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A Proatherogenic Role for Cgmp-Dependent Protein Kinase in Vascular Smooth Muscle Cells

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A Proatherogenic Role for Cgmp-Dependent Protein Kinase in Vascular Smooth Muscle Cells A proatherogenic role for cGMP-dependent protein kinase in vascular smooth muscle cells Wiebke Wolfsgruber*, Susanne Feil*, Sabine Brummer, Oliver Kuppinger, Franz Hofmann, and Robert Feil† Institut fu¨r Pharmakologie und Toxikologie, Technische Universita¨t, Biedersteiner Strasse 29, 80802 Munich, Germany Communicated by Joseph A. Beavo, University of Washington School of Medicine, Seattle, WA, September 18, 2003 (received for review May 19, 2003) Nitric oxide (NO) exerts both antiatherogenic and proatherogenic cGKI modulates the properties of aortic SMCs in vitro and in vivo effects, but the cellular and molecular mechanisms that contribute and promotes atherosclerosis. to modulation of atherosclerosis by NO are not understood com- pletely. The cGMP-dependent protein kinase I (cGKI) is a potential Materials and Methods mediator of NO signaling in vascular smooth muscle cells (SMCs). Experimental Animals. The generation of mice carrying a condi- Postnatal ablation of cGKI selectively in the SMCs of mice reduced tional loxP-flanked cGKI allele (L2) or a recombined cGKI-null atherosclerotic lesion area, demonstrating that smooth muscle allele (LϪ) and the detection of the cGKI WT(ϩ), L2, and LϪ cGKI promotes atherogenesis. Cell-fate mapping indicated that alleles by PCR have been described (14). Mice carrying the cGKI is involved in the development of SMC-derived plaque cells. SM-CreERT2 knock-in allele (Cre) were genotyped as described Activation of endogenous cGKI in primary aortic SMCs resulted in (15). Mice carrying the ROSA26 Cre reporter (R26R) (16) or cells with increased levels of proliferation; increased levels of ApoE-null (17) alleles were obtained from The Jackson Labo- vascular cell adhesion molecule-1, peroxisome proliferator-acti- ratory and genotyped according to published protocols (avail- vated receptor ␥, and phosphatidylinositol 3-kinase͞Akt signaling; able at www.jax.org). Mice with modified cGKI alleles were and decreased plasminogen activator inhibitor 1 mRNA, which all crossed with SM-CreERT2 knock-in mice to generate ‘‘pro- are potentially proatherogenic properties. Taken together, these mutant’’ mice (genotype cGKILϪ/L2; SM-CreERT2(Cre/ϩ)) and results highlight the pathophysiologic significance of vascular control mice (genotype cGKIϩ/L2; SM-CreERT2(Cre/ϩ)). For SMCs in atherogenesis and identify a key role for cGKI in the analysis of atherosclerosis, all mice had an ApoEϪ/Ϫ genotype. development of atherogenic SMCs in vitro and in vivo. We suggest To induce the Cre-mediated conversion of the cGKI L2 allele that activation of smooth muscle cGKI contributes to the into the LϪ allele in SMCs, mice were injected i.p. with 1 mg of proatherogenic effect of NO and that inhibition of cGKI might be tamoxifen (Sigma) for 5 consecutive days (15). For experiments, a therapeutic option for treating atherosclerosis in humans. litter-matched female control and pro-mutant mice on a mixed 129Sv͞C57BL6 genetic background were used with the investi- therosclerosis causes heart attack and stroke, the major gator unaware of the genotype of the mice. Experiments had Acauses of death in industrial nations. The pathophysiology been approved by the local government’s committee on animal of atherogenesis is complex. It is considered to be a chronic care and welfare in Munich. inflammatory condition that results from the interaction be- tween modified lipoproteins and various cell types, including 5-Bromo-4-chloro-3-indolyl ␤-D-Galactoside (X-Gal) Staining and Im- leukocytes, platelets, and cells of the vessel wall (1). The munohistochemistry. LacZ activity was detected by staining aortas development of an atherosclerotic plaque involves, in addition to with X-Gal as described (15). For immunohistochemistry, ani- inflammation, the phenotypic modulation of vascular smooth mals were deeply anesthetized and perfused with 10% phos- muscle cells (SMCs) to proliferating and dedifferentiated cells. phate-buffered formalin. Aortas were dissected and postfixed However, the mechanisms that contribute to the development of overnight in the same fixative, embedded in paraffin, and cut at plaque SMCs and their pathophysiologic significance are not 8-␮m thickness. Sections were stained by using rabbit antisera to well understood (2). cGKI (18) or proliferating cell nuclear antigen (PCNA; Santa The signaling molecule nitric oxide (NO) has critical roles in Cruz Biotechnology), goat anti-CD68 (Santa Cruz Biotechnol- the pathogenesis of atherosclerosis (3). Analysis of transgenic ogy), and a mouse monoclonal antibody to ␣-smooth muscle mice that lack or overexpress NO synthases indicated that NO actin (Sigma). For detection of primary antibodies, we used the exerts both protective (4, 5) and atherogenic (6–9) effects. The avidin–biotin method with diaminobenzidine or Vector blue double role of NO might explain why NO-generating drugs (e.g., substrate as a chromogen (Vector Laboratories). For detection