Los inhibidores de la HMG-COA reductasa regulan la sntesis del factor de crecimiento de tejido conectivo (CTGF) inducido por la angiotensina

ll. Un efecto adicional vasoprotector?

HMG-COA reductase inhibitors regulate angotensin ll-induced connective tissue growth synthesis. An additional vasoprotective effect?
Ruprez M., Blanco-Colio Rodrguez-Vita L., Snchez-Lpez E, Esteban J., Egido J., Ruiz-Ortega M. V.,

factor

RESUMEN
La AngII participa en el acmulo de matriz extracelular en la pared va\cular, proce\o que ocurre en sltuaclones patol:lca\ como la hlperttmin arterIal y la atcro\clero\l\ El factor de creclmlento conecttvo tt\ular (CTGF) est tmphcado en proce\os de fibro\ls A\. se han de\crlto nIvele\ elevados de CTGF en placas de ateroma humana\ y tra\ mfarto agudo de mlocardlo en la\ zonas de fibrosl\ Los InhIbIdores de la HMGCOA (3-htdrox~-3-met~lglutnl coenzuna A) reductasa (e\tatmaa) han demostrado ser benehclo\os en el tratamenta de dIvena\ enfermedades cardiovaxulares E\to\ efecto\ \on debldos d la regulacin del colesterol y a accIones celulares, como la mtublcln de la isoprendacln de protenas y cambloa en los mveles de actlvacln de protenas G. Nuestro obJetlvo fue estudiar la poalble relacin entre loa InhIbIdores de la ISOpremlacln protetca y las acctone de la AngII en el dao vaxular, uvesngando en clulas de mxulo IIFO vascular (CMLV) FU efecto sobre la modulacin del CTGF En CMLV, la AnglI. a travs del receptor AT,. Induce la exprestn y <ntesIs del CTGF (Northern y We%ern blot). La mcubacln con un ollgonucleotldo annsenttdo de CTGF, que bloquea IU produccin endgena, dl\mmuy la +xe\ls de fibronectma InducIda por AngII, lo que qugtere que el CTGF es un mediador de la fibrosl\ causa da por AngII. La premcubacin de las CMLV con atorvastatina o \mvastatma (IO- mol/L a 10m9 mol/L) mhlbl de forma dosla dependiente la produccin de CTGF causada por AnglI, efecto que fue revertIdo por el mevalonato y por metabohtos mtermedtartos. La Angll, va AT,, activa Ia5 protena5 Rho. El bloqueo de RhoA, mediante transfecctones transttortas con un vector dommante negativo de RhoA, mhibl la produccln de CTGF causada por AngII InhIbIdores de la protena qumasa de Rho (Y-27632 y fasudd) dlsmmuyeron de fornxa dosis dependiente la produccln de CTGF InducIda por AnglI. Conclusiones: En CMLV, la AngII, medtante la unin a receptore AT, y acnvacln de protena\ qumasas (como la quinasa de Rh). regula la produccin de CTGF. Las e\tatmas. a travs de la mhilxcln de ISOprenoldes y modulando la acttvtdad de las protenas Rho, InhIben la produccln de CTGF causada por AngII. Nuestros resultados sugleren que alguno\ de lo efecto5 beneficIoso\ de las estatina pueden \er atrIbuIdo\ a sus acciones celulares sobre la regulacin de las respuestas de la AngII en el dao vaacular, exphcando efecto\ protectores mdependlente\ de la regulacin del colesterol.

ABSTRACT
Angtotemm II (AnglI) participate\ in the development of fibro\t\ durmg vaxular damage, mcludmg hypertenslon and athero\cleroal\ Connectivc tl\\ue growth factor (CTGF) ha\ been dc\crlbed u\ a novel flbrotx medlator Elevated CTGF level\ have been reported tn human atherosclerotlc plaques and after myocardlal mfarcnon. The 3.hydroxy3-methylglutaryl coenzyme A (HMG-COA) reductaae mhlbltor\ (\tatm\) have shown beneficIaI eftects tn the treatment ot cardtovascular dlaease\ a\ hpld-lowenng drugs. Recent studles suggeat choleaterol-mdependent acttons \uch ag mhilxnon of prenylatlon and acnvauon of G protem5. Our ann was to mvesngate whether HMG-COA reductae mhtbttorc could modulate AngII responses, mveangatmg m vascular amooth muscle cell\ (VSMCs) the effect on CTGF expre$\!on. Result\. m growth-arre\ted VSMC\. AngII, via AT, receptor, mduced CTGF mRNA expresslon (Northern blot) and protem producnon (We\tern blot). A CTGF ann\ense oltgonucleottde decreaed Angll-tnduced fibronectm \yntheFi\. \uggestmg that CTGF could be a medlator of Rbrogenx effects of AngII Pretreatment of VSMC\ wnh HMG-COA reductaFe mhlbnors (\nnva\tatin and atorvastatm: IO- mol/l to IO- mol/l) time- and dose-dependently mhlbned AngII-mduced CTGF producnon The mhlbltlon ot CTGF expresalon was prevented when cella were mcubated wnh mevalonate and other tsoprenolds. AngII actIvate\ Rho protem vla ATI Overexpre%lon of domnxant neganve vector of RhoA suppressed CTGF mductlon by AngII. Inhibttor\ of Rho kinase (Y-27632 and fasudll) do\e-dependently decreased AnglI-mduced CTGF production. ConcIusions: In VSMC\, AnglI, vla AT, receptor and acnvauon of kmase\ ($uch as Rho kmase), regulate CTGF producnon. Our data \ugge\t that statms. through tsoprenotd mhlbitton and therefore mhlbmon of Rho protems, regulate AngII-mduced CTGF producnon These data augget that some of the beneficIaI actiom of rtatm\ could be due to modulation of AngII-medlated vascular damage, accounting for choleterolmdependent protecnve effects.

Los inhibidores de la HMG-COA reductasa regulan la sntesis del factor de crecimiento de tejido conectivo (CTGF) inducido por la angiotensina II. Un efecto adtcional vasoprotector? Ruprez M, Blanco-Colio L, Snchez-Lpez E, Esteban V, Rodrguez-Vita J, Egido J, Ruiz-Ortega M Iwestigncirz Cndiovrrvc~tlnr, 2004; 7: 60-80

HMG-COA reductase inhibitors regulate angiotensin II-induced connective tissue growth factor synthesis. An additional vasoprotective effect? Ruprez M, Blanco-Colio L, Snchez-Lpez E, Esteban V, Rodrguez-Vita J, Egido J, Ruiz-Ortega M hwestigctcir~ C~rr-rliovnscular; 2004; 7: 60-80

Correspondencia / correspondence: M. Ruiz-Ortega Laboratorio de Patologa Vascular y Renal Fundacin Jimnez Daz Avda. Reyes Catlicos, 2 28040 Madrid Email: mruizo@fjd.es

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Angll,

estatinas y

datio

vascular

Angiotensin

II, statins

and

vascular

damage

INTRODUCCIN El estudio del papel del sistema retina angiotensina (SRA) y de su pptido efector, la angiotensina II (Angll) en la progresin de las enfermedades cardiovasculares (aterosclerosis, hipertensin y remodelamiento cardiaco) es un campo de investigacin muy activo en biomedicina (1). La Angll es una verdadera citoquina, capaz de regular el crecimiento celular y los procesos inflamatorios y fibrticos (2-4). Se ha demostrado que frmacos que bloquean su accin (inhibidores del enzima de conversin de angiotensina, iECAs, y antagonistas de los receptores AT,) mejoran la evolucin clnica de estos pacientes (5). Otro tratamiento con resultados beneficiosos en estas patologas es el empleo de los inhibidores de la HMG-COA (3-hdroxi-3-metilglutaril coenzyma A) reductasa (tambin denominados estatinas). Los ensayos clnicos han mostrado que estos frmacos disminuyen la incidencia de episodios coronarios agudos y mejoran la supervivencia (6,7). Los inhibidores de la HMG-COA reductasa actan inhibiendo la enzima limitante en el metabolismo del colesterol, disminuyendo la sntesis heptica de lipoprotenas de baja densidad (LDL). Adems de su eficacia como agentes reductores del colesterol, las estatinas actan a escala intracelular, inhibiendo la proliferacin, migracin y seales intracelulares de las clulas de msculo liso vascular (CMLV) (8,9). Algunos de estos efectos celulares son debidos, al menos en parte, a la inhibicin de la isoprenilacin y a modificaciones postraduccionales de las protenas, que regulan su actividad y localizacin en la membrana. Entre las protenas diana de estas inhibiciones destaca la supetfamilia de protenas G pequeas, que incluyen las protenas Ras, Rho, Rac y Cdc42. Estas GTPasas intracelulares

INTRODUCTION The study of the role of the renin-angiotensin system IRAS) and its effector peptide, angiotensin II (Anglll, in the progression of cardiovascular diseases fatherosclerosis, hypertension and cardiac remodeling) constitutes a very active field for research in biomedicine (1). Angll is a true cytokine, capable of regulating cell growth and inflammatory and fibrotc processes (2-4). It has been shown that drugs blocking its action (angiotensin-converting enzyme inhibitors -ACElsand AT, receptor antagonists) improve the clinical course of these diseases (51. Another treatment with beneficial effects in patents with these conditions is represented by the HMG-COA (3-hydroxy-3-methylglutaryl coenzyme A) reductase inhibitors (also called statinsl. Clinical trials have shown these drugs to decrease the incidence of acute coronary events and to improve survival(6, 7). Statins act by inhibiting the rate-limiting enzyme n cholesterol metabolism, thus decreasing the synthess of low density lipoproteins (LDL) in the liver. In addtion to their efficacy as cholesterol-lowering drugs, statins act at intracellular level, inhibiting vascular smooth muscle cell (VSMC) proliferaton, migration and intracellular signals (8, 9). Some of these cellular effects are at least pattly due to inhibition of isoprenylation and post-translational changes in proteins, regulating their activity and location in the membrane. The target proteins of these inhibitions patticularly include the superfamily of small G proteins, including the proteins Ras, Rho, Rac and Cdc42. These intracellular

Abreviaturas: Angiotensina II: Angll. Receptores de tipo 1 de angiotensina: AT,. Receptores de tipo 2 de angiotensina: AT,. Clulas de msculo liso vascular: CMLV. Enzima de conversin de angiotensina: ECA. Estatinas: inhibidores de la HMG-COA (3.hidroxi-3-metilglutaril coenzyma A) reductasa. Ester de forbol miristato acetato: PMA. Factor de crecimiento de tejido conectivo: CTGF. Factor de crecimiento transformante: TGF-13. Farnesilpirofosfato: FPP. Fibronectina: FN. Geranilgeranilpirofosfato: GGPP. Sistema renina angiotensina: SRA.

