Regulation of cGMP-dependent Protein Kinase Expression by Soluble Guanylyl Cyclase in Vascular Smooth Muscle Cells

Abstract

Vascular smooth muscle cells (VSMC) undergo many phenotypic changes when placed in culture. Several studies have shown that the levels of expression of soluble guanylyl cyclase (sGC) or cGMP-dependent protein kinase (PKG) are altered in cultured VSMC. In this study the mechanisms involved in the coordinated expression of sGC and PKG were examined. Pro-inflammatory cytokines that increase the expression of type II NO synthase (inducible NO synthase, or iNOS) decreased PKG expression in freshly isolated, non-passaged bovine aortic SMC. However, in several passaged VSMC lines (i.e. bovine aortic SMC, human aortic SMC, and A7r5 cells), PKG protein expression was not suppressed by cytokines or NO. sGC was highly expressed in non-passaged bovine aortic SMC but not in passaged cell lines. Restoration of expression of sGC to passaged bovine SMC using adenovirus encoding the α1 and β1 subunits of sGC restored the capacity of the cells to increase cGMP in response to NO. Furthermore, treatment of these sGC-transduced cells with NO donors for 48 h resulted in decreased PKG protein expression. In contrast, passaged rat aortic SMC expressed high levels of NO-responsive sGC but demonstrated reduced expression of PKG. Adenovirus-mediated expression of the PKG catalytically active domain in rat aortic SMC caused a reduction in the expression of sGC in these cells. These results suggest that there is a mechanism for the coordinated expression of sGC and PKG in VSMC and that prolonged activation of sGC down-regulates PKG expression. Likewise, the loss of PKG expression appears to increase sGC expression. These effects may be an adaptive mechanism allowing growth and survival of VSMC in vitro. Vascular smooth muscle cells (VSMC) undergo many phenotypic changes when placed in culture. Several studies have shown that the levels of expression of soluble guanylyl cyclase (sGC) or cGMP-dependent protein kinase (PKG) are altered in cultured VSMC. In this study the mechanisms involved in the coordinated expression of sGC and PKG were examined. Pro-inflammatory cytokines that increase the expression of type II NO synthase (inducible NO synthase, or iNOS) decreased PKG expression in freshly isolated, non-passaged bovine aortic SMC. However, in several passaged VSMC lines (i.e. bovine aortic SMC, human aortic SMC, and A7r5 cells), PKG protein expression was not suppressed by cytokines or NO. sGC was highly expressed in non-passaged bovine aortic SMC but not in passaged cell lines. Restoration of expression of sGC to passaged bovine SMC using adenovirus encoding the α1 and β1 subunits of sGC restored the capacity of the cells to increase cGMP in response to NO. Furthermore, treatment of these sGC-transduced cells with NO donors for 48 h resulted in decreased PKG protein expression. In contrast, passaged rat aortic SMC expressed high levels of NO-responsive sGC but demonstrated reduced expression of PKG. Adenovirus-mediated expression of the PKG catalytically active domain in rat aortic SMC caused a reduction in the expression of sGC in these cells. These results suggest that there is a mechanism for the coordinated expression of sGC and PKG in VSMC and that prolonged activation of sGC down-regulates PKG expression. Likewise, the loss of PKG expression appears to increase sGC expression. These effects may be an adaptive mechanism allowing growth and survival of VSMC in vitro. The nitric oxide (NO)-cGMP signaling pathway plays a critical role in vascular function (1Wong S.K. Garbers D.L. J. Clin. Investig. 