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• W2012047843 abstract "CCN1 is an angiogenic factor that promotes cell adhesion, proliferation, and differentiation. CCN1-deficient mice suffer embryonic death because of vascular defects, demonstrating that CCN1 is required for vessel development. Because mechanical stretch may act as a trigger for vessel development, we investigated the impact of mechanical stretch on the regulatory mechanism of CCN1 expression. Mechanical stretch rapidly enhances CCN1 expression and release in vascular smooth muscle cells (VSMC) in vitro and CCN1 expression in murine aortic segments in vivo. Transfection experiments of VSMC with deletion constructs of the CCN1 promoter revealed the regulatory region responsible for the stretch-induced CCN1 expression in the ∼200-bp promoter region upstream of the TATA-box containing potential binding sites for early growth response-1 (Egr-1), nuclear factor of activated T-cells and cAMP response element binding protein. Decoy oligonucleotides to Egr-1, but not to nuclear factor of activated T-cells or cAMP response element binding protein, abolished the stretch-induced transcription of CCN1. In addition, mutagenesis of the Egr-1 binding site within the CCN1 promoter completely blunted the stretch-induced activation of the promoter. Furthermore, mechanical stretch induced the expression and DNA-binding activity of Egr-1 in VSMC as demonstrated by Western blot and electromobility shift assay. Moreover, a pressure overload-dependent de novo synthesis of Egr-1 was observed after aortic banding. These findings indicate that mechanical stretch leads to enhanced expression of CCN1 via the mechanosensitive transcription factor Egr-1, suggesting a central role for mechanical stretch in the regulation of CCN1-dependent pro-angiogenic potency. CCN1 is an angiogenic factor that promotes cell adhesion, proliferation, and differentiation. CCN1-deficient mice suffer embryonic death because of vascular defects, demonstrating that CCN1 is required for vessel development. Because mechanical stretch may act as a trigger for vessel development, we investigated the impact of mechanical stretch on the regulatory mechanism of CCN1 expression. Mechanical stretch rapidly enhances CCN1 expression and release in vascular smooth muscle cells (VSMC) in vitro and CCN1 expression in murine aortic segments in vivo. Transfection experiments of VSMC with deletion constructs of the CCN1 promoter revealed the regulatory region responsible for the stretch-induced CCN1 expression in the ∼200-bp promoter region upstream of the TATA-box containing potential binding sites for early growth response-1 (Egr-1), nuclear factor of activated T-cells and cAMP response element binding protein. Decoy oligonucleotides to Egr-1, but not to nuclear factor of activated T-cells or cAMP response element binding protein, abolished the stretch-induced transcription of CCN1. In addition, mutagenesis of the Egr-1 binding site within the CCN1 promoter completely blunted the stretch-induced activation of the promoter. Furthermore, mechanical stretch induced the expression and DNA-binding activity of Egr-1 in VSMC as demonstrated by Western blot and electromobility shift assay. Moreover, a pressure overload-dependent de novo synthesis of Egr-1 was observed after aortic banding. These findings indicate that mechanical stretch leads to enhanced expression of CCN1 via the mechanosensitive transcription factor Egr-1, suggesting a central role for mechanical stretch in the regulation of CCN1-dependent pro-angiogenic potency. CCN1 (formerly known as CYR61) is a cysteine-rich heparin-binding protein that is encoded by an immediate early gene and belongs to the novel CCN gene family (connective tissue growth factor (CTGF); cysteine-rich angiogenic protein 61 (CYR61); nephroblastoma overexpressed (Nov) (1Lau L.F. Lam S.C. Exp. Cell Res. 1999; 248: 44-57Crossref PubMed Scopus (580) Google Scholar). The expression of CCN1 is rapidly and transiently induced in response to growth factors (2O'Brien T.P. Yang G.P. Sanders L. Lau L.F. Mol. Cell. Biol. 1990; 10: 3569-3577Crossref PubMed Scopus (271) Google Scholar). CCN1 is expressed by all types of vascular cells and is associated with the extracellular matrix (ECM). 