Cyclic Strain Inhibits Notch Receptor Signaling in Vascular Smooth Muscle Cells In Vitro
http://www.100md.com
循环研究杂志 2005年第3期
the Vascular Health Research Centre (D.M., C.S., Y.A.B., P.M.C., P.A.C.), Faculty of Science and Health
the School of Biotechnology and the National Centre for Sensor Research (D.W.), Dublin City University, Ireland
Department of Surgery (E.M.R.), University of Rochester Medical Center, Rochester, NY.
Abstract
Notch signaling has been shown recently to regulate vascular cell fate in adult cells. By applying a uniform equibiaxial cyclic strain to vascular smooth muscle cells (SMCs), we investigated the role of strain in modulating Notch-mediated growth of SMCs in vitro. Rat SMCs cultured under conditions of defined equibiaxial cyclic strain (0% to 15% stretch; 60 cycles/min; 0 to 24 hours) exhibited a significant temporal and force-dependent reduction in Notch 3 receptor expression, concomitant with a significant reduction in Epstein Barr virus latency C promoter-binding factor-1/recombination signal-binding protein of the J immunoglobulin geneeCdependent Notch target gene promoter activity and mRNA levels when compared with unstrained controls. The decrease in Notch signaling was Gi-proteineC and mitogen-activated protein kinaseeCdependent. In parallel cultures, cyclic strain inhibited SMC proliferation (cell number and proliferating cell nuclear antigen expression) while significantly promoting SMC apoptosis (annexin V binding, caspase-3 activity and bax/bcl-xL ratio). Notch 3 receptor overexpression significantly reversed the strain-induced changes in SMC proliferation and apoptosis to levels comparable to unstrained control cells, whereas Notch inhibition further potentiated the changes in SMC apoptosis and proliferation. These findings suggest that cyclic strain inhibits SMC growth while enhancing SMC apoptosis, in part, through regulation of Notch receptor and downstream target gene expression.
Key Words: notch cyclic strain apoptosis proliferation vascular G-proteins
Introduction
Hemodynamic forces associated with the flow of blood play an important role in the physiological control of vascular tone, remodeling, and associated vascular pathologies. These forces include cyclic circumferential strain, which is caused by a transmural force acting perpendicular to the vessel wall.1eC4 Mechanotransduction is known to play a central role in the highly coordinated cellular response of the vasculature to changes in hemodynamic stimulation. Transduction of biomechanical stimuli leads to activation of cellular signaling mechanisms that ultimately lead to adaptive, and sometimes maladaptive, changes in cell and tissue fate.5,6 The ultimate arbiter of vascular cell fate (growth, migration, differentiation, and apoptosis) in response to hemodynamic stimulation is unclear but considered fundamental to the pathogenesis of vascular disease. Strain-induced changes in smooth muscle cell (SMC) growth, defined as the balance between SMC proliferation and apoptosis, participates in the local vascular reaction to hypertension,3,7 late lumen loss, and restenosis after vascular interventions, as well as plaque vulnerability during athersosclerosis.1,8
Because changes in vascular cell fate are also apparent during vascular morphogenesis and modeling of the embryonic vasculature,9,10 the control of these cell fate decisions in adult cells may share similar signaling patterns. Notch receptoreCligand interactions are a highly conserved mechanism, originally described in developmental studies using Drosophila, that regulate intercellular communication and direct individual vascular cell fate decisions.9,10 Notch receptors and ligands are transmembrane proteins that have been identified in mammalian cells (Notch receptors 1 through 4; Notch ligands Delta, Serrate, and Jagged). Studies using constitutively activated Notch receptors missing their extracellular domains (Notch intracellular [IC]) have shown that Notch signaling determines proliferation, differentiation, and, more recently, apoptosis in several mammalian cell types.11eC13 Notch IC is translocated to the nucleus, where it interacts with the CSL family of transcription factors (Epstein Barr virus latency C promoter-binding factor-1/recombination signal-binding protein of the J immunoglobulin gene [CBF-1/RBP-J], suppressor of hairless, and longevity assurance gene-1) to become a transcriptional activator that can then modulate the expression of Notch target genes that regulate cell fate decisions. These include the "Hairy Enhancer of Split" (hes) gene and HES-related transcription factors (Hrts) that are critically involved in mammalian cell differentiation.12,14 Recent studies demonstrate that Notch receptors and hrt genes are coordinately upregulated in neointimal cells but downregulated in medial cells after vascular injury, an effect that is mimicked by addition of serum mitogens (platelet-derived growth factor) to cultured cells in vitro.15 Furthermore, Notch signaling may be a critical determinant of SMC survival and vascular structure by modulating the expression of downstream mediators of apoptosis.16eC19
Biomechanical signals induce a highly restricted transcriptional response in vascular SMCs that include genes that can modify vascular structure.20 Because components of the Notch-signaling pathway are regulated coordinately in vascular tissue after injury,13,15,16,19,21 the present study examined the specific role of uniform equibiaxial cyclic strain on endogenous Notch-signaling components in SMCs and their contributory role in controlling the growth response of these cells after strain.
Materials and Methods
Materials
All items were of the highest purity commercially available and purchased from Sigma Aldrich unless otherwise stated.
Cell Culture
Rat vascular SMCs (R354-05) were purchased from Cell Applications Inc. For cyclic strain studies, cells were seeded into 6-well Bioflex plates (Dunn Labortechnik) at a density of 6x105 cells/well. When cells had reached 80% confluence, a Flexercell Tension Plus FX-4000T system (Flexcell International Corp.) was subsequently used to apply a physiological level of cyclic strain to each plate (0% to 15% strain; 60 cycles/min; 0 to 24 hours using the Heartbeat simulation).
Notch-Expressing Vectors/Luciferase Reporter Plasmids
Notch 3 IC expression vectors (cytomegalovirus X-Notch 3 IC) were a kind gift from Prof Urban Lendahl, Karolinska Institute, Stockholm, Sweden. Notch 1 IC delta RAM domain (mNotch 1 IC) was a kind gift of Prof Bettina Kempkes, GSF Institute of Clinical Molecular Biology, Neuherberg, Germany. R218H was a kind gift from Prof Tasuku Honjo, Department of Medical Chemistry, Kyoto University Faculty of Medicine, Japan. Epstein Barr viruseCencoded gene product that binds CBF-1 (RPMS-1) was a gift from Prof Paul J. Farrell, Ludwig Institute for Cancer Research, Imperial College School of Medicine, London, UK.18
Plasmid Preparation, Transient Transfection, Luciferase, and -Galactosidase Assays
Plasmids were prepared for transfection according to manufacturer instructions using a Qiagen plasmid Midi kit (Qiagen) as described previously.18
Western Blot Analysis
Proteins from cell lysates (12 to 15 e) were resolved on SDS-PAGE (12% resolving; 5% stacking) before transfer onto nitrocellulose membrane (Amersham Biosciences). Membranes were stained in Ponceau S to ensure equal protein loading and rinsed in wash buffer (PBS containing 0.05% Tween 20) before being probed, as described previously.18
Caspase-3 Assay
Activation of the caspase cascade was determined using a colorimetric assay based on the cleavage of acetyl-Asp-Glu-Val-Asp-pNitroanaline (Ac-DEVD-pNA) as described previously.18
Acridine Orange/Ethidium Bromide and Annexin V-Fluos/Propidium Iodide Cell Staining
Apoptotic cells were stained and visualized using the annexin VeCfluos/propidium iodide (AV/PI) stain (Roche Diagnostics) and acridine orange/ethidium bromide (AO/EtBr) stain as described previously.18
Quantitative Real-Time RT-PCR
Quantitative real-time RT-PCR was performed using the Rotor Gene (RG-3000; Corvett Research) and the SYBR green PCR kit (Qiagen) as described previously.18
Small Interfering RNA Transfection
For gene silencing studies, Lipofectamine 2000 reagent (Invitrogen) was used for transient transfection of SMCs with gene-specific small interfering RNA (siRNA) duplexes as described previously.22 The siRNA duplexes for Hrt-2 corresponded to position 123 to 143 in the rat Hrt-2 sequence (Accession number: XM.344806). All duplexes were acquired from MWG Biotech.
Data Analysis
Results are expressed as mean±SEM. Experimental points were performed in triplicate, with a minimum of 3 independent experiments. An unpaired Student’s t test and a Wilcoxon signed rank test were used for comparison of two groups. A value of P<0.05 was considered significant. More information is available online at http://circres.ahajournals.org.