1992; 90: 299-305Crossref PubMed Scopus (110) Google Scholar, 2Denninger J.W. Marletta M.A. Biochim. Biophys. Acta. 1999; 1411: 334-350Crossref PubMed Scopus (881) Google Scholar). Cyclic GMP is a second messenger that is generated by cytoplasmic soluble guanylyl cyclases (sGCs), 1The abbreviations used are: sGC, soluble guanylyl cyclase; VSMC, vascular smooth muscle cells (VSM); PKG, cGMP-dependent protein kinase; Cat, active catalytic domain of PKG-I; LPS, lipopolysaccharide; iNOS, inducible NO synthase; TNF-α, tumor necrosis factor-α; IL-Iβ, interleukin Iβ; DETA NONOate, (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate; DEA NONOate, 2-(N,N-diethylamino)-diazenolate-2-oxide; HuAoSMC, human aortic smooth muscle cells; A7r5, rat embryonic thoracic aortic smooth muscle cells; FBS, fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium; Ad, adenovirus; SNAP, S-nitroso-N-acetylpenicillamine; SNP, sodium nitroprusside; m.o.i., multiplicity of infection. which are activated by NO, or particulate guanylyl cyclases, which are activated by peptide hormones such as atrial natriuretic peptide. sGC is a heterodimer that consists of two subunits, termed α and β, which are linked by disulfide bonds, and has a prosthetic heme group covalently bound to the heterodimer (1Wong S.K. Garbers D.L. J. Clin. Investig. 1992; 90: 299-305Crossref PubMed Scopus (110) Google Scholar, 2Denninger J.W. Marletta M.A. Biochim. Biophys. Acta. 1999; 1411: 334-350Crossref PubMed Scopus (881) Google Scholar). NO activates cGMP synthesis by binding to the heme, leading to the conversion of GTP to cGMP. Although two isoforms of each subunit have been cloned from rat and bovine lung, the a1b1 heterodimer has been found in most tissues and cell types, including vascular smooth muscle cells (VSMC) (3Harteneck C. Koesling D. Soling A. Schultz G. Bohme E. FEBS Lett. 1990; 272: 221-223Crossref PubMed Scopus (142) Google Scholar, 4Buechler W.A. Nakane M. Murad F. Biochem. Biophys. Res. Commun. 1991; 174: 351-357Crossref PubMed Scopus (140) Google Scholar, 5Nakane M. Saheki S. Kuno T. Ishii K. Murad F. Biochem. Biophys. Res. Commun. 1988; 157: 1139-1147Crossref PubMed Scopus (123) Google Scholar, 6Koesling D. Herz J. Gausepohl H. Niroomand F. Hinsch K.D. Mulsch A. Bohme E. Schultz G. Frank R. FEBS Lett. 1988; 239: 29-34Crossref PubMed Scopus (116) Google Scholar). NO and cGMP are involved in many physiological processes such as VSMC relaxation, apoptosis, proliferation, migration, and extracellular matrix production (7Garg U.C. Hassid A. J. Clin. Investig. 1989; 83: 1774-1777Crossref PubMed Scopus (1997) Google Scholar, 8Sarkar R. Meinberg E.G. Stanley J.C. Gordon D. Webb R.C. Circ. Res. 1996; 78: 225-230Crossref PubMed Scopus (356) Google Scholar, 9Pollman M.J. Yamada T. Horiuchi M. Gibbons G.H. Circ. Res. 1996; 79: 748-756Crossref PubMed Scopus (314) Google Scholar, 10Murad F. Circulation. 1997; 95: 1101-1103Crossref PubMed Scopus (51) Google Scholar). An important cGMP receptor protein expressed in VSMC is cGMP-dependent protein kinase (PKG), which is ultimately responsible for mediating the effects of cGMP in vascular smooth muscle relaxation (11Schlossmann J. Feil R. Hofmann F. Ann. Med. 2003; 35: 21-27Crossref PubMed Scopus (140) Google Scholar). There are two distinct genes encoding PKG in mammalian cells, the type I and type II genes (12Wernet W. Flockerzi V. Hofmann F. FEBS Lett. 1989; 251: 191-196Crossref PubMed Scopus (162) Google Scholar, 13Francis S.H. Woodford T.A. Wolfe L. Corbin J.D. Second Messengers Phosphoproteins. 1988; 12: 301-310PubMed Google Scholar). Type I, which is the gene product expressed in VSMC, exists as two isoforms, PKG Iα and PKG Iβ, generated by alternate mRNA splicing of the type I gene (13Francis S.H. Woodford T.A. Wolfe L. Corbin J.D. Second Messengers Phosphoproteins. 1988; 12: 301-310PubMed Google Scholar). There is much current interest in the regulation of gene expression by NO and cGMP (14Pilz R.B. Casteel D.E. Circ. Res. 2003; 93: 1034-1046Crossref PubMed Scopus (247) Google Scholar). However, less is known about the regulation of PKG and sGC gene expression in VSMC. This is particularly important because several studies demonstrate that sGC and PKG expression are altered in VSMC in culture. Indeed, we have suggested that the loss of PKG expression in cultured rat aortic VSMC may be responsible in part at least for the phenotypic modulation of the cells from a more differentiated, contractile phenotype to a dedifferentiated, synthetic phenotype (15Boerth N.J. Dey N.B. Cornwell T.L. Lincoln T.M. J. Vasc. Res. 1997; 34: 245-259Crossref PubMed Scopus (154) Google Scholar). Several studies show that PKG expression is decreased in VSMC after balloon catheter injury in vivo (16Sinnaeve P. Chiche J.D. Gillijns H. Van Pelt N. Wirthlin D. Van De Werf F. Collen D. Bloch K.D. Janssens S. Circulation. 