1The abbreviations used are: ECM, extracellular matrix; VSMC, vascular smooth muscle cell; TAC, transverse aortic constriction; RT, reverse transcriptase; ODN, oligonucleotide; NFAT, nuclear factor of activated T cells; CREB, cAMP response element-binding protein; SMC, smooth muscle cell; Egr-1, early growth response-1.1The abbreviations used are: ECM, extracellular matrix; VSMC, vascular smooth muscle cell; TAC, transverse aortic constriction; RT, reverse transcriptase; ODN, oligonucleotide; NFAT, nuclear factor of activated T cells; CREB, cAMP response element-binding protein; SMC, smooth muscle cell; Egr-1, early growth response-1. CCN1 mediates cell adhesion, migration, proliferation, and neovascularization through cell-type specific binding to different integrins (1Lau L.F. Lam S.C. Exp. Cell Res. 1999; 248: 44-57Crossref PubMed Scopus (580) Google Scholar). It is noteworthy that CCN1-deficient mice suffer embryonic death because of placental vascular insufficiency and compromised vessel integrity. The knockout is further characterized by a specific defect in vessel bifurcation development and an impaired expression of vascular endothelial growth factor-C (3Mo F.E. Muntean A.G. Chen C.C. Stolz D.B. Watkins S.C. Lau L.F. Mol. Cell. Biol. 2002; 22: 8709-8720Crossref PubMed Scopus (342) Google Scholar). These observations point to CCN1 as a novel and essential regulator of vessel development. In addition, recombinant CCN1 protein was shown to induce genes involved in angiogenesis, inflammation, ECM remodeling, and cell-matrix interactions (4Chen C.C. Mo F.E. Lau L.F. J. Biol. Chem. 2001; 276: 47329-47337Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). Besides its ability to promote tumor growth, enhanced CCN1 expression has been associated with pathophysiological settings in atherosclerotic plaques and in neointima formation after vascular injury by balloon angioplasty (5Babic A.M. Kireeva M.L. Kolesnikova T.V. Lau L.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6355-6360Crossref PubMed Scopus (431) Google Scholar, 6Hilfiker A. Hilfiker-Kleiner D. Fuchs M. Kaminski K. Lichtenberg A. Rothkotter H.J. Schieffer B. Drexler H. Circulation. 2002; 106: 254-260Crossref PubMed Scopus (89) Google Scholar, 7Wu K.J. Yee A. Zhu N.L. Gordon E.M. Hall F.L. Int. J. Mol. Med. 2000; 6: 433-440Crossref PubMed Google Scholar, 8Grzeszkiewicz T.M. Lindner V. Chen N. Lam S.C. Lau L.F. Endocrinology. 2002; 143: 1441-1450Crossref PubMed Scopus (133) Google Scholar). A common feature of these conditions is an augmented mechanical stress within the vessel wall, primarily sensed by vascular smooth muscle cells (VSMC) as one of the main sources of CCN1 synthesis. In this regard, maladaptive remodeling of the vessel wall is a characteristic feature of vascular diseases such as arterial hypertension or atherosclerosis and seems to involve vasoactive peptides (e.g. angiotensin II) (9Naftilan A.J. Curr. Opin. Nephrol. Hypertens. 1994; 3: 218-227Crossref PubMed Scopus (26) Google Scholar), and mechanical stretch (10Dzau V.J. Curr. Opin. Nephrol. Hypertens. 1993; 2: 27-32Crossref PubMed Scopus (104) Google Scholar). However, little is known about the mechanism by which mechanical stretch is linked to angiogenesis. Recent observations by our group that pointed to mechanical stretch as a critical regulator of vessel remodeling (11Grote K. Flach I. Luchtefeld M. Akin E. Holland S.M. Drexler H. Schieffer B. Circ. Res. 2003; 92: E80-E86Crossref PubMed Google Scholar) led us to investigate the regulation of the CCN1 expression by mechanical stretch as a possible trigger mechanism for pro-angiogenic effects. To analyze expression, release, and transcriptional regulation of CCN1, mechanical stretch was applied to cultured VSMC in vitro by using the Flexercell Strain Unit. The role of increased vascular stretch on CCN1 expression in vivo was achieved by inducing pressure overload by transverse aortic constriction (TAC) in mice. Cell Culture—Murine aortic VSMC were isolated from C57BL/6 mice by enzymatic dispersion as described previously (12Blank R.S. Thompson M.M. Owens G.K. J. Cell Biol. 1988; 107: 299-306Crossref PubMed Scopus (83) Google Scholar). Cells were maintained in Dulbecco's modified Eagle's medium (Biochrom) supplemented with 10% fetal calf serum (Invitrogen), 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). Cells