Results
Notch 3 Receptor Promotes SMC Proliferation and Inhibits Apoptosis Through Activation of Notch Target Genes in a CBF-1/RBP-JeCDependent Manner
Expression of RPMS-1 to inhibit CBF-1/RBP-JeCdependent signaling resulted in a significant increase in the number of apoptotic nuclei when compared with mock controls (Figure 1a). Overexpression of constitutively active Notch 3 IC in serum-deprived cells resulted in a significant decrease in the percentage of apoptotic nuclei (67.75±4.5% in mock compared with 38.53±2.0% in cells overexpressing Notch 3 IC; n=3; P<0.05), an effect that was reversed after coexpression with RPMS-1 (data not shown). In addition, inhibition of endogenous CBF-1/RBP-JeCdependent Notch signaling after expression of RPMS-1, R218H, and mNotch 1 IC in cycling SMCs significantly increased caspase-3 activity when compared with mock controls (Figure 1b). Overexpression of constitutively active Notch 3 IC also resulted in a significant reduction in the bax/bcl-xL ratio such that steady-state bax mRNA levels decreased, whereas bcl-xL mRNA levels increased when compared with mock controls (Figure 1c and 1d). Moreover, inhibition of endogenous CBF-1/RBP-JeCdependent signaling after expression of RPMS-1 resulted in an increase in the proapoptotic bax mRNA levels while concomitantly inhibiting antiapoptotic bcl-xL mRNA levels (Figure 1c and 1d).
Furthermore, the changes in the bax/bcl-xL ratio after overexpression of constitutively active Notch 3 IC were reversed after coexpression with RPMS-1 (Figure 1c). In addition, selective knockdown of Notch target gene Hrt-2 with siRNA resulted in a significant increase in bax mRNA levels while significantly decreasing bcl-xL mRNA when compared with the scrambled siRNA control (Figure 1c).
Cyclic Strain Induces Changes in SMC Growth In Vitro
Cyclic strain (10%) significantly decreased expression of proliferating cell nuclear antigen (pCNA), a marker for cell proliferation,18 after 24 hours while concomitantly decreasing the proliferation rate of SMC for up to 10 days after strain (Figure 2a). The lack of an effect of cyclic strain on SMC viability was confirmed by measuring LDH levels in media from strained cells (data not shown).
In parallel cultures, cyclic strain promoted SMC apoptosis because annexin V positive binding was significantly increased after strain when compared with unstrained controls (Figure 2b). This was confirmed using an AO/EtBr stain of apoptotic nuclei (8.25±1.25% in the unstrained controls to 30.75±8.98%; n=3; P<0.05 in the strained cells). Serum deprivation of SMCs for 48 hours, a known ubiquitous proapoptotic stimulus for SMCs, caused a marked increase in SMC bax protein expression while concurrently inhibiting bcl-xL expression (Figure 2c). Cyclic strain also increased the bax/bcl-xL ratio by increasing the expression of bax and decreasing the expression of bcl-xL, respectively, when compared with unstrained cells (Figure 2c). The change in the bax/bcl-xL ratio was further confirmed by examining bax and bcl-xL mRNA levels after strain. Strain significantly increased the levels of proapoptotic bax mRNA and decreased the levels of bcl-xL mRNA when compared with unstrained cells (Figure 2d). The levels of caspase-3 activity were also significantly increased after exposure to cyclic strain when compared with unstrained controls (Figure 2d).
Cyclic Strain Inhibits Notch Signaling in SMC In Vitro
Cyclic strain (10%; 24 hours) caused a significant decrease in Notch 3 protein expression (Notch IC and the full-length receptor [Notch extracellular]) concomitant with a significant decrease in the expression of Notch ligand jagged-1 and the Notch target gene products Hrt-1 and Hrt-3 (Figure 3a). Cyclic strain also significantly decreased Notch 1 and Notch 3 receptor and Jagged-1 steady-state mRNA levels concomitant with a significant decrease in hrt-1, hrt-2, and hrt-3, and hes-1 and hes-5 mRNA levels when compared with unstrained controls (Figure 3a).
The effects of cyclic strain on the Notch-signaling pathway were force and time dependent. Notch 3 mRNA levels were temporally reduced after exposure to strain for up to 24 hours when compared with the unstrained controls (Figure 3b). Similarly, hes-5 mRNA levels were significantly decreased in a force- and time-dependent manner in SMCs subjected to 0, 5, 10, and 15% cyclic strain for up to 24 hours when compared with the unstrained controls (Figure 3b). For subsequent studies, regulation of Notch signaling was examined after exposure to 10% cyclic strain for 24 hours. There was a significant temporal decrease in baseline transactivation CBF-1/RBP-JeCdependent promoter activity in cells exposed to strain when compared with unstrained controls at all times examined (6, 10, and 24 hours) with strain maximally decreasing CBF-1/RBP-J transactivation after 6 hours (Figure 3e). Furthermore, the strain-induced reduction in transactivation of CBF-1/RBP-JeCdependent promoter activity was maintained after overexpression of Notch 3 IC when compared with unstrained controls (Figure 3c). Cyclic strain also significantly decreased Notch target gene promoter activity (hrt-1 and hes-5) at all times examined after strain, with maximal inhibition occurring after 8 hours (Figure 3c).
Cyclic Strain Inhibits Notch Signaling in a GieCMitogen-Activated Protein KinaseeCDependent Manner
Because cyclic strain regulates SMC fate through various mechanosensitive pathways,5,6,20,23,24 the role of inhibitory Gi-proteins and mitogen-activated protein kinase (MAPK) in mediating the strain-induced response was examined. Pretreatment of cells with the Gi-protein inhibitor pertussis toxin (PTX; 100 ng/mL for 24 hours) significantly inhibited baseline levels of Notch3 IC protein expression and Hrt-2 mRNA in unstrained cells (Figure 4a). Moreover, the strain-induced decrease in Notch 3 IC expression and Hrt-2 mRNA levels was blocked after PTX treatment (Figure 4a). Furthermore, pretreatment of cells with the MAPK inhibitor PD98059 significantly inhibited baseline levels of Notch 3 IC protein expression while concurrently inhibiting the strain-induced decrease in Notch IC expression (Figure 4b).
In parallel studies, pretreatment of cells with PTX significantly inhibited the strain-induced decrease in pCNA expression (Figure 4c) while concomitantly inhibiting the strain-induced increase in caspase-3 activity in these cells (Figure 4d).
Effect of Notch Signaling on Cyclic StraineCInduced Changes in SMC Proliferation and Apoptosis In Vitro
Overexpression of constitutively active Notch 3 IC was confirmed using an anti-hemagglutinin (HA) antibody specific for cells expressing the HA-tagged plasmid encoding Notch 3 IC (Figure 5a, inset). The recovery from strain-induced decreases in Notch signaling was confirmed by demonstrating that overexpression of Notch 3 IC recovered the strain-induced decrease in Notch 3 and hrt-2 mRNA to levels that were comparable to unstrained cells (Figure 5a and 5b). In parallel cultures, cyclic strain decreased pCNA expression in mock controls, an effect that was significantly attenuated after overexpression of Notch 3 IC (Figure 5c). Moreover, the cyclic straineCinduced increase in the percentage of apoptotic nuclei in mock controls was significantly attenuated after overexpression of Notch 3 IC (Figure 5d).
The effect of Notch 3 IC overexpression on cyclic straineCinduced changes in caspase-3 and Bcl-2 family mRNA levels was also investigated. The cyclic straineCinduced increase in caspase-3 activity was attenuated after overexpression of constitutively active Notch 3 IC (Figure 6a). Cyclic strain significantly decreased bcl-xL mRNA levels when compared with mock controls, an effect that was significantly attenuated after overexpression of Notch 3 IC (Figure 6b). In contrast, the cyclic straineCinduced increase in bax mRNA levels in mock controls was further enhanced after overexpression of constitutively active Notch 3 IC (Figure 6d). In parallel studies, inhibition of Notch IC by coexpression with RPMS-1 further enhanced the strain-induced increase in bax mRNA while concomitantly potentiating the strain-induced decrease in bcl-xL mRNA levels when compared with mock controls (Figure 6b). The inhibition of Notch IC also further enhanced the strain-induced decrease in pCNA expression compared with mock controls (Figure 6b).
Discussion
Biomechanical signals can induce a highly restricted transcriptional response in vascular SMCs that include genes that can modify vascular structure.5eC8 The current study examined the effect of equibiaxial cyclic strain on Notch signaling in adult SMCs and determined the contributory role of Notch receptors in regulating strain-induced changes in SMC proliferation and apoptosis. We established that cyclic strain induces a significant decrease in the proproliferative and antiapoptotic effects of Notch by decreasing the expression and activity of components of the Notch 3eCsignaling pathway in a force- and time-dependent manner. This decrease in Notch signaling was Gi-proteineC and MAPK-dependent. Moreover, overexpression of constitutively active Notch 3 IC reversed the strain-induced inhibition of SMC proliferation to levels comparable to that of unstrained cells while attenuating strain-induced SMC apoptosis in vitro. Furthermore, Notch inhibition potentiated the strain-induced decrease in Notch signaling. Collectively, these studies suggest for the first time that biomechanical stimulation of SMC inhibits endogenous Notch receptor signaling, resulting in fundamental changes in vascular SMC proliferation and apoptosis in vitro. Understanding these responses may provide new insights into the pathogenesis and treatment of vascular diseases such as atherosclerosis and intimal hyperplasia.