2002; 105: 2911-2916Crossref PubMed Scopus (58) Google Scholar, 17Anderson P.G. Boerth N.J. Liu M. McNamara D.B. Cornwell T.L. Lincoln T.M. Arteriosclerosis, Arterioscler. Thromb. Vasc. Biol. 2000; 20: 2192-2197Crossref PubMed Scopus (70) Google Scholar). Recently our laboratory demonstrated that inflammatory cytokines and lipopolysaccharide (LPS), biological modulators associated with inflammation, suppressed PKG I gene expression in VSMC through a mechanism involving the expression of inducible, type II NO synthase (iNOS). Other studies show that sGC is down-regulated in various cells in response to various stimuli (18Ujiie K. Hogarth L. Danziger R. Drewett J.G. Yuen P.S. Pang I.H. Star R.A. J. Pharmacol. Exp. Ther. 1994; 270: 761-767PubMed Google Scholar, 19Takata M. Filippov G. Liu H. Ichinose F. Janssens S. Bloch D.B. Bloch K.D. Am. J. Physiol. Lung Cell. Mol. Physiol. 2001; 280: 272-278Crossref PubMed Google Scholar, 20Filippov G. Bloch D.B. Bloch K.D. J. Clin. Investig. 1997; 100: 942-948Crossref PubMed Scopus (133) Google Scholar). In rat pulmonary artery SMC, for instance, NO donors were found to reduce sGC subunit mRNA levels, and this effect was inhibited by the addition of a selective sGC inhibitor (20Filippov G. Bloch D.B. Bloch K.D. J. Clin. Investig. 1997; 100: 942-948Crossref PubMed Scopus (133) Google Scholar). Additionally, sGC has been shown to be down-regulated in young and aging rats that are spontaneously hypertensive (21Ruetten H. Zabel U. Linz W. Schmidt H.H. Circ. Res. 1999; 85: 534-541Crossref PubMed Scopus (189) Google Scholar). The down-regulation of sGC and/or PKG would be expected to impair the ability of NO to regulate VSMC functions, potentially contributing to the excessive proliferation and secretory activity of VSMC in vascular disorders. In the current study we have examined the role of sGC activity on PKG expression in cultured VSMC. We found in several lines of VSMC expressing high levels of PKG that pro-inflammatory cytokines and NO donor compounds were unable to induce cGMP synthesis or reduce PKG expression. This lack of responsiveness was determined to be due to loss of sGC activity and protein expression. Adenovirus-mediated gene transfer of α1 and β1 sGC subunits with NO treatment decreased PKG protein expression in VSMC. bovine Dulbecco's modified Eagle's and were from and were from was from and were from aortic vascular smooth muscle cells were from embryonic thoracic aortic VSMC were from Type were from or from freshly were by and aortic VSMC were from the the aortic cells were in FBS, and and in a at were using passaged bovine aortic VSMC were to the and A7r5 cells were cultured in and in a at aortic VSMC were and cultured as N.B. Boerth N.J. Lincoln T.M. Circ. Res. PubMed Scopus Google Scholar). aortic VSMC were in FBS, and in a at The was to the cells using and to the cells at as was after each using and VSMC were with and the were with an of and The were by in and to The were in and with or as N.B. Boerth N.J. Lincoln T.M. Circ. Res. PubMed Scopus Google Scholar). of and was from cell using as by the was on and was as N.B. Boerth N.J. Lincoln T.M. Circ. Res. PubMed Scopus Google Scholar). were with and at and at and with at were to which as the gene for The for bovine and were generated as and using the of expressing the α1 and β1 subunits of sGC or the catalytic domain of PKG were generated as P. Chiche J.D. Van Pelt N. Gillijns H. Collen D. Bloch K.D. Janssens S. Circ. Res. 2001; PubMed Scopus Google Scholar). and are α1 the encoding the α1 subunit of β1 the encoding the β1 subunit of the for the catalytic domain of the α the gene encoding a was on a by T. Biol. 1994; PubMed Scopus Google Scholar). were and bovine was The was by and were at to of aortic VSMC were at a and to were with or with and for h in at was to each and cells were to for 48 was using the in and by for the β1 subunit of to studies using rat aortic SMC with adenovirus (15Boerth N.J. Dey N.B. Cornwell T.L. Lincoln T.M. J. Vasc. Res. 1997; 34: 245-259Crossref PubMed Scopus (154) Google we found that for sGC subunit an of was to of the cells. of rat aortic SMC with an of was to of the cells. Cyclic levels were using the as T.L. E. Boerth N.J. Lincoln T.M. Am. J. Physiol. 