De-differentiated Vascular Smooth Muscle Cells Research Articles

Plaque-targeted, proteolysis-resistant, activatable and MRI-visible nano-GLP-1 receptor agonist targets smooth muscle cell differentiation in atherosclerosis.

Background: Glucagon-like peptide-1 receptor (GLP-1R) agonists are powerful glycemia-lowering agents, which have systematically been shown to lower cardiovascular events and mortality. These beneficial effects were difficult to pinpoint within atherosclerotic plaque due to lack of particular specificity of such agonists to the vascular cells and an inadequate understanding of the GLP-1R expression in atherosclerosis. Here, we hypothesized that the direct engagement of the GLP-1R in atherosclerosis by targeted agonists will alleviate vascular inflammation and plaque burden, even at a very low dose.Methods: The expression of GLP-1 receptor (GLP-1R, Glp1r mRNA) in human lesions with pathologic intimal thickening, Apoe-/- mouse atheroma and cultured immune/non-immune cells was investigated using genetic lineage tracing, Southern blotting and validated antisera against human GLP-1R. Protease-resistant and “activatable” nanoparticles (NPs) carrying GLP-1R agonist liraglutide (GlpNP) were engineered and synthesized. Inclusion of gadolinium chelates into GlpNP allowed for imaging by MRI. Atherosclerotic Apoe-/- mice were treated intravenously with a single dose (30 µg/kg of liraglutide) or chronically (1 µg/kg, 6 weeks, 2x/week) with GlpNP, liraglutide or control NPs, followed by assessment of metabolic parameters, atheroma burden, inflammation and vascular function.Results: Humal plaque specimens expressed high levels of GLP-1R within the locus of de-differentiated smooth muscle cells that also expressed myeloid marker CD68. However, innate immune cells under a variety of conditions expressed very low levels of Glp1r, as seen in lineage tracing and Southern blotting experiments examining full-length open reading frame mRNA transcripts. Importantly, de-differentiated vascular smooth muscle cells demonstrated significant Glp1r expression levels, suggesting that these could represent the cells with predominant Glp1r-positivity in atherosclerosis. GlpNP resisted proteolysis and demonstrated biological activity including in vivo glycemia lowering at 30 µg/kg and in vitro cholesterol efflux. Activatable properties of GlpNP were confirmed in vitro by imaging cytometry and in vivo using whole organ imaging. GlpNP targeted CD11b+/CD11c+ cells in circulation and smooth muscle cells in aortic plaque in Apoe-/- mice when assessed by MRI and fluorescence imaging. At a very low dose of 1 µg/kg, previously known to have little effect on glycemia and weight loss, GlpNP delivered i.v. for six weeks reduced triglyceride-rich lipoproteins in plasma, plaque burden and plaque cholesterol without significant effects on weight, glycemia and plasma cholesterol levels.Conclusions: GlpNP improves atherosclerosis at weight-neutral doses as low as 1 µg/kg with the effects independent from the pancreas or the central nervous system. Our study underlines the importance of direct actions of GLP-1 analogs on atherosclerosis, involving cholesterol efflux and inflammation. Our findings are the first to suggest the therapeutic modulation of vascular targets by GlpNP, especially in the context of smooth muscle cell inflammation.

Resident multipotent vascular stem cells exhibit amplitude dependent strain avoidance similar to that of vascular smooth muscle cells

Atherosclerosis is one of the leading causes of mortality worldwide, and presents as a narrowing or occlusion of the arterial lumen. Interventions to re-open the arterial lumen can result in re-occlusion through intimal hyperplasia. Historically only de-differentiated vascular smooth muscle cells were thought to contribute to intimal hyperplasia. However recent significant evidence suggests that resident medial multipotent vascular stem cells (MVSC) may also play a role. We therefore investigated the strain response of MVSC since these resident cells are also subjected to strain within their native environment. Accordingly, we applied uniaxial 1 Hz cyclic uniaxial tensile strain at three amplitudes around a mean strain of 5%, (4–6%, 2–8% and 0–10%) to either rat MVSC or rat VSMC before their strain response was evaluated. While both cell types strain avoid, the strain avoidant response was greater for MVSC after 24 h, while VSMC strain avoid to a greater degree after 72 h. Additionally, both cell types increase strain avoidance as strain amplitude is increased. Moreover, MVSC and VSMC both demonstrate a strain-induced decrease in cell number, an effect more pronounced for MVSC. These experiments demonstrate for the first time the mechano-sensitivity of MVSC that may influence intimal thickening, and emphasizes the importance of strain amplitude in controlling the response of vascular cells in tissue engineering applications.