were growth-arrested in Dulbecco's modified Eagle's medium containing 0.5% fetal calf serum for 48 h before use, experiments were performed under serum-free conditions. Mechanical Stretch—VSMC were plated on 6-well silicone elastomer plates coated with collagen type I (Bioflex; Flexcell, Hillsborough, NC). The cells were exposed to continuous cycles of stretch and relaxation (0.5 Hz) by use of the Flexercell Strain Unit FX-3000 (Flexcell) for the indicated times; a maximum of 15% radial stretch of the membrane was applied. Transverse Aortic Constriction (TAC)—Twelve-week-old male C57BL/6 mice were sedated with propofol (10 mg/kg, i.v.), anesthetized with 3% isoflurane, and connected to a rodent ventilator (Harvard, Holliston, MA). Transverse aortic constriction was performed between the left and right carotid arteries. Expression levels in pressure overload segments proximal to the site of constriction were compared with expression levels in distal control segments. Arterial blood pressure recordings were obtained using a 1.4 farad micromanometer conductance catheter (Millar Instruments, Houston, TX) inserted via the right carotid artery as described previously (13Fuchs M. Hilfiker A. Kaminski K. Hilfiker-Kleiner D. Guener Z. Klein G. Podewski E. Schieffer B. Rose-John S. Drexler H. FASEB J. 2003; 17: 2118-2120Crossref PubMed Google Scholar). TAC-operated mice exhibited a significant increase in left ventricular systolic pressure (167 ± 23 mm Hg) versus sham-operated mice (123 ± 7 mm Hg). Semiquantitative RT-PCR—Total RNA from VSMC exposed to mechanical stretch was isolated using TriFast-Reagent (peqLAB, Erlangen, Germany). Total RNA was reverse transcribed using Superscript reverse transcriptase (RT) (Invitrogen), oligo(dT) primers and deoxynucleoside triphosphates. The RT products were amplified using Taq DNA polymerase (Invitrogen). PCR for CCN1 was carried out for 26 cycles (forward primer, 5′-CAA CCC TGC GAC CAC AC-3′; reverse primer, 5′-TGC CCT TTT TTA GGC TGC-3′, 643 bp) and normalized to the expression of the 18 S rRNA (18 cycles, forward primer, 5′-CCT GCG GCT TAA TTT GAC TC-3′; reverse primer, 5′-GGC CTC ACT AAA CCA TCC AA-3′, 510 bp) using oligonucleotides obtained from MWGBiotech (Ebersberg, Germany). PCR products were separated by 1% agarose gel electrophoresis and quantified densitometrically using a Gel Doc image analysis system (Bio-Rad). Western Blotting—Protein extracts (30 μg) were separated by denaturing SDS (10%) PAGE and then transferred to polyvinylidene difluoride membrane (Amersham Biosciences). Transferred proteins were probed with a goat polyclonal anti-CCN1 antibody (1:1000) or with a rabbit polyclonal anti-Egr-1 antibody (1:5000) (Santa Cruz Biotechnology, Santa Cruz, CA) and visualized using a horseradish peroxidase conjugated secondary anti-goat (1:3000; Dianova, Hamburg, Germany) or an anti-rabbit (1:3000; Amersham Biosciences) antibody and ECL solution. Equal protein loading was verified by reprobing the membrane with a mouse monoclonal anti-α-smooth muscle cell actin antibody (α-SMC actin; Sigma-Aldrich). Immunohistochemistry—Serial cryostat sections were used for immunohistochemical analysis. After blocking endogenous peroxidase activity with 3% H2O2/phosphate-buffered saline, sections were incubated with 4% blocking serum (Vector Laboratories, Burlingame, CA). Sections were stained with a goat polyclonal CCN1 antibody (1:200; Santa Cruz Biotechnology) or a mouse monoclonal anti-α-smooth muscle cell actin antibody (1:1000; Sigma-Aldrich). Incubation with primary antibody was followed by application of the biotin conjugated second antibody (1:200), containing 4% species-appropiate normal serum and the streptavidin-biotin-peroxidase-complex (Vector Laboratories). The final reaction was visualized with either diaminobenzidine or 3-amino-9-ethylcarbazole (DakoCytomation, Hamburg, Germany), followed by counterstaining with hematoxylin (Sigma-Aldrich). Promoter Luciferase Assay—A 2.0-kb fragment of the mouse CCN1 gene promoter (full length, -2062 to +1; GenBank accession number X56790) was cloned into the luciferase reporter vector pGL3-basic (Invitrogen) at the KpnI/NheI-site by PCR using Pfu polymerase (Promega). Serial 5′-deletion constructs of the CCN1 promoter at position -1561 (del1), -1032 (del2), -512 (del3), -262 (del4), and -32 (TATABox) were also produced by PCR and restriction enzyme digestions. Two different constructs with point-mutations in the Egr-1 binding site of the full-length CCN1 promoter fused to the luciferase reporter vector pGL3-basic were produced using the site-directed mutagenesis kit (Stratagene, Heidelberg, Germany) and the specific primers Egr-1 (mut1) (sense, 5′-CGT CAC TGC AAC ACG CTA CGC CTA GGC AGG C-3′) and Egr-1 (mut2) (sense, 5′-CGT CAC TGC AAC ACG CGG ATC CTA GGC AGG C-3′) (underlined sequence denote the CCN1 promoter-specific Egr-1 binding site; substituted nucleotides are bold). VSMC were plated on 6-well Bioflex plates and transiently transfected with 2 μg/well of the different CCN1 promoter/pGL3 constructs using Lipofectamine (Invitrogen) according to the manufacturer's protocol. VSMC were subjected to stretch regimes for 24 h. Firefly luciferase activity was normalized to whole protein content, quantified and expressed as relative luciferase light units using a luminometer (Lumat; Berthold, Bad Wildbad, Germany). Potential responsive elements within the CCN1 promoter were determined using the TRANSFAC data base (14Wingender E. Chen X. Hehl R. Karas H. Liebich I. Matys V. Meinhardt T. Pruss M. Reuter I. Schacherer F. Nucleic Acids Res. 2000; 28: 316-319Crossref PubMed Scopus (1025) Google Scholar). Electrophoretic Mobility Shift Assay—Nuclear extracts from cultured VSMC exposed to mechanical stretch were isolated, and nondenaturing 4% acrylamide containing PAGE was performed as described previously (15Bavendiek U. Libby P. Kilbride M. Reynolds R. Mackman N. Schon-beck U. J. Biol. Chem. 2002; 277: 25032-25039Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Double-stranded gel shift oligonucleotides for Egr-1 (sense, 5′-CCC GGC GCG GGG GCG ATT TCG AGT CA-3′; underlined sequence denotes the Egr-1 consensus binding site) were end-labeled with [γ-32P]ATP using T4 kinase and incubated with nuclear extracts (10 μg) for 30 min at ambient temperature. The specificity of the Egr-1 gel-shift oligonucleotide was tested by using a 50-fold excess of the unlabeled competitive oligonucleotide and a labeled unspecific noncompetitive hypoxia-inducible factor-1α oligonucleotide and by supershift analyses using an anti-Egr-1 antibody (Santa Cruz Biotechnology). Decoy ODN Technique—Double-stranded decoy oligonucleotides (ODN) containing the conserved promoter-binding site of NFAT, CREB, Egr-1, and a scrambled sequence were prepared from complementary single-stranded phosphorothioate-bonded ODN obtained from EUROGENTEC (Köln, Germany) by melting at 95 °C for 5 min, followed by a cool-down phase over night. The efficiency of the hybridization reaction was controlled in 2.5% agarose gels and was almost 100%. Decoy ODN were added in 2.5 μm concentration to the cell culture supernatant and incubated for 4 h. The single-stranded sequences of the decoy ODN were as follows (* denotes the phosphorothioate bonds; underlined sequences denote the transcription factor binding sites): NFAT (consensus): sense, 5′-T*, C*, T*, AAA GTG GAG GAA AAT TTG GAA*, C*, T*, C-3′; CREB (consensus): sense, 5′-A*, G*, A*, GAT TGC CTG ACG TCA GAG AGC*, T*, A*, G-3′; Egr-1 (consensus): sense, 5′-C*, C*, C*, GGC GCG GGG GCG ATT TCG AG*, T*, C*, A-3′; Egr-1 (CCN1-specific), 5′-G*, C*, A*, ACA CGC GGC GCC TAG GCA GG*, C*, A*, T-3′; and scrambled: sense, 5′-A*, A*, C*, AGA AGC CAG GAA CCC TCC*, T*, C*, T-3′. Statistical Analysis—All data are presented as mean ± S.E. of at least four independent experiments. Differences were evaluated by analysis of variance. Statistical significance was defined as p < 0.05. Mechanical Stretch Enhances CCN1 Expression and Release by Vascular Smooth Muscle Cells in Vitro—Mechanical stretch rapidly enhanced CCN1 mRNA expression in cultured VSMC with a peak at 0.5 h (1.9 ± 0.4-fold) and 1 h (2.1 ± 0.5-fold) declining after 24 h of mechanical stretch (Fig. 1A). Mechanical stretch of VSMC enhanced CCN1 protein expression after the CCN1 mRNA expression with a delayed kinetic. CCN1 protein expression in cell lysates was transiently increased at 1 h (2.2 ± 0.2-fold) and 3 h (2.1 ± 0.3-fold), and reached expression levels of the unstretched control thereafter (Fig. 1B). It is noteworthy that mechanical stretch induced the release of CCN1 protein into the