Abbreviatons: Angiotensin converting enzyme: ACE. Angiotensin II: Angll. Angiotensin type 1 receptors: AT,. Angiotensin type 2 receptors: AT,. Connective tissue growth factor: CTGF. Famesylpyrophosphate: FPP. Fibronectin: FN. Geranylgeranylpyrophosphate: GGPP. Phorbol myristate acetate ester: PMA. Renin-angiotensin system: RAS. Statins: HMG-COA (3.hydroxy-3-methylglutaryl enzyme Al reductase inhibitors. Transforming growth factor: TGF-/3. Vascular smooth muscle cells: VSMCs.

co-

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M. Ruprez,

L. Blanco-Colio,

E. Snchez-Lpez,

et al.

tienen un papel regulador importante en la transmisin de seales desde la membrana celular hacia el ncleo. Cuando se activan las protenas Ras estimulan una cascada de protenas serina-treoninaquinasa que conduce a un aumento en la transcripcin de algunos genes. Las protenas Rho estn implicadas en la regulacin de diversas funciones celulares, como cambios en el citoesqueleto de actina (contraccin, movilidad, adhesin), activacin de factores de transcripcin, progresin del ciclo celular, apoptosis y transformaciones celulares (10). El acmulo de la matriz extracelular en el sistema cardiovascular desempea un papel muy importante en el desarrollo de la hipertrofia vascular y ventricular y de la insuficiencia cardaca. La hipertensin provoca cambios estructurales en las arterias que incluyen el aumento del depsito de colgenos, la destruccin de las fibras elsticas y la hipertrofia de CMLV (1). Este aumento de la matriz extracelular se ha atribuido a efectos hemodinmicos asociados con el estrs mecnico y a factores como Angll y TGF-13 (1). Recientemente se ha descrito un nuevo y potente factor profibrtico: el factor de crecimiento conectivo tisular (CTGF). Este factor es miembro de la familia CCN (CYRGI, CTGF y NOV) de genes de respuesta tempranos (ll), y ha sido implicado en el desarrollo de varias patologas asociadas a fibrosis, como esclerodermia, queloides y otros desrdenes de la piel, desarrollo de tumores y enfermedades pulmonares y renales (Il, 12). El CTGF es un factor de crecimiento ubicuo que regula la proliferacin de fibroblastos, la adhesin celular, la angiognesis y la estimulacin de la produccin de matriz extracelular (1 I-13). Su importancia en el dao vascular ha sido demostrada al observar que su expresin est aumentada en lesiones aterosclerticas (13) y en infarto de miocardio (14). El CTGF regula el crecimiento, la migracin y la produccin de matriz extracelular en CMLV (12, 15). Por otro lado, la sobreexpresin de CTGF induce apoptosis de CMLV (161, las principales clulas productoras de matriz extracelular. Recientemente se estn realizando terapias combinadas con inhibidores de la HMG-COA reductasa y frmacos que bloquean la Angll. Nuestro objetivo ha sido estudiar la posible relacin entre los inhibidores de la isoprenilacin proteica y las acciones de la angiotensina ll en el dao vascular, estudiando en concreto su efecto sobre la modulacin del CTGF. El conocimiento de los mecanismos moleculares implicados en estos procesos puede ayudar a comprender la patologa cardiovascular y a mejorar las estrategias teraputicas.
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GTPases play a significant regulating role in signa1 transmission from the cell membrane to the nucleus. Activated Ras proteins stimulate a cascade of serine-treonine kinase proteins leading to an increased transcription of some genes. The Rho proteins are implicated in the regulation of different cell functions, such as changes in the actin cytoskeleton (contraction, mobility, adhesion), activation of transcription factors, cell cycle progression, and apoptosis and cell transformations (70). Extracellular matrix accumulation in the cardiovascular system plays a very important role in the development of vascular and ventricular hypertrophy, and heart failure. Hypertension causes structural changes in the arteries, including an increased collagen depost, destruction of elastic fibers, and hypertrophy of VSMCs (7). This increase in extracellular matrix has been attributed to hemodynamic effects associated with mechanical stress and factors such as Angll and TGF-l3 (7). A new and potent profibrotic factor has recently been repotted: the connective tissue growth factor (CTGF). This factor is a member of the CCN family (CYR67, CTGF and NOV) of early response genes (7 7), and has been implicated in the development of different conditions associated to fibrosis, such as scleroderma, keloids and other skin disorders, the development of tumors and lung and kidney diseases (7 7, 72). CTGF is an ubiquitous growth factor that regulates fibroblast proliferation, cell adhesion, angiogenesis and stimulation of extracellular matrix production (7 7- 73). Its significance in vascular damage has been shown by the fact that CTGF expression is increased in atherosclerotic lesions (73) and in myocardial infarction (741. CTGF regulates the growth, migration and production of extracellular matrix in VSMCs 172, 75). On the other hand, CTGF over-expression induces the apoptosis of VSMCs (761, the main cell producing extracellular matrix. Combined therapies with HMG-COA reductase and Angll blockers have recently been introduced. Our purpose was to study the potential relationship between protein isoprenylation inhibtors and angiotensin ll actions in relation to vascular damage, specifically evaluating the effect on CTGF modulation. Knowledge of the molecular mechanisms implicated in these processes may help to understand cardiovascular disease and improve treatment strategies.
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dario

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Angiotensin

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damage

MTODOS Cultivos celulares El cultivo de CMLV se realiz a partir de aortas de ratas Wistar mediante digestin con colagenasa (17). Las clulas se cultivaron en medio DMEM con 10% STF y se usaron entre los pases segundo y sptimo. Las CMLV se caracterizaron por microscopia de contraste de fase, tincin positiva para cc-actina y tincin negativa para el antgeno relacionado con el factor VIII. Cell cultures

METHODS

VSMC culture was petformed using Wistar rat aortas with collagenase digestion (17). The cells were cultured in DMEM medium with 10% FCS, and were harvested between the second and seventh passages. The VSMCs were characterized using phase contrast microscopy, positive staining for a-actin, and negative staining for factor VIII related antigen.

Reactivos y anticuerpos Todos los reactivos para cultivo celular se obtuvieron de Gibco BRL (Paisley, Scotland, UK). El Losar-tan (antagonista del receptor AT,) fue donado por MSD (Madrid, Espaa) y el inhibidor Y27632 es de Tocris (UK). Los istopos radiactivos se obtuvieron de Amersham (Buckinghamshire, UK). Los anticuerpos frente a CTGF son de Torrey Pines (San Diego, USA) y el resto de DAKO. Los anticuerpos secundarios conjugados con HRPO o FITC son de The Binding Site Inc (Birmingham, UK). Los inhibidores de seales intracelulares son de Calbiochem. El resto de los compuestos qumicos empleados provienen de Sigma Chemical (St Louis, MO, USA). Ninguno de los reactivos utilizados fue txico a las dosis empleadas (datos no mostrados). En algunos experimentos se utilizan metabolitos intermedios de la ruta del colesterol (mevalonato, geranilgeranilpirofosfato 0 farnesilpirofosfato).

Reagen ts and an tibodies All reagents for cell culture were obtained from Gibco BRL (Paisley, Scotland, UK). Losattan (an AT, receptor antagonist) was donated by MSD (Madrid, Spain), and the Y-27632 inhibitor was supplied by Tocris (UK). The radioactive isotopes were obtained from Amersham (Buckinghamshire, UK). The antibodies against CTGF were from Torre y Pines (San Diego, USA), and the rest from DAKO. The secondary antibodes conjugated with HRPO or FITC were obtaned from The Binding Site, Inc (Birmingham, UK). The intracellular signa1 inhibitors were from Calbiochem. All other chemicals used were obtained from Sigma Chemical (St Louis, MO, USA). None of the reagents were toxic at the doses used (data not shown). In some experiments, intermediate metabolites in the cholesterol pathwa y were used (mevalonate, geran ylgeran ylp yrophosphate or farnesylpyrophosphate).

Determinacin

de protenas Protein determination Protein levels were determined by immunocytochemistty and/or Western blot using specific antibodies. For immunocytochemistty, the cells were cultured in 8-well plates, fixed with methanol: acetone at -20C and 3% paraformaldeh yde, and permeatedby treatment with 0.5% Triton-X100. For Western blot, proteins were separated by 12% SDS-PAGE electrophoresis under reducing conditions and transferred to polyvin ylidene difluoride membranes. The samples were first incubated for one hour at room temperature in blocking buffer (0.1 mmol/l NaCI, 0.01 mmol/ Tris pH 7.5, 0.1% Tween-20 and 5% semi-skimmed milk), and then for 18 hours at
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Los niveles de protenas se estudiaron por inmunocitoqumica y/o Western blot empleando anticuerpos especficos. Para inmunocitoqumica, las clulas se cultivaron en placas de ocho pocillos, se fijaron con metanol:acetona a -20 C y con 3% paraformaldehdo, y se permebilizaron tratando con 0,5% Tritn-X100. Para Western blot, las protenas se separaron por SDS-PAGE electroforesis al 12% en condiciones reductoras y se transfirieron a membranas de difluorido de polivinilideno. Las muestras se incubaron primero durante una hora a temperatura ambiente en tampn de bloqueo (0,l mmol/L NaCI, 0,Ol mmol/L Tris pH 7,5, O,l% Tween-20 y 5% leche semidesnatada) y despus durante 18 horas a 4 C con el anticuerpo primario correspondiente en el misINVESTIGACIN CARDIOVASCULAR, 2004; val. 7, n.' 1

M. Ruprez, L. Blanco-Colio, E. Snchez-Lpez, et al.

mo tampn. Tras lavarlas, se realiz la deteccin incubando durante una hora a temperatura ambiente con un anticuerpo secundario marcado con peroxidasa, y se revel empleando tcnicas de quimioluminiscencia de ECL (Amershan Pharmacia Biotech, Buckinghamshire, UK). La especificidad de los anticuerpos se realiz por ensayos de bloqueo con pptido control (Western) o incubando las clulas sin el anticuerpo primario (inmunocitoqumica).

4 C with the corresponding primaty antibody in the same buffer. After washing, detection was carried out by incubation for one hour at room temperature with a peroxidase-labeled secondary antibody, followed by development with ECL chemoluminiscence techniques (Amersham Pharmacia Biotech, Buckinghamshire, UKI. Antibody specificity was established by blocking tests with control peptide (Western blotl, or incubating the cells without the primary antibody (immunocytochemistry).

Expresin gnica El RNA total se aisl por el mtodo del Trizo1 (Gibco). Los niveles del mRNA del CTGF se estudiaron por Northern blot (18). La calidad del RNA se determin por electroforesis en geles del 1% agarosa-formaldehdo y tincin con bromuro de etidio. Los cebadores usados fueron: CTGF (sentido: 5-GAGTGGGTGTGTGACGAGCCCAAGG -3, antisentido 5-ATGTCTCCGTACATCTTCCTGTAGT -3) y G3PDH (sentido: 5-AATGCATCCTGCACCACCAA-3, antisentido: 5-GTAGCCATAlTCAlTGTCATA-31, que dan lugar a productos de 378 pb y 515 pb, por PCR (un minuto a 94, dos minutos a 63/54 C y un minuto a 68; 25 ciclos) respectivamente. Para los estudios de Northern blot las membranas fueron prehibridadas durante cuatro horas a 42 C (50% formamida, 1% SDS, 5x SSC, Ix Denhardt, 0,25 mg/ml DNA y 50 mmol/L fosfato sdico pH 6,5), y la hibridacin se realiz a 42 C durante la noche con 20% sulfato de dextrano y a-i3Pl-sonda desnaturalizada. Las membranas se lavaron con 2x SSC, O,l% SDS. Los resultados se expresan como unidades arbitrarias de densitometrado relativas a la G3PDH.

Gene expression Total RNA was isolated by the Trizo1 method (Gibco). CTGF mRNA levels were evaluated by Northern blot (18). RNA quality was assessed by electrophoresis in 1% agarose-formaldeh yde gels and staining with ethidium bromide. The primers used were: CTGF (sense: 5-GAGTGGGTGTGTGACGAGCCCAAGG-3: antisense 5-ATGTCTCCGTACATCTTGTAGT-3) and G3PDH (sense: r-AATGCATCCTGCACCACCA-AS; antisense: 5-GTAGCCATATTCATTGTCATA-3), gving rise to 378 bp and 515 bp products by PCR (ene minute at 94 C, two minutes at 63 OC/54 C, and one minute at 68 C; 25 cycles), respectively. For the Northern blot studies, the membranes were pre-h ybridized for four hours at 42 C (50% formamide, 1% SOS, 5x SSC, Ix Denhardt, 0.25 mg/ml DNA and 50 mmol/ sodium phosphate pH 6.51, and hybrdization was performed at 42C with 20% dextran sulfate and a-f2PI-denatured probe overnight. The membranes were washed with 2x SSC, 0.1% SDS. The results are expressed as arbitrary densitometry units relative to G3PDH.