1994; PubMed Google Scholar). cells were cultured in and with cytokines or NO were with of The were and in of for of cGMP were for in a and were using was with and for protein by the were with protein and expressed as of cGMP of activity of sGC was using a modified by Murad and W.A. Nakane M. Murad F. Biochem. Biophys. Res. Commun. 1991; 174: 351-357Crossref PubMed Scopus (140) Google Scholar). were with and with of were for at and at for at were and protein was determined using the of in a of of and of was in the or of for at were by the addition of and The were at for and by the addition of of sodium were placed on for were and a cGMP was are expressed as the was in with were by or of was determined by using with at of on PKG studies were to smooth muscle cell for the PKG expression and in cGMP In bovine aortic SMC, such as and TNF-α, as as cGMP levels and decreased PKG mRNA and protein expression T.L. E. Boerth N.J. Lincoln T.M. Am. J. Physiol. 1994; PubMed Google Scholar). However, studies with bovine aortic SMC that been passaged several shown in treatment with IL-Iβ, TNF-α, or effect on PKG protein expression in passaged bovine aortic SMC. results were when this was in and A7r5 cells the for the and passaged bovine aortic SMC, the effects of cytokines and on the pathway were more examined. shown in TNF-α, IL-Iβ, or mRNA expression in passaged bovine aortic SMC. The in mRNA expression was to because cells these of and demonstrated that the signaling pathway that is responsible for expression was in the and the passaged VSMC T.L. E. Boerth N.J. Lincoln T.M. Am. J. Physiol. 1994; PubMed Google Scholar, D. M. Am. J. Physiol. 1994; Google of cytokines and on mRNA expression. bovine aortic VSMC were with or for and mRNA levels were by of and NO on cGMP examined the effects of cytokines on cGMP levels in and passaged bovine aortic SMC. were with cytokines and for and cGMP levels were shown in cytokines and cGMP levels in non-passaged bovine aortic SMC, in the passaged of bovine aortic SMC the cytokines effect on cGMP levels these results have been due to sGC that was to the effects of cytokines bovine aortic SMC were with NO and cGMP levels were shown in and NO donors and and effect on cGMP levels in passaged bovine aortic SMC or HuAoSMC, In contrast, treatment of passaged rat aortic SMC with these NO donors resulted in a more in cGMP levels The of and on cell to NO, DEA NO, that the lack of response to the various NO donor was not due to in the capacity of the cells to NO from NO donors from in guanylyl of NO donors on cGMP production in VSMC. were for with DEA or or for with or and cGMP levels were using passaged bovine aortic SMC passaged human aortic SMC and passaged rat aortic SMC the in the of the in and with that of C. are expressed as of at least DEA and in shown in high levels of sGC activity were in passaged rat aortic SMC and bovine aortic SMC, but sGC activity was in passaged bovine aortic SMC and this in sGC we sGC protein expression by shown in using that the β1 of that high levels of sGC protein were expressed in rat lung, passaged rat aortic SMC, bovine and bovine aortic SMC. However, sGC protein expression was in passaged bovine aortic SMC and is important to that the expression of PKG was high in and passaged bovine aortic SMC and but was in passaged rat aortic SMC These results suggest that for freshly of bovine aortic SMC, the expression of PKG and sGC may be in cultured VSMC. Restoration of activity of sGC is expression of the α and subunits of the expressing each and were to sGC protein expression in passaged bovine aortic SMC. with and restored sGC expression in passaged bovine aortic SMC of sGC protein expression an active cells were with for and cGMP levels were shown in treatment of cells with cGMP production more the of (16Sinnaeve P. Chiche J.D. Gillijns H. Van Pelt N. Wirthlin D. Van De Werf F. Collen D. Bloch K.D. Janssens S. Circulation. 