glyceryl trinitrate) have not been reported to limit the progres- of ␣-smooth muscle actin, the MOM Basic immunodetection kit MEDICAL SCIENCES sion of atherosclerosis in humans. The opposing actions of NO (Vector Laboratories) was used. Control sections were pro- on atherogenesis might depend on the spatiotemporal profile of cessed in the absence of primary antibodies. Stained sections its production and are likely mediated by different cellular and were mounted in 80% glycerol containing 1 ␮g͞ml Hoechst molecular mechanisms. Signaling pathways in SMCs that con- 33258 (Sigma) to visualize cell nuclei. To determine the per- tribute to NO modulation of atherogenesis have not been centage of cGKI- or cGKI͞PCNA-positive SMCs, immuno- identified. In vascular SMCs, NO is thought to exert many of its effects by activation of soluble guanylyl cyclase, synthesis of the second messenger cGMP, and activation of cGMP-dependent Abbreviations: cGKI, cGMP-dependent protein kinase I; DETA-NO, diethylenetriamine protein kinase I (cGKI) (10). The analysis of the functional NONOate; ko, knockout; MAPK, mitogen-activated protein kinase; p-FKHR, phospho- significance of cGKI in NO͞cGMP signaling is complicated by forkhead transcription factor; PAI-1, plasminogen activator inhibitor 1; PCNA, proliferating cell nuclear antigen; PDGF, platelet-derived growth factor; PI3K, phosphatidylinositol the existence of multiple receptors for cGMP (11) and by the lack 3-kinase; PPAR-␥, peroxisome proliferator-activated receptor ␥; SMC, smooth muscle cell; of highly specific agonists and inhibitors of cGKI (12, 13). Here, VCAM-1, vascular cell adhesion molecule 1; X-Gal, 5-bromo-4-chloro-3-indolyl we have studied the consequences of postnatal SMC-specific ␤-D-galactoside. inactivation of the cGKI gene by using a spatiotemporally *W.W. and S.F. contributed equally to this work. controlled Cre͞lox system for combined gene targeting and †To whom correspondence should be addressed. E-mail: [email protected]. cell-fate mapping in mice. Our results show that smooth muscle © 2003 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.1936024100 PNAS ͉ November 11, 2003 ͉ vol. 100 ͉ no. 23 ͉ 13519–13524 Downloaded by guest on September 26, 2021 stained cells and total cells (Hoechst 33258-stained) were counted in sections from five mice (three sections per mouse). Analysis of Atherosclerosis. Pro-mutant and control mice on an ApoE-deficient genetic background (genotype cGKILϪ/L2;SM- CreERT2(Cre/ϩ); ApoEϪ/Ϫ and cGKIϩ/L2; SM-CreERT2(Cre/ϩ); ApoEϪ/Ϫ, respectively) were fed an atherogenic diet (20% fat, 1.5% cholesterol by weight; Altromin, Lage, Germany) begin- ning at 5 weeks of age. All animals were injected with tamoxifen at 4 and 8 weeks of age. After 8 and 16 weeks on the atherogenic diet, plasma lipid profiles (Medical Service Labo- ratorium, Munich) and atherosclerotic lesions in the aorta were analyzed. Animals were deeply anesthetized and per- fused with 10% phosphate-buffered formalin. Aortas were isolated, postfixed in the same fixative overnight, and stained with oil red O (Sigma) as described (19). Two different methods were used for quantitative estimation of lesion area. In one method, aortas were mounted between two glass slides Fig. 1. Tamoxifen-induced ablation of cGKI in aortic SMCs. Four-week-old and photographed from each side. In the other method, aortas mice were injected with either vehicle (ctr) or tamoxifen (tam) for 5 consec- were cut open longitudinally, pinned to Sylgard 184 (Dow- utive days and then analyzed at an age of 8 weeks. (a) PCR analysis of Corning), and photographed. A 5-mm scale bar was used for Cre-mediated recombination in tissues from cGKIϩ/L2; SM-CreERT2(Cre/ϩ) mice. calibration. Images were scanned, and the oil red O-positive PCR products amplified from the cGKI L2, WT (ϩ), and LϪ alleles are indicated. area in the aortic arch was determined by using UTHSCSA In the corresponding diagrams, exon 10 of the cGKI (■) and loxP (‹) sites are IMAGE TOOL, version 2.00 (University of Texas Health Science indicated. (b) Detection of ␤-galactosidase activity by X-Gal (Left) and immu- ϩ Ϫ Center, San Antonio, TX). The mean lesion areas obtained by nohistochemical staining of cGKI (Right) on aortic sections from R26R / ; T2(Cre/ϩ) LϪ/L2 T2(Cre/ϩ) using each method were not significantly different. Blood SM-CreER mice and from cGKI ; SM-CreER mice, respectively. (Original magnification of photomicrographs was ϫ400.) pressure and heart rate were measured by using the tail-cuff method on conscious restrained mice. carrying a conditional cGKI allele (L2) with two loxP sites Analysis of Primary Aortic SMCs. Aortic SMCs were isolated from flanking the critical exon 10 of the cGKI gene, Cre-mediated Ϫ/Ϫ WT mice and cGKI mice (14) and grown in DMEM supple- recombination of the loxP sites results in the excision of exon mented with 10% FCS as described (15). Cell numbers were 10 and thus in a cGKI-null allele (LϪ) (14). Conversion of the determined from photomicrographs of 12-well plates by using cGKI L2 into the LϪ allele takes place only in cells expressing 3 UTHSCSA IMAGE TOOL, version 2.00. For measuring [ H]thymi- active Cre recombinase.
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