and de-differentiation, but the SUMOlyation of PML that mediates these changes, and points to the PML-mediated protein SUMOylation pathway as a target for anti-atherosclerotic therapeutic strategies. This pathway is summarized in Figure 1.

PML SUMOylation, VSMCs differentiation and atherosclerosis

Figure 1
PML SUMOylation, VSMCs differentiation and atherosclerosis

The left side of the panel depicts PML and VSMC under normal, non-inflammatory conditions, where PML exists in its native, un-SUMOlyated form and VSMCs exist in a contractile, quiescent phenotype. These non-synthetic, contractile VSMCs generally reside in the media of non-atherosclerotic arteries. The right side of the panel depicts PML in the inflammatory state, where it is SUMOlyated and the number of PML-NBs increase, with ensuing alterations in gene expression. In this situation, VSMC exist in a synthetic, proliferative, de-differentiated phenotype, and generally exist in the plaque shoulder and cap regions of atherosclerotic plaque.

Figure 1
PML SUMOylation, VSMCs differentiation and atherosclerosis

The left side of the panel depicts PML and VSMC under normal, non-inflammatory conditions, where PML exists in its native, un-SUMOlyated form and VSMCs exist in a contractile, quiescent phenotype. These non-synthetic, contractile VSMCs generally reside in the media of non-atherosclerotic arteries. The right side of the panel depicts PML in the inflammatory state, where it is SUMOlyated and the number of PML-NBs increase, with ensuing alterations in gene expression. In this situation, VSMC exist in a synthetic, proliferative, de-differentiated phenotype, and generally exist in the plaque shoulder and cap regions of atherosclerotic plaque.

Limitations and future directions

As novel as this study is, there are areas which need be developed to more definitively ascribe a role for PML SUMOylation in regulation of VSMC homeostasis and atherogenesis. Most promising would be development of an appropriate genetically modified animal model with which to determine causality. PML knockout mice are viable, fertile and phenotypically indistinguishable from wildtype and heterozygous littermates, but demonstrated hematopoietic irregularities and were tumorigenic [19]. It would be informative to generate a SMC-specific PML deletion moue, or a dominant-negative PML mutant lacking SUMOylation residues crossed with one of the accepted athero-prone mouse strains (ApoE−/− or LDLR−/−) to demonstrate PML causality. As the authors correctly point out, macrophages other cell types can express SMC markers, and in fact, VSMCs often lose typical VSMC markers in the presence of cholesterol and inflammatory cytokines in plaques [1,20]. In this regard, we cannot be absolutely sure that the PML contribution to SUMOlyation and phenotype modulation are occurring in VSMC. This limitation being pointed out, it does open up an intriguing possibility that the phenotype of other cell types relevant to plaque development, such as the M1-M2 paradigm in macrophages, could also be regulated by PML and contribute to atherosclerotic plaque development. Regardless of these limitations, this study points to PML SUMOylation as a previously unrecognized cellular event in development of atherosclerosis, and provides us with novel targets for pharmaceutical intervention and future investigation.

Competing Interests

The authors declare that there are no competing interests associated with the manuscript.

Funding

This work was supported by the National Heart, Lung, and Blood Institute of the National Institutes of Health [grant numbers HL141108, HL117724 (to M.V.A.)].

Abbreviations

     
  • PML

    promyelocytic leukemia protein

  •  
  • PML-NB

    PML-nuclear body

  •  
  • SUMO

    small ubiquitin-like modifier

  •  
  • VSMC

    vascular smooth muscle cell

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Author notes

*

These authors equally contributed as first author to this work.