2002; 105: 2911-2916Crossref PubMed Scopus (58) Google Scholar). cells sGC activity and NO of NO on PKG the role of sGC and NO in the regulation of PKG expression in VSMC, the SMC with the for the sGC subunits were with DETA for 48 and PKG expression was using shown in passaged bovine aortic SMC with which as a demonstrated in PKG protein expression in the of a NO However, PKG expression was decreased more in cells with and and with NO. These results suggest that sGC and cGMP production PKG protein expression in passaged bovine aortic SMC and that sGC expression PKG in these cells. of PKG on sGC the role of PKG in expression we used passaged rat aortic SMC that high levels of sGC but levels of PKG of these cells expressing the active catalytic domain of resulted in the expression of the β1 subunit of sGC, the α1 subunit was reduced by the of active PKG which as a effect on sGC subunit expression. These results suggest that active sGC activity by the expression of the β1 The of this study was to the role of sGC in PKG levels in VSMC. our laboratory has shown that passaged rat aortic SMC or PKG but are highly to NO donors with in cGMP T.L. E. Boerth N.J. Lincoln T.M. Am. J. Physiol. 1994; PubMed Google Scholar, T.L. Lincoln T.M. J. Vasc. Res. 1994; PubMed Scopus Google Scholar). However, the of these we found that several VSMC passaged bovine aortic SMC, HuAoSMC, and A7r5 cells, high levels of PKG. In this when to PKG in these cell lines by cytokines or NO donor compounds we were unable to increase cGMP levels Furthermore, of these cell with NO donors cGMP such as not increase cGMP C. N. K. D. and T. M. These have to that in passaged VSMC PKG expression is not reduced but sGC of sGC activity and protein expression that several lines of passaged VSMC levels of Adenovirus-mediated gene transfer of and sGC subunits restored expression and cyclase activity in passaged bovine aortic SMC. These results suggest that the for the loss in the capacity of these cell lines to to cytokines and NO donors with in cGMP is due to the loss in expression of sGC of the current study is that after of sGC expression to passaged bovine aortic SMC, we the expression of PKG in these cells by sGC In bovine aortic SMC that have been in for less sGC and PKG are the these two However, sGC expression is PKG expression is in rat aortic SMC PKG expression is but sGC expression and activity are of active PKG down-regulates sGC β1 subunit expression. L. G. J.W. S. G. Circ. Res. 2000; PubMed Scopus Google found that decreased expression of the β1 subunit of sGC was to cGMP in SMC from The mechanism of such is not but may of sGC subunit mRNA synthesis (18Ujiie K. Hogarth L. Danziger R. Drewett J.G. Yuen P.S. Pang I.H. Star R.A. J. Pharmacol. Exp. Ther. 1994; 270: 761-767PubMed Google Scholar, 19Takata M. Filippov G. Liu H. Ichinose F. Janssens S. Bloch D.B. Bloch K.D. Am. J. Physiol. Lung Cell. Mol. Physiol. 2001; 280: 272-278Crossref PubMed Google Scholar, 20Filippov G. Bloch D.B. Bloch K.D. J. Clin. Investig. 1997; 100: 942-948Crossref PubMed Scopus (133) Google or of sGC S. M. D. M. Biochim. Biophys. Acta. 2003; PubMed Scopus Google found that natriuretic peptide and decreased sGC β1 subunit in human cells by a and Mulsch S. R. Mulsch A. Mol. Pharmacol. PubMed Scopus Google have shown that activation of protein kinase kinase the expression of in rat aortic smooth effects be for the down-regulation of the current study has a the expression of sGC and PKG. In rat aortic SMC PKG expression but sGC activity and protein A7r5 cells, HuAoSMC, and passaged bovine aortic SMC PKG expression but sGC expression. These suggest that in at VSMC may these adaptive mechanisms to the pathway to increase survival of the cells in culture. is that an active signaling pathway be growth for VSMC D. Res. 1999; PubMed Scopus Google or in VSMC M.J. Yamada T. Horiuchi M. Gibbons G.H. Circ. Res. 1996; 79: 748-756Crossref PubMed Scopus (314) Google Scholar, J.D. Bloch D.B. J.D. Filippov G. Janssens A. Bloch K.D. J. Biol. PubMed Scopus Google Scholar). mechanisms that the pathway in may growth and survival of cultured VSMC. that sGC expression is decreased vascular S. A. Mulsch A. 2000; 35: PubMed Scopus Google found that sGC expression is decreased in and hypertensive of rats and that was expression of the β1 subunit of sGC of synthesis from VSMC from rats L. G. J.W. S. G. Circ. Res. 2000; PubMed Scopus Google Scholar). of NO synthase in was found to sGC activity T. S. M. N. K. H. M. 2000; PubMed Scopus Google and sGC was found to be by NO J. J. U. S. A. 2000; PubMed Scopus Google Scholar). Filippov that NO sGC mRNA and protein levels in pulmonary SMC by a cGMP-dependent mechanism (20Filippov G. Bloch D.B. Bloch K.D. J. Clin. Investig. 