Previous studies have presented conflicting reports on the effects of cyclic strain on SMC growth in vitro.25eC30 Depending on the species of SMC, the phenotype studied, the extracellular matrix environment, the cell cycle status (whether quiesced or cycling), and the type of cyclical strain regime applied, SMCs can either increase26,28,29,31 or decrease25,27,30 their proliferative capacity. In the current study, cyclic strain decreased SMC proliferation while concomitantly increasing SMC apoptosis. This concurs with several other studies that report increased SMC apoptosis vitro32,33 and in vivo34,35 in response to strain or pressure. The cyclic straineCinduced SMC apoptosis is, at least in part, associated with an increase in caspase-3 activity and a change in the bax/bcl-xL ratio in favor of apoptosis. It is clear that strain-induced regulation of Bcl-2 proteins is consistent with strain-induced changes in caspase-3 activity in these cells, and mirrors changes during vascular remodeling in vivo.36eC40 The stimulus for the observed increase in bax expression after strain is likely attributable to increased p53 activity because bax is a direct transcriptional target for p53, and two recent studies have shown increases in p53 activity and bax expression attributable to cyclic strain.33,41 The strain-induced decrease in bcl-xL is consistent with previous studies in vitro42 and validates previous in vivo studies that demonstrate a correlation between downregulation of bcl-xL and increased SMC apoptosis.38eC40 Together, this study has clearly established that cyclic strain decreases proliferation and increases SMC apoptosis through a mechanism involving modulation of the Bcl-2 family and activation of caspase-3.
We and others have established a functional coupling between Notch signaling, SMC proliferation, and apoptosis in SMCs in vitro and in vivo.15eC19 The current study demonstrated for the first time that constitutively active Notch 3 IC increased bcl-xL expression while concomitantly inhibiting bax expression, an effect that was fully reversed after inhibition of CBF-1/RBP-J with expression of RPMS-1. These data suggest that bax and bcl-xL are downstream targets of CBF-1/RBP-JeCdependent Notch signaling in these cells. This is further reinforced by studies that demonstrate constitutively active Notch 1 modulates expression of bcl-xL.43 Moreover, we have shown using specific siRNA targeted against an individual Notch target gene (Hrt-2) selective hrt-2 knockdown resulted in a significant increase in bax and a decrease in bcl-xL expression in SMCs, further confirming the importance of Notch IC regulation of Bcl-2 family of proteins in these cells. At present, it is unclear why bax mRNA levels are enhanced in strained cells after constitutive Notch activation despite being significantly inhibited in unstrained cells. Additional factors affected by cyclic strain may act to regulate and potentiate bax expression in strained cells. This possibility is further reinforced because bax expression is significantly enhanced in strained cells irrespective of whether Notch IC is overexpressed or inhibited (after RPMS-1 expression).
Because cyclic strain can induce a highly restricted transcriptional response in vascular SMCs that regulates vascular structure,2,5 we hypothesized that Notch could represent a novel pathway for contributing to strain-induced changes in vascular fate. The current study demonstrates a functional role for cyclic straineCinduced decreases in Notch-mediated CBF-1/RBP-JeCdependent signaling and subsequent changes in vascular cell fate. The level of inhibition of Notch signaling after exposure to cyclic strain suggests that Notch is a major target for biomechanical regulation of SMCs in vitro. Indeed, previous in vivo studies concur by demonstrating an acute decrease in Notch receptor and Notch target gene expression after balloon injury and subsequent cellular distension, with the most dramatic decrease evident for the Notch 3 receptor and the hrt-1 target gene.15 This response to strain appears to be temporal.15,16 Platelet-derived growth factor and angiotensin II also induced a coordinate downregulation of Notch signaling in SMCs through an extracellular signal-regulated kinaseeCdependent pathway.15,16,19 In the current study, the decrease in the expression of Notch components is consistent with the observed changes in SMC fate after strain. Moreover, selective inhibition of Gi-proteins and MAPK, respectively, which are known signaling pathways involved in mechanotransduction in vascular cells,22,23,24 despite decreasing baseline Notch signaling, blocked the strain-induced changes in Notch and corresponding changes in SMC fate. To our knowledge, this is the first demonstration that the expression of Notch receptors and downstream target genes are directly modulated by strain in mammalian cells in vitro. This finding further suggests a possible nexus by which the activation of a biomechanical signaling pathway is coupled to the Notch cellular fate program. Additional studies will be required to delineate the mechanism(s) by which cyclic strain regulates the expression of Notch in vitro.
The regulation of Notch signaling is known to occur at multiple levels, including patterns of ligand and receptor expression, Notch-ligand interactions, trafficking of the receptor and ligands, and covalent modifications, including glycosylation, phosphorylation, and ubiquitination.10,11,44,45 A possible mechanistic explanation for the effect of cyclic strain on Notch 3 IC activity may be post-translational modifications, such as phosphorylation, that may influence its transactivation capacity.45,46 Indeed, glycogen synthase kinase-3 (GSK3) is known to modulate Notch signaling through phosphorylation of Notch IC,45eC47 such that inhibition of GSK3 shortened the half life of Notch1 IC, whereas, conversely, activated GSK3 reduced the quantity of Notch IC that was degraded by the proteasome.46 Moreover, the regulatory phosphorylation of GSK3 and hence its activity is under the control of MAPK-dependent signaling pathways.48 Therefore, it is tempting to speculate that cyclic strain modulates this process, thereby influencing the translocation of Notch IC into the nucleus. However, because capacity of Notch 3 IC to transactivate various CBF-1/RBP-JeCdependent promoters is inhibited by cyclic strain and because strain does not affect constitutively active Notch 3 IC expression, these data collectively suggest that other strain-induced events are critical to the ability of Notch IC (whether endogenous or vector encoded) to transactivate CBF-1/RBP-J target promoters. Indeed, it is known that Notch IC interacts with coactivating proteins, including histone acetyltransferase proteins, which play a role in chromatin opening and initiation of transcription.49,50 Moreover, strain may inhibit endogenous inhibitors of Notch IC, such as Numb, that result in a loss of Notch IC functionality after strain. However, because Notch 3 mRNA levels and the expression of full-length Notch 3 receptors are also decreased after strain, it is clear that cyclic strain inhibits the expression of the Notch 3 receptor in addition to any post-translation modifications that may occur to shorten the half life of Notch IC.
In conclusion, we have shown for the first time that equibiaxial cyclic strain induces apoptosis and inhibits proliferation of rat SMC in vitro. Cyclic straineCinduced changes in fate are attributable, at least in part, to a cyclic straineCinduced decrease in expression and activity of Notch signaling pathway components. These studies provide further evidence that Notch receptors acting through CBF-1/RBP-JeCdependent signaling pathways are an important determinant of SMC fate in vitro.
Acknowledgments
This research was supported by grants from the Higher Education Authority (HEA) of Ireland, Wellcome Trust, Science Foundation Ireland, and the Health Research Board of Ireland (to P.A.C.) and by grants from the National Institutes of Health (HL59696 and AA-12610; to P.A.C. and E.M.R.).
The first 2 authors contributed equally to this work.
References
Schwartz RS, Henry TD. Pathophysiology of coronary artery restenosis. Rev Cardiovasc Med. 2002; 3: (suppl 5): S4eCS9.
Kleinstreuer C, Hyun S, Buchanan JR Jr, Longest PW, Archie JP Jr, Truskey GA. Hemodynamic parameters and early intimal thickening in branching blood vessels. Crit Rev Biomed Eng. 2001; 29: 1eC64.
Ferguson JE III, Patterson C. Break the cycle: the role of cell-cycle modulation in the prevention of vasculoproliferative diseases. Cell Cycle. 2003; 2: 211eC219.
Manabe I, Nagai R. Regulation of smooth muscle phenotype. Curr Atheroscler Rep. 2003; 5: 214eC222.
Osol G. Mechanotransduction by vascular smooth muscle. J Vasc Res. 1995; 32: 275eC292.
Lehoux S, Tedgui A. Cellular mechanics and gene expression in blood vessels. J Biomech. 2003; 36: 631eC643.
Koller A. Signaling pathways of mechanotransduction in arteriolar endothelium and smooth muscle cells in hypertension. Microcirculation. 2002; 9: 277eC294.
Brooks AR, Lelkes PI, Rubanyi GM. Gene expression profiling of vascular endothelial cells exposed to fluid mechanical forces: relevance for focal susceptibility to atherosclerosis. Endothelium. 2004; 11: 45eC57.
Majesky MW. Vascular smooth muscle diversity: insights from developmental biology. Curr Atheroscler Rep. 2003; 5: 208eC213.
Weinstein BM, Lawson ND. Arteries, veins, Notch, and VEGF. Cold Spring Harb Symp Quant Biol. 2002; 67: 155eC162.
Iso T, Hamamori Y, Kedes L. Notch signaling in vascular development. Arterioscler Thromb Vasc Biol. 2003; 23: 543eC553.
