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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.

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.

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.

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.

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.

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.

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.

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.

Atherosclerosis and restenosis after angioplasty are associated with exaggerated proliferation of vascular smooth muscle cells (VSMCs). Pioglitazone (PIO), an antidiabetic drug that belongs to the family of peroxisome proliferator-activated receptor gamma (PPARg) agonists, reduces intimal hyperplasia after coronary angioplasty in diabetic and nondiabetic subjects. However, the molecular mechanisms by which PIO regulates VSMC phenotype remain unclear. The objective of the current study is to determine the role of AMP-activated protein kinase (AMPK, a cellular energy sensor) versus PPARg in PIO-induced changes in VSMC phenotype. Exposure of human aortic VSMCs to PIO followed by immunoblot analysis revealed a robust increase in the phosphorylation of AMPK (Thr172) and its downstream effector, Acetyl-CoA carboxylase (p < 0.05, n = 3), as a function of concentration (3 to 30 μM) and time (1 to 48 hr). Notably, PIO (30 μM, 48 hr) activation of AMPK remained essentially the same after PPARg downregulation (~90%) by target-specific siRNA (500 pmoles), suggesting lack of dependency on PPARg. In addition, PIO activation of AMPK was accompanied by induction of mitochondrial membrane depolarization with a trend toward an increase in AMP/ATP ratio (~40%), as revealed by tetramethylrhodamine methyl ester (TMRM) fluorescent redistribution & LC-MS/MS MRM analysis, respectively. Importantly, PIO pretreatment (30 μM, 30 min) resulted in significant inhibition of PDGF (30 ng/ml, 48 hr)-induced VSMC proliferation by ~85% (cell count) and the associated increases in proliferative markers (cyclin D1 and pRb) by > 95% (p < 0.05, n = 3). Downregulation of AMPKa1 (~80%) by target-specific AMPKa1 siRNA (500 pmoles) revealed that PIO-mediated AMPK activation inhibited mTORC1 signaling by > 95% & upregulated cell cycle inhibitor p27Kip1 by ~40%, thus accounting for PIO-mediated changes in VSMC phenotype. In vivo, PIO administration (10 mg/Kg/d, oral gavage) for 3 weeks in CJ57BL6 mice mitigated neointimal growth by ~60% in guide wire-injured femoral artery (p < 0.05, n = 8). In conclusion, the present study provides the first direct evidence for VSMC-specific AMPK activation by PIO, which may in part contribute to its vasoprotective effects in nondiabetic state.

Objective The role of glucose transporters (GLUTs) in the phenotypic regulation of vascular smooth muscle cells (VSMCs) has not been fully characterized. The present study has examined the effects of PDGF ( vs insulin) on GLUT isoform expression/localization, GLUT1 glycosylation, glucose uptake and ATP formation during VSMC proliferation. Methods Human aortic VSMCs were exposed to PDGF (30 ng/ml) or insulin (1 μM) for different time intervals. GLUT isoform expression was determined by qPCR and immunoblot analysis. GLUT isoform distribution/localization was determined by subcellular fractionation studies and immunocytochemical analysis. Glucose utilization and ATP formation were determined by glucose oxidation assay and luminescent ATP detection assay, respectively. The role of GLUT1 in VSMCs was determined by GLUT1 siRNA knockdown studies. Results Exposure of VSMCs to PDGF resulted in significant increases in glucose uptake and cell proliferation, compared with insulin. In addition, PDGF and insulin enhanced the mRNA expression levels of total GLUT isoforms (GLUT1 through 14) to ~4317/10 5 B2M transcripts and ~8,174 transcripts/10 5 B2M transcripts, respectively, compared with control (~3259 transcripts/10 5 B2M transcripts). In particular, PDGF and insulin enhanced GLUT1 mRNA to 1711 ± 38/10 5 B2M transcripts and 3387 ± 58 transcripts/10 5 B2M transcripts, respectively. Immunoblot analysis using whole cell homogenates showed a significant increase in GLUT1 protein expression in PDGF or insulin-treated VSMCs. Importantly, subcellular localization studies using PDGF-treated VSMCs revealed enhanced GLUT1 protein expression and its glycosylation in the plasma membrane. Furthermore, PDGF enhanced glucose metabolism as revealed by increased glucose utilization and ATP formation. Down regulation of GLUT1 using target-specific siRNA significantly attenuated PDGF-induced glucose uptake. Conclusions Enhanced expression and glycosylation of plasma membrane-localized GLUT1 isoform plays a major role in promoting VSMC proliferation, which may contribute to accelerated atherosclerosis.

Objective Metformin, a widely used antidiabetic drug, has been shown to exert protective effects in the vasculature by improving endothelium-dependent vasodilation. However, the beneficial effect of metformin toward smooth muscle contraction and the molecular events involved in this process remain unclear. Using endothelium-denuded aorta, the present study has therefore examined the extent of metformin accumulation and the likely changes in AMP-activated protein kinase phosphorylation during smooth muscle contraction induced by G-protein coupled receptor agonists, including phenylephrine (PE) and serotonin (5-HT). Methods Endothelium-denuded rat aortic ring preparations were mounted in organ baths for isometric tension measurements. The contractile responses to cumulative concentrations of PE or 5-HT were determined before and after pretreatments with metformin or AICAR (a direct AMPK activator). The changes in AMPK phosphorylation were determined by immunoblot analysis. Metformin accumulation was determined by LC-MS/MS. Results Concentration dependency studies revealed that pretreatment with 3 mM metformin resulted in significant inhibition of PE- or 5-HT-induced contractility (p < 0.05). In parallel, pretreatment with 1 mM AICAR led to a similar inhibition of agonist-induced contractility (p < 0.05). Notably, the inhibitory effects of metformin on smooth muscle contractility persisted even after the washout of this drug. In contrast, washout of AICAR restored the contractile responses to PE or 5-HT. Pretreatment of aortic rings with 40 μM compound C, an AMPK inhibitor, resulted in significant reversal of metformin and AICAR-mediated decrease in agonist-induced contractility (p < 0.05). An association between metformin/AICAR treatment and AMPK phosphorylation was confirmed by immunoblot analysis. Importantly, metformin treatment resulted in its significant accumulation in the aortic tissue as revealed by LC-MS/MS analysis. Conclusion In conclusion, metformin accumulation contributes to sustained inhibition of arterial smooth muscle contractility via activation of AMPK. The present findings suggest that metformin has the potential to ameliorate chronic hypertension in patients with type 2 diabetes.