1997; 100: 942-948Crossref PubMed Scopus (133) Google Scholar). These results suggest that PKG may sGC to the effects of sGC to PKG expression. There are a of studies using cultured VSMC to that may be involved in the expression of genes that of the cells. In for instance, rat aortic SMC contractile function and highly and with this in there is a in the expression of PKG. Restoration of PKG expression to these cells by or gene transfer the cells to a more contractile phenotype (15Boerth N.J. Dey N.B. Cornwell T.L. Lincoln T.M. J. Vasc. Res. 1997; 34: 245-259Crossref PubMed Scopus (154) Google Scholar). the cultured bovine aortic SMC and contractile phenotype such as smooth muscle Likewise, these cells PKG and have levels of cGMP sGC expression is is to that the PKG, which has been shown by Corbin and R.B. S.H. K. Corbin J.D. J. Biol. 2000; PubMed Scopus Google to in a active may be responsible in part at least for the expression of contractile phenotype in these cells. of PKG on the would to the loss in ability of the cells to contractile phenotype with sGC activity Other studies have demonstrated the of regulation of PKG expression in the of vascular A. P. S. L. M. C. M. A. F. A. P. R. Hofmann F. J. PubMed Scopus Google have shown that of the PKG gene in resulted in and and Other studies have shown that PKG expression is decreased after vascular injury (16Sinnaeve P. Chiche J.D. Gillijns H. Van Pelt N. Wirthlin D. Van De Werf F. Collen D. Bloch K.D. Janssens S. Circulation. 2002; 105: 2911-2916Crossref PubMed Scopus (58) Google Scholar, 17Anderson P.G. Boerth N.J. Liu M. McNamara D.B. Cornwell T.L. Lincoln T.M. Arteriosclerosis, Arterioscler. Thromb. Vasc. Biol. 2000; 20: 2192-2197Crossref PubMed Scopus (70) Google Scholar). P.G. Boerth N.J. Liu M. McNamara D.B. Cornwell T.L. Lincoln T.M. Arteriosclerosis, Arterioscler. Thromb. Vasc. Biol. 2000; 20: 2192-2197Crossref PubMed Scopus (70) Google demonstrated PKG expression in P.G. Boerth N.J. Liu M. McNamara D.B. Cornwell T.L. Lincoln T.M. Arteriosclerosis, Arterioscler. Thromb. Vasc. Biol. 2000; 20: 2192-2197Crossref PubMed Scopus (70) Google Scholar). These were to by (16Sinnaeve P. Chiche J.D. Gillijns H. Van Pelt N. Wirthlin D. Van De Werf F. Collen D. Bloch K.D. Janssens S. Circulation. 2002; 105: 2911-2916Crossref PubMed Scopus (58) Google in rat Furthermore, gene transfer of the PKG I catalytic domain reduced in rats after balloon catheter injury (16Sinnaeve P. Chiche J.D. Gillijns H. Van Pelt N. Wirthlin D. Van De Werf F. Collen D. Bloch K.D. Janssens S. Circulation. 2002; 105: 2911-2916Crossref PubMed Scopus (58) Google Scholar). loss of sGC or PKG to of vascular is that of sGC or PKG function effect Although the results of these studies are with an role for the pathway in vascular SMC, a by W. Feil S. S. Hofmann F. Feil R. U. S. A. 2003; 100: PubMed Scopus Google that the pathway may be for vascular SMC. an in vivo and in studies with cultured aortic SMC were used to demonstrate such a role for PKG. is not the high of cGMP used in this study expression of in this the cells more to growth the results of this study many studies the role of the pathway in vascular SMC more studies to be to more the biological role of cGMP and PKG on SMC growth and gene In the of the current study was to mechanism involved in the coordinated expression of sGC and PKG in VSMC. We found that pro-inflammatory cytokines and PKG protein expression in bovine aortic VSMC. However, PKG protein expression was not suppressed by cytokines or in several lines of passaged VSMC. of these passaged cell lines that these expression and activity of sGC with cell In contrast, rat aortic VSMC expression of PKG but sGC activity and expression. gene sGC expression was restored in passaged bovine aortic VSMC. Additionally, NO treatment of sGC-transduced cells resulted in a in PKG expression. These results demonstrate that there is a of sGC and PKG expression in VSMC, and our suggest an important role in the regulation of the signaling pathway in the of survival mechanism of VSMC in vitro.