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Phenotype-Specific Induced Pluripotent Stem Cell–Derived Vascular Smooth Muscle Cells to Model Vascular Disease: Implications of Differentiation Protocols

Originally publishedhttps://doi.org/10.1161/HYPERTENSIONAHA.123.20871Hypertension. 2023;80:754–756

See related article, pp 740–753

Vascular smooth muscle cells (VSMCs) are central components of vascular biology as they are responsible for maintaining structural integrity of blood vessels and are the functional elements responsible for controlling vascular tone through changes in contraction and dilation. VSMCs are regarded as highly specialized contractile cells that also possess remarkable plasticity changing their phenotype in response to injury in attempt to maintain or repair vascular function.1 However, this extensive plasticity is at the core of pathological processes of several cardiovascular diseases such as hypertension, atherosclerosis, aneurysm formation, intimal hyperplasia, transplant vasculopathy, and abnormal tumor vasculature.2 Extensive research in the field identified factors that influence the VSMC phenotype; however, genetic and environmental factors regulating VSMC plasticity are still incompletely understood. Investigation of molecular processes underlying phenotypic switching is critical for mechanism- and disease-specific therapeutic targeting of VSMC-related cardiovascular diseases. In the present issue of Hypertension, Liu et al3 deeply characterized 2 human induced pluripotent stem cell (iPSC)–derived smooth muscle cell (SMC) models representing quiescent/procontractile and proliferative/synthetic VSMCs and shed light on epigenetic and transcriptional properties involved in VSMC differentiation and plasticity and the implications in pathological conditions (Figure).

In healthy vessels, VSMCs are quiescent contractile cells that express high levels of a unique set of contractile machinery proteins such as SM-MHC (smooth muscle myosin heavy chain), α-SMA (alpha smooth muscle actin), SM22 (smooth muscle protein 22), CNN1 (calponin-1), and smoothelin. Under pathological conditions, VSMCs differentiate into a synthetic and migratory phenotype with reduction of expression of contractile proteins and increase in proliferation, migration, and extracellular matrix (ECM) production.1 Advances in the field with the development of high-throughput single-cell multiomics and fate tracking add to the complexity of these processes and have revealed that there is VSMC diversity in healthy arteries and that during diseases, such as atherosclerosis, VSMCs can assume multiple phenotypes including macrophage-like, mesenchymal stem cell–like, chondrocyte-like cells, myofibroblasts, and beige adipocyte-like cells.4 Several factors influencing VSMC phenotypic switching including transcriptional factors (KLF4 [Krüppel-like factor 4], NOTCH3, Hippo/YAP1 [Yes-associated protein 1]) and epigenetic regulation (DNA methylation, histone deacetylation, and microRNAs), as well as signals from growth factors, cytokines, mechanical forces, ECM, and oxidative stress were identified.2 However, molecular mechanisms regulating VSMC transition and commitment to specific phenotypes are still incompletely understood.

Differentiation of VSMCs from iPSCs is a promising approach to investigate genetic and epigenetic regulation of VSMC plasticity. Advances in iPSC biology have facilitated generation of lineage-specific VSMCs from iPSCs, overcoming some of the challenges of primary human VSMC culture, such as limited tissue access, phenotypic alteration in culture, and heterogeneity of VSMC culture.5 However, despite progress in the field, generation of iPSC-VSMCs that recapitulate relevant phenotypes in cardiovascular disease is still challenging. Most protocols of VSMC differentiation rely on factors that modulate the VSMC phenotype such as transforming growth factor beta (TGF-β)6 and platelet-derived growth factor (PDGF)7 and expression of markers of mature VSMCs such as SM-MHC and smoothelin are variable.5 New approaches to address these challenges are urgently needed so that iPSC-VSMCs truly recapitulate native VSMCs. The field is advancing with novel strategies to differentiate VSMCs without having to expose cells to different growth factors that may variably influence the phenotype of VSMCs, as highlighted in the current issue.3

Liu et al3 implemented a protocol using the small molecule RepSox, identified as a promoter of SM-MHC positive cell differentiation via activation of NOTCH signaling,8 as a model for development of procontractile VSMCs (R-SMCs). The authors compared this model with the classical TGF-β/PDGF model of VSMC differentiation (TP-SMCs; Figure). When compared with TP-SMCs, R-VSMCs showed high levels of SM-MHC, lower migration and proliferation rates, and higher Ca2+ influx in response to procontractile agents angiotensin II, a potent vasoactive peptide, and carbachol, an activator of muscarinic receptors. The cellular characterization performed in the study supports the different VSMC phenotypes; however, further characterization of contractile properties of this model would be interesting to assess the functional relevance of procontractile iPSC-VSMCs in the context of cardiovascular disease. In the present study,3 the authors used the collagen contraction assay as an index of contraction. A potential limitation is that this is an indirect assay, and, therefore, there is no definitive proof that contraction of VSMCs is actually measured. The collagen contraction assay readout is not always accurate or reliable, and at best, the responses recorded are qualitative.9 Additional patch-clamp studies to directly measure VSMC contraction would complement characterization of the model as contractile VSMCs and would help overcome the challenges of studying contractile cells in vitro.

