RESUMEN
Optimization of ICH safety guideline studies for inclusion into regulatory submissions is critical for resource conservation, animal use reduction, and efficient drug development. The ICH S7A guidance for Safety Pharmacology (SP) studies adopted in 2001 identified the core battery of studies to evaluate the acute safety of putative pharmaceutical molecules prior to First in Human (FIH) trials. To assess the utility of respiratory studies in predicting clinical AE's, seven pharmaceutical companies pooled preclinical and clinical respiratory findings. A large database of novel molecules included all relevant data from standard S7A respiratory (n = 459) and FIH studies (n = 309). The data were analyzed with respect to the progression of these molecules, clinical adverse event reporting of these same molecules, and achieved exposures. These S7A respiratory assay findings had no impact on compound progression, and only 12 of 309 drug candidates were 'positive' preclinically and reported a respiratory-related AE in clinical trials (i.e. cough, dyspnea, etc.), an overall incidence rate of 3.9%. Contingency tables/statistics support a lack of concordance of these preclinical assays. Overall, our extensive analysis clearly indicated that the preclinical respiratory assay fails to provide any prognostic value for detecting clinically relevant respiratory adverse events.
RESUMEN
Extracellular matrix signaling via integrin receptors is important for smooth muscle cell (SMC) differentiation during vasculogenesis and for phenotypic modulation of SMCs during atherosclerosis. We previously reported that the noncatalytic carboxyl-terminal protein binding domain of focal adhesion kinase (FAK) is expressed as a separate protein termed FAK-related nonkinase (FRNK) and that ectopic expression of FRNK can attenuate FAK activity and integrin-dependent signaling (A. Richardson and J. T. Parsons, Nature 380:538-540, 1996). Herein we report that in contrast to FAK, which is expressed ubiquitously, FRNK is expressed selectively in SMCs, with particularly high levels observed in conduit blood vessels. FRNK expression was low during embryonic development, was significantly upregulated in the postnatal period, and returned to low but detectable levels in adult tissues. FRNK expression was also dramatically upregulated following balloon-induced carotid artery injury. In cultured rat aortic smooth muscle cells, overexpression of FRNK attenuated platelet-derived growth factor (PDGF)-BB-induced migration and also dramatically inhibited [(3)H]thymidine incorporation upon stimulation with PDGF-BB or 10% serum. These effects were concomitant with a reduction in SMC proliferation. Taken together, these data indicate that FRNK acts as an endogenous inhibitor of FAK signaling in SMCs. Furthermore, increased FRNK expression following vascular injury or during development may alter the SMC phenotype by negatively regulating proliferative and migratory signals.
Asunto(s)
Músculo Liso Vascular/citología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/biosíntesis , Adenoviridae/metabolismo , Animales , Northern Blotting , Western Blotting , Arterias Carótidas/metabolismo , Adhesión Celular , Ciclo Celular , División Celular , Movimiento Celular , Células Cultivadas , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Matriz Extracelular/metabolismo , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Proteínas Fluorescentes Verdes , Inmunohistoquímica , Proteínas Luminiscentes/metabolismo , Fenotipo , Fosforilación , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Pruebas de Precipitina , Ratas , Factores de Tiempo , Distribución Tisular , Regulación hacia ArribaRESUMEN
While it is well established that phenotypic modulation of vascular smooth muscle cells (VSMCs) contributes to the development and progression of vascular lesions, little is known regarding the molecular mechanisms of phenotypic modulation in vivo. Here we show that vascular injury reduces transcription of VSMC differentiation marker genes, and we identify cis regulatory elements that may mediate this decrease. Using a carotid wire-injury model in mice carrying transgenes for smooth muscle alpha-actin, smooth muscle myosin heavy chain, or a SM22alpha promoter-beta-gal reporter, we collected arteries 7 and 14 days after injury and assessed changes in endogenous protein and mRNA levels and in beta-gal activity. Endogenous levels for all markers were decreased 7 days after injury and returned to nearly control levels by 14 days. beta-gal staining in all lines followed a similar pattern, suggesting that transcriptional downregulation contributed to the injury-induced decreases. To begin to dissect this response, we mutated a putative G/C-rich repressor in the SM22alpha promoter transgene and found that this mutation significantly attenuated injury-induced downregulation. Hence, transcriptional downregulation contributes to injury-induced decreases in VSMC differentiation markers, an effect that may be partially mediated through a G/C-rich repressor element.
