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Background: Vascular homeostasis is maintained by the differentiated phenotype of vascular smooth muscle cells (VSMCs). The landscape of protein coding genes comprising the transcriptome of differentiated VSMCs has been intensively investigated but many gaps remain including the emerging roles of noncoding genes. Methods: We reanalyzed large-scale, publicly available bulk and single-cell RNA sequencing datasets from multiple tissues and cell types to identify VSMC-enriched long noncoding RNAs. The in vivo expression pattern of a novel smooth muscle cell (SMC)–expressed long noncoding RNA, Carmn (cardiac mesoderm enhancer-associated noncoding RNA), was investigated using a novel Carmn green fluorescent protein knock-in reporter mouse model. Bioinformatics and quantitative real-time polymerase chain reaction analysis were used to assess CARMN expression changes during VSMC phenotypic modulation in human and murine vascular disease models. In vitro, functional assays were performed by knocking down CARMN with antisense oligonucleotides and overexpressing Carmn by adenovirus in human coronary artery SMCs. Carotid artery injury was performed in SMC-specific Carmn knockout mice to assess neointima formation and the therapeutic potential of reversing CARMN loss was tested in a rat carotid artery balloon injury model. The molecular mechanisms underlying CARMN function were investigated using RNA pull-down, RNA immunoprecipitation, and luciferase reporter assays. Results: We identified CARMN , which was initially annotated as the host gene of the MIR143/145 cluster and recently reported to play a role in cardiac differentiation, as a highly abundant and conserved, SMC-specific long noncoding RNA. Analysis of the Carmn GFP knock-in mouse model confirmed that Carmn is transiently expressed in embryonic cardiomyocytes and thereafter becomes restricted to SMCs. We also found that Carmn is transcribed independently of Mir143/145 . CARMN expression is dramatically decreased by vascular disease in humans and murine models and regulates the contractile phenotype of VSMCs in vitro. In vivo, SMC-specific deletion of Carmn significantly exacerbated, whereas overexpression of Carmn markedly attenuated, injury-induced neointima formation in mouse and rat, respectively. Mechanistically, we found that Carmn physically binds to the key transcriptional cofactor myocardin, facilitating its activity and thereby maintaining the contractile phenotype of VSMCs. Conclusions: CARMN is an evolutionarily conserved SMC-specific long noncoding RNA with a previously unappreciated role in maintaining the contractile phenotype of VSMCs and is the first noncoding RNA discovered to interact with myocardin.

Introduction: Restenosis after percutaneous intervention is predominantly attributed to proliferation and migration of vascular smooth muscle cells (VSMCs). However, the key regulators responsible for VSMC proliferation and migration remain to be identified. Hypothesis: We previously reported that the novel high mobility group (HMG) nuclear protein HMGXB4 (HMG-Box containing 4) plays a critical role in the de-differentiation of vascular smooth muscle cells in vitro and in acute inflammatory response to septic shock. We hypothesize that HMGXB4 is critical for neointimal hyperplasia in response to inflammatory stimuli. Methods and Results: We found that the expression of HMGXB4 is dramatically induced in ligation or wire injury-induced neointimal hyperplasia and correlated with the activation of inflammatory signaling in mice. Using an inducible smooth muscle-specific Hmgxb4 KO (knockout) mice model, we found specific KO of Hmgxb4 in VSMCs ameliorates ligation- or wire- injury induced neointimal formation. Among an array of growth factors and inflammation cytokines, we found that TNFα and INFγ effectively induces the expression of HMGXB4 in VSMCs and correlates with the VSMC proliferation in vitro. Furthermore, we found deletion of HMGXB4 attenuates while over-expression of HMGXB4 promotes inflammation cytokines-induced VSMC proliferation in vitro. These results suggest injury-induced inflammatory signal triggers HMGXB4 induction, which, in turn, promotes the VSMC proliferation and neointimal formation. Conclusions: Our study not only demonstrates a critical role of HMGXB4 in promoting neointimal hyperplasia in response the arterial injury, but also suggests HMGXB4 is a potential novel target for the management of restenosis in human.

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.

