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The Hippo signaling effector, TEAD1 plays an essential role in cardiovascular development. However, a role for TEAD1 in postmitotic cardiomyocytes (CMs) remains incompletely understood. Herein we reported that TEAD1 is required for postmitotic CM survival. We found that adult mice with ubiquitous or CM-specific loss of Tead1 present with a rapid lethality due to an acute-onset dilated cardiomyopathy. Surprisingly, deletion of Tead1 activated the necroptotic pathway and induced massive cardiomyocyte necroptosis, but not apoptosis. In contrast to apoptosis, necroptosis is a pro-inflammatory form of cell death and consistent with this, dramatically higher levels of markers of activated macrophages and pro-inflammatory cytokines were observed in the hearts of Tead1 knockout mice. Blocking necroptosis by administration of necrostatin-1 rescued Tead1 deletion-induced heart failure. Mechanistically, genome-wide transcriptome and ChIP-seq analysis revealed that in adult hearts, Tead1 directly activates a large set of nuclear DNA-encoded mitochondrial genes required for assembly of the electron transfer complex and the production of ATP. Loss of Tead1 expression in adult CMs increased mitochondrial reactive oxygen species, disrupted the structure of mitochondria, reduced complex I-IV driven oxygen consumption and ATP levels, resulting in the activation of necroptosis. This study identifies an unexpected paradigm in which TEAD1 is essential for postmitotic CM survival by maintaining the expression of nuclear DNA-encoded mitochondrial genes required for ATP synthesis.

We have previously demonstrated that the transcription co-factor yes-associated protein 1 (YAP1) promotes vascular smooth muscle cell (VSMC) de-differentiation. Yet, the role and underlying mechanisms of YAP1 in neointima formation in vivo remain unclear. The goal of this study was to investigate the role of VSMC-expressed YAP1 in vascular injury-induced VSMC proliferation and delineate the mechanisms underlying its action. Experiments employing gain- or loss-of-function of YAP1 demonstrated that YAP1 promotes human VSMC proliferation. Mechanistically, we identified platelet-derived growth factor receptor beta (PDGFRB) as a novel YAP1 target gene that confers the YAP1-dependent hyper-proliferative effects in VSMCs. Furthermore, we identified TEA domain transcription factor 1 (TEAD1) as a key transcription factor that mediates YAP1-dependent PDGFRβ expression. ChIP assays demonstrated that TEAD1 is enriched at a PDGFRB gene enhancer. Luciferase reporter assays further demonstrated that YAP1 and TEAD1 co-operatively activate the PDGFRB enhancer. Consistent with these observations, we found that YAP1 expression is upregulated after arterial injury and correlates with PDGFRβ expression and VSMC proliferation in vivo. Using a novel inducible SM-specific Yap1 knockout mouse model, we found that the specific deletion of Yap1 in adult VSMCs is sufficient to attenuate arterial injury-induced neointima formation, largely due to inhibited PDGFRβ expression and VSMC proliferation. Our study unravels a novel mechanism by which YAP1/TEAD1 promote VSMC proliferation via transcriptional induction of PDGFRβ, thereby enhancing PDGF-BB downstream signaling and promoting neointima formation.

Objective: While GFAP (glial fibrillary acidic protein) is commonly used as a classical marker for astrocytes in the central nervous system, GFAP-expressing progenitor cells give rise to other cell types during development. The goal of this study was to investigate whether GFAP-expressing progenitor cells contribute to the development of vascular cells in major arteries. Approach and Results: To label GFAP-expressing progenitor cells and their progeny, we crossed GFAP promoter-driven Cre recombinase mice ( GFAP - Cre ) with transgenic mice expressing the Cre -dependent mTmG dual fluorescent reporter gene. Using this genetic fate-mapping approach, here we demonstrate that GFAP-positive progenitor cells contribute to the development of vascular smooth muscle cells in both neural crest- and non-neural crest-derived vascular beds. In addition, GFAP-positive progenitor cells contribute to a subset of endothelial cells in some vasculature. Furthermore, fate-mapping analyses at multiple time points of mouse development demonstrate a time-dependent increase in the contribution of GFAP-positive progenitors to vascular smooth muscle cells, which mostly occurs in the postnatal period. Conclusions: Our study demonstrates that vascular smooth muscle cells and endothelial cells within the same vascular segment are developmentally heterogeneous, where varying proportions of vascular smooth muscle cells and endothelial cells are contributed by GFAP-positive progenitor cells.