The gene expression profile of the iPSC-VSMCs also showed striking differences between R-SMCs and T-SMCs. The R-SMC gene expression profile was associated with genes involved in muscular contraction and had a higher correlation with arterial tissues. Open chromatin analysis by assay of transposase accessible chromatin sequencing (ATAC-seq) revealed enrichment in binding sites for transcription factors FOX (Fork-head box) and HAND1 (Heart And Neural Crest Derivatives Expressed 1). This model could be a useful tool to investigate the mechanisms related to maintenance of the contractile phenotype and vascular homeostasis in health and cardiovascular diseases. Conversely, the TP-SMC gene profile was associated with endoplasmic reticulum stress response, macroautophagy, and protein secretion. Endoplasmic reticulum stress, a response to accumulation of misfolded proteins in the endoplasmic reticulum, plays an important role in VSMC dysfunction associated with hypertension and other cardiovascular diseases.10 It has been shown that endoplasmic reticulum stress participates in VSMC phenotypic modulation in atherosclerosis via induction of the transcription factor KLF4.11 Additionally, enrichment of RUNX family transcription factor in TP-SMCs could be considered reassuring that the model is relevant to VSMC disease modeling, as it is also involved in VSMC phenotypic switching toward an osteogenic-like phenotype.12 These findings demonstrated that the synthetic VSMC model resembles some of the characteristics observed in human disease and provide a potential platform for study of novel regulatory mechanisms.

The iPSC-SMC models implemented by Liu et al3 also showed the potential to study regulation of noncoding variants associated with cardiovascular diseases. The authors fully characterized the transcriptomic profile of the iPSC-SMC models and compared open chromatin regions of both models to primary VSMC from coronary arteries and whole coronary arteries. Variants associated with systolic blood pressure were found in both models; however, variants associated with intracranial aneurysm were enriched only in R-SMCs while TP-SMCs were associated with peripheral artery disease.

Taken together, the work from Liu et al3 has revealed new models to study genetic and epigenetic factors relevant to cardiovascular diseases. In addition to deeply characterizing iPSC-SMCs using different cell differentiation approaches, this study3 further raises important questions as to why gene profiles of apparent contractile and proliferative VSMCs differ depending on the experimental strategies used to differentiate cells. Moreover, these differential findings raise concerns regarding how closely iPSC-VSMCs recapitulate apparent VSMC changes in vascular changes associated with cardiovascular disease. It is only when VSMC contraction is directly measured, for example, using electrophysiology approaches, that the phenotype of VSMCs can definitively be identified as functionally contractile. Future studies refining protocols that truly resemble contractile VSMC and other VSMC phenotypes would advance the understanding of VSMC phenotypic modulation and the relevance of different VSMC phenotypes in cardiovascular diseases.

Figure

Figure Generation of procontractile and synthetic vascular smooth muscle cells (VSMCs) from induced pluripotent stem cells (iPSCs). Differentiation of VSMCs using the small molecule RepSox generated procontractile VSMCs characterized by reduced proliferation and migration and increased Ca2+ in response to contractile agents. Further transcriptomic profiling revealed enrichment of genes related to muscle contraction and cardiovascular disease (CAD)–associated variants related to blood pressure and intracranial aneurism. On the contrary, differentiation of VSMCs using PDGF (platelet-derived growth factor)-BB and TGF-β (transforming growth factor beta) generated cells that resemble the synthetic VSMC phenotype, showing higher rates of proliferation and migration and a transcriptomic profile associated with endoplasmic reticulum (ER) stress and peripheral artery disease.

Article Information

Disclosures None.

Footnotes

For Sources of Funding and Disclosures, see page 756.

The opinions expressed in this article are not necessarily those of the editors nor the American Heart Association.