Transfecciones

transitorias

Transent transfections The cells were transiently transfected with Fugene 6 (Roche, Basel, Swtzerland) in the presente of different expresson vectors; pcDNA-NlSRhoa: negative domnant of RhoA, pcDNA-Q63LhoA: constitutive form of RhoA, pcDNA-wtRhoA: wild form of RhoA, pcDNA3B: e,mpty vector (kindly donated by doctor Angel Pascual, of the Instituto de Investigaciones Biomdicas, Madrid, Spain). Following transfection, the cells were maintained for 24 hours without serum before stimulation.
INVESTIGACIN CARDIOVASCULAR, 2004; val. 7, II. 1

Las clulas fueron transfectadas de forma transitoria con Fugene 6 (Roche, Base1 Switzerland) en presencia de los diferentes vectores de expresin; pcDNA-N19RhoA: dominante negativo de RhoA, pcDNA-Q63LhoA: constitutivo de RhoA, pcDNAwtRhoA: forma salvaje de RhoA, pcDNA3B: vectpr vaco (donados amablemente por el doctor Angel Pascual del Instituto de Investigaciones Biomdicas, Madrid). Despus de la transfeccin, las clulas se mantuvieron 24 horas sin suero antes de ser estimuladas.
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Angll, estatinas y dao vascular

Angiotensin II, statins and vascular damage

Anlisis estadstico de los resultados

y valoracin

Las pelculas de autoradiografa fueron escaneadas usando el densitmetro GS-800 Calibrated Densitometer (Quantity One, BioRad, ). Los datos se expresan en unidades arbitrarias de densitometrado como incremento respecto al control (nveces) de media k error estndar de la media (EEM) o como resultado representativo de varios experimentos. El anlisis de significacin se realiz empleando el programa GraphPad Instat (GraphPad Software, San Diego, USA). Las poblaciones se compararon utilizando el test de Wilcoxon y el de Student-Newman-Keuls, considerando diferencias significativas cuando p < 0,05.

Statistical analysis and assessment of the results Autoradiographic films were scanned using a GS-800 Calibrated Densitometer (Quality One, BioRadl. The data are expressed in arbitrary densitometry unts as increase over the control fn-fold) in the mean 2 standard error of the mean (SEnll), oras a representative res& of severa/ experiments. Statistical significance was evaluated using the GraphPad Instat program (GraphPad Software, San Diego, USA). The populations were compared using the Wilcoxon test and the Student-Newman-Keuls test, considering as signficant a value of p < 0.05.

RESULTADOS La angiotensina ll aumenta la expresin gnica y la sntesis proteica de CTGF en clulas de msculo liso vascular En primer lugar, investigamos si la Angll era capaz de regular la expresin y sntesis de CTGF en CMLV. Las clulas, en fase de reposo, se estimularon con IO- mol/L Angll durante l-24 horas. Transcurrido el tiempo de incubacin, el RNA se aisl y se analiz la expresin gnica de CTGF por Northern blot. Como controles positivos de la induccin del CTGF se emplearon TGF-C3(1 ng/mL) y el ester de forbol PMA (IO- mol/L). Las CMLV en reposo presentaron niveles muy bajos de expresin de CTGF (Figura IA). El tratamiento con Angll aument los niveles del mRNA de CTGF con una respuesta muy rpida, mxima a las tres horas y disminuy, aunque sin alcanzar niveles control, a las seis horas (Figura IB). La estimulacin con Angll a tiempos ms largos (18 y 24 horas) tambin aument la expresin gnica del CTGF. Esta cintica es similar a la observada en respuesta al TGF-13, aunque la respuesta del TGF-13 es mayor a tiempos ms cortos. Para determinar si el aumento en la expresin gnica del CTGF se corresponda con un aumento en su produccin proteica realizamos estudios de Western blot, utilizando un anticuerpo que reconoce los aminoacidos 247-260 del CTGF. En CMLV quiescentes se observ en la fraccin celular, pero no en la fraccin liberada al sobrenadante, la expresin de una nica banda de unos 38kDa correspondiente al peso molecular aparente del CTGF (Figura 2A). La estimulacin con Angll durante 24 horas caus un aumento en los niveles
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RESULTS Angiotensin II increases gene expression and protein synthesis of CTGF in VSMCs

In the present study we first evaluated whether Angll was able to regulate the expression and synthesis of CTGF in VSMCs. Growth-arrested cells were stimulated with 1p7 mol/L Angll for 1 to 24 hours. Following incubation, RNA was isolated and genic expression of CTGF was assessed by Northern blot. TGF-13 (1 ng/ml) and phorbol myristate acetate ester (PMAl (lp7 mal/) were used as positive CTGF induction controls. Resting VSMCs showed very low levels of CTGF expression (Figure IA). Treatment with Angll increased the levels of CTGF mRNA, with a very rapid response that reached a maximum at three hours and decreased at six hours but did not return to control levels (Figure IB). Stimulation with Angll for longer time periods (18 and 24 hours) likewise increased gene expression of CTGF. These kinetics are similar to those seen in response to TGF-13, though the response to TGF-B was greater afier shotter time perods. In order to determine whether an increased genic expression of CTGF was associated to an increased protein synthesis, Western blot studies were conducted using an antibody that recognizes amino acids 247-260 of CTGF. In quiescent VSMCs, expression of a single band of approximately 38 kDa, corresponding to the apparent molecular weight of CTGF, was seen in the cellular fraction, but not in the fraction released into the supernatant (Figure ZA). Stmulation with Angll for 24 hours resulted in
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M.

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et al.

Figura

1. La Angll aumenta la expresin gnica del CTGF en clulas de msculo liso vascular. Las clulas en reposo fueron tratadas con Angll IO- mol/L desde l-24 horas. El TGF-13 (1 ng/mL) y el PMA (IO- mol/L) fueron utilizados como controles positivos. La figura A muestra un Northern blot representativo de ocho realizados, donde se puede observar que las CMLV en estado de reposo expresan constitutivamente el mRNA del CTGF, correspondiente a una banda de 2,4 Kb, y en respuesta a la estimulacin con Angll se induce una sobreexpresin de este gen. La figura B muestra los valores de las medias + EEM. * p < 0,05 VS control. Las barras negras representan los valores de Angll, las blancas de TGF-13 y las rayadas de PMA. Los valores del mRNA del CTGF, obtenidos por anlisis densitomtrico, se expresan como la relacin CTGF/GBPDH en n-veces respecto al incremento del control.

A .

II

.-

> --

:t

- 1. -.

iaI

1. Angll Icreases the genc expression of CTGF n VSMCs. Resting cells were treated with Angll l7 mol/l for 1 to 24 hours. TGF-B (1 ng/ml) and PMA (1p7 mal/) were used as positive controls. A. The figure shows a representative Northern blot of the eight tests petformed, showing that resting VSMCs constitutively express CTGF mRNA, corresponding to a 2.4 Kb band, and that overexpression of this gene is induced in response to stimulation with Angll. Figure B shows the means + SEM. * p < 0.05 VS control. The black bars represent the values of Angll, while the white bars correspond to TGF-8 and the striped bars to PMA. The CTGF mRNA values, obtained by densitometric analysis, are expressed as the CTGF/G3PDH ratio in n-fold compared to the control increase.

Figure

de CTGF en la fraccin celular, similares a los causados por el TGF-13 y PMA (Figura 2A). Adems, indujo la liberacin de CTGF al medio extracelular (Figura 2A). Este aumento en la produccin de CTGF (celular y soluble) slo se observ despus de 24 horas, y se mantuvo elevado hasta las 72 horas (Figura 2B). Mediante immunocitoqumica hemos localizado la tincin para CTGF. Las CMLV en reposo apenas presentan tincin frente a CTGF, pero al tratarlas con Angll o 10% STF (control positivo) durante 48 horas se observ un claro aumento de la tincin a nivel citoplasmtico (Figura 2C), lo que confirma el aumento en la produccin de CTGF causado por la Angll. Como control negativo de la tcnica, algunas muestras se incubaron en ausencia de anticuerpo primario (no mostrado).

an increase in CTGF levels in the cellular fraction similar to that caused by TGF-l3 and PMA (Figure 2A). CTGF release into the extracellular medium was also induced (Figure 2A). The increase on CTGF production (cellular and soluble) was only seen after 24 hours, and was sustained for 72 hours (Figure 2B). CTGF staining was detected by immunohistochemistry. Resting VSMCs barely showed CTGF staining, but after treating them with Angll or 10% FCS (positive control) for 48 hours, a clear increase in staining was noted at cytoplasmic level (Figure 2C), confirming the increase in CTGF production caused by Angll. As negative control for the technique, some samples were incubated in the absence of primary antibody (not shown).

Los receptores AT, estn implicados en la regulacin de CTGFcausada por angiotensina

en clulas de msculo liso vascular

II

AT, receptors are implicated in CTGF regulaton caused by angiotensin II in VSMCs In earlier studies we have shown that VSMCs, under our culture conditions, express AT, and AT, receptors (17). The next objective was to determine the type of receptor involved
INVESTIGACION CARDIOVASCULAR, 2004, val. 7, n. 1

En estudios previos hemos demostrado que las CMLV, en nuestras condiciones de cultivo, expresan los receptores AT, y AT, (17). El siguiente 66

Angll,

estatinas

y dao

vascular

Angiotensin

II, statins

and

vascular

damage

Figura 2. La Angll aumenta la produccin de CTGF citoslica y liberada al sobrenadante, en clulas de msculo liso vascular. Las clulas quiescentes fueron tratadas con Angll 1w7 mol/L desde 3 a 72 horas y se analiz la produccin de CTGF por Western blot. Se observ una banda de alrededor de 40 kDa, correspondiente al tamao molecular del CTGF. La Angll aumenta la produccin de CTGF celular (fraccin citoslica) y soluble (liberada al sobrenadante) despus de 24, pero no de seis horas de tratamiento. La figura A muestra un Western blot representativo de cinco realizados, la tubulina se utiliz como control de carga. B. Los resultados de la produccin del CTGF se obtuvieron por anlisis densitomtrico y se expresan como la relacin CTGF/ tubulina en n-veces respecto al incremento sobre control. Las barras negras representan los valores de Angll y las blancas de TGF-13. * p c 0,05 vs control. C. Localizacin del CTGF por inmunocitoqumica. Las clulas en reposo no expresan CTGF, sin embargo, al tratarlas durante 48 horas con Angll o 10% STF se observa un claro aumento de la tincin citoplasmtica. B

Figure 2. Angll

increases the production of cytosoli and supernatant-released CTGF in VSMCs. Quiescent cells were treated with Angll 1O-7 mol/l for 3 to 72 hours, and CTGF production was analyzed by Western blot. A band of approximately 40 kDa was seen, corresponding to the molecular size of CTGF. Angll increases the production of cellular (cytosolic fraction) and soluble CTGF (released into the supernatant) after 24 hours, but not after six hours of treatment. Figure A is a representative Western blot of the five tests petformed; tubulin was used as loading control. B. The results of CTGFproduction were obtained by densitometric analysis, and are expressed as the CTGF/ubulin ratio in n-fold compared to the control increase. The black bars represent the values of Angll, while the white bars correspond to TGF-13. * p < 0.05 VS control. C. Localization of CTGF by immunocytochemistty. Resting cells do not express CTGF, but after treating them for 48 hours with Angll or 10% FCS, a marked increase in cytoplasmic staining was seen.

objetivo fue determinar el tipo de receptor implicado en la induccin de CTGF mediante el empleo farmacolgico de antagonistas no peptdicos especficos para cada subtipo de receptor. Los antagonistas del receptor AT, son bifenilimidazoles, representados por losartan, mientras que los del AT, son tetrahidroimidazopiridinas, como PD123319. Las clulas vasculares, en estado de reposo, fueron preincubadas durante 30 minutos con los anINVESTIGACIN CARDIOVASCULAR, 2004; val. 7, n. 1

in CTGF induction, based on the pharmacological use of non-peptide antagonists specific for each receptor subtype. The AT, receptor antagonists are biphenylimidazoles, represented by losartan, while the AT, receptor antagonists are tetrahydroimidazopyridines such as PD123379. The vascular cells, under resting conditions, were preincubated for 30 minutes with the AT, (losartan) and AT, antagonists (PD1233791
67

M. Ruprez,

L. Blanco-Colio,

E. Snchez-Lpez,

et al.

tagonistas AT, (losartan) y AT, (PD1233191 (lO$ mol/L y lOe5 mol/L, respectivamente), y posteriormente se estimularon con Ic mol/L Angll durante 24 horas. A ambos tiempos, el antagonista AT, caus una disminucin significativa de la expresin del mRNA del CTGF inducida por la Angll, mientras que el antagonista AT, no tuvo efecto (Figura 3A). La sntesis de CTGF inducida tras 24 horas de tratamiento con Angll slo fue inhibida por el antagonista AT,, pero no por el AT, (Figura 3B). Ninguno de estos antagonistas por s mismo afect significativamente la expresin de CTGF en clulas control (datos no mostrados).