Lai EC. Keeping a good pathway down: transcriptional repression of Notch pathway target genes by CSL proteins. EMBO Rep. 2002; 3: 840eC845.
Harper JA, Yuan JS, Tan JB, Visan I, Guidos CJ. Notch signaling in development and disease. Clin Genet. 2003; 64: 461eC472.
Iso T, Kedes L, Hamamori Y. HES and HERP families: multiple effectors of the Notch signaling pathway. J Cell Physiol. 2003; 194: 237eC255.
Wang W, Campos AH, Prince CZ, Mou Y, Pollman MJ. Coordinate Notch 3-hairy-related transcription factor pathway regulation in response to arterial injury. Mediator role of platelet-derived growth factor and ERK. J Biol Chem. 2002; 277: 23165eC23171.
Campos AH, Wang W, Pollman MJ, Gibbons GH. Determinants of Notch-3 receptor expression and signaling in vascular smooth muscle cells: implications in cell-cycle regulation. Circ Res. 2002; 91: 999eC1006.
Wang W, Prince CZ, Hu X, Pollman MJ. HRT1 modulates vascular smooth muscle cell proliferation and apoptosis. Biochem Biophys Res Commun. 2003; 308: 596eC601.
Sweeney C, Morrow D, Birney YA, Coyle S, Hennessy C, Scheller A, Cummins PM, Walls D, Redmond EM, Cahill PA. Notch 1 and 3 receptor signaling modulates vascular smooth muscle cell growth, apoptosis, and migration via a CBF-1/RBP-J-dependent pathway. FASEB J. 2004; 18: 1421eC1423
Wang W, Prince CZ, Mou Y, Pollman MJ. Notch3 signaling in vascular smooth muscle cells induces c-FLIP expression via ERK/MAPK activation. Resistance to Fas ligand-induced apoptosis. J Biol Chem. 2002; 277: 21723eC21729.
Xu Q. Biomechanical-stress-induced signaling and gene expression in the development of arteriosclerosis. Trends Cardiovasc Med. 2000; 10: 35eC41.
Lindner V, Booth C, Prudovsky I, Small D, Maciag T, Liaw L. Members of the Jagged/Notch gene families are expressed in injured arteries and regulate cell phenotype via alterations in cell matrix and cell-cell interaction. Am J Pathol. 2001; 159: 875eC883.
Von Offenberg Sweeney N, Cummins PM, Birney YA, Redmond EM, Cahill PA. Cyclic strain-induced endothelial MMP-2: role in vascular smooth muscle cell migration. Biochem Biophys Res Commun. 2004; 320: 325eC333.
Cullen JP, Sayeed S, Sawai RS, Theodorakis NG, Cahill PA, Sitzmann JV, Redmond EM. Pulsatile flow-induced angiogenesis: role of G(i) subunits. Arterioscler Thromb Vasc Biol. 2002; 22: 1610eC1616.
Shaw A, Xu Q. Biomechanical stress-induced signaling in smooth muscle cells: an update. Curr Vasc Pharmacol. 2003: 41eC58.
Schulze PC, de Keulenaer GW, Kassik KA, Takahashi T, Chen Z, Simon DI, Lee RT. Biomechanically induced gene iex-1 inhibits vascular smooth muscle cell proliferation and neointima formation. Circ Res. 2003; 93: 1210eC1217.
Sedding DG, Seay U, Fink L, Heil M, Kummer W, Tillmanns H, Braun-Dullaeus RC. Mechanosensitive p27Kip1 regulation and cell cycle entry in vascular smooth muscle cells. Circulation. 2003 108: 616eC622.
Hipper A, Isenberg G. Cyclic mechanical strain decreases the DNA synthesis of vascular smooth muscle cells. Pfle筭ers Arch. 2000; 440: 19eC27.
Birukov KG, Shirinsky VP, Stepanova OV, Tkachuk VA, Hahn AW, Resink TJ, Smirnov VN. Stretch affects phenotype and proliferation of vascular smooth muscle cells. Mol Cell Biochem. 1995; 144: 131eC139.
Wilson E, Mai Q, Sudhir K, Weiss RH, Ives HE. Mechanical strain induces growth of vascular smooth muscle cells via autocrine action of PDGF. J Cell Biol. 1993; 23: 741eC747.
Sumpio BE, Banes AJ. Response of porcine aortic smooth muscle cells to cyclic tensional deformation in culture. J Surg Res. 1988; 44: 696eC701.
Cappadona C, Redmond EM, Theodorakis NG, McKillop IH, Hendrickson R, Chhabra A, Sitzmann JV, Cahill PA. Phenotype dictates the growth response of vascular smooth muscle cells to pulse pressure in vitro. Exp Cell Res. 1999; 250: 174eC186.
Kakisis JD, Liapis CD, Sumpio BE. Effects of cyclic strain on vascular cells. Endothelium. 2004; 11: 17eC28.
Mayr M, Hu Y, Hainaut H, Xu Q. Mechanical stress-induced DNA damage and rac-p38MAPK signal pathways mediate p53-dependent apoptosis in vascular smooth muscle cells. FASEB J. 2002; 16: 1423eC1425.
Mayr M, Li C, Zou Y, Huemer U, Hu Y, Xu Q. Biomechanical stress-induced apoptosis in vein grafts involves p38 mitogen-activated protein kinases. FASEB J. 2000; 14: 261eC270.
Wernig F, Xu Q. Mechanical stress-induced apoptosis in the cardiovascular system. Prog Biophys Mol Biol. 2002; 78: 105eC137.
Nhan TQ, Liles WC, Chait A, Fallon JT, Schwartz SM. The p17 cleaved form of caspase-3 is present within viable macrophages in vitro and in atherosclerotic plaque. Arterioscler Thromb Vasc Biol. 2003; 23: 1276eC1282.
Assuncao Guimaraes C, Linden R. Programmed cell deaths. Apoptosis and alternative death styles. Eur J Biochem. 2004; 271: 1638eC1650.
Kim HS, Hwang KK, Seo JW, Kim SY, Oh BH, Lee MM, Park YB. Apoptosis and regulation of Bax and Bcl-X proteins during human neonatal vascular remodeling. Arterioscler Thromb Vasc Biol. 2000; 20: 957eC963.
Pollman MJ, Hall JL, Mann MJ, Zhang L, Gibbons GH. Inhibition of neointimal cell bcl-x expression induces apoptosis and regression of vascular disease. Nat Med. 1998; 4: 222eC227.
Pollman MJ, Hall JL, Gibbons GH. Determinants of vascular smooth muscle cell apoptosis after balloon angioplasty injury. Influence of redox state and cell phenotype. Circ Res. 1999; 84: 113eC121.
Mayr U, Mayr M, Li C, Wernig F, Dietrich H, Hu Y, Xu Q. Loss of p53 accelerates neointimal lesions of vein bypass grafts in mice. Circ Res. 2002; 90: 197eC204.
Birney YB, Sweeney CH, Cappadona CR, Sitzmann JVS, Cummins PM, Redmond EM Cahill PA. Pulse pressure induced transmural fluid flux increases bovine aortic smooth muscle cell apoptosis in a mitogen activated protein kinase-dependent manner. J Vasc Res. 2004; 41: 364eC374.
Jang MS, Miao H, Carlesso N, Shelly L, Zlobin A, Darack N, Qin JZ, Nickoloff BJ, Miele L. Notch-1 regulates cell death independently of differentiation in murine erythroleukemia cells through multiple apoptosis and cell cycle pathways. J Cell Physiol. 2004; 199: 418eC433.
Baron M, Aslam H, Flasza M, Fostier M, Higgs JE, Mazaleyrat SL, Wilkin MB. Multiple levels of Notch signal regulation. Mol Membr Biol. 2002; 19: 27eC38.
Nie L, Xu M, Vladimirova A, Sun XH. Notch-induced E2A ubiquitination and degradation are controlled by MAP kinase activities. EMBO J. 2003; 22: 5780eC5792.
Foltz DR, Santiago MC, Berechid BE, Nye JS. Glycogen synthase kinase-3beta modulates notch signaling and stability. Curr Biol. 2002; 12: 1006eC1011.
Espinosa L, Ingles-Esteve J, Aguilera C, Bigas A. Phosphorylation by glycogen synthase kinase-3 beta down-regulates Notch activity, a link for Notch and Wnt pathways. J Biol Chem. 2003; 278: 32227eC32235.
Liu F, Schaphorst KL, Verin AD, Jacobs K, Birukova A, Day RM, Bogatcheva N, Bottaro DP, Garcia JG. Hepatocyte growth factor enhances endothelial cell barrier function and cortical cytoskeletal rearrangement: potential role of glycogen synthase kinase-3beta. FASEB J. 2002; 16: 950eC962.
Utley RT, Cote J. The MYST family of histone acetyltransferases. Curr Top Microbiol Immunol. 2003; 274: 203eC236.