Label-free discrimination analysis of de-differentiated vascular smooth muscle cells, mesenchymal stem cells and their vascular and osteogenic progeny using vibrational spectroscopy

The accumulation of vascular smooth muscle (SMC)-like cells and stem cell-derived myogenic and osteogenic progeny contributes significantly to arteriosclerotic disease. This study established whether label-free vibrational spectroscopy can discriminate de-differentiated ‘synthetic’ SMCs from undifferentiated stem cells and their myogenic and osteogenic progeny in vitro, compared with conventional immunocytochemical and genetic analyses. TGF-β1- and Jagged1-induced myogenic differentiation of CD44+ mesenchymal stem cells was confirmed in vitro by immunocytochemical analysis of specific SMC differentiation marker expression (α-actin, calponin and myosin heavy chain 11), an epigenetic histone mark (H3K4me2) at the myosin heavy chain 11 locus, promoter transactivation and mRNA transcript levels. Osteogenic differentiation was confirmed by alizarin red staining of calcium deposition. Fourier Transform Infrared (FTIR) maps facilitated initial screening and discrimination while Raman spectroscopy of individual cell nuclei revealed specific spectral signatures of each cell type in vitro, using Principal Components Analysis (PCA). PCA fed Linear Discriminant Analysis (LDA) enabled quantification of this discrimination and the sensitivity and specificity value was determined for all cell populations based on a leave-one-out cross validation method and revealed that de-differentiated SMCs and stem-cell derived myogenic progeny in culture shared the greatest similarity. FTIR and Raman spectroscopy discriminated undifferentiated stem cells from both their myogenic and osteogenic progeny. The ability to detect stem cell-derived myogenic progeny using label-free platforms in situ may facilitate interrogation of these important phenotypes during vascular disease progression.

Abstract P313: Alterations in Notch Signaling in Diabetic Coronary Microvascular Smooth Muscle and Endothelial Cells

Notch signaling between vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) is crucial during normal vascular development and homeostasis and may contribute to vasculopathies. Notch signaling is known to regulate VSMC phenotypic mechanisms, including proliferation and differentiation. We previously reported differences in VSCM Notch3 and EC Jagged1 mRNA expression, as well as a de-differentiated VSMC phenotype, in diabetic coronary resistance microvascular (CRM) cells as a potential mechanism for the inward remodeling observed in those vessels. The current study tested the hypothesis that VSMC-EC dependent Notch signaling accounts for phenotypic changes in VSMCs that may promote adverse remodeling observed in diabetic CRMs. Low-passage primary coronary VMSCs were isolated from normal (n=6) and type 2 diabetic db/db mice (n=4) and were either cultured alone or co-cultured with normal or type 2 diabetic human coronary microvascular endothelial cells (hCMECs), respectively. Notch2 expression was significantly attenuated in diabetic CRM VSMCs in both mono- and co-culture conditions (0.53±0.12 vs. 1.00±0.06, p <0.01 and 0.59±0.09 vs. 0.94±0.04, p <0.01, respectively). There were no significant differences in Hes1, and Hrt3 was increased in only normal VSMCs that were co-cultured with ECs. We further found that proliferation inhibitor p53 expression was significantly lower in diabetic CRM VSMCs in when co-cultured with ECs (0.78±0.15 vs. 1.34±0.13, p <0.01), and Smad3 was significantly reduced in diabetic co-cultured CRM VSMCs (0.90±0.02 vs. 1.14±0.06, p <0.05). Collectively, these data implicate that Notch signaling through p53 and Smad3 may account for phenotypic alterations of diabetic CRM VSMCs.