(106 mol/l and 1@ mal/, respectively), and subsequently stimulated with l7 mol/l Angll for 24 hours. At both timepoints, the AT, antagonist caused a significant decrease in CTGF mRNA expression induced by Angll, while the AT, antagonist had no effect (Figure 3A). CTGF synthesis induced after 24 hours of treatment with Angll was inhibited by the AT, antagonist but not by the AT, antagonist (Figure 36). Neither of these antagonists alone significantly affected CTGF expression in control cells (data not shown).

Los inhibidores de la HMG-COA reductasa disminuyen la produccin de CTGF causada por Angll
Para determinar si las estatinas pueden regular las respuestas de la Angll, las CMLV fueron pretratadas durante una hora con dos estatinas: atorvastatina y simvastatina (1t7 mol/L to lOmg mol/L). La atorvastatina caus una inhibicin, de forma dosis dependiente, de la produccin de CTGF inducida por Angll (Figura 4). Un efecto inhibitorio similar se observ con las simvastatina, indican-

HMG-COA reductase inhibitors decrease angiotensin Il-induced CTGF producton In order to determine whether statins are able to regulate Angll responses, VSMCs were pretreated for one hour with two statins: atorvastatin and simvastatin (IU- mal/ to lPg mol/l). Atorvastatin caused a dose-dependent inhibition of Angll-induced CTGF production (Figure 4). A similar inhibitory effect was seen with simvastatin, suggesting of a class effect of these agents in the regulation of CTGF. In control cells, statins, at the doses

Figura 3. El CTGF est regulado por los receptores AT, en clulas de msculo liso vascular. Las clulas fueron pretratadas durante 30 minutos con IO+ mol/L losartan (antagonista AT,) o IO5 mol/L PD123319 (antagonista AT,), y despus estimuladas con Ir7 mol/L Angll durante 24 horas. La figura A muestra un Northern blot representativo y B muestra un Western blot representativo de seis realizados.

CTGF

L mRNA de CTGF (n-veces) CTGFmRN.4 (-fdd, n.. Produccvm de CTGF/ CTGF,xodc,,on * . a

Figure 3. CTGF (AT, antagonist) Figure A shows

is regulated by AT, receptors in VSMCs. The cells were pretreated for 30 minutes with lo-6 ml/ losartan or 1P5 mal/ PD123319 (AT, antagonist), and subsequently stimulated with 1P7 mol/l Angll for 24 hours. a representative Northern blot, while B shows a representative Western blot of the six tests performed.

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CARDIOVASCULAR,

2004, VO. 7, n o 1

68

Angll,

estatinas

y dao

vascular

Angiotensin

II, statins

and

vascular

damage

Figura 4. Efecto de inhibidores de la HMG-COA reductasa en la produccin de CTGF inducida por Angll. Las CMLV fueron preincubadas con dos estatinas: la atorvastatina (A) y simvastatina (BI (1Od mol/L a lOe9 mol/L) y posteriormente estimuladas con Angll (IO- mol/L) durante 24 y 48 horas. Algunas clulas fueron preincubadas con mevalonato y de otros metabolitos de la ruta del colesterol; el geranilgeranilpirofosfato (GGPP) y el farnesilpirofosfato (FPP) (16 mol/L). La figura muestra un Western blot representativo de tres. Como control de carga se utiliz el rojo ponceau (Al y la tubulina (B).

Figure 4. Effect of HMG-COA reductase inhibitors on Angll-induced CTGF production. VSMCs were preincubated with two statins: atorvastatin (A) and simvastatin (B) (Ie mol/l to 1cg mol/l), and subsequently stimulated with Angll (1W7 mol/l) for 24 and 48 hours. Some cells were preincubated with mevalonate and other metabolites of the cholesterol pathway: geranylgeranylpyrophosphate (GGPP) and farnesylpyrophosphate (FPP) (1p5 mol/l). The figure shows a representative Western blot of the three tests performed. Ponceau red (Al and tubulin (B) were used as loading control.

do un efecto de clase de estos agentes en la regulacin del CTGF. En clulas control, las estatinas, a las dosis estudiadas, no modificaron los niveles proteicos del CTGF ni indujeron apoptosis (no mostrado). Evaluamos el efecto del mevalonato, el metabolito directo de la HMG-COA reductasa, para demostrar que el efecto de las estatinas es debido a la inhibicin de esta enzima. En presencia de L-mevalonato (10m4 mol/L) el efecto inhibitorio se revirti. Tambin determinamos el papel de los isoprenoides geranilgeranilpirofosfato (GGPP) y farnesilpirofosfato (FPP) en la regulacin del CTGF. El cotratamiento de las clulas con GGPP (5 x 10m5 mol/L) completamente revirti la induccin de CTGF causada por Angll, mientras que el FPP slo caus una ligera disminucin, lo que sugiere que protenas geranilgeraniladas regulan la produccin de CTGF.

studied, did not modify the protein levels of CTGF or induce apoptosis (not shown). We evaluated the effect of mevalonate, the direct metabolite of HMG-COA reductase, to show that the efect of statins is due to inhibition of this enzyme. In the presente of L-mevalonate (IO4 mol/l), the inhibitory effect was reversed. We also determined the role of the isoprenoids geranylgeranylpyrophosphate (GGPP) and farnesylpyrophosphate (FPPI in CTGF regulation. Co-treatment of the cells with GGPP (5 x 1O5 mol/l) completely reversed CTGF induction caused by Angll, while FPP only caused a slight decrease, which suggests that geranylgeranylated proteins regulate CTGF production.

Small G proteins participate induced CTGF production Las protenas G pequeas participan en la produccin de CTGF inducida por Angll

in angiotensin

II

Diferentes receptores acoplados a protenas G, incluidos los AT,, activan el sistema p2lras y Rho/quinasa Rho (19, 20). Hemos evaluado el papel de las protenas Rho en la sntesis de CTGF inducida por Angll, mediante varias estrategias: 1) el uso de inhibidor de la actividad GTPasa de Rho,

Different receptors coupled to G proteins, including AT, receptors, activate the p2lras and Rho/Rho kinase system (19, 20). We evaluated the role of Rho proteins in CTGF synthesis induced by Angll by severa/ approaches: 1) the use of an inhibitor of Rho GTPase activity; 2) the use of transient transfections of expression vectors that overexpress different
69

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M. Ruprez,

L. Blanco-Colio,

E. Snchez-Lpez,

et al.

2) el empleo de transfecciones transitorias de vectores de expresin, que sobreexpresan diferentes formas mutadas de las protena RhoA, como el dominante negativo y el constitutivamente activo, lo que conduce a su bloqueo o a su activacin, 3) y el uso de inhibidores de la protena quinasa de Rho. En CMLV utilizamos la exoenzima C3 de Closticfium botulinum, que es un inhibidor de la actividad GTPasa de Rho. La preincubacin de CMLV con la exoenzima C3 durante 48 horas, tiempo necesario para inhibir la actividad GTPasa, disminuy la produccin de CTGF causada por Angll (Figura 5). El bloqueo de RhoA, mediante transfecciones transitorias con un vector dominante negativo de RhoA (NI9 RhoA), inhibi la produccin de CTGF causada por Angll (Figura 6). En las clulas transfectadas con el vector constitutivo de RhoA (Q63L-RhoA) se observ un aumento en la produccin de CTGF. Como controles del experimento se utilizaron el vector wi/d type (forma salvaje) de RhoA y el vaco que no aumentaron la sntesis de CTGF. Adems, en las clulas transfectadas con el vector vaco y estimuladas con Angll existi un aumento en la produccin de CTGF (Figura 6). Los inhibidores de la protena quinasa de Rho, la cual transmite la sealizacin mediada por Rho: el Y-27632 y el fasudil, disminuyeron de forma dosis dependiente la produccin de CTGF inducida por Angll (Figura 71, demostrando la participacin de las protenas Rho en la regulacin de CTGF.

mutated forms of the RhoA protein, such as the dominant negative and the constitutively active forms, leading to its blockade or activation; and 3) the use of inhibitors of the Rho kinase. In VS/VKs, the exoenzyme C3 of Clostridium botulinum, which is an inhibitor of Rho GTPase activity, was used. Preincubation of VSMCs with exoenzyme C3 for 48 hours, the time required to inhibit GTPase activity, decreased the production of CTGF induced by Angll (Figure 5). RhoA blockade by transient transfections with a negative dominant vector of RhoA (NI9 RhoA) inhibited Angll-induced CTGF production (Figure 6). In the cells transfected with the constitutive vector of RhoA (Q63L-RhoA), an increased CTGF production was seen. As controls for the experiment, the wild type vector of RhoA and the empty vector were used, and did not increase CTGF production. Moreover, in cells transfected with the empty vector and stimulated with Angll, CTGF production was increased (Figure 6). The inhibitors of the Rho protein kinase, which transmits signaling mediated by Rho, i.e., Y-27632 and fasudil, dose-dependently decreased Angll-induced CTGF production (Figure 7), which shows the involvement of Rho proteins in CTGF regulation.
These results suggest that Angll, through the AT, receptor, and by means of the activation of Rho and Rho kinase, regulates CTGF synthesis.

Estos resultados sugieren que la Angll a travs del receptor AT, y mediante la activacin de Rho y de la protena quinasa de Rho regula la sntesis de CTGF.

CTGF mediates fibrosis caused by angiotensin II CTGF is able to induce the expression of extracellular matrix proteins, such as type I collagen, fibronectin and a5 integrin (ll). CTGF has also been reported to be a mediator of fibrosis caused by TGF-13 (21). Our last objective was to assess whether CTGF was

El CTGF es un mediador por la Angll

de la fibrosis causada

El CTGF es capaz de inducir protenas de matriz extracelular,

la expresin de como colgeno

Figura 5. Papel de las protenas G pequeas (Rho) en la produccin de CTGF inducida por Angll. Las CMLV fueron con preincubadas con la exoenzima C3 (5 pg/mL) durante 48 horas, y se estimularon con Angll (IO- mol/L) durante otras 24 horas. La figura muestra un Western blot representativo de tres realizados.

% 2 2 P

t $2 EE! .ij $ sF

i: Ou EE .N B SS

Figure 5. Role of small G proteins (Rho) in Angll-nduced CTGF production. VSMCs were preincubated with exoenzyme C3 (5 pg/ml) for 48 hours, and stimulated with Angll(1~7mol/ll for another 24 hours. The figure shows a representative Western blot of the three tests performed.