Asunto(s)
Músculo Liso Vascular/lesiones , Músculo Liso Vascular/metabolismo , Actinas/genética , Actinas/metabolismo , Animales , Secuencia de Bases , Biomarcadores , Diferenciación Celular , Cartilla de ADN/genética , Expresión Génica , Genes Reporteros , Hibridación in Situ , Operón Lac , Ratones , Ratones Transgénicos , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Liso Vascular/patología , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Fenotipo , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismo , beta-Galactosidasa/genéticaRESUMEN
The use of genetically modified mice has been an important model system to study gene function in cardiovascular development and under pathophysiological conditions. Although conventional gene knockout studies have provided important insights into gene function in the cardiovascular system, they may be limited by upregulation of compensatory pathways and the inability to differentiate direct versus indirect functions in vivo. As a first step in developing systems that can target gene activation or inactivation specifically to smooth muscle cells (SMCs), we coupled the smooth muscle myosin heavy chain (SMMHC) promoter to the cre recombinase gene and generated transgenic mice that express cre in SMCs. In addition, we used these mice to address whether the heterogeneous staining observed in SMMHC-LacZ mice was due to subsets of SMCs that required different regulatory cassettes of the promoter or if it reflected episodic expression of the transgene. To address both the feasibility of SMC targeting and the apparent heterogeneous expression, we bred SMMHC-cre mice to indicator mice containing a cre-activated LacZ gene. Results showed high-level expression in SMCs at various embryonic time points and in adult tissues. Because breeding of SMMHC-cre mice to an indicator line provided an integration of cre activity over time, results of this study revealed that expression of the SMMHC promoter fragment more closely resembled the expression of the endogenous gene, both with respect to the onset of activation during development and uniformity of staining among individual cells within tissues. Overall, these mice will provide a powerful tool to researchers to study gene function in vascular development/disease by using cre/lox technology to direct smooth muscle-specific gene activation or inactivation in vivo.
Asunto(s)
Integrasas/genética , Músculo Liso Vascular/metabolismo , Cadenas Pesadas de Miosina/genética , Proteínas Virales , beta-Galactosidasa/biosíntesis , Animales , Aorta Torácica/embriología , Arterias Carótidas/embriología , Cruzamientos Genéticos , Embrión de Mamíferos/metabolismo , Regulación de la Expresión Génica , Marcación de Gen , Edad Gestacional , Corazón/embriología , Ratones , Ratones Transgénicos , Músculo Liso Vascular/embriología , Músculo Liso Vascular/enzimología , Regiones Promotoras Genéticas , Recombinación Genética , Activación Transcripcional , Transgenes , beta-Galactosidasa/análisis , beta-Galactosidasa/genéticaRESUMEN
Transforming growth factor beta (TGF-beta) is implicated in the regulation of smooth muscle cell (SMC) differentiation. We previously identified a novel TGF-beta control element (TCE) in the promoters of SMC differentiation marker genes, including alpha-smooth muscle actin and SM22alpha. In this study, the importance of the TCE in regulation of SM22alpha gene expression in vivo was investigated by mutating it within the context of a mouse SM22alpha promoter-lacZ transgenic construct. Mutation of the TCE completely abolished SM22alpha promoter activity in arterial SMCs as well as in developing heart and skeletal muscle. To identify the transcription factor(s) binding to the TCE, we performed yeast one-hybrid cloning analysis and identified gut-enriched Krüppel-like factor (GKLF). However, cotransfection studies in cultured cells showed that GKLF repressed the TGF-beta-dependent increases in SM22alpha and alpha-smooth muscle actin promoter activities. Furthermore, GKLF was not highly expressed in differentiated SMCs in vivo, and TGF-beta down-regulated GKLF expression in dedifferentiated cultured SMCs. In contrast, overexpression of a related factor (BTEB2) transactivated SM22alpha promoter activity. Thus, our findings suggest a reciprocal role for related Krüppel-like transcription factors in the regulation of SMC differentiation through a TCE-dependent mechanism.