MiR-18a-5p MicroRNA Increases Vascular Smooth Muscle Cell Differentiation by Downregulating Syndecan4.

Background and ObjectivesDifferentiation and de-differentiation of vascular smooth muscle cells (VSMCs) are important events in atherosclerosis and restenosis after angioplasty. MicroRNAs are considered a key regulator in cellular processes such as differentiation, proliferation, and apoptosis. Here, we report the role of new miR-18a-5p microRNA and its downstream target genes in VSMCs and in a carotid balloon injury model.Materials and MethodsExpression of miR-18a-5p and its candidate genes was examined in VSMCs and in a carotid artery injury model by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and microRNA microarray analysis. VSMC differentiation marker genes including smooth muscle (SM) α-actin and SM22α were determined by Western blot, qRT-PCR, and a SM22α promoter study. Gene overexpression or knockdown was performed in VSMCs.ResultsmiR-18a-5p was upregulated in the rat carotid artery at the early time after balloon injury. Transfection of the miR-18a-5p mimic promoted the VSMC differentiation markers SM α-actin and SM22α. In addition, miR-18a-5p expression was induced in differentiated VSMCs, whereas it decreased in de-differentiated VSMCs. We identified syndecan4 as a downstream target of miR-18-5p in VSMCs. Overexpression of syndecan4 decreased Smad2 expression, whereas knockdown of syndecan4 increased Smad2 expression in VSMCs. Finally, we showed that Smad2 induced the expression of VSMC differentiation marker genes in VSMCs.ConclusionThese results indicate that miR-18a-5p is involved in VSMC differentiation by targeting syndecan4.

Abstract P341: MRTF-A, a Rho-Dependent Coactivator of SRF, Plays a Critical Role in Vascular Remodeling

Objective: Rho-ROCK pathway is involved in the progression of pathological vascular remodeling through the regulation of cell proliferation and migration by controlling gene transcription. While a transcriptional co-activator Myocardin-related transcription factor(MRTF)-A transduces Rho-actin signaling to SRF activation in the nucleus, roles of MRTF-A in the process underlying vascular diseases remain unknown. Methods and Results: We found that MRTF-A mRNA and protein expression in femoral arteries 2weeks after wire injury was significantly higher than that in sham-operated arteries of mice. In contrast, the expression of myocardin or MRTF-B was significantly decreased in injured arteries. We then evaluated the role played by MRTF-A in pathological vascular remodeling by creating several vascular disease models of mice lacking MRTF-A (MRTF-A -/- ). Neointima formation induced by ligation in carotid arteries of MRTF-A -/- was significantly smaller than that of MRTF-A +/- . Neointima formation induced by wire injury in femoral arteries of MRTFA -/- was significantly smaller than that of MRTF-A +/+ . Atherosclerotic lesions of MRTF-A -/- ;ApoE -/- was also significantly smaller than those of MRTF-A +/+ ;ApoE -/ . The expression of SRF-target genes involved in cellular migration, such as MMP9 and vinculin,within injured arteries of MRTF-A -/- was significantly attenuated compared to that of MRTF-A +/+ . In cultured, de-differentiated rat aortic vascular smooth muscle cells (RAVSMC), the expression of these genes was decreased by knocking down MRTF-A. Indeed, the promoter activity of vinculin gene was controlled by MRTF-A in RAVSMC. In addition, knocking down MRTF-A in RAVSMC resulted in a significant impairment in migration capacity. Furthermore, treatment with a small molecule CCG1423 inhibiting MRTF-A significantly reduced neointima formation induced by wire injury in femoral arteries of mice. Conclusions: Our study revealed that in de-differentiated vascular smooth muscle cells, in which myocardin and MRTTF-B expression is decreased, increased expression of MRTF-A contributes to the acquisition of migration capacity by maintaining SRF-target genes transcription, thereby promoting stress-induced vascular remodeling.