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Angll,

estatinas

y dao

vascular

Angiotensin

Il, statins

and

vascular

damage

Figura 6. La Angll va RhoA regula la produccin de CTGF. Las CMLV fueron transfectadas de forma transitoria con los diferentes vectores de expresin: NlSRhoA: dominante negativo de RhoA, wtRhoa: wilt type de RhoA, Q63L-RhoA: constitutivo de RhoA, 38: vector vaco, durante 24 horas y se estimularon con Angll (Ir7 mol/L) durante otras 24 horas. La figura muestra un Western blot representativo de cinco realizados.

E i= CTGF [G L!~.LdL~]

< 0

s 4 + 4, e

Figure 6. Angll via RhoA regulates CTGF production through RhoA. VSMCs were transiently transfected with dif. golf .

Tubulina I Tbh

-rrmlD~-~~-~, -

hours, and were then stimulated with Angll ( l7 mol/l) for another 24 hours. The figure shows a representative Western blot of the five tests per-

Figura 7. Los inhibidores de la protena quinasa de Rho disminuyen la produccin de CTGF inducida por Angll. Las CMLV fueron preincubadas con Y27632 (A) o fasudil (B) y posteriormente estimuladas con Angll (lOe7 mol/L) durante 24 horas. La figura muestra un Western blot representativo de tres. Como control de carga se utiliz la tubulina.

Figure 7. Rho protein kinase inhibitors decrease Angll-induced CTGF production. VSMCs were preincubated with Y-27632 (A) or fasudil (B), and subsequently stimulated with Angll (l mol/U for 24 hours. The figure shows a representative Western blot of the three tests performed. Tubulin was used as loading control.

tipo 1,fibronectina e integrina cx5 (11). Adems se ha descrito que es un mediador de la fibrosis causada por TGF-B (21). Nuestro ltimo objetivo fue evaluar si el CTGF estaba implicado en la fibrosis causada por la Angll, utilizando un oligonucleotido antisentido de CTGF, construido por 16 bases derivadas del sitio de inicio de la transcripcin, que contiene el sitio de inicio ATG y cuya secuencia es 5-TACTGGCGGCGGTCAT-3. La incubacin en presencia del oligonucleotido antisentido de CTGF (20 pg/mL), ariadido directamente al medio sin re-

involved in fibrosis caused by Angll by using an antisense oligonucleotide of CTGF composed of 16 bases derived from the transcription starting site, which contains the ATG starting site and whose sequence is 5- TACTGGCGGCGGTCAT-3. Incubation in the presente of the antisense oligonucleotide of CTGF (O pg/ml), directly added to the medium without transfection reagents, reduced Angll-induced fibronectin synthesis (Figure 8). The antisense oligonucleotide of CTGF was also
71

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7, n. 1

M.

Ruprez,

L. Blanco-Colio,

E. Snchez-Lpez,

et

al.

participa en la induccin de fibronectina causada por la Angll. Las clulas fueron estimuladas con Angll (IO- mol/L) durante 48 horas. Para neutralizar el CTGF se emple un oligonucletido antisentido. Como control negativo se utiliz el oligonucletido sentido de CTGF y como control positivo de produccin de FN se emple 10% STF. B. Papel del CTGF en la induccin de CTGF causada por la Angll. Las clulas fueron estimuladas durante 24 horas. Las figuras muestran un Western blot (A) y una RT-PCR (B) representativos de tres realizados (panel superior) y los valores de las medias + EEM obtenidos por anlisis densitomtrico se muestran en el panel inferior.

Figura 8. A. El CTGF

figure

8. A. CTGF is mvolved N, tibronectin induction caused by Angll. The cells were stmulated with Angll (l moVI) for 48 hours. An antisense oligonucleotide was used to neutralize CTGF. The CTGF sense oligonucleotide was used as negative control, while 10% FCS was used as positive control for FN production. B. Role of CTGF in CTGF induction caused by Angll. The cells were stimulated for 24 hours . The figures show a Western blot (A) and a RT-PCR (B) representative of the three performed (top panel); the means 2 SEM obtained by densitometric analysis are shown in the lower panel.

activos de transfeccin, disminuy la sntesis de fibronectina inducida por Angll (Figura 8). Adems, hemos observado que el oligonucleotido antisentido de CTGF disminuy la expresin gnica de CTGF inducido por la Angll (Figura 8).

seen to decrease the gene expression of CTGF induced by Angll (Figure 8).

DISCUSSION DISCUSIN El CTGF es un mediador inducida por Angll de la fibrosis vascular CTGF mediates angiotensin vascular fibrosis Il-induced

En este trabajo hemos demostrado que en CMLV la Angll, mediante su unin a los recepto72

This study has demostrated that Angll, by binding to AT, receptors, increases CTGF expression and synthesis in VSMCs, with a

INVESTIGACIN

CARDIOVASCULAR,

2004; val. 7, n.O 1

Angll,

estatinas

y dao

vascular

Angiotensin

Il, statins

and

vascular

damage

Figura 9. Hiptesis: las estatinas, va protenas Rho, regulan la produccin de CTGF inducida por Angll. La Angll va AT, y activacin de diversas seales intracelulares como protenas como Rho, regula la expresin y sntesis de CTGF. En este trabajo hemos demostrado que la inhibicin de las protenas Rho (transfecciones de vector dominante negativo y uso del inhibidor de la quinasa de Rho) disminuye la produccin de CTGF causada por Angll. Adems, el CTGF participa en la acumulacin de la matriz extracelular causada por la Angll. Las estatinas actan a nivel intracelular inhibiendo la ruta de sntesis del colesterol y la produccin de mevalonato y otros metabolitos implicados en la isoprenilacin de protenas. Las estatinas inhiben las respuestas celulares causadas por la Angll (produccin de CTGF) al inhibir la activacin de protenas Rho.

, 2 I

AT,

1 7

\
Seales intracelulares by AT,

Acetil-CoA / Acetyl COA

HMG-COA reductasa / HMG-COA

activated

reductase

Mevaionato Mevalonate

Sntesis de protenas Protein synthesis

Farn&il-PP Farnesyl-PP

Regulacin de la matriz extracelular / Extracellular matrix

Geranil-GPP Geranyl-GPP

Coleziterol Cholesterol Figure tivation shown

9, Hypothesis: statins, through Rho proteins, regulate Angll-induced CTGF production. Angll, through AT, and acregulates CJGF expression and synthesis. Jhis study has of various intracellular signals such as Rho, proteins, that inhibition of Rho proteins (transfections of dominant negative vector and use of Rho kinase inhibitor) decreases Angll-induced CTGF production. Moreover, CTGF is involved in the accumulation of extracellular matrix caused by Angll. Statins act at an intracellular leve/ by inhibiting the cholesterol pathway and the production of mevalonate and other metabolites implicated in protein prenylation. Statins inhibit the cellular responses caused by Angll (CTGF production) by inhibiting Rho protein activation.

res AT,, aumenta la expresin y sntesis de CTGF, presentando una respuesta similar a la descrita con potentes inductores del CTGF, como TGF-13 y PMA (Il). Adems, hemos observado que el bloqueo del CTGF, mediante oligonucleotidos antisentido, inhibe la produccin de fibronectina inducida por la Angll. Todos estos datos demuestran que el CTGF es un mediador de la fibrosis inducida por Angll. Se ha descrito un aumento de la Angll local en diversas situaciones de dao vascular, como en placas aterosclerticas humanas (22). En estas lesiones se ha demostrado la presencia de CTGF, localizado principalmente en CMLV y en algunas clulas endoteliales, situadas
INVESTIGACIN CARDIOVASCULAR, 2004: WI. 7, no 1

response similar to that reported with potent CTGF inducers, such as TGF-13 and PMA (ll). We also noted that CTGF blockade using antisense oligonucleotides inhibits Angll-induced fibronectin production. All these data show that CTGF is a mediator of Angll-induced fibrosis. An increase in local Angll has been reported in different situations of vascular damage, such as in human atherosclerotic plaques (22). In these lesions the presente of CTGF has been demonstrated, mainly in the VSMCs and in some endothelial cells, located in areas with an accumulation of extracellular matrix and fibrosis ( 13). In the 73

M. Ruprez, L. Blanco-Colio, E. Snchez-Lpez, et al.

en reas con acmulo de matriz extracelular y fibrosis (13). En corazones de pacientes con isquemia cardaca y de ratas sometidas a infarto de miocardio se ha descrito un aumento de CTGF (14). La hipertensin es un factor de riesgo en el desarrollo de enfermedades coronarias y arteriosclerosis (1). En un modelo de hipertensin en ratas inducido por ciclosporina (CsAl y dieta alta en sodio, la expresin de CTGF est elevada en arterias epicardicas, correlacionada con una elevacin en la presin sangunea (23). El estrs mecnico puede ser responsable del aumento de CTGF en los animales hipertensos, ya que el estiramiento mecnico cclico induce CTGF, aumenta la produccin de Angll en el medio de cultivo y la produccin de matriz extracelular (18, 24). Sin embargo, en el modelo de CsA el antagonista AT, slo disminuy de forma parcial la presin sangunea y el desarrollo de la hipertrofia ventricular izquierda, mientras que normaliz la expresin cardaca de CTGF (23). El tratamiento con frmacos que bloquean el SRA ha demostrado efectos beneficiosos en diversas patologas cardiovasculares, independientemente de la presencia de hipertensin. Nuestros datos sugieren que los efectos beneficios del bloqueo del SRA podran atribuirse al bloqueo de las acciones directas de la Angll sobre las clulas diana del dao vascular, en concreto a su efecto en la sobreexpresin de CTGF y de matriz extracelular que se observa en estas enfermedades.

hearts of patients with cardiac ischemia and rats subjected to myocardial infarction, increased CTGF levels have been reported (14). Hypertension is a risk factor for the development of coronary dscase and arteriosclerosis (1). In a rat model of hypertension induced by cyclosporine (CsA) and a high sodium diet, CTGF expression is increased in epicardial arteries, in correlation to a rise in blood pressure (231. Mechanical stress can be responsible for the increase in CTGF in hypertensive animals, since cyclic mechanical stretching induces CTGF and increases Angll production in culture medium and the production of extracellular matrix (18, 24). However, in the CsA model, the AT, antagonist only partially decreased blood pressure and the development of left ventricular hypertrophy, while the cardiac expression of CTGF normalized (23). Treatment with drugs that block the RAS has been shown to have beneficial effects in various cardiovascular diseases, regardless of the presente of hypertension. Our data suggest that the beneficial effects of RAS blockade could be attrbuted to blockade of the direct actions of Angll upon the target cells for vascular damage, and specifically to its effect on the overexpression of CTGF and extracellular matrix seen in these diseases.