Pioglitazone (PIO), a PPARγ agonist that improves glycemic control in type 2 diabetes through its insulin-sensitizing action, has been shown to exhibit beneficial effects in the vessel wall. For instance, it inhibits vascular smooth muscle cell (VSMC) proliferation, a major event in atherosclerosis and restenosis after angioplasty. Although PPARγ-dependent and PPARγ-independent mechanisms have been attributed to its vasoprotective effects, the signaling events associated with PIO action in VSMCs are not fully understood. To date, the likely intermediary role of AMP-activated protein kinase (AMPK) toward PIO inhibition of VSMC proliferation has not been examined. Using human aortic VSMCs, the present study demonstrates that PIO activates AMPK in a sustained manner thereby contributing in part to inhibition of key proliferative signaling events. In particular, PIO at 30μM concentration activates AMPK to induce raptor phosphorylation, which diminishes PDGF-induced mTOR activity as evidenced by decreased phosphorylation of p70S6K, 4E-BP1, and S6 and increased accumulation of p27(kip1), a cell cycle inhibitor. In addition, PIO inhibits the basal phosphorylation of ERK in VSMCs. Downregulation of endogenous AMPK by target-specific siRNA reveals an AMPK-independent effect for PIO inhibition of ERK, which contributes in part to diminutions in cyclin D1 expression and Rb phosphorylation and the suppression of VSMC proliferation. Furthermore, AMPK-dependent inhibition of mTOR/p70S6K and AMPK-independent inhibition of ERK signaling occur regardless of PPARγ expression/activation in VSMCs as evidenced by gene silencing and pharmacological inhibition of PPARγ. Strategies that utilize nanoparticle-mediated PIO delivery at the lesion site may limit restenosis after angioplasty without inducing PPARγ-mediated systemic adverse effects.

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.

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.

Insulin resistance is associated with accelerated atherosclerosis. Although high fructose is known to induce insulin resistance, it remains unclear as to how fructose regulates insulin receptor signaling and proliferative phenotype in vascular smooth muscle cells (VSMCs), which play a major role in atherosclerosis. Using human aortic VSMCs, we investigated the effects of high fructose treatment on insulin receptor substrate-1 (IRS-1) serine phosphorylation, insulin versus platelet-derived growth factor (PDGF)-induced phosphorylation of Akt, S6 ribosomal protein, and extracellular signal-regulated kinase (ERK), and cell cycle proteins. In comparison with PDGF (a potent mitogen), neither fructose nor insulin enhanced VSMC proliferation and cyclin D1 expression. D-[14C(U)]fructose uptake studies revealed a progressive increase in fructose uptake in a time-dependent manner. Concentration-dependent studies with high fructose (5 to 25 mM) showed marked increases in IRS-1 serine phosphorylation, a key adapter protein in insulin receptor signaling. Accordingly, high fructose treatment led to significant diminutions in insulin-induced phosphorylation of downstream signaling components including Akt and S6. In addition, high fructose significantly diminished insulin-induced ERK phosphorylation. Nevertheless, high fructose did not affect PDGF-induced key proliferative signaling events including phosphorylation of Akt, S6, and ERK and expression of cyclin D1 protein. Together, high fructose dysregulates IRS-1 phosphorylation state and proximal insulin receptor signaling in VSMCs, but does not affect PDGF-induced proliferative signaling. These findings suggest that systemic insulin resistance rather than VSMC-specific dysregulation of insulin receptor signaling by high fructose may play a major role in enhancing atherosclerosis and neointimal hyperplasia.

Previous studies have shown that PDGF-induced activation of mTOR/p70S6kinase signaling is associated with dysregulation of insulin receptor substrates (IRS1 and IRS2) in vascular smooth muscle cells (VSMCs). In addition, pioglitazone (PIO, an antidiabetic drug) has been shown to inhibit mTOR/p70S6kinase signaling thereby suppressing PDGF-induced VSMC proliferation. The role of IRS1/IRS2 on proliferative signaling events has not yet been examined in VSMCs treated with fructose (an agent that induces insulin resistance) or PIO (an insulin sensitizer). In the present study, unlike PDGF treatment (48 hr) that enhanced IRS serine phosphorylation and diminished IRS2 expression, exposure of human aortic VSMCs to D-fructose (5.5 to 25 mM) for 48 hr resulted in a concentration-dependent increase in IRS1 serine phosphorylation (up to ~5-fold , n = 3), but without any changes in IRS2 protein expression. In D-fructose-treated VSMCs, Akt phosphorylation in response to acute insulin exposure (6 min) was markedly decreased (~69%, n = 3), suggesting that IRS1 serine phosphorylation alone is sufficient to dysregulate insulin receptor signaling. In addition, D-fructose treatment led to a further increase in PDGF-induced VSMC proliferation (~40%, n = 3). Furthermore, we examined the effects of PIO on PDGF-induced dysregulation of IRS in VSMCs. Notably, PIO treatment (30 μM, 48 hr) by itself led to an upregulation of IRS-2 expression (~2.5-fold) and also diminished PDGF-induced IRS1 serine phosphorylation (~63%). Under these conditions, PIO treatment resulted in significant inhibition of PDGF-induced cyclin D1 expression, Rb phosphorylation, and VSMC proliferation (~83%, n = 3). In conclusion, the present study reveals that dysregulated insulin signaling (by PDGF and fructose) is associated with enhanced VSMC proliferation, whereas insulin sensitization (by PIO) is associated with inhibition of VSMC proliferation. Together, these findings underscore the significance of intact insulin signaling toward limiting exaggerated VSMC proliferation in the vessel wall.