Background/Aims: Pioglitazone (PIO), an antidiabetic drug, has been shown to attenuate vascular smooth muscle cell (VSMC) proliferation, which is a major event in atherosclerosis and restenosis after angioplasty. Till date, the likely contributory role of AMP-activated protein kinase (AMPK) toward PIO inhibition of VSMC proliferation has not been examined in vivo. This study is aimed at determining whether pharmacological inhibition of AMPK would prevent the inhibitory effect of PIO on neointima formation in a mouse model of arterial injury. Methods: Male CJ57BL/6J mice were subjected to femoral artery injury using guidewire. PIO (20 mg/kg/day) was administered orally 1 day before surgery and for 3 weeks until sacrifice in the absence or presence of compound C (an AMPK inhibitor). Injured femoral arteries were used for morphometric analysis of neointima formation. Aortic tissue lysates were used for immunoblot analysis of phosphorylated AMPK. Results: PIO treatment resulted in a significant decrease in intima-to-media ratio by ∼50.3% (p < 0.05, compared with vehicle control; n = 6), which was accompanied by enhanced phosphorylation of AMPK by ∼85% in the vessel wall. Compound C treatment led to a marked reduction in PIO-mediated inhibition of neointima formation. Conclusion: PIO attenuates injury-induced neointima formation, in part, through the activation of AMPK.

TEAD1 (TEA domain transcription factor 1), a transcription factor known for the functional output of Hippo signaling, is important for tumorigenesis. However, the role of TEAD1 in the development of vascular smooth muscle cell (VSMC) is unknown. To investigate cell-specific role of Tead1, we generated cardiomyocyte (CMC) and VSMC-specific Tead1 knockout mice. We found CMC/VSMC-specific deletion of Tead1 led to embryonic lethality by E14.5 in mice due to hypoplastic cardiac and vascular walls, as a result of impaired CMC and VSMC proliferation. Whole transcriptome analysis revealed that deletion of Tead1 in CMCs/VSMCs downregulated expression of muscle contractile genes and key transcription factors including Pitx2c and myocardin. In vitro studies demonstrated that PITX2c and myocardin rescued TEAD1-dependent defects in VSMC differentiation. We further identified Pitx2c as a novel transcriptional target of TEAD1, and PITX2c exhibited functional synergy with myocardin by directly interacting with myocardin, leading to augment the differentiation of VSMC. In summary, our study reveals a critical role of Tead1 in cardiovascular development in mice, but also identifies a novel regulatory mechanism, whereby Tead1 functions upstream of the genetic regulatory hierarchy for establishing smooth muscle contractile phenotype.

In response to vascular injury, vascular smooth muscle cells (VSMCs) may switch from a contractile to a proliferative phenotype thereby contributing to neointima formation. Previous studies showed that the long noncoding RNA (lncRNA) NEAT1 is critical for paraspeckle formation and tumorigenesis by promoting cell proliferation and migration. However, the role of NEAT1 in VSMC phenotypic modulation is unknown. Herein we showed that NEAT1 expression was induced in VSMCs during phenotypic switching in vivo and in vitro. Silencing NEAT1 in VSMCs resulted in enhanced expression of SM-specific genes while attenuating VSMC proliferation and migration. Conversely, overexpression of NEAT1 in VSMCs had opposite effects. These in vitro findings were further supported by in vivo studies in which NEAT1 knockout mice exhibited significantly decreased neointima formation following vascular injury, due to attenuated VSMC proliferation. Mechanistic studies demonstrated that NEAT1 sequesters the key chromatin modifier WDR5 (WD Repeat Domain 5) from SM-specific gene loci, thereby initiating an epigenetic “off” state, resulting in down-regulation of SM-specific gene expression. Taken together, we demonstrated an unexpected role of the lncRNA NEAT1 in regulating phenotypic switching by repressing SM-contractile gene expression through an epigenetic regulatory mechanism. Our data suggest that NEAT1 is a therapeutic target for treating occlusive vascular diseases.