supernatant of cultured VSMC (∼10 ± 4-fold at 1 and 3 h; ∼20 ± 7-fold at 6 and 24 h) (Fig. 1C). Pressure Overload Enhances CCN1 Expression in Vivo—CCN1 mRNA expression was found to be up-regulated in aortic segments exposed to pressure overload after aortic banding (overload) at 0.5 h (2.4 ± 0.5-fold) and at 1 h (1.9 ± 0.3-fold) compared with control segments (Fig. 2A). Mechanical stretch in vivo also resulted in an increased expression of the CCN1 protein. OCT-embedded aortic tissue rings of pressure overload segments revealed an enhanced CCN1 staining 6 h after TAC in the medial layer of the aorta verified by staining for α-SMC actin compared with control segments. Unspecific IgG showed no staining in these sections (Fig. 2B). Enhanced CCN1 staining was already observed 3 h after TAC and still enhanced after 24 h. Sham operation did not enhance CCN1 protein expression (data not shown). Mechanical Stretch Up-regulates CCN1 Promoter Activity by the Transcription Factor Egr-1—According to TRANSFAC analysis (14Wingender E. Chen X. Hehl R. Karas H. Liebich I. Matys V. Meinhardt T. Pruss M. Reuter I. Schacherer F. Nucleic Acids Res. 2000; 28: 316-319Crossref PubMed Scopus (1025) Google Scholar), the promoter of the mouse CCN1 gene (GenBank accession number X56790) contains several putative binding sites for transcription factors (e.g. activator protein-1, NFAT, signal transducer and activator of transcription, Egr-1, etc.) (Fig. 3A). To investigate the transcriptional regulation of the CCN1 gene by mechanical stretch in VSMC, the full-length CCN1 promoter was cloned into the promoterless luciferase reporter vector pGL3-basic. To identify promoter regions sensitive to mechanical stretch, we performed transfection studies employing 5′-deletion constructs of the CCN1 promoter (del1, del2, del3, and del4). Cultured VSMC transfected with the different CCN1 promoter constructs were subsequently stretched and assayed for luciferase activity. All reporter constructs employed resulted in a strong stretch-induced luciferase activity (9-10-fold) compared with unstretched VSMC (Fig. 3B). No loss of luciferase activity was observed employing 5′-deletion constructs deleted up to bp -262 of the CCN1 promoter compared with the full-length construct. In contrast, the use of a construct containing the 30 bp of the CCN1 promoter including the TATA-box (bp -32 to +1) led to a complete loss of luciferase activity, indicating that the regulatory elements responsible for the induction of the CCN1 promoter by mechanical stretch are located in the promoter region downstream of position -262 (= del4). Putative binding sites for transcription factors in the remaining region are NFAT (bp -212 to -201), CREB (bp -63 to -56), and Egr-1 (bp -48 to -41). Using neutralizing consensus decoy ODN against these transcription factors revealed that only the decoy ODN against Egr-1 abolished the up-regulation of CCN1 mRNA by mechanical stretch (1.3 ± 0.2-fold versus 2.8 ± 0.3), which is shown in Fig. 3C. Likewise, a decoy ODN against the CCN-1 promoter-specific Egr-1 binding site abolished stretch-induced CCN1 mRNA expression (1.25 ± 0.2-fold versus 2.8 ± 0.3-fold), demonstrating the specific binding of Egr-1 to the CCN1 promoter after mechanical stretch. NFAT, CREB, and scrambled ODN had no inhibitory effect on CCN1 mRNA expression (Fig. 3C). In addition, site-directed mutagenesis of the Egr-1 binding site in the CCN1 promoter was performed to further demonstrate the impact of Egr-1 on the stretch-induced CCN1 expression. Two different substitutions in the Egr-1 binding site were generated based on the full-length CCN1 promoter fused to the luciferase reporter gene (Egr-1/mut1, GG→ TA; Egr-1/mut2, CG→ AT). Cultured VSMC transfected with the different CCN1 promoter constructs were subsequently stretched and assayed for luciferase activity. As expected, the full-length reporter constructs resulted in a strong stretch-induced luciferase activity (9.5-fold) compared with unstretched VSMC, whereas both Egr-1/mut1 and Egr-1/mut2 reporter constructs showed completely abrogated stretch-induced luciferase activity (Fig. 3D). Mechanical Stretch Enhances Egr-1 Activation in Vitro and in Vivo—To confirm the role of Egr-1 in the stretch-dependent regulation of the CCN1 gene promoter, Egr-1 de novo synthesis by