Las estatinas regulan la produccin causada por Angll

de CTGF

Statins regulate CTGFprodmtion angiotensn II

caused by

En este trabajo hemos observado que dos estatinas, atorvastatina y simvastatina, disminuyen de forma dosis dependiente la produccin de CTGF inducida por Angll. Estudios en modelos experimentales han demostrado que los inhibidores de la HMG-COA reductasa son beneficiosos en el tratamiento de diversas enfermedades cardiovasculares, disminuyendo, entre otros parmetros, la fibrosis (25). En modelos de aterosclerosis se ha demostrado que estos frmacos disminuyen la formacin de neontima y fibrosis y estabilizan las placas aterosclerticas (26, 27). En un modelo de infarto de miocardio en ratas se ha observado que la fluvastatina atena la hipertrofia de miocitos y la fibrosis intersticial, asociado a una disminucin en la actividad de metaloproteinasas (26). Estudios in vitre tambin demuestran que las estatinas son capaces de regular la matriz extracelular. En clulas mesangiales tratadas con suero, glucosa o LDL, las estatinas disminuyen la expresin de prote74

This study showed that two statins, atorvastatn and simvastatin, dose-dependently decrease Angll-induced CTGF production. Studies in experimental models have shown HMG-COA reductase inhibitors to be beneficial in the treatment of various cardiovascular diseases by decreasing fibrosis, among other parameters 125). In models of atherosclerosis, these drugs have been shown to reduce fibrosis and neointima formation, and to stabilize the atherosclerotic plaques (26, 27). In a myocardial infarction model in rats, fluvastatin has been shown to attenuate myocyte hypertrophy and interstitial fibrosis, effects associated to a decreased metalloproteinase activity (26). In vitro studies also demonstrate that statins are able to regulate the extracellular matrix. In mesangial cells treated with serum, glucose or LDL, statins reduce the expression of extracellular matrix proteins (28-30). However, in VSMCs they only regulate the mRNA of
INVESTIGACIN CARDIOVASCULAR, 2004; val. 7, n. 1

Angll, estatinas y daiio vascular

Angiotensin II, statins and vascular damage

nas de matriz extracelular (28-30). Sin embargo, en CMLV slo regulan el mRNA de protenas de matriz extracelular a concentraciones txicas (31). En relacin a las respuestas de Angll, las estatinas inhiben la expresin de c-Fos y c-Jun y la sntesis de protenas (32, 33). En un modelo de dao cardiaco mediado por Angll, la cerivastatina atenu la hipertrofia cardaca y la fibrosis (reduciendo los niveles de fibronectina, laminina y colgeno tipo 1) (34). Nuestros datos demuestran que las estatinas disminuyen la produccin de CTGF inducida por Angll, sugiriendo una protena diana de la accin de las estatinas. La relacin entre inhibidores de la HMG-COA reductasa y CTGF se ha observado en otros tipos celulares. As, en clulas mesangiales y fibroblastos renales, varias estatinas inhiben la induccin del mRNA del CTGF causado por TGF-13 (35, 361, lo que explicara los efectos beneficiosos observados en enfermedades renales, donde las estatinas son capaces de prevenir la glomeruloesclerosis y la fibrosis intersticial (30). Estos datos demuestran que la inhibicin de la HMG-COA reductasa regula la produccin de CTGF, mostrando un posible mecanismo implicado en la disminucin de la fibrosis causada por las estatinas. Diferentes ensayos clnicos han demostrado que las estatinas ejercen efectos beneficiosos sobre la morbilidad y mortalidad cardiovascular. Sin embargo, los beneficios clnicos observados tras el tratamiento con estos frmacos parecen ser mayores a los esperables en relacin con la disminucin de colesterol, sugiriendo que las estatinas poseen efectos independientes de la disminucin de colesterol. En este trabajo hemos observado efectos celulares directos (regulacin de CTGF) que apoyan esta hiptesis. La relacin potencial entre frmacos bloqueantes de la Angll y estatinas se ha sugerido recientemente (37). En ratas dobles transgnicas para los genes de la reina y angiotensingeno el tratamiento con cerivastatina disminuye el dao causado por la Angll (38). Nuestros datos demuestran que las estatinas regulan de forma directa la respuesta de Angll en CMLV, al ser capaces de inhibir la produccin de CTGF. Todas estas evidencias demuestran la existencia de una interaccin directa entre los inhibidores de la HMG-COA reductasa y las seales intracelulares activadas por la Angll, lo que podra tener importantes implicaciones teraputicas. Por otro lado, la hipercolesterolemia est asociada a niveles elevados del receptor AT,, lo que implica una mayor respuesta biolgica a Angll, hecho con importantes consecuencias en la patogenia de la aterosclerosis e hipertensin. Adems, las estatinas disminuyen los niveles de AT, (39). Nuestros datos apoyan la nueINVESTIGACIN CARDIOVASCULAR, 2004; val. 7, n. 1

extracellular matrix proteins at toxic concentrations (31). As regards Angll responses, statins inhibit the expression of c-Fos and c-Jun, and protein synthesis (32, 33). In a model of Angll-mediated cardiac damage, cerivastatin attenuated cardiac h ypertroph y and fibrosis - reducing the levels of fibronectin, laminin and type I collagen (341. Our data show that statins decrease Angll-induced CTGF production, suggesting the existence of a target protein for statin action. The relationship between HMG-COA reductase inhibitors and CTGF has been seen in other cell types. Thus, in mesangial cells and renal fibroblasts, severa1 statins inhibit the TGF-B-mediated induction of CTGF mRNA (35, 36/, which would account for the beneficial effects seen in kidney diseases, where statins are able to prevent glomerulosclerosis and interstitial fibrosis (301. These data show that HMG-COA reductase inhibition regulates CTGF production, pointing to a possible mechanism implicated in the reduction of fibrosis caused by statins. Different clinical trials have shown statins to have beneficial effects on cardiovascular morbidity and mortal@. However, the clinical benefits seen after treatment with these drugs seem to be greater than expected in relation to cholesterol reduction, suggesting that statins have effects independent of cholesterol reduction. In this study we have found direct cellular effects (CTGF regulation) that support this hypothesis. The potential relationship between Angll blocking drugs and statins has recently been suggested (37). In double transgenic rats for the renin and angiotensinogen genes, treatment with cerivastatin decreased the damage caused by Angll(38). Our data show that statins directly regulate Angll response in VSMCs, as they are capable of inhibiting CTGF production. All this evidente points to the existence of a direct interaction between HMG-COA reductase inhibitors and intracellular signals activated by Angll, which could have significant therapeutic implications. On the other hand, hypercholesterolemia is associated to increased AT, receptor levels, which implies a greater biological response to Angll; this has significant consequences for the pathogenesis of atherosclerosis and hypertension. Moreover, statins decrease AT, levels (39). Our results support the novel idea of the use of combined therapies of RAS blockers and statins, which could have an increased beneficial effect in certain cardiovascular diseases.

M. Ruprez, L. Blanco-Colio, E. Snchez-Lpez, et al

va idea del empleo de terapias combinadas de bloqueantes del SRA y estatinas, que podran tener un efecto beneficioso mayor en algunas patologas cardiovasculares.

Intracellolar mechanisms statin effects

implicated

in

Mecanismos intracelulares implicados los efectos de las estatinas

en

La inhibicin de la HMG-COA reductasa no slo provoca una deprivacin de mevalonato, sino tambin de una gran variedad de isoprenoides de la ruta de sntesis del colesterol, como dolicol, ubiquinona, el farnesil pirofosfato (FPP) y el gerenilgeranil pirofosfato (GGPP) (8). El FPP y el GGPP son utilizados para la modificacin postraduccional de diferentes protenas, incluidas las lminas nucleares, la subunidad g de las protenas G heterotrimricas y las protenas G pequeas como Ras y las relacionadas con ella (como Rac, Rab y Rho, entre otras) (40). Las protenas G pequeas estn implicadas en diferentes funciones celulares como la regulacin de la expresin gnica, organizacin del citoesqueleto celular, trfico de membranas en el interior de la clula, proliferacin, diferenciacin y muerte celular programada o apoptosis (41, 42). La unin del isoprenoide a estas protenas es necesaria para su anclaje a la membrana plasmtica y su correcta funcionalidad. Previamente hemos demostrado que en CMLV el tratamiento con las estatinas, atorvastatina y simvastatina regula la localizacin celular de las protenas Ras y Rho, ya que impide su anclaje a la membrana celular, proceso necesario para que estas protenas sean funcionales (43, 44). En este estudio hemos visto que el efecto de la atorvastatina y la simvastatina sobre el CTGF producido por la Angll fue revertido por el mevalonato y GGPP, y en menor medida por FPP. Diversos estudios han demostrado que las estatinas son capaces de inhibir la isoprenilacin de las protenas Ras, inhiben la ruta AP-IiFias y disminuyen la proliferacin celular. En CMLV hemos observado que la atorvastatina disminuye la activacin de los factores de transcripcin AP-1 y NF-KB y la induccin de citoquinas causada por la Angll (45). Este efecto se revirti en presencia de diferentes compuestos de la ruta de sntesis del colesterol como el mevalonato y los isoprenoides FPP y GGPP. Adems, la manumicina A, un inhibidor de la isoprenilacin proteica, tambin disminuy la actividad de NFKB inducida por Angll y TNF-a, indicando que los isoprenoides participan en la activacin de este factor de transcripcin (45). Otros factores de transcripcin tambin se regulan con estatinas. El tratamiento con diferentes estatinas puede modular
76

Inhibition of HMG-COA reductase not only causes deprivation of mevalonate, but also of a great variety of isoprenoids of the cholesterol synthetic pathway, such as dolichol, ubiquinone, farnesylpyrophosphate (FPP) and gerenylgeranylpyrophosphate (GGPP) (8). FPP and GGPP are used for the post-translational modification of different proteins, including nuclear laminins, the subunit g of heterotrimeric G proteins and small G proteins such as Ras and those related to it fsuch as Rac, Rab and Rho, among others) (40). The small G proteins are involved in different cellular functions such as regulation of gene expression, organization of cell cytoskeleton, membrane traffic within the cell, cell proliferation and differentiation, and programmed cell death or apoptosis (4 1, 42). Isoprenoid binding to these proteins is necessary for their anchoring to the plasma membrane and adequate function. We have previously shown that in VSMCs, treatment with statins atorvastatin and simvastatin regulates the cellular localization of Ras and Rho proteins by preventing their anchoring to the cell membrane, a process required for these proteins to be functional(43, 44). In this study we have seen that the effect of atorvastatin and simvastatin on CTGF produced by Angll was reversed by mevalonate and GGPP, and to a lesser extent by FPP. Different studies have shown that statins are able to inhibit Ras protein prenylation, inhibit the AP-l/Ras pathway and decrease cell proliferation. In VSMCs, it was seen that atorvastatin reduces activation of the transcrption factors AP-1 and NF-~6, and cytokine induction caused by Angll(45). This effect was reversed in the presente of different components of the cholesterol synthesis pathway, such as mevalonate and the isoprenoids FPP and GGPP. In addition, manumycin A, a protein isoprenylation inhibitor, also decreased NF-KB activity induced by Angll and TNF-l3, suggesting that isoprenoids are involved in the activation of this transcription factor (45). Other transcription factors are also regulated by statins. Treatment with different statns can modulate the expression of different proin flammatory cytokines such as IL- IB, IL-6 and COX- by increasing PPARcl in endothelial cells (46). Our data support the idea that HMG-COA reductase inhibitors are directly involved in intracellular signaling mechanisms
INVESTIGACIN CARDIOVASCULAR, 2004; val. 7, n. 1

Angll, estatinas y dario vascular

Angiotensin

II, statins

and vascular

damage

la expresin de diferentes citocinas proinflamatorias como la IL-l& la IL-6 y la COX-2, mediante el incremento del PPARI en clulas endoteliales (46). Nuestros datos apoyan la idea de que los inhibidores de la HMG-COA reductasa participan de forma directa en mecanismos de sealizacin intracelular implicados en la isoprenilacin de protenas de bajo peso molecular las cuales que juegan un papel clave en la transduccin de seales al ncleo.

implicated in the prenylation of low molecular weight proteins that play a key role in signa/ transduction to the cell nucleus.