Correspondence to: Livia L. Camargo, PhD, Research Institute of the McGill University Health Centre, McGill University, Site Glen Block E, Bureau/Office E01.3362, 1001 Decarie Blvd, Montreal, Quebec H4A3J1, Canada. Email

References

  • 1. Owens GK, Kumar MS, Wamhoff BR. Molecular regulation of vascular smooth muscle cell differentiation in development and disease.Physiol Rev. 2004; 84:767–801. doi: 10.1152/physrev.00041.2003CrossrefMedlineGoogle Scholar
  • 2. Chakraborty R, Chatterjee P, Dave JM, Ostriker AC, Greif DM, Rzucidlo EM, Martin KA. Targeting smooth muscle cell phenotypic switching in vascular disease.JVS Vasc Sci. 2021; 2:79–94. doi: 10.1016/j.jvssci.2021.04.001CrossrefMedlineGoogle Scholar
  • 3. Liu L, Jouve C, Henry J, Berrandou TE, Hulot JS, Georges A, Bouatia-Naji N. Genomic, transcriptomic, and proteomic depiction of induced pluripotent stem cell–derived smooth muscle cells as emerging cellular models for arterial diseases.Hypertension. 2023; 80:740–753. doi: 10.1161/HYPERTENSIONAHA.122.19733LinkGoogle Scholar
  • 4. Liu M, Gomez D. Smooth muscle cell phenotypic diversity.Arterioscler Thromb Vasc Biol. 2019; 39:1715–1723. doi: 10.1161/atvbaha.119.312131LinkGoogle Scholar
  • 5. Shen M, Quertermous T, Fischbein MP, Wu JC. Generation of vascular smooth muscle cells from induced pluripotent stem cells: methods, applications, and considerations.Circ Res. 2021; 128:670–686. doi: 10.1161/circresaha.120.318049LinkGoogle Scholar
  • 6. Tang Y, Urs S, Boucher J, Bernaiche T, Venkatesh D, Spicer DB, Vary CP, Liaw L. Notch and transforming growth factor-beta (TGFbeta) signaling pathways cooperatively regulate vascular smooth muscle cell differentiation.J Biol Chem. 2010; 285:17556–17563. doi: 10.1074/jbc.M109.076414CrossrefMedlineGoogle Scholar
  • 7. Thomas JA, Deaton RA, Hastings NE, Shang Y, Moehle CW, Eriksson U, Topouzis S, Wamhoff BR, Blackman BR, Owens GK. PDGF-DD, a novel mediator of smooth muscle cell phenotypic modulation, is upregulated in endothelial cells exposed to atherosclerosis-prone flow patterns.Am J Physiol Heart Circ Physiol. 2009; 296:H442–H452. doi: 10.1152/ajpheart.00165.2008CrossrefMedlineGoogle Scholar
  • 8. Zhang J, McIntosh BE, Wang B, Brown ME, Probasco MD, Webster S, Duffin B, Zhou Y, Guo LW, Burlingham WJ, et al. A human pluripotent stem cell-based screen for smooth muscle cell differentiation and maturation identifies inhibitors of intimal hyperplasia.Stem Cell Rep. 2019; 12:1269–1281. doi: 10.1016/j.stemcr.2019.04.013CrossrefMedlineGoogle Scholar
  • 9. Bravo DD, Chernov-Rogan T, Chen J, Wang J. An impedance-based cell contraction assay using human primary smooth muscle cells and fibroblasts.J Pharmacol Toxicol Methods. 2018; 89:47–53. doi: 10.1016/j.vascn.2017.10.006CrossrefMedlineGoogle Scholar
  • 10. Camargo LL, Harvey AP, Rios FJ, Tsiropoulou S, Da Silva RNO, Cao Z, Graham D, McMaster C, Burchmore RJ, Hartley RC, et al. Vascular Nox (NADPH Oxidase) compartmentalization, protein hyperoxidation, and endoplasmic reticulum stress response in hypertension.Hypertension. 2018; 72:235–246. doi: 10.1161/HYPERTENSIONAHA.118.10824LinkGoogle Scholar
  • 11. Chattopadhyay A, Kwartler CS, Kaw K, Li Y, Kaw A, Chen J, LeMaire SA, Shen YH, Milewicz DM. Cholesterol-induced phenotypic modulation of smooth muscle cells to macrophage/fibroblast-like cells is driven by an unfolded protein response.Arterioscler Thromb Vasc Biol. 2021; 41:302–316. doi: 10.1161/ATVBAHA.120.315164. Epub 2020 Oct 8LinkGoogle Scholar
  • 12. da Silva RA, da S Feltran G, da C Fernandes CJ, Zambuzzi WF. Osteogenic gene markers are epigenetically reprogrammed during contractile-to-calcifying vascular smooth muscle cell phenotype transition.Cell Signal. 2020; 66:109458. doi: 10.1016/j.cellsig.2019.109458CrossrefMedlineGoogle Scholar

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