mechanical stretch was investigated by Western blotting. Mechanical stretch of VSMC enhanced Egr-1 protein expression at 0.5 h (1.9 ± 0.3-fold), peaked at 1 h (2.5 ± 0.5-fold), and declined thereafter (Fig. 4A). In addition, Egr-1 translocation and DNA-binding after mechanical stretch was investigated in gel shift experiments. Mechanical stretch-induced Egr-1 activation in VSMC was already detectable at 15 min (7-fold) and peaked at 30 min (10-fold) (Fig. 4B). The DNA/protein complex was displaced by a 50-fold molar excess of unlabeled competitive Egr-1 oligonucleotides, not influenced by a 50-fold molar excess of labeled unspecific noncompetitive hypoxia-inducible factor-1α oligonucleotides, and supershifted in the presence of an anti-Egr-1 antibody, showing the specificity of the detected complex. In addition, pressure overload-induced mechanical stretch in vivo increased the de novo-synthesis of Egr-1 protein. Therefore, Egr-1 protein expression was investigated after TAC by Western blotting and was found to be up-regulated in the aortic segments exposed to pressure overload peaks at 30 min (2.7 ± 0.6-fold) compared with control segments (Fig. 4C). Sham-operation did not enhance Egr-1 protein expression (data not shown). The present study demonstrates that mechanical stretch enhances the expression and release of the angiogenic factor CCN1 from smooth muscle cells in vitro. Moreover, we demonstrated in this study that CCN1 expression in vivo is induced in vascular segments exposed to pressure overload and that the induction of CCN1 expression is mediated by the mechanosensitive activation of the transcription factor Egr-1. Maladaptive remodeling of the vessel wall is a characteristic feature of vascular diseases such as arterial hypertension or atherosclerosis and seems to involve vasoactive peptides (e.g. angiotensin II (9Naftilan A.J. Curr. Opin. Nephrol. Hypertens. 1994; 3: 218-227Crossref PubMed Scopus (26) Google Scholar)) and mechanical stretch (10Dzau V.J. Curr. Opin. Nephrol. Hypertens. 1993; 2: 27-32Crossref PubMed Scopus (104) Google Scholar). Among a variety of pathophysiological settings such as excessive pulmonary ventilation and bladder-obstructive disorders, mechanical stretch plays a major role in triggering maladaptive mechanisms (e.g. abnormal ECM deposition), especially involving smooth muscle cells (16Wyncoll D.L. Evans T.W. Lancet. 1999; 354: 497-501Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 17Brading A.F. Scand. J. Urol. Nephrol. Suppl. 1997; 184: 51-58PubMed Google Scholar). However, little is known about the mechanism by which mechanical stretch is linked to angiogenesis. In this regard, CCN1 was first reported by Lau and colleagues as a growth factor-inducible immediate early gene associated with the ECM (2O'Brien T.P. Yang G.P. Sanders L. Lau L.F. Mol. Cell. Biol. 1990; 10: 3569-3577Crossref PubMed Scopus (271) Google Scholar, 18Latinkic B.V. O'Brien T.P. Lau L.F. Nucleic Acids Res. 1991; 19: 3261-3267Crossref PubMed Scopus (75) Google Scholar). Furthermore, CCN1 was associated with cell adhesion, cell migration, and cell proliferation of vascular cells. Thereafter, CCN1 was shown to induce neovascularization in the rat cornea model through cell-type-specific binding to different integrins (1Lau L.F. Lam S.C. Exp. Cell Res. 1999; 248: 44-57Crossref PubMed Scopus (580) Google Scholar, 5Babic A.M. Kireeva M.L. Kolesnikova T.V. Lau L.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6355-6360Crossref PubMed Scopus (431) Google Scholar). These observations suggest an important role of CCN1 in vascular remodeling and angiogenesis. Beside the direct effect of CCN1 on various cells of the vessel wall, recombinant CCN1 protein induces a genetic program that contributes to angiogenesis, inflammation, remodeling, and cell-matrix interactions (4Chen C.C. Mo F.E. Lau L.F. J. Biol. Chem. 2001; 276: 47329-47337Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). In fact, the essential role of CCN1 in vessel development was recently demonstrated in the CCN1 knock-out mouse suffering from embryonic death because of impaired placental development and vascular integrity. CCN1 deficiency results in a specific defect in vessel bifurcation development, leading to a disordered vascularization of the placenta, which is correlated