Kurooka H, Honjo T. Functional interaction between the mouse notch1 intracellular region and histone acetyltransferases PCAF and GCN5. J Biol Chem. 2000; 275: 17211eC17220.(David Morrow, Catherine S)
the School of Biotechnology and the National Centre for Sensor Research (D.W.), Dublin City University, Ireland
Department of Surgery (E.M.R.), University of Rochester Medical Center, Rochester, NY.
Abstract
Notch signaling has been shown recently to regulate vascular cell fate in adult cells. By applying a uniform equibiaxial cyclic strain to vascular smooth muscle cells (SMCs), we investigated the role of strain in modulating Notch-mediated growth of SMCs in vitro. Rat SMCs cultured under conditions of defined equibiaxial cyclic strain (0% to 15% stretch; 60 cycles/min; 0 to 24 hours) exhibited a significant temporal and force-dependent reduction in Notch 3 receptor expression, concomitant with a significant reduction in Epstein Barr virus latency C promoter-binding factor-1/recombination signal-binding protein of the J immunoglobulin geneeCdependent Notch target gene promoter activity and mRNA levels when compared with unstrained controls. The decrease in Notch signaling was Gi-proteineC and mitogen-activated protein kinaseeCdependent. In parallel cultures, cyclic strain inhibited SMC proliferation (cell number and proliferating cell nuclear antigen expression) while significantly promoting SMC apoptosis (annexin V binding, caspase-3 activity and bax/bcl-xL ratio). Notch 3 receptor overexpression significantly reversed the strain-induced changes in SMC proliferation and apoptosis to levels comparable to unstrained control cells, whereas Notch inhibition further potentiated the changes in SMC apoptosis and proliferation. These findings suggest that cyclic strain inhibits SMC growth while enhancing SMC apoptosis, in part, through regulation of Notch receptor and downstream target gene expression.
Key Words: notch cyclic strain apoptosis proliferation vascular G-proteins
Introduction
Hemodynamic forces associated with the flow of blood play an important role in the physiological control of vascular tone, remodeling, and associated vascular pathologies. These forces include cyclic circumferential strain, which is caused by a transmural force acting perpendicular to the vessel wall.1eC4 Mechanotransduction is known to play a central role in the highly coordinated cellular response of the vasculature to changes in hemodynamic stimulation. Transduction of biomechanical stimuli leads to activation of cellular signaling mechanisms that ultimately lead to adaptive, and sometimes maladaptive, changes in cell and tissue fate.5,6 The ultimate arbiter of vascular cell fate (growth, migration, differentiation, and apoptosis) in response to hemodynamic stimulation is unclear but considered fundamental to the pathogenesis of vascular disease. Strain-induced changes in smooth muscle cell (SMC) growth, defined as the balance between SMC proliferation and apoptosis, participates in the local vascular reaction to hypertension,3,7 late lumen loss, and restenosis after vascular interventions, as well as plaque vulnerability during athersosclerosis.1,8
Because changes in vascular cell fate are also apparent during vascular morphogenesis and modeling of the embryonic vasculature,9,10 the control of these cell fate decisions in adult cells may share similar signaling patterns. Notch receptoreCligand interactions are a highly conserved mechanism, originally described in developmental studies using Drosophila, that regulate intercellular communication and direct individual vascular cell fate decisions.9,10 Notch receptors and ligands are transmembrane proteins that have been identified in mammalian cells (Notch receptors 1 through 4; Notch ligands Delta, Serrate, and Jagged). Studies using constitutively activated Notch receptors missing their extracellular domains (Notch intracellular [IC]) have shown that Notch signaling determines proliferation, differentiation, and, more recently, apoptosis in several mammalian cell types.11eC13 Notch IC is translocated to the nucleus, where it interacts with the CSL family of transcription factors (Epstein Barr virus latency C promoter-binding factor-1/recombination signal-binding protein of the J immunoglobulin gene [CBF-1/RBP-J], suppressor of hairless, and longevity assurance gene-1) to become a transcriptional activator that can then modulate the expression of Notch target genes that regulate cell fate decisions. These include the "Hairy Enhancer of Split" (hes) gene and HES-related transcription factors (Hrts) that are critically involved in mammalian cell differentiation.12,14 Recent studies demonstrate that Notch receptors and hrt genes are coordinately upregulated in neointimal cells but downregulated in medial cells after vascular injury, an effect that is mimicked by addition of serum mitogens (platelet-derived growth factor) to cultured cells in vitro.15 Furthermore, Notch signaling may be a critical determinant of SMC survival and vascular structure by modulating the expression of downstream mediators of apoptosis.16eC19
Biomechanical signals induce a highly restricted transcriptional response in vascular SMCs that include genes that can modify vascular structure.20 Because components of the Notch-signaling pathway are regulated coordinately in vascular tissue after injury,13,15,16,19,21 the present study examined the specific role of uniform equibiaxial cyclic strain on endogenous Notch-signaling components in SMCs and their contributory role in controlling the growth response of these cells after strain.
Materials and Methods
Materials
All items were of the highest purity commercially available and purchased from Sigma Aldrich unless otherwise stated.
Cell Culture
Rat vascular SMCs (R354-05) were purchased from Cell Applications Inc. For cyclic strain studies, cells were seeded into 6-well Bioflex plates (Dunn Labortechnik) at a density of 6x105 cells/well. When cells had reached 80% confluence, a Flexercell Tension Plus FX-4000T system (Flexcell International Corp.) was subsequently used to apply a physiological level of cyclic strain to each plate (0% to 15% strain; 60 cycles/min; 0 to 24 hours using the Heartbeat simulation).
Notch-Expressing Vectors/Luciferase Reporter Plasmids
Notch 3 IC expression vectors (cytomegalovirus X-Notch 3 IC) were a kind gift from Prof Urban Lendahl, Karolinska Institute, Stockholm, Sweden. Notch 1 IC delta RAM domain (mNotch 1 IC) was a kind gift of Prof Bettina Kempkes, GSF Institute of Clinical Molecular Biology, Neuherberg, Germany. R218H was a kind gift from Prof Tasuku Honjo, Department of Medical Chemistry, Kyoto University Faculty of Medicine, Japan. Epstein Barr viruseCencoded gene product that binds CBF-1 (RPMS-1) was a gift from Prof Paul J. Farrell, Ludwig Institute for Cancer Research, Imperial College School of Medicine, London, UK.18
Plasmid Preparation, Transient Transfection, Luciferase, and -Galactosidase Assays
Plasmids were prepared for transfection according to manufacturer instructions using a Qiagen plasmid Midi kit (Qiagen) as described previously.18
Western Blot Analysis
Proteins from cell lysates (12 to 15 e) were resolved on SDS-PAGE (12% resolving; 5% stacking) before transfer onto nitrocellulose membrane (Amersham Biosciences). Membranes were stained in Ponceau S to ensure equal protein loading and rinsed in wash buffer (PBS containing 0.05% Tween 20) before being probed, as described previously.18
Caspase-3 Assay
Activation of the caspase cascade was determined using a colorimetric assay based on the cleavage of acetyl-Asp-Glu-Val-Asp-pNitroanaline (Ac-DEVD-pNA) as described previously.18
Acridine Orange/Ethidium Bromide and Annexin V-Fluos/Propidium Iodide Cell Staining
Apoptotic cells were stained and visualized using the annexin VeCfluos/propidium iodide (AV/PI) stain (Roche Diagnostics) and acridine orange/ethidium bromide (AO/EtBr) stain as described previously.18
Quantitative Real-Time RT-PCR
Quantitative real-time RT-PCR was performed using the Rotor Gene (RG-3000; Corvett Research) and the SYBR green PCR kit (Qiagen) as described previously.18
Small Interfering RNA Transfection
For gene silencing studies, Lipofectamine 2000 reagent (Invitrogen) was used for transient transfection of SMCs with gene-specific small interfering RNA (siRNA) duplexes as described previously.22 The siRNA duplexes for Hrt-2 corresponded to position 123 to 143 in the rat Hrt-2 sequence (Accession number: XM.344806). All duplexes were acquired from MWG Biotech.
Data Analysis
Results are expressed as mean±SEM. Experimental points were performed in triplicate, with a minimum of 3 independent experiments. An unpaired Student’s t test and a Wilcoxon signed rank test were used for comparison of two groups. A value of P<0.05 was considered significant. More information is available online at http://circres.ahajournals.org.