Angiotensn proteins

II regdates

CTGF via Rho

La Angll regula CTGF va protenas

Rho

Los receptores AT, estn acoplados a protenas G y se ha descrito que activan las protenas Ras, estimulan la formacin de Ras-GTP e inducen la localizacin de Ras y Rho en la membrana celular. La Angll aumenta p21 Ras, Racl, Rho Ay el sistema Rho/quinasa Rho (19, 47, 48). Estas protenas regulan diversas funciones celulares y procesos de dao vascular, como activacin de plaquetas, transmigracin de leucocitos, formacin de neontima e hipertrofia cardaca (10, 41, 42). Estos datos muestran similitudes entre los mecanismos de actuacin de Angll y protenas Ras/Rho y sugieren que estas protenas podran mediar algunas acciones de la Angll. Recientemente se ha descrito que las protenas Rho participan en la vasoconstriccin y la hipertrofia inducida por Angll en cardiomiocitos y CMLV (33, 47, 49). Otras repuestas inducidas por Angll tambin estn mediadas por las protenas Rho, como la expresin de MCP-1 (50) y la reorganizacin de actina (51). En clulas mesangiales, las protenas Rho son esenciales para la expresin, basal e inducida por LPS y TGF-13, del CTGF (52). En este trabajo hemos visto que el empleo de inhibidores de la quinasa de Rho (Y-27632 y fasudil) en CMLV disminuy la produccin de CTGF causada por Angll. El bloqueo de RhoA mediante transfecciones transitorias con un vector dominante negativo de RhoA inhibi el CTGF inducido por Angll. Adems, en las clulas transfectadas con el vector constitutivo de RhoA se observ un aumento en la produccin de CTGF. Estos datos demuestran que la activacin de las protenas Rho est implicada en la sntesis de CTGF inducida por Angll. La inhibicin de Rho y por lo tanto de su quinasa, la Rho quinasa, es un posible mecanismo que puede mediar los llamados efectos pleiotrpicos de las estatinas en la pared vascular dado que cambios en Rho afectan al transporte intracelular, la estabilidad del RNA mensajero de algunos genes y la transcripcin gnica (9). Como se ha comentado anteriormente, las estatinas disminuyen el CTGF causado por Angll, los datos anINVESTIGACIN CARDIOVASCULAR, 2004; val. 7, no 1

AT, receptors are coupled to G proteins, and have been reported to activate Ras proteins, stimulate Ras-GTP formation, and induce Ras and Rho localization to the cell membrane. Angll increases p21 Ras, Racl, Rho A and the Rho/Rho kinase system (19, 47, 48). These proteins regulate various cell functions and vascular damage processes, such as platelet activation, leukocyte transmigration, neointimal formation and cardiac hypertrophy (10, 41, 42). These data show similarities between the mechanisms of action of Angll and Ras/Rho proteins, and suggest that these proteins could mediate some of the actions of Angll. It has recently been reported that Rho proteins are involved in Angll-induced vasoconstriction and hypertrophy in cardiomyocytes and VSMCs 133, 47, 49). Other responses induced by Angll are also mediated by Rho proteins, such as MCP- 1 expression (50) and actin reorganization (51). In mesangial cells, Rho proteins are essential for CTGF expression. both basal and induced by LPS and TGF-/3 (52). In this study we have observed that the use of Rho kinase inhibitors (Y-27632 and fasudill in VSMCs decreased Angll-mediated CTGF production. RhoA blockade by transient transfections with a Rho negative dominant vector inhibited Angll-induced CTGF. Moreover, in cells transfected with the Rho constitutive vector, an increase was seen in CTGF production. These data show that activation of Rho proteins is implicated in Angll-induced CTGF synthesis. Inhbtion of Rho, and therefore of its kinase (Rho kinase), is a potential mechanism that could mediate the so-called pleiotropic effects of statins on the vascular Wall, since changes in Rho affect intracellular transpori, the stability of mRNA of some genes, and gene transcription 191.As discussed above, statins decrease Angll-induced CTGF, and the above data support the hypothesis that these compounds regulate CTGF by interfering with RhoA isoprenylation.
77

M. Ruprez, L. Blanco-Colio, E. Snchez-Lpez, et al.

teriores apoyan la hiptesis de que estos compuestos regulan el CTGF al interferir en la isoprenilacin de Rho A. Varios estudios experimentales han demostrado que la inhibicin de protenas Rho provoca desorganizacin del citoesqueleto e induccin del sistema fibrinoltico (10, 40). En un modelo hipertensin en ratas causado por administracin de LNAME, el inhibidor de la quinasa de Rho (Y-276321, disminuy la inflamacin vascular, la expresin de MCP-1 y TGF-13, y la progresin de la aterosclerosis (531, como tambin ocurri en un modelo de aterosclerosis en cerdos 1541. Resultados similares en prevencin del remodelamiento vascular se han observado con fasudil, otro inhibidor de la quinasa de Rho (55). En este modelo se haba descrito previamente que las estatinas mejoraban el dao vascular sin modificar los niveles lipdieos, mostrando efectos independientes del colesterol (56). Por otro lado, un posible mecanismo que podra explicar los cambios vasculares en la hipertensin es el aumento del sistema Rho/quinasa de Rho que provoca vasoconstriccin (57). Estos resultados sugieren que el dao vascular inducido por Angll est mediado por la activacin de protenas Rho, y sugieren que el empleo de tratamientos que inhiben su activacin, como estatinas o inhibidores de la actividad de Rho, es una buena eleccin como estrategia teraputica en enfermedades asociadas a un aumento de produccin de Angll, independientemente de la presencia de hipertensin. En resumen, nuestros datos demuestran que los inhibidores de la HMG-COA reductasa a travs de la inhibicin de la sntesis de isoprenoides y mediante la regulacin de la actividad de las protenas Rho regulan la produccin de CTGF causada por Angll. Nuestros resultados sugieren que los efectos beneficiosos de estos frmacos pueden ser atribuidos a sus acciones celulares, regulando efectos patolgicos de la Angll en el dao vascular, y apoyan el empleo de la terapia combinada, de frmacos que modulan la generacin de Angll y estatinas, demostrando un mecanismo molecular que podra explicar estos efectos beneficiosos.

Severa/ experimental studies have shown that inhibition of Rho proteins causes disorganization of the cytoskeleton and induction of the fibrinolytic system (10, 40). In a rat model of h ypertension produced by administering L-NAME, the Rho kinase inhibitor (Y-27632) decreased vascular inflammation, MCP- 1 and TGF-r3 expression, and progression of atherosclerosis (531 in a way similar to the results obtained in a model of atherosclerosis in swine 1541. Similar results in the prevention of vascular remodeling have also been found with fasudil, another Rho kinase inhibitor (55). In this model, it had previously been reported that statins improved vascular damage without modifying lipd levels, showed cholesterol-independent effects 156). On the other hand, a possible mechanism that could explain the vascular changes in hypertension is the increase in the Rho/Rho kinase system, causing vasoconstriction (57). These results suggest that the vascular damage induced by Angll s mediated by Rho protein activation, and that the use of treatments that inhibit such activation, such as statins or Rho activity inhibitors, is a good choice as a therapeutic approach in diseases assocated with increased Angll production, regardless of the presente of hypertension. To summarize, our results demonstrate that HMG-COA reductase inhibitors, through inhibition of isoprenod synthesis and by regulating Rho protein activity, regulate Angll-induced CTGF producton. Our results suggest that the beneficial effects of these drugs may be attributed to their cellular actions, with regulation of pathological Angll effects in vascular damage, and support the use of combined therapy wth drugs that modulate Angll generation and statins. Our data demonstrate molecular mechanism that could explain these beneficial effects.

Acknowledgements Agradecimientos Este trabajo ha sido financiado por una ayuda de la Comunidad Autnoma de Madrid (08.4/0017/ 20001, del Fondo de Investigacin Sanitaria Fis (01/3130), y de la Fundacin MAPFRE Medicina. M. R., V. E. y J. R-V son becarios del FIS.
78

This study was supported by grants from the Autonomous Community of Madrid (08.4/00 17/2000), the Fondo de Investigacin Sanitaria (FIS) (01/3130), and the Fundacin MAPFRE Medicina. M. R., V.E. and J. R-V are FIS scholarship holders.
INVESTIGACION CARDIOVASCULAR, 2004; val. 7, n. 1

Angll,

estatinas

y dao vascular

Angiotensin

II, statins

and

vascular

damage

BIBLIOGRAFA
1. TURN J, RUIZ-ORTEGA M, EGIDO J. Regulation of matrix proteins and impact on vascular structure. Current Hyperten Repotts. 2000; 2: 106-I 13. RUIZ-ORTEGA M, LORENZO 0, SUZUKI Y, RUPREZ M, EGIDO J. Proinflammatoty actions of Angiotensin ll. Current Opin Nephrol Hypertens. 2001; 10: 321-329. TOUYZ R M, SCHIFFRIN E L.Signal transduction mechanisms mediating the physiological and pathophysiological actions of angiotensin ll in vascular smooth muscle cells. Pharmacol Rev. 2000; 52: 639-672. RUIZ-ORTEGA M, RUPREZ M, ESTEBAN V, EGIDO J. Molecular Mechanisms of Angiotensin Il-lnduced Vascular Injury. Current Hypertension Reporfs. 2003: 5: 73-79. LARAGH J H. The renin system and new understanding of the complications of hypertension and their treatment. Atzneim/Forsch/Drug Res. 1993; 43: 247-254. CARO J, KLIlTICH W, MCGUIRE A, FORD 1, PElTIlT D, NORRIE J, SHEPHERD J. International economic analysis of primary prevention of cardiovascular disease with pravastatin in WOSCOPS. West of Scotland Coronary Prevention Study. Eur Heart J. 1999; 20: 263. 268. The 4s Investigators. Randomized trial of cholesterol lowering in 4444 patients with coronary heart disease: The Scandinavian Simvastatin Survival Study (4s). Lancet. 1994; 344: 1383-I 389. GOLDSTEIN J L, BROWN M S. Regulation of the mevalonate pathway. Nature. 1990; 343: 425-430. TAKEMOTO M, LIA0 J. Pleiotropic effects of 3-Hydroxy3-Methylglutaryl coenzyme A reductase. Arterioscler Thromb Vasc Biol. 2001; 21: 1712-1719. KNAUS. Rho GTPase signaling in inflammation and transformation. /mmuno/ Res. 2000; 21: 103-109. LAU LF, L4M S C T. The CCN family of angiogenic regulators: The integrin connection. fxp Ce// Res. 1999; 248: 44-57. OEMAR B S, LUSCHER T F. Connective tissue growth factor. Friend or foe? Arterioscler Thromb Vasc Biol. 1997; 17: 1483-1489. OEMAR B S, WERNER A, GARNIER J M, DO D D, GODOY N, NAUCK M, MARZ W, RUPP J, PECH M, LUSCHER T F. Human connective tissue growth factor is expressed in advanced atherosclerotic lesions. Circulation. 1997; 18; 831-839. OHNISHI H, OKA T, KUSACHI S, NAKANISHI T, TAKEDA K, NAKAHAMA M, DOI M, MURAKAMI T, NINOMIYA Y, TAKIGAWA M, TSUJI T. Increased expression of connective tissue growth factor in the infarct zone of experimentally induced myocardial infarction in rats. J Mo/ Ce// Cardiol. 1998; 30: 241 l-2422. FAN W H, PECH M, KARNOVSKY M J. Connective tissue growth factor (CTGF) stimulates vascular smooth muscle cell growth and migration in vitro. Eur J Ce// Biol. 2000; 79: 915-923. HISHIKAWA K, OEMAR B S, TANNER F C, NAKAKI T, FUJI1 T, LUSCHER T F. Overexpression of connective tissue growth factor gene induces apoptosis in human aortic smooth muscle cells. Circulation. 1999; 100: 2108. 2112. RUIZ-ORTEGA M, LORENZO 0, RUPREZ M, KONIG S, WIlTIG B, EGIDO J. Angiotensin ll activates nuclear transcription factor KB through AT, and AT, recep-