Metformin, a widely prescribed antidiabetic drug, has been shown to reduce the risk of cardiovascular disease, including hypertension. Its beneficial effect toward improved vasodilation results from its ability to activate AMPK and enhance nitric oxide formation in the endothelium. To date, metformin regulation of AMPK has not been fully studied in intact arterial smooth muscle, especially during contraction evoked by G protein-coupled receptor (GPCR) agonists. In the present study, ex vivo incubation of endothelium-denuded rat aortic rings with 3 mM metformin for 2 hours resulted in significant accumulation of metformin (~600 pmoles/mg tissue), as revealed by LC-MS/MS MRM analysis. However, metformin did not show significant increase in AMPK phosphorylation under these conditions. Exposure of aortic rings to a GPCR agonist (e.g., phenylephrine) resulted in enhanced AMPK phosphorylation by ~2.5-fold. Importantly, in metformin-treated aortic rings, phenylephrine challenge showed an exaggerated increase in AMPK phosphorylation by ~9.7-fold, which was associated with an increase in AMP/ATP ratio. Pretreatment with compound C (AMPK inhibitor) prevented AMPK phosphorylation induced by phenylephrine alone and also that induced by phenylephrine after metformin treatment. However, pretreatment with STO-609 (CaMKKβ inhibitor) diminished AMPK phosphorylation induced by phenylephrine alone but not that induced by phenylephrine after metformin treatment. Furthermore, attenuation of phenylephrine-induced contraction (observed after metformin treatment) was prevented by AMPK inhibition but not by CaMKKβ inhibition. Together, these findings suggest that, upon endothelial damage in the vessel wall, metformin uptake by the underlying vascular smooth muscle would accentuate AMPK phosphorylation by GPCR agonists independent of CaMKKβ to promote vasorelaxation.

Hypoadiponectinemia is associated with an increased risk of coronary artery disease. Although adiponectin replenishment mitigates neointimal hyperplasia and atherosclerosis in mouse models, adiponectin therapy has been hampered in a clinical setting due to its large molecular size. Recent studies demonstrate that AdipoRon (a small-molecule adiponectin receptor agonist) improves glycemic control in type 2 diabetic mice and attenuates postischemic cardiac injury in adiponectin-deficient mice, in part, through activation of AMP-activated protein kinase (AMPK). To date, it remains unknown as to whether AdipoRon regulates vascular smooth muscle cell (VSMC) proliferation, which plays a major role in neointima formation. In the present study, oral administration of AdipoRon (50mg/kg) in C57BL/6J mice significantly diminished arterial injury-induced neointima formation by ∼57%. Under in vitro conditions, AdipoRon treatment led to significant inhibition of platelet-derived growth factor (PDGF)-induced VSMC proliferation, DNA synthesis, and cyclin D1 expression. While AdipoRon induced a rapid and sustained activation of AMPK, it also diminished basal and PDGF-induced phosphorylation of mTOR and its downstream targets, including p70S6K/S6 and 4E-BP1. However, siRNA-mediated AMPK downregulation showed persistent inhibition of p70S6K/S6 and 4E-BP1 phosphorylation, indicating AMPK-independent effects for AdipoRon inhibition of mTOR signaling. In addition, AdipoRon treatment resulted in a sustained and transient decrease in PDGF-induced phosphorylation of Akt and ERK, respectively. Furthermore, PDGF receptor-β tyrosine phosphorylation, which controls the phosphorylation state of Akt and ERK, was diminished upon AdipoRon treatment. Together, the present findings suggest that orally-administered AdipoRon has the potential to limit restenosis after angioplasty by targeting mTOR signaling independent of AMPK activation.