Results
Notch 3 Receptor Promotes SMC Proliferation and Inhibits Apoptosis Through Activation of Notch Target Genes in a CBF-1/RBP-JeCDependent Manner
Expression of RPMS-1 to inhibit CBF-1/RBP-JeCdependent signaling resulted in a significant increase in the number of apoptotic nuclei when compared with mock controls (Figure 1a). Overexpression of constitutively active Notch 3 IC in serum-deprived cells resulted in a significant decrease in the percentage of apoptotic nuclei (67.75±4.5% in mock compared with 38.53±2.0% in cells overexpressing Notch 3 IC; n=3; P<0.05), an effect that was reversed after coexpression with RPMS-1 (data not shown). In addition, inhibition of endogenous CBF-1/RBP-JeCdependent Notch signaling after expression of RPMS-1, R218H, and mNotch 1 IC in cycling SMCs significantly increased caspase-3 activity when compared with mock controls (Figure 1b). Overexpression of constitutively active Notch 3 IC also resulted in a significant reduction in the bax/bcl-xL ratio such that steady-state bax mRNA levels decreased, whereas bcl-xL mRNA levels increased when compared with mock controls (Figure 1c and 1d). Moreover, inhibition of endogenous CBF-1/RBP-JeCdependent signaling after expression of RPMS-1 resulted in an increase in the proapoptotic bax mRNA levels while concomitantly inhibiting antiapoptotic bcl-xL mRNA levels (Figure 1c and 1d).
Furthermore, the changes in the bax/bcl-xL ratio after overexpression of constitutively active Notch 3 IC were reversed after coexpression with RPMS-1 (Figure 1c). In addition, selective knockdown of Notch target gene Hrt-2 with siRNA resulted in a significant increase in bax mRNA levels while significantly decreasing bcl-xL mRNA when compared with the scrambled siRNA control (Figure 1c).
Cyclic Strain Induces Changes in SMC Growth In Vitro
Cyclic strain (10%) significantly decreased expression of proliferating cell nuclear antigen (pCNA), a marker for cell proliferation,18 after 24 hours while concomitantly decreasing the proliferation rate of SMC for up to 10 days after strain (Figure 2a). The lack of an effect of cyclic strain on SMC viability was confirmed by measuring LDH levels in media from strained cells (data not shown).
In parallel cultures, cyclic strain promoted SMC apoptosis because annexin V positive binding was significantly increased after strain when compared with unstrained controls (Figure 2b). This was confirmed using an AO/EtBr stain of apoptotic nuclei (8.25±1.25% in the unstrained controls to 30.75±8.98%; n=3; P<0.05 in the strained cells). Serum deprivation of SMCs for 48 hours, a known ubiquitous proapoptotic stimulus for SMCs, caused a marked increase in SMC bax protein expression while concurrently inhibiting bcl-xL expression (Figure 2c). Cyclic strain also increased the bax/bcl-xL ratio by increasing the expression of bax and decreasing the expression of bcl-xL, respectively, when compared with unstrained cells (Figure 2c). The change in the bax/bcl-xL ratio was further confirmed by examining bax and bcl-xL mRNA levels after strain. Strain significantly increased the levels of proapoptotic bax mRNA and decreased the levels of bcl-xL mRNA when compared with unstrained cells (Figure 2d). The levels of caspase-3 activity were also significantly increased after exposure to cyclic strain when compared with unstrained controls (Figure 2d).
Cyclic Strain Inhibits Notch Signaling in SMC In Vitro
Cyclic strain (10%; 24 hours) caused a significant decrease in Notch 3 protein expression (Notch IC and the full-length receptor [Notch extracellular]) concomitant with a significant decrease in the expression of Notch ligand jagged-1 and the Notch target gene products Hrt-1 and Hrt-3 (Figure 3a). Cyclic strain also significantly decreased Notch 1 and Notch 3 receptor and Jagged-1 steady-state mRNA levels concomitant with a significant decrease in hrt-1, hrt-2, and hrt-3, and hes-1 and hes-5 mRNA levels when compared with unstrained controls (Figure 3a).
The effects of cyclic strain on the Notch-signaling pathway were force and time dependent. Notch 3 mRNA levels were temporally reduced after exposure to strain for up to 24 hours when compared with the unstrained controls (Figure 3b). Similarly, hes-5 mRNA levels were significantly decreased in a force- and time-dependent manner in SMCs subjected to 0, 5, 10, and 15% cyclic strain for up to 24 hours when compared with the unstrained controls (Figure 3b). For subsequent studies, regulation of Notch signaling was examined after exposure to 10% cyclic strain for 24 hours. There was a significant temporal decrease in baseline transactivation CBF-1/RBP-JeCdependent promoter activity in cells exposed to strain when compared with unstrained controls at all times examined (6, 10, and 24 hours) with strain maximally decreasing CBF-1/RBP-J transactivation after 6 hours (Figure 3e). Furthermore, the strain-induced reduction in transactivation of CBF-1/RBP-JeCdependent promoter activity was maintained after overexpression of Notch 3 IC when compared with unstrained controls (Figure 3c). Cyclic strain also significantly decreased Notch target gene promoter activity (hrt-1 and hes-5) at all times examined after strain, with maximal inhibition occurring after 8 hours (Figure 3c).
Cyclic Strain Inhibits Notch Signaling in a GieCMitogen-Activated Protein KinaseeCDependent Manner
Because cyclic strain regulates SMC fate through various mechanosensitive pathways,5,6,20,23,24 the role of inhibitory Gi-proteins and mitogen-activated protein kinase (MAPK) in mediating the strain-induced response was examined. Pretreatment of cells with the Gi-protein inhibitor pertussis toxin (PTX; 100 ng/mL for 24 hours) significantly inhibited baseline levels of Notch3 IC protein expression and Hrt-2 mRNA in unstrained cells (Figure 4a). Moreover, the strain-induced decrease in Notch 3 IC expression and Hrt-2 mRNA levels was blocked after PTX treatment (Figure 4a). Furthermore, pretreatment of cells with the MAPK inhibitor PD98059 significantly inhibited baseline levels of Notch 3 IC protein expression while concurrently inhibiting the strain-induced decrease in Notch IC expression (Figure 4b).
In parallel studies, pretreatment of cells with PTX significantly inhibited the strain-induced decrease in pCNA expression (Figure 4c) while concomitantly inhibiting the strain-induced increase in caspase-3 activity in these cells (Figure 4d).
Effect of Notch Signaling on Cyclic StraineCInduced Changes in SMC Proliferation and Apoptosis In Vitro
Overexpression of constitutively active Notch 3 IC was confirmed using an anti-hemagglutinin (HA) antibody specific for cells expressing the HA-tagged plasmid encoding Notch 3 IC (Figure 5a, inset). The recovery from strain-induced decreases in Notch signaling was confirmed by demonstrating that overexpression of Notch 3 IC recovered the strain-induced decrease in Notch 3 and hrt-2 mRNA to levels that were comparable to unstrained cells (Figure 5a and 5b). In parallel cultures, cyclic strain decreased pCNA expression in mock controls, an effect that was significantly attenuated after overexpression of Notch 3 IC (Figure 5c). Moreover, the cyclic straineCinduced increase in the percentage of apoptotic nuclei in mock controls was significantly attenuated after overexpression of Notch 3 IC (Figure 5d).
The effect of Notch 3 IC overexpression on cyclic straineCinduced changes in caspase-3 and Bcl-2 family mRNA levels was also investigated. The cyclic straineCinduced increase in caspase-3 activity was attenuated after overexpression of constitutively active Notch 3 IC (Figure 6a). Cyclic strain significantly decreased bcl-xL mRNA levels when compared with mock controls, an effect that was significantly attenuated after overexpression of Notch 3 IC (Figure 6b). In contrast, the cyclic straineCinduced increase in bax mRNA levels in mock controls was further enhanced after overexpression of constitutively active Notch 3 IC (Figure 6d). In parallel studies, inhibition of Notch IC by coexpression with RPMS-1 further enhanced the strain-induced increase in bax mRNA while concomitantly potentiating the strain-induced decrease in bcl-xL mRNA levels when compared with mock controls (Figure 6b). The inhibition of Notch IC also further enhanced the strain-induced decrease in pCNA expression compared with mock controls (Figure 6b).
Discussion
Biomechanical signals can induce a highly restricted transcriptional response in vascular SMCs that include genes that can modify vascular structure.5eC8 The current study examined the effect of equibiaxial cyclic strain on Notch signaling in adult SMCs and determined the contributory role of Notch receptors in regulating strain-induced changes in SMC proliferation and apoptosis. We established that cyclic strain induces a significant decrease in the proproliferative and antiapoptotic effects of Notch by decreasing the expression and activity of components of the Notch 3eCsignaling pathway in a force- and time-dependent manner. This decrease in Notch signaling was Gi-proteineC and MAPK-dependent. Moreover, overexpression of constitutively active Notch 3 IC reversed the strain-induced inhibition of SMC proliferation to levels comparable to that of unstrained cells while attenuating strain-induced SMC apoptosis in vitro. Furthermore, Notch inhibition potentiated the strain-induced decrease in Notch signaling. Collectively, these studies suggest for the first time that biomechanical stimulation of SMC inhibits endogenous Notch receptor signaling, resulting in fundamental changes in vascular SMC proliferation and apoptosis in vitro. Understanding these responses may provide new insights into the pathogenesis and treatment of vascular diseases such as atherosclerosis and intimal hyperplasia.