Nuclear Import Mechanism for Myocardin Family Members and Their Correlation with Vascular Smooth Muscle Cell Phenotype

Myocardin (Mycd), which is essential for the differentiation of the smooth muscle cell lineage, is constitutively located in the nucleus, although its family members, myocardin-related transcription factors A and B (MRTF-A/B), mostly reside in the cytoplasm and translocate to the nucleus in response to Rho signaling. The mechanism for their nuclear import is unclear. Here we investigated the mechanism for the nuclear import of Mycd family members and demonstrated any correlation between such mechanism and the phenotype of vascular smooth muscle cells (VSMCs). In cultured VSMCs, the knockdown of importin β1 inhibited the nuclear import of Mycd and MRTF-A/B. Their NH(2)-terminal basic domain was identified as a binding site for importin α/β1 by in vitro analyses. However, Mycd had a higher affinity for importin α/β1 than did MRTF-A/B, even in the absence of G-actin, and Mycd affinity for importin α1/β1 was stronger than for any other importin α/β1 heterodimers. The binding of Mycd to importin α/β1 was insensitive to G-actin, whereas that of MRTF-A/B was differently inhibited by G-actin. In dedifferentiated VSMCs, the levels of importins α1 and β1 were reduced concomitant with down-regulation of Mycd, serum response factor, and smooth muscle cell markers. By contrast, in differentiated VSMCs, their expressions were up-regulated. Thus, the nuclear import of Mycd family members in VSMCs depends on importin α/β1, and their relative affinities for importin α/β1 heterodimers determine Mycd nuclear import. The expression of Mycd nuclear import machineries is related to the expression levels of VSMC phenotype-dependent smooth muscle cell markers.

MicroRNA and vascular smooth muscle cell phenotype: new therapy for atherosclerosis?

MicroRNAs (miRNAs) represent a class of small, non-coding RNAs that negatively regulate gene expression via degradation or translational inhibition of their target mRNAs. Recent studies have identified that miR-145 is the most abundant miRNA in normal arteries and vascular smooth muscle cells (VSMCs), and its expression is significantly downregulated in dediffer-entiated VSMCs and atherosclerotic arteries. miR-145 plays a critical role in modulating VSMC phenotype. Because phenotypic modulation of VSMCs is an initial cellular event in the development of atherosclerosis, miRNAs, and miR-145 in particular, may represent new therapeutic targets for atherosclerosis.

α8 Integrin overexpression in de-differentiated vascular smooth muscle cells attenuates migratory activity and restores the characteristics of the differentiated phenotype

Loss of the differentiated (contractile) phenotype of vascular smooth muscle cells (VSMCs) heightens their migratory activity. Integrins, as the main integrators of cell-extracellular matrix, regulate different aspects of cell behavior including migration and differentiation. α8β1 Integrin being expressed in cell types with contractile abilities is downregulated during VSMC phenotype modulation. In this report the ability of α8β1 integrin to induce the characteristics of the contractile phenotype as well as suppression of VSMC migratory activity was investigated. Forced expression of α8 integrin in passage-5 rat VSMCs resulted in lower migratory activity. Western blot and immunoconfocal studies revealed that α8 integrin overexpression was associated with the reappearance of VSMC contractile hallmarks including upregulation of contractile markers, assembly of stress fibres, and increased number of focal adhesions. α8 Integrin overexpression in fibroblast-like Rat1 cells also induced SMC-like characteristics. α8 Integrin-induced reappearance of the contractile hallmarks in de-differentiated VSMCs was impaired by RhoA inhibitors. These results provide evidences that α8 integrin overexpression may assist phenotype-modulated VSMCs to revert to the contractile phenotype possibly via RhoA signaling pathway. Our findings suggest a dynamic role for α8β1 integrin to induce contractile phenotype as well as suppression of VSMC migration, a key player during arterial stenosis.