Asunto(s)
Biomarcadores , Diferenciación Celular , Proteínas de Unión al ADN/metabolismo , Proteínas de Microfilamentos/genética , Proteínas Musculares/genética , Músculo Liso/citología , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Aorta Torácica/citología , Secuencia de Bases , ADN , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel , Ratones , Mutagénesis Sitio-Dirigida , Regiones Promotoras Genéticas , Ratas , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Ácido NucleicoRESUMEN
The smooth muscle myosin heavy chain (SM-MHC) gene encodes a major contractile protein whose expression exclusively marks the smooth muscle cell (SMC) lineage. To better understand smooth muscle differentiation at the transcriptional level, we have initiated studies to identify those DNA sequences critical for expression of the SM-MHC gene. Here we report the identification of an SM-MHC promoter-intronic DNA fragment that directs smooth muscle-specific expression in transgenic mice. Transgenic mice harboring an SM-MHC-lacZ reporter construct containing approximately 16 kb of the SM-MHC genomic region from -4.2 to + 11.6 kb (within the first intron) expressed the lacZ transgene in all smooth muscle tissue types. The inclusion of the intronic sequence was required for transgene expression, since 4.2 kb of the 5'-flanking region alone was not sufficient for expression. In the adult mouse, transgene expression was observed in both arterial and venous smooth muscle, in airway smooth muscle of the trachea and bronchi, and in the smooth muscle layers of all abdominal organs, including the stomach, intestine, ureters, and bladder. During development, transgene expression was first detected in airway SMCs at embryonic day 12.5 and in vascular and visceral SMC tissues by embryonic day 14.5. Of interest, expression of the SM-MHC transgene was markedly reduced or absent in some SMC tissues, including the pulmonary circulation. Moreover, the transgene exhibited a heterogeneous pattern between individual SMCs within a given tissue, suggesting the possibility of the existence of different SM-MHC gene regulatory programs between SMC subpopulations and/or of episodic rather than continuous expression of the SM-MHC gene. To our knowledge, results of these studies are the first to identify a promoter region that confers complete SMC specificity in vivo, thus providing a system with which to define SMC-specific transcriptional regulatory mechanisms and to design vectors for SMC-specific gene targeting.
Asunto(s)
Intrones , Músculo Liso Vascular/metabolismo , Músculo Liso/metabolismo , Miocardio/metabolismo , Cadenas Pesadas de Miosina/biosíntesis , Cadenas Pesadas de Miosina/genética , Regiones Promotoras Genéticas , Animales , ADN/química , ADN/genética , Genes Reporteros , Inmunohistoquímica , Ratones , Ratones Transgénicos , Especificidad de Órganos , Ratas , Proteínas Recombinantes de Fusión/biosíntesis , beta-Galactosidasa/biosíntesisRESUMEN
We have previously shown that maximal expression of the rat smooth muscle myosin heavy chain (SM-MHC) gene in cultured rat aortic smooth muscle cells (SMCs) required the presence of a highly conserved domain (nucleotides -1321 and -1095) that contained two positive-acting serum response factor (SRF) binding elements (CArG boxes 1 and 2) and a negative-acting GC-rich element that was recognized by Sp1 (Madsen, C. S., Hershey, J. C., Hautmann, M. B., White, S. L., and Owens, G. K. (1997) J. Biol. Chem. 272, 6332-6340). In this study, to better understand the functional role of these three cis elements, we created a series of SM-MHC reporter-gene constructs in which each element was mutated either alone or in combination with each other and tested them for activity in transient transfection assays using primary cultured rat aortic SMCs. Results demonstrated that the most proximal SRF binding element (CArG-box1) was active in the absence of CArG-box2, but only upon removal of the GC-rich repressor. In contrast, regardless of sequence context, CArG-box2 was active only when CArG-box1 was present. We further demonstrated using electrophoretic mobility shift assays that Sp1 binding to the GC-rich repressor element did not prevent SRF binding to the adjacent CArG-box2. Thus, unlike other proteins reported to inhibit SRF activity, the repressor activity associated with the GC-rich element does not appear to function through direct inhibition of SRF binding. As a first step toward understanding the importance of these elements in vivo, we performed in vivo footprinting on the intact rat aorta. We demonstrated that both CArG boxes and the GC-rich element were bound by protein within the animal. Additionally, using the rat carotid injury model we showed that Sp1 protein was significantly increased in SMCs located within the myointimal lesion, suggesting that increased expression of this putative repressor factor may contribute to the decreased SM MHC expression within SMCs found in myointimal lesions.