Previous studies have presented conflicting reports on the effects of cyclic strain on SMC growth in vitro.25eC30 Depending on the species of SMC, the phenotype studied, the extracellular matrix environment, the cell cycle status (whether quiesced or cycling), and the type of cyclical strain regime applied, SMCs can either increase26,28,29,31 or decrease25,27,30 their proliferative capacity. In the current study, cyclic strain decreased SMC proliferation while concomitantly increasing SMC apoptosis. This concurs with several other studies that report increased SMC apoptosis vitro32,33 and in vivo34,35 in response to strain or pressure. The cyclic straineCinduced SMC apoptosis is, at least in part, associated with an increase in caspase-3 activity and a change in the bax/bcl-xL ratio in favor of apoptosis. It is clear that strain-induced regulation of Bcl-2 proteins is consistent with strain-induced changes in caspase-3 activity in these cells, and mirrors changes during vascular remodeling in vivo.36eC40 The stimulus for the observed increase in bax expression after strain is likely attributable to increased p53 activity because bax is a direct transcriptional target for p53, and two recent studies have shown increases in p53 activity and bax expression attributable to cyclic strain.33,41 The strain-induced decrease in bcl-xL is consistent with previous studies in vitro42 and validates previous in vivo studies that demonstrate a correlation between downregulation of bcl-xL and increased SMC apoptosis.38eC40 Together, this study has clearly established that cyclic strain decreases proliferation and increases SMC apoptosis through a mechanism involving modulation of the Bcl-2 family and activation of caspase-3.
We and others have established a functional coupling between Notch signaling, SMC proliferation, and apoptosis in SMCs in vitro and in vivo.15eC19 The current study demonstrated for the first time that constitutively active Notch 3 IC increased bcl-xL expression while concomitantly inhibiting bax expression, an effect that was fully reversed after inhibition of CBF-1/RBP-J with expression of RPMS-1. These data suggest that bax and bcl-xL are downstream targets of CBF-1/RBP-JeCdependent Notch signaling in these cells. This is further reinforced by studies that demonstrate constitutively active Notch 1 modulates expression of bcl-xL.43 Moreover, we have shown using specific siRNA targeted against an individual Notch target gene (Hrt-2) selective hrt-2 knockdown resulted in a significant increase in bax and a decrease in bcl-xL expression in SMCs, further confirming the importance of Notch IC regulation of Bcl-2 family of proteins in these cells. At present, it is unclear why bax mRNA levels are enhanced in strained cells after constitutive Notch activation despite being significantly inhibited in unstrained cells. Additional factors affected by cyclic strain may act to regulate and potentiate bax expression in strained cells. This possibility is further reinforced because bax expression is significantly enhanced in strained cells irrespective of whether Notch IC is overexpressed or inhibited (after RPMS-1 expression).
Because cyclic strain can induce a highly restricted transcriptional response in vascular SMCs that regulates vascular structure,2,5 we hypothesized that Notch could represent a novel pathway for contributing to strain-induced changes in vascular fate. The current study demonstrates a functional role for cyclic straineCinduced decreases in Notch-mediated CBF-1/RBP-JeCdependent signaling and subsequent changes in vascular cell fate. The level of inhibition of Notch signaling after exposure to cyclic strain suggests that Notch is a major target for biomechanical regulation of SMCs in vitro. Indeed, previous in vivo studies concur by demonstrating an acute decrease in Notch receptor and Notch target gene expression after balloon injury and subsequent cellular distension, with the most dramatic decrease evident for the Notch 3 receptor and the hrt-1 target gene.15 This response to strain appears to be temporal.15,16 Platelet-derived growth factor and angiotensin II also induced a coordinate downregulation of Notch signaling in SMCs through an extracellular signal-regulated kinaseeCdependent pathway.15,16,19 In the current study, the decrease in the expression of Notch components is consistent with the observed changes in SMC fate after strain. Moreover, selective inhibition of Gi-proteins and MAPK, respectively, which are known signaling pathways involved in mechanotransduction in vascular cells,22,23,24 despite decreasing baseline Notch signaling, blocked the strain-induced changes in Notch and corresponding changes in SMC fate. To our knowledge, this is the first demonstration that the expression of Notch receptors and downstream target genes are directly modulated by strain in mammalian cells in vitro. This finding further suggests a possible nexus by which the activation of a biomechanical signaling pathway is coupled to the Notch cellular fate program. Additional studies will be required to delineate the mechanism(s) by which cyclic strain regulates the expression of Notch in vitro.
The regulation of Notch signaling is known to occur at multiple levels, including patterns of ligand and receptor expression, Notch-ligand interactions, trafficking of the receptor and ligands, and covalent modifications, including glycosylation, phosphorylation, and ubiquitination.10,11,44,45 A possible mechanistic explanation for the effect of cyclic strain on Notch 3 IC activity may be post-translational modifications, such as phosphorylation, that may influence its transactivation capacity.45,46 Indeed, glycogen synthase kinase-3 (GSK3) is known to modulate Notch signaling through phosphorylation of Notch IC,45eC47 such that inhibition of GSK3 shortened the half life of Notch1 IC, whereas, conversely, activated GSK3 reduced the quantity of Notch IC that was degraded by the proteasome.46 Moreover, the regulatory phosphorylation of GSK3 and hence its activity is under the control of MAPK-dependent signaling pathways.48 Therefore, it is tempting to speculate that cyclic strain modulates this process, thereby influencing the translocation of Notch IC into the nucleus. However, because capacity of Notch 3 IC to transactivate various CBF-1/RBP-JeCdependent promoters is inhibited by cyclic strain and because strain does not affect constitutively active Notch 3 IC expression, these data collectively suggest that other strain-induced events are critical to the ability of Notch IC (whether endogenous or vector encoded) to transactivate CBF-1/RBP-J target promoters. Indeed, it is known that Notch IC interacts with coactivating proteins, including histone acetyltransferase proteins, which play a role in chromatin opening and initiation of transcription.49,50 Moreover, strain may inhibit endogenous inhibitors of Notch IC, such as Numb, that result in a loss of Notch IC functionality after strain. However, because Notch 3 mRNA levels and the expression of full-length Notch 3 receptors are also decreased after strain, it is clear that cyclic strain inhibits the expression of the Notch 3 receptor in addition to any post-translation modifications that may occur to shorten the half life of Notch IC.
In conclusion, we have shown for the first time that equibiaxial cyclic strain induces apoptosis and inhibits proliferation of rat SMC in vitro. Cyclic straineCinduced changes in fate are attributable, at least in part, to a cyclic straineCinduced decrease in expression and activity of Notch signaling pathway components. These studies provide further evidence that Notch receptors acting through CBF-1/RBP-JeCdependent signaling pathways are an important determinant of SMC fate in vitro.
Acknowledgments
This research was supported by grants from the Higher Education Authority (HEA) of Ireland, Wellcome Trust, Science Foundation Ireland, and the Health Research Board of Ireland (to P.A.C.) and by grants from the National Institutes of Health (HL59696 and AA-12610; to P.A.C. and E.M.R.).
The first 2 authors contributed equally to this work.
References
Schwartz RS, Henry TD. Pathophysiology of coronary artery restenosis. Rev Cardiovasc Med. 2002; 3: (suppl 5): S4eCS9.
Kleinstreuer C, Hyun S, Buchanan JR Jr, Longest PW, Archie JP Jr, Truskey GA. Hemodynamic parameters and early intimal thickening in branching blood vessels. Crit Rev Biomed Eng. 2001; 29: 1eC64.
Ferguson JE III, Patterson C. Break the cycle: the role of cell-cycle modulation in the prevention of vasculoproliferative diseases. Cell Cycle. 2003; 2: 211eC219.
Manabe I, Nagai R. Regulation of smooth muscle phenotype. Curr Atheroscler Rep. 2003; 5: 214eC222.
Osol G. Mechanotransduction by vascular smooth muscle. J Vasc Res. 1995; 32: 275eC292.
Lehoux S, Tedgui A. Cellular mechanics and gene expression in blood vessels. J Biomech. 2003; 36: 631eC643.
Koller A. Signaling pathways of mechanotransduction in arteriolar endothelium and smooth muscle cells in hypertension. Microcirculation. 2002; 9: 277eC294.
Brooks AR, Lelkes PI, Rubanyi GM. Gene expression profiling of vascular endothelial cells exposed to fluid mechanical forces: relevance for focal susceptibility to atherosclerosis. Endothelium. 2004; 11: 45eC57.
Majesky MW. Vascular smooth muscle diversity: insights from developmental biology. Curr Atheroscler Rep. 2003; 5: 208eC213.
Weinstein BM, Lawson ND. Arteries, veins, Notch, and VEGF. Cold Spring Harb Symp Quant Biol. 2002; 67: 155eC162.
Iso T, Hamamori Y, Kedes L. Notch signaling in vascular development. Arterioscler Thromb Vasc Biol. 2003; 23: 543eC553.
Lai EC. Keeping a good pathway down: transcriptional repression of Notch pathway target genes by CSL proteins. EMBO Rep. 2002; 3: 840eC845.