Blockade of Nuclear Factor of Activated T Cells Activation Signaling Suppresses Balloon Injury-induced Neointima Formation in a Rat Carotid Artery Model

We have previously reported that nuclear factor of activated T cells (NFATs) play an important role in the regulation of vascular smooth muscle cell migration and proliferation by receptor tyrosine kinase and G protein-coupled receptor agonists, platelet-derived growth factor-BB and thrombin, respectively. To understand the role of NFATs in vascular disease, we have now studied the involvement of these transcription factors in neointima formation in a rat carotid artery balloon injury model. The levels of NFATc1 in injured right common carotid arteries were increased at 72 h, 1 week, and 2 weeks after balloon injury compared with its levels in uninjured left common carotid arteries. Intraperitoneal injection of cyclosporine A (CsA), a pharmacological inhibitor of the calcineurin-NFAT activation pathway, suppressed balloon injury-induced neointima formation by 40%. Similarly, adenoviral-mediated expression of GFPVIVIT, a competent peptide inhibitor of the calcineurin-NFAT activation pathway, in injured arteries also reduced neointima formation by about 40%. Furthermore, CsA and GFPVIVIT attenuated balloon injury-induced neointimal smooth muscle cell proliferation as determined by bromodeoxyuridine staining. Platelet-derived growth factor-BB induced the expression of COX-2 in cultured VSMC in a time- and NFAT-dependent manner. COX-2 expression was also increased in the right common carotid artery in a time-dependent manner after balloon injury as compared with its levels in uninjured left common carotid artery and both CsA and GFPVIVIT negated this response. Together these results for the first time demonstrate that NFATs play a critical role in neointima formation via induction of expression of COX-2.

ERK1/2-dependent contractile protein expression in vascular smooth muscle cells.

In vivo, vascular smooth muscle (VSM) cells change their contractile phenotype toward a more proliferative phenotype during the pathogenesis of vascular diseases. Because these dedifferentiated VSM cells may gradually regain contractile functions, we aimed to identify signaling pathways that result in an increased expression of contractile proteins in VSM cells. In vitro, serum and thrombin induced a reversible upregulation of smooth muscle myosin heavy-chain (SM-MHC) in cultured neonatal rat VSM cells. Cotransfection of a SM-MHC-promoter chloramphenicol acetyltransferase-construct with dominant-negative N17Ras or N17Raf or treatment with the mitogen-activated/ERK-activating kinase (MEK) inhibitor PD 98059 concentration dependently decreased the serum- or thrombin-induced SM-MHC promoter activity. Consistently, the serum- or thrombin-induced phosphorylation of MEK and extracellular signal-regulated kinase 1/2 (ERK1/2) coincided with a MEK-dependent nuclear accumulation of phosphorylated ERK1/2 and subsequent nuclear phosphorylation of the transcription factors c-myc and Elk-1. A 5'-deletion analysis of cis-elements within the SM-MHC promoter demonstrated that a conserved region (nucleotide -1346 to -1102) was required for both cell type-specific expression and serum- or thrombin-induced upregulation of the SM-MHC promoter in VSM cells. Within this region, 2 CArG-boxes, a GC-rich element, and a CTF/NF-1 site are critical positively acting cis-elements for the serum- or thrombin-induced upregulation of SM-MHC. We conclude that the serum- or thrombin-induced differentiation requires an intact Ras/Raf/MEK/ERK signaling cascade, nuclear translocation of activated ERK1/2, phosphorylation of transcription factors, and several cis-elements within the SM-MHC promoter.