with impaired vascular endothelial growth factor-C expression (3Mo F.E. Muntean A.G. Chen C.C. Stolz D.B. Watkins S.C. Lau L.F. Mol. Cell. Biol. 2002; 22: 8709-8720Crossref PubMed Scopus (342) Google Scholar). These observations established CCN1 as an essential regulator of vascular development. Genes involved in differentiation and angiogenesis such as vascular endothelial growth factor and platelet derived growth factor have been shown to be up-regulated by mechanical stretch in smooth muscle cells pointing to the important impact of mechanical stretch on vessel development (19Smith J.D. Davies N. Willis A.I. Sumpio B.E. Zilla P. Endothelium. 2001; 8: 41-48Crossref PubMed Scopus (48) Google Scholar, 20Wilson E. Mai Q. Sudhir K. Weiss R.H. Ives H.E. J. Cell Biol. 1993; 123: 741-747Crossref PubMed Scopus (337) Google Scholar). CCN1 was first linked to augmented mechanical stretch by the observation that its expression is up-regulated under pathophysiological conditions (e.g. after balloon angioplasty or outlet obstructed bladders (7Wu K.J. Yee A. Zhu N.L. Gordon E.M. Hall F.L. Int. J. Mol. Med. 2000; 6: 433-440Crossref PubMed Google Scholar, 8Grzeszkiewicz T.M. Lindner V. Chen N. Lam S.C. Lau L.F. Endocrinology. 2002; 143: 1441-1450Crossref PubMed Scopus (133) Google Scholar, 21Chaqour B. Whitbeck C. Han J.S. Macarak E. Horan P. Chichester P. Levin R. Am. J. Physiol. 2002; 283: E765-E774Crossref PubMed Scopus (50) Google Scholar). Herein, we first demonstrated that mechanical stretch induces a fast and transient up-regulation of the CCN1 mRNA (maximum at 0.5 and 1 h) and protein expression (maximum at 1 and 3 h) in cultured vascular smooth muscle cells. In addition, we could show that the CCN1 protein, which contains an N-terminal secretory signal peptide, is released by VSMC in response to mechanical stretch. The time-dependent CCN1 release follows the intracellular CCN1 protein expression with a delayed kinetic and accumulates after 6 and 24 h of mechanical stretch. The fact that only small amounts of CCN1 are detectable in the supernatant of quiescent VSMC may be attributed to a minor release under resting conditions suggesting an important role of mechanical stretch in the induction of CCN1-dependent effects in the vessel. However, the new finding suggests that stretch-dependent CCN1 release may act as an essential trigger mechanism for the biological function of this ECM associated protein. To induce elevated mechanical stretch in vivo, we used the murine aortic banding model. This technique was first established to mimic pressure overload in the heart (22Rockman H.A. Ross R.S. Harris A.N. Knowlton K.U. Steinhelper M.E. Field L.J. Ross Jr., J. Chien K.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8277-8281Crossref PubMed Scopus (632) Google Scholar). To investigate stretch-dependent CCN1 expression in vivo, we compared aorta segments exposed to pressure overload proximal to the aortic stenosis with control segments located distal of the stenosis. We here show that pressure overload enhances both the vascular mRNA and protein expression of CCN1, supporting the notion that enhanced mechanical stretch in vivo may trigger the CCN1 expression. Recently Tamura et al. (23Tamura I. Rosenbloom J. Macarak E. Chaqour B. Am. J. Physiol. 2001; 281: C1524-C1532Crossref PubMed Google Scholar) reported that mechanical stretch enhanced CCN1 expression in fetal bovine bladder smooth muscle cells involves protein kinase C, phosphatidylinositol 3-kinase (PI3K), Rho-kinase, and the actin cytoskeleton. In this regard the transcriptional regulation of CCN1 remained unclear. Latinkic et al. (18Latinkic B.V. O'Brien T.P. Lau L.F. Nucleic Acids Res. 1991; 19: 3261-3267Crossref PubMed Scopus (75) Google Scholar) demonstrated that serum enhanced CCN1 promoter activation in murine fibroblasts depends on a serum responsive element (SRE). Furthermore, Han et al. reported a CREB and AP-1 dependent activation of the differently organized human CCN1 promoter in bladder smooth muscle cells by sphingosine 1-phosphate (24Han J.S. Macarak E. Rosenbloom J. Chung K.C. Chaqour B. Eur. J. Biochem. 