/ REFERENCES
tors in cultured vascular smooth muscle cells. Molecular mechanisms. Cir Res. 2000; 23: 1266-1272. RISER B L, DENICHILLO M, CORTES P, BAKER C, GRODIN J M, YEE J, NARINS R G. Regulation of connective tissue growth factor activity in cultured rat mesangial cells and its expression in experimental diabetic glomeruloesclrerosis. J Am Soc Nephrol. 2000; Il : 25-38. SCHIEFFER B, PAXTON W G, CHAI 0, MARRERO M B, BERNSTEIN K E. Angiotensin ll controls p21 ras activity via pp6Oc-ser. J Biol Chem. 1996; 271: 10329. 10333. JOHNS D G, DORRANCE A M, LEITE R, WEBER D S, WEBB R C. Novel signaling pathways contributing to vascular changes in hypertension. J Biomed Sc;. 2000; 7: 431-433. DUNCAN M R, FRAZIER K S, ABRAMSON S, WILLIAMS S, KLAPPER H, HUANG X, GROTENDORST G R. Connective tissue growth factor mediates transforming growth factor beta-induced collagen synthesis: down-regulation by cAMP. FASEB J. 1999; 13: 1774 1786. SCHIEFFER B, SCHIEFFER E, HILFIKER-KLEINER D, HILFIKER A, KOVANEN P T, KAARTINEN M, NUSSBERGER J, HARRINGER W, DREXLER H. Expression of angiotensin ll and interleukin 6 in human coronary atherosclerotic plaques: potential implications for inflammation and plaque instability. Circulation. 2000; 101: 1372-1378. FINCKENBERG P, LASSILA M, INKINEN K, et al. Cyclosporine induces myocardial connective tissue growth factor in spontaneously hypertensive rats on high-sodium diet. Transplantation. ?OOl; 71: 951-958. TAMURA K, CHEN Y E, LOPEZ-LLASACA M, et al. Molecular mechanism of fibronectin gene activation by cyclic stretch in vascular smooth muscle cells. J Biol Chem. 2000; 275: 34619-34627. LIBBY P, RIDKER P M, MASERI A. Inflammation and atherosclerosis. Circulation. 2002; 5: 1051135-1143. HAYASHIDANI S, TSUTSUI H, SHIOMI T, SUEMATSU N, KINUGAWA S, IDE T, WEN J, TAKESHITA A. Fluvastatin, a 3-hydroxy-3-methylglutaryl coenzyme a reductase inhibitor, attenuates left ventricular remodeling and failure after experimental myocardial infarction. Circulation. 2002; 19 (105): 868-873. TAKEMOTO M, NODE K, NAKAGAMI H, LIA0 Y, GRIMM M, TAKEMOTO Y, KITAKAZE M, LIA0 J K. Statins as antioxidant therapy for preventing cardiac myocyte hypertrophy. J Clin Invest. 2001; 108: 1429. 1437. NOGAKI F, MUS0 E, YASHIRO M, KASUNO K, KAMATA T, ONO T, SASAYAMA S. Direct inhibitory effects of simvastatin on matrix accumulation in cultured murine mesangial cells. Kidney Int Suppl. 1999; 71: S198-201. CHEN H C, GUH J Y, SHIN S J, LAI Y H. Pravastatin suppress superoxide and fibronectin production of glomerular mesangial cells induced by oxidized-LDL and high glucose. Atherosclerosis. 2002; 160: 141-146. ODA H, KEANE W F. Recent advances in statins and the kidney. Kidney Int Suppl. 1999; 71: S2-5. RIESSEN R, AXEL D 1, FENCHEL M, HERZOG U U, ROSSMANN H, KARSCH K R. Effect of HMG-COA re-

18.

2.

3.

19.

4.

20.

5.

21.

6.

22.

7.

8. 9.

23.

10. ll.

24.

12.

25. 26.

13.

14.

27.

28.

15.

16.

29.

30. 31.

17.

INVESTIGACIN

CARDIOVASCULAR,

2004; val. 7, n. 1

79

M. Ruprez,

L. Blanco-Colio,

E. Snchez-Lpez,

et al.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41. 42.

43.

44.

45.

ductase inhibitors on extracellular matrix expression in human vascular smooth muscle cells. Basic Res Cardio/. 1999;94: 322-332. KREUZER J, WATSON L, HERDEGEN T, LOEBE M, WENDE P, KUBLER K. Effects of HMG-COA reductase inhibition on PDGF- and angiotensin II- mediated signal transduction: suppression of c-Jun and c-Fos in human smooth muscle cells in vitro. Eur J Med Res. 1999; 4: 135-143. YAMAKAWA T, TANAKA S, NUMAGUCHI K, YAMAKAWA Y, MOTLEY E D, ICHIHARA S, INAGAMI T. Involvement of Rho-kinase in angiotensin Il-induced hypertrophy of rat vascular smooth muscle cells. Hypertension. 2000;35: 313-318. DECHEND R, FIEBELER A, PARK J K, MULLER D N, THEUER J, MERVAALA E, BIERINGER M, GULBA D, DIETZ R, LUFT F C, HALLER H. Amelioration of angiotensin Il-induced cardiac injury by a 3-hydroxy-3-methylglutaryl coenzyme a reductase inhibitor. Circulation. 2001;104:576-581. EBERLEIN M, HEUSINGER-RIBEIRO J, GOPPELTSTRUEBE M. Rho-dependent inhibition of the induction of connective tissue growth factor (CTGF) by HMG COA reductase inhibitors (statins). Br J Pharmacol. 2001; 133: 1172-11780. GOPPELT-STRUEBE M, HAHN A, IWANCIW D, REHM M, BANAS B. Regulation of connective tissue growth factor (ccn2; ctgf) gene expression in human mesangial cells: modulation by HMG COA reductase inhibitors (statins). Mo/Patho/. 2001;54: 176-179. FAGGIOlTO A, PAOLElTI R. State-of-the-At-t lecture. Statins and blockers of the renin angiotensin systemvascular protection beyond their primary mode of action. Hypertension. 2000;34: 987-996. MULLER D N, DECHEND R, MERVAALA E M, PARK J K, SCHMIDT F, FIEBELER A, THEUER J, BREU V, GANTEN D, HALLER H, LUFT F C. Statins ameliorates angiotensin Il-induced inflammatory damage in rats Hypertension.2000;35: 193-201. NICKENIG G, BAUMER AT, TEMUR Y, KEBBEN D, JOCKENHOVEL F, BOHM M. Statin-sensitive dysregulated AT1 receptor function and density in hypercholesterolemic men. Circulation. 1999;23 (100): 2131-2134. VAN AELST L, DSOUZASCHOREY C. Rho GTPases and signaling networks. Genes Dev. 1997; 11:22952322. MACKAY D J, HALL A. Rho GTPases. J Biol Chem. 1998;273:20685-20688. ZOHN I M, CAMPBELL S L, KHOSRAVI-FAR R, et al. Rho family proteins and Ras transformation: the RHOad less traveled gets congested. Oncogene. 1998; 17: 1415. 1438. BLANCO-COL10 LM, VILLA A, ORTEGO M, et al. 3Hydroxy-3-methyl-glutaryl Coenzyme A reductase inhibitors atorvastatin and simvastatin induce apoptosis in vascular smooth muscle cells by downregulation of Bel-2 expression and RhoA prenylation. Atherosclerosis. 2002; 161: 17-26. GUIJARRO C, BLANCO-COL10 L M, ORTEGO M, et al. 3-Hydroxy-3-methy-glutaryl coenzyme A redcurase and isoprenylation inhibitors induce apoptosis in vascular smooth muscle cells in culture. Circ Res. 1998; 83: 490. 500. ORTEGO M, BUSTOS B, HERNANDEZ-PRESA M A, et al. Atorvastatin reduces NF-kB activation and chemo-

46.

47.

48.

49.

50.

51.

52.

53.

54.

55.

56.

57.

kine expression in vascular smooth muscle cells and mononuclear cells. Atherosclerosis. 1999; 147: 253-261. INOUE 1, GOTO S, MIZOTANI K, et al. Lipophilic HMGCOA reductase inhibitor has an anti-inflammatory effect: reduction of mRNA levels for interleukin-1 b, interleukin-6, cyclooxygenase 2, and p22phox by regulation of peroxisome proliferator-activated receptor-a (PPAR-a) in primary endothelial cells. Life Sci. 2000;67: 863-876. AOKI H, IZUMO S, SADOSHIMA J. Angiotensin ll activates RhoA in cardiac myocytes: a critica1 role of RhoA in angiotensin Il-induced premyofibril formation. Circ Res. 1998 Apr 6; 82 (6): 666-676. SCHMITZ U, THOMMES K, BEIER 1, WAGNER W, SACHINIDIS A, DUSING R, VElTER H. Angiotensin IIinduced stimulation of p21-activated kinase and c-Jun NH2-terminal kinase is mediated by Racl and Nck. J Biol Chem. 2001;22 (276): 22003-22010. AIKAWA R, KOMURO 1, NAGAI R, YAZAKI Y. Rho plays an important role in Angll-induced hypertrophic responses in cardiac myocites. Mo// Ce// Biochem. 2000; 212: 177-182. FUNAKOSHI Y, ICHIKI T, SHIMOKAWA H, EGASHIRA K, TAKEDA K, KAIBUCHI K, TAKEYA M, YOSHIMURA T, TAKESHITA A. Rho-kinase mediates angiotensin IIinduced monocyte chemoattractant protein-1 expression in rat vascular smooth muscle cells. Hypertension. 200;38: 100-104. KUWAHARA M. Involvement of Rho and tyrosine kinase in angiotensin Il-induced actin reorganization in mesothelial cells. EurJ Pharmacol. 2002; 436: 15-21. HAHN A, HEUSINGER-RIBEIRO J, LANZ T, ZENKEL S, GOPPELT-STRUEBE M. Inductir.I of connective tissue growth factor by activation of heptahelical receptors. Modulation by Rho proteins and the actin cytoskeleton. JBio/Chem.2000;275: 37429-37435. KATAOKA C, EGASHIRA K, INOUE S, TAKEMOTO M, NI W, KOYANAGI M, KITAMOTO S, USUI M, KAIBUCHI K, SHIMOKAWA H, TAKESHITA A. Important role of Rho-kinase in the pathogenesis of cardiovascular inflammation and remodeling induced by long-term blockade of nitric oxide synthesis in rats. Hypertension. 2002;39: 245-250. SHIMOKAWA H, MORISHIGE K, MIYATA K, KANDABASHI T, ET0 Y, IKEGAKI 1, ASAN0 T, KAIBUCHI K, TAKESHITA A. Long-term inhibition of Rho-kinase induces a regression of arteriosclerotic coronary lesions in a porcine model in vivo. Cardiovasc Res. 2001; 51: 169-177. IKEGAKI 1, HATTORI T, YAMAGUCHI T, SASAKI Y, SATOH S, ASAN0 T, SHIMOKAWA H. Involvement of Rho-kinase in vascular remodeling caused by longterm inhibition of nitric oxide synthesis in rats. Eur J Pharmacol.2001;427: 69-475. NI W, EGASHIRA K, KATAOKA C, KITAMOTO S, KOYANAGI M, INOUE S, TAKESHITA A. Antiinflammatory and antiarteriosclerotic actions of HMG-COA reductase inhibitors in a rat model of chronic inhibition of nitric oxide synthesis. Circ Res.2001;89: 415-421. MUKAI Y, SHIMOKAWA H, MATOBA T, KANDABASHI T, SATOH S, HIROKI J, KAIBUCHI K, TAKESHITA A. Involvement of Rho-kinase in hypertensive vascular disease: a novel therapeutic target in hypertension FASEBJ. 2001: 15: 1062-1066.

80

INVESTIGACIN

CARDIOVASCULAR,

2004; WI. 7, n. 1