A potential role for sterol 27-hydroxylase in atherogenesis

Objective: 27-hydroxycholesterol is the product of the mitochondrial cytochrome P450 sterol 27-hydroxylase, a key enzyme in cholesterol metabolism present in most tissues of the body. 27-hydroxycholesterol increases in abundance with progression of human atherosclerotic lesions, therefore the aim of this study was to determine the pattern of sterol 27-hydroxylase gene expression in normal and diseased arteries and to identify the cell types responsible for its expression. Methods: Reverse transcription-polymerase chain reaction (RT-PCR) analysis and in situ hybridisation, utilising a sterol 27-hydroxylase cDNA probe, and immunohistochemistry, utilising an antibody to sterol 27-hydroxylase, together with an antibody to smooth muscle cell α-actin and an antibody to CD68, a marker for macrophages, were used to study expression of 27-hydroxylase in arterial specimens. In addition, RT-PCR was used to study expression of 27-hydroxylase in cultured macrophages and smooth muscle cells. Results: Semi-quantitative RT-PCR analysis of normal and atherosclerotic human aortas showed that 27-hydroxylase is constitutively expressed in the normal artery wall, and is substantially up-regulated in atherosclerosis. RT-PCR analysis of 27-hydroxylase expression in vitro demonstrated that macrophages constitutively express high levels throughout their differentiation in culture whilst de-differentiated vascular smooth muscle cells express very low levels. In situ hybridisation revealed that in normal artery and fatty streaks, expression of mRNA for 27-hydroxylase was low in the media, but higher in intimal smooth muscle cells. The macrophages of fatty streaks expressed low or undetectable levels of 27-hydroxylase. However in advanced lesions the highest expression of 27-hydroxylase was detectable in macrophages. Immunohistochemistry demonstrated that high levels of 27-hydroxylase protein occurred in macrophages near the shoulder region of plaques, at the edge of the lipid core. Conclusions: 27-hydroxylase may constitute a protective mechanism for removing cholesterol from macrophages and smooth muscle cells. Genetic heterogeneity resulting in differences in sterol 27-hydroxylase activity between individuals may affect their ability to deal with accumulated cholesterol in the arterial intima, and hence their relative degree of predisposition to atherosclerosis.

Genomic Cloning and Promoter Analysis of Aortic Preferentially Expressed Gene-1: IDENTIFICATION OF A VASCULAR SMOOTH MUSCLE-SPECIFIC PROMOTER MEDIATED BY AN E BOX MOTIF

Aortic preferentially expressed gene-1 (APEG-1) was originally identified as a 1.4-kilobase (kb) transcript preferentially expressed in differentiated vascular smooth muscle cells (VSMC). Its expression is markedly down-regulated in de-differentiated VSMC, suggesting a role for APEG-1 in VSMC differentiation. We have now determined that APEG-1 is a single-copy gene in the human, rat, and mouse genomes and have mapped human APEG-1 to chromosome 2q34. To study the molecular mechanisms regulating its expression, we characterized the genomic organization and promoter of mouse APEG-1. APEG-1 spans 4.5 kb in the mouse genome and is composed of five exons. Using reporter gene transfection analysis, we found that a 2. 7-kb APEG-1 5'-flanking sequence directed a high level of promoter activity only in VSMC. Its activity was minimal in five other cell types. A repressor region located within an upstream 685-base pair sequence suppressed the activity of this 2.7-kb promoter. Further deletion and mutation analyses identified an E box motif as a positive regulatory element, which was bound by nuclear protein prepared from VSMC. In conjunction with its flanking sequence, this E box motif confers VSMC-specific enhancer activity to a heterologous SV40 promoter. To our knowledge, this is the first demonstration of an E box motif that mediates gene expression restricted to VSMC.

Collagen VIII is expressed by vascular smooth muscle cells in response to vascular injury.

To identify genes involved in vascular remodeling, we applied differential mRNA display analysis to the rat carotid artery balloon injury model. One polymerase chain reaction product showing increased expression at days 2 to 14 after vascular injury was nearly identical to the mouse alpha 1 chain of type VIII collagen, a heterotrimeric short-chain collagen of uncertain function expressed by a limited number of cell types. By Northern analysis, expression of both chains of the type VIII collagen heterotrimer increased: collagen alpha 1 (VIII) mRNA expression was almost 4-fold higher than control by 7 days after vascular injury, and collagen alpha 2 (VIII) mRNA expression reached a maximum of almost 6-fold above baseline by 3 days after injury. By immunohistochemical analysis, type VIII collagen expression increased in the media and neointima in a localized pattern consistent with the distribution of activated dedifferentiated vascular smooth muscle cells (VSMCs). Cultured VSMCs expressed higher levels of type VIII collagen in response to serum and growth factors, notably platelet-derived growth factor (PDGF)-BB. VSMCs adhered significantly less to type VIII collagen than to type I collagen substrata and showed greater PDGF-BB-stimulated migration (by 2.2-fold) on type VIII collagen than on type I collagen. We hypothesize that increased expression of type VIII collagen by VSMCs after arterial injury may contribute to vascular remodeling through the promotion of VSMC migration.