2003; 270: 3408-3421Crossref PubMed Scopus (75) Google Scholar). Here we addressed the activation of mechano-sensitive transcription factors linking mechanical stretch of VSMC to CCN1 promoter activation. We observed that mechanical stretch-induced CCN1 expression is regulated by a single binding site for Egr-1. In contrast to the previously described SRE which is located ∼2 kb (bp -1912 to -1933) upstream of the transcription start the novel stretch responsive element identified in this study is located directly in front of the TATA-box in the CCN1 promoter (bp -48 to -41). Egr-1 is a zinc finger transcription factor first identified because of its characteristic immediate-early expression pattern during growth and differentiation (25Sukhatme V.P. Cao X.M. Chang L.C. Tsai-Morris C.H. Stamenkovich D. Ferreira P.C. Cohen D.R. Edwards S.A. Shows T.B. Curran T. et al.Cell. 1988; 53: 37-43Abstract Full Text PDF PubMed Scopus (1025) Google Scholar). In addition, Egr-1 regulates the expression of genes known to be important for differentiation and angiogenesis, the vascular endothelial growth factor receptor Flt-1 (26Akuzawa N. Kurabayashi M. Ohyama Y. Arai M. Nagai R. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 377-384Crossref PubMed Scopus (40) Google Scholar). It is noteworthy that the messenger RNA of Egr-1 was found to be up-regulated in pressure overloaded rat hearts linking Egr-1 to elevated wall stress in left ventricle hypertrophy (27Schunkert H. Weinberg E.O. Bruckschlegel G. Riegger A.J. Lorell B.H. J. Clin. Investig. 1995; 96: 2768-2774Crossref PubMed Scopus (62) Google Scholar). In addition, the ability of mechanical stretch to increase Egr-1 transcript levels was demonstrated in endothelial cells and smooth muscle cells (28Stula M. Orzechowski H.D. Gschwend S. Vetter R. von Harsdorf R. Dietz R. Paul M Mol. Cell Biochem. 2000; 210: 101-108Crossref PubMed Google Scholar, 29Morawietz H. Ma Y.H. Vives F. Wilson E. Sukhatme V.P. Holtz J. Ives H.E. Circ. Res. 1999; 84: 678-687Crossref PubMed Scopus (69) Google Scholar). In this regard, it is likely that the SRE acts as a regulatory element important for the basal activity of the CCN1 promoter, whereas Egr-1 binding is important for enhanced CCN1 promoter activity under certain conditions, e.g. during differentiation and angiogenesis, potentially in response to enhanced mechanical stretch. As mentioned previously, stretch enhanced CCN1 expression in bladder smooth muscle cells involves the protein kinase C/PI3K/Rho-kinase signaling pathway (23Tamura I. Rosenbloom J. Macarak E. Chaqour B. Am. J. Physiol. 2001; 281: C1524-C1532Crossref PubMed Google Scholar). Inhibition of this pathway abrogates Egr-1 expression and activity (30Inuzuka H. Nanbu-Wakao R. Masuho Y. Muramatsu M. Tojo H. Wakao H. Biochem. Biophys. Res. Commun. 1999; 265: 664-668Crossref PubMed Scopus (75) Google Scholar, 31Guillemot L. Levy A. Raymondjean M. Rothhut B. J. Biol. Chem. 2001; 276: 39394-39403Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 32Liu Y. Suzuki Y.J. Day R.M. Fanburg B.L. Circ. Res. 2004; 95: 579-586Crossref PubMed Scopus (176) Google Scholar) providing the possibility of an involvement of the protein kinase C/PI3K/Rhokinase signaling pathway on the stretch dependent activation of Egr-1. Furthermore, Yamaguchi et al. (30Inuzuka H. Nanbu-Wakao R. Masuho Y. Muramatsu M. Tojo H. Wakao H. Biochem. Biophys. Res. Commun. 1999; 265: 664-668Crossref PubMed Scopus (75) Google Scholar) reported that stretch-dependent Egr-1 activation enhances the expression of membrane type 1 matrix metalloproteinase in rat microvascular endothelial cells with a significant impact on angiogenesis by affecting the activity of matrix metalloproteinase-2. In this regard, we recently demonstrated that mechanical stretch enhances expression and release of matrix metalloproteinase-2 in a redox-sensitive manner (11Grote K. Flach I. Luchtefeld M. Akin E. Holland S.M. Drexler H. Schieffer B. Circ. Res. 2003; 92: E80-E86Crossref PubMed Google Scholar). In summary, we could show that mechanical stretch enhances the angiogenic factor CCN1 via the activation of the mechanosensitive transcription factor Egr-1. These findings suggest a central role for mechanical stretch in the regulation of CCN1-dependent functions in angiogenesis, cell adhesion, proliferation, and differentiation. We thank Tanja Sander, Birgit Brandt, and Silke Pretzer for excellent technical assistance." @default.
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