Harper JA, Yuan JS, Tan JB, Visan I, Guidos CJ. Notch signaling in development and disease. Clin Genet. 2003; 64: 461eC472.
Iso T, Kedes L, Hamamori Y. HES and HERP families: multiple effectors of the Notch signaling pathway. J Cell Physiol. 2003; 194: 237eC255.
Wang W, Campos AH, Prince CZ, Mou Y, Pollman MJ. Coordinate Notch 3-hairy-related transcription factor pathway regulation in response to arterial injury. Mediator role of platelet-derived growth factor and ERK. J Biol Chem. 2002; 277: 23165eC23171.
Campos AH, Wang W, Pollman MJ, Gibbons GH. Determinants of Notch-3 receptor expression and signaling in vascular smooth muscle cells: implications in cell-cycle regulation. Circ Res. 2002; 91: 999eC1006.
Wang W, Prince CZ, Hu X, Pollman MJ. HRT1 modulates vascular smooth muscle cell proliferation and apoptosis. Biochem Biophys Res Commun. 2003; 308: 596eC601.
Sweeney C, Morrow D, Birney YA, Coyle S, Hennessy C, Scheller A, Cummins PM, Walls D, Redmond EM, Cahill PA. Notch 1 and 3 receptor signaling modulates vascular smooth muscle cell growth, apoptosis, and migration via a CBF-1/RBP-J-dependent pathway. FASEB J. 2004; 18: 1421eC1423
Wang W, Prince CZ, Mou Y, Pollman MJ. Notch3 signaling in vascular smooth muscle cells induces c-FLIP expression via ERK/MAPK activation. Resistance to Fas ligand-induced apoptosis. J Biol Chem. 2002; 277: 21723eC21729.
Xu Q. Biomechanical-stress-induced signaling and gene expression in the development of arteriosclerosis. Trends Cardiovasc Med. 2000; 10: 35eC41.
Lindner V, Booth C, Prudovsky I, Small D, Maciag T, Liaw L. Members of the Jagged/Notch gene families are expressed in injured arteries and regulate cell phenotype via alterations in cell matrix and cell-cell interaction. Am J Pathol. 2001; 159: 875eC883.
Von Offenberg Sweeney N, Cummins PM, Birney YA, Redmond EM, Cahill PA. Cyclic strain-induced endothelial MMP-2: role in vascular smooth muscle cell migration. Biochem Biophys Res Commun. 2004; 320: 325eC333.
Cullen JP, Sayeed S, Sawai RS, Theodorakis NG, Cahill PA, Sitzmann JV, Redmond EM. Pulsatile flow-induced angiogenesis: role of G(i) subunits. Arterioscler Thromb Vasc Biol. 2002; 22: 1610eC1616.
Shaw A, Xu Q. Biomechanical stress-induced signaling in smooth muscle cells: an update. Curr Vasc Pharmacol. 2003: 41eC58.
Schulze PC, de Keulenaer GW, Kassik KA, Takahashi T, Chen Z, Simon DI, Lee RT. Biomechanically induced gene iex-1 inhibits vascular smooth muscle cell proliferation and neointima formation. Circ Res. 2003; 93: 1210eC1217.
Sedding DG, Seay U, Fink L, Heil M, Kummer W, Tillmanns H, Braun-Dullaeus RC. Mechanosensitive p27Kip1 regulation and cell cycle entry in vascular smooth muscle cells. Circulation. 2003 108: 616eC622.
Hipper A, Isenberg G. Cyclic mechanical strain decreases the DNA synthesis of vascular smooth muscle cells. Pfle筭ers Arch. 2000; 440: 19eC27.
Birukov KG, Shirinsky VP, Stepanova OV, Tkachuk VA, Hahn AW, Resink TJ, Smirnov VN. Stretch affects phenotype and proliferation of vascular smooth muscle cells. Mol Cell Biochem. 1995; 144: 131eC139.
Wilson E, Mai Q, Sudhir K, Weiss RH, Ives HE. Mechanical strain induces growth of vascular smooth muscle cells via autocrine action of PDGF. J Cell Biol. 1993; 23: 741eC747.
Sumpio BE, Banes AJ. Response of porcine aortic smooth muscle cells to cyclic tensional deformation in culture. J Surg Res. 1988; 44: 696eC701.
Cappadona C, Redmond EM, Theodorakis NG, McKillop IH, Hendrickson R, Chhabra A, Sitzmann JV, Cahill PA. Phenotype dictates the growth response of vascular smooth muscle cells to pulse pressure in vitro. Exp Cell Res. 1999; 250: 174eC186.
Kakisis JD, Liapis CD, Sumpio BE. Effects of cyclic strain on vascular cells. Endothelium. 2004; 11: 17eC28.
Mayr M, Hu Y, Hainaut H, Xu Q. Mechanical stress-induced DNA damage and rac-p38MAPK signal pathways mediate p53-dependent apoptosis in vascular smooth muscle cells. FASEB J. 2002; 16: 1423eC1425.
Mayr M, Li C, Zou Y, Huemer U, Hu Y, Xu Q. Biomechanical stress-induced apoptosis in vein grafts involves p38 mitogen-activated protein kinases. FASEB J. 2000; 14: 261eC270.
Wernig F, Xu Q. Mechanical stress-induced apoptosis in the cardiovascular system. Prog Biophys Mol Biol. 2002; 78: 105eC137.
Nhan TQ, Liles WC, Chait A, Fallon JT, Schwartz SM. The p17 cleaved form of caspase-3 is present within viable macrophages in vitro and in atherosclerotic plaque. Arterioscler Thromb Vasc Biol. 2003; 23: 1276eC1282.
Assuncao Guimaraes C, Linden R. Programmed cell deaths. Apoptosis and alternative death styles. Eur J Biochem. 2004; 271: 1638eC1650.
Kim HS, Hwang KK, Seo JW, Kim SY, Oh BH, Lee MM, Park YB. Apoptosis and regulation of Bax and Bcl-X proteins during human neonatal vascular remodeling. Arterioscler Thromb Vasc Biol. 2000; 20: 957eC963.
Pollman MJ, Hall JL, Mann MJ, Zhang L, Gibbons GH. Inhibition of neointimal cell bcl-x expression induces apoptosis and regression of vascular disease. Nat Med. 1998; 4: 222eC227.
Pollman MJ, Hall JL, Gibbons GH. Determinants of vascular smooth muscle cell apoptosis after balloon angioplasty injury. Influence of redox state and cell phenotype. Circ Res. 1999; 84: 113eC121.
Mayr U, Mayr M, Li C, Wernig F, Dietrich H, Hu Y, Xu Q. Loss of p53 accelerates neointimal lesions of vein bypass grafts in mice. Circ Res. 2002; 90: 197eC204.
Birney YB, Sweeney CH, Cappadona CR, Sitzmann JVS, Cummins PM, Redmond EM Cahill PA. Pulse pressure induced transmural fluid flux increases bovine aortic smooth muscle cell apoptosis in a mitogen activated protein kinase-dependent manner. J Vasc Res. 2004; 41: 364eC374.
Jang MS, Miao H, Carlesso N, Shelly L, Zlobin A, Darack N, Qin JZ, Nickoloff BJ, Miele L. Notch-1 regulates cell death independently of differentiation in murine erythroleukemia cells through multiple apoptosis and cell cycle pathways. J Cell Physiol. 2004; 199: 418eC433.
Baron M, Aslam H, Flasza M, Fostier M, Higgs JE, Mazaleyrat SL, Wilkin MB. Multiple levels of Notch signal regulation. Mol Membr Biol. 2002; 19: 27eC38.
Nie L, Xu M, Vladimirova A, Sun XH. Notch-induced E2A ubiquitination and degradation are controlled by MAP kinase activities. EMBO J. 2003; 22: 5780eC5792.
Foltz DR, Santiago MC, Berechid BE, Nye JS. Glycogen synthase kinase-3beta modulates notch signaling and stability. Curr Biol. 2002; 12: 1006eC1011.
Espinosa L, Ingles-Esteve J, Aguilera C, Bigas A. Phosphorylation by glycogen synthase kinase-3 beta down-regulates Notch activity, a link for Notch and Wnt pathways. J Biol Chem. 2003; 278: 32227eC32235.
Liu F, Schaphorst KL, Verin AD, Jacobs K, Birukova A, Day RM, Bogatcheva N, Bottaro DP, Garcia JG. Hepatocyte growth factor enhances endothelial cell barrier function and cortical cytoskeletal rearrangement: potential role of glycogen synthase kinase-3beta. FASEB J. 2002; 16: 950eC962.
Utley RT, Cote J. The MYST family of histone acetyltransferases. Curr Top Microbiol Immunol. 2003; 274: 203eC236.
Kurooka H, Honjo T. Functional interaction between the mouse notch1 intracellular region and histone acetyltransferases PCAF and GCN5. J Biol Chem. 2000; 275: 17211eC17220.(David Morrow, Catherine S)