Asunto(s)
Regulación de la Expresión Génica , Músculo Liso Vascular/metabolismo , Cadenas Pesadas de Miosina/genética , Secuencias Reguladoras de Ácidos Nucleicos , Proteínas Represoras/genética , Animales , Aorta/metabolismo , Secuencia de Bases , ADN/metabolismo , Huella de ADN , Análisis Mutacional de ADN , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Ratas , Proteínas Represoras/metabolismo , Factor de Transcripción Sp1/metabolismo , Transcripción GenéticaRESUMEN
To determine the role of angiotensin II-receptor blockade on cardiovascular remodeling in a pressure-overload model of cardiac hypertrophy, a subdiaphragmatic aortic band was placed in adult male Sprague-Dawley rats. Aortic-banded (AB) rats were left untreated or were losartan (Los; 250 mg/l) treated (AB-Los). Sham-operated (S) controls were either left untreated or treated with Los (S-Los). After 4 wk, rats were catheterized for measurement of mean arterial pressures [carotid (CMAP) and femoral (FMAP), in mmHg]. Hearts were perfused on a modified Langendorff system, and minimal coronary resistance (MCR) was determined. Hearts were then perfusion fixed, total and regional heart weights were recorded, and sections were processed for morphology. Changes in coronary artery medial thickness and perivascular fibrosis were assessed by quantitative image analysis. CMAP was significantly higher in AB and AB-Los than in S or S-Los (P < 0.05). There was no difference in FMAP in AB vs. S, but AB-Los and S-Los had lower FMAPs than S. Total heart weight and left ventricular weight-to-body weight ratios were increased in AB and AB-Los compared with S and S-Los (P < 0.05). MCR of AB was greater than S and S-Los. MCR of AB-Los was significantly lower than AB and was not significantly different from S and S-Los. In coronary vessels, medial thickness was greatest in AB, whereas there was no difference among AB-Los, S, and S-Los. Similarly, the increase in perivascular fibrosis was greatest in AB, and there was no difference among AB-Los, S, and S-Los. These data suggest that angiotensin II, independent of increased arterial pressure, is critical for the development of the vascular and fibrotic changes that occur in this model of pressure-overload hypertrophy.
Asunto(s)
Angiotensina II/fisiología , Antagonistas de Receptores de Angiotensina , Cardiomegalia/fisiopatología , Animales , Aorta , Compuestos de Bifenilo/farmacología , Presión Sanguínea , Cardiomegalia/patología , Constricción , Circulación Coronaria , Vasos Coronarios/anatomía & histología , Corazón/anatomía & histología , Imidazoles/farmacología , Losartán , Masculino , Tamaño de los Órganos , Ratas , Ratas Sprague-Dawley , Receptor de Angiotensina Tipo 1 , Receptor de Angiotensina Tipo 2 , Receptores de Angiotensina/fisiología , Tetrazoles/farmacología , Resistencia VascularRESUMEN
The purpose of the current study was to determine if early, short-term treatment of spontaneously hypertensive rats (SHR) with captopril would cause a persistent attenuation of the structural alterations of the heart, aorta, and coronary arteries that are commonly seen in adult SHR. Therefore, mating pairs of SHR were treated with captopril and the pups were kept on captopril (SHRC) or were taken off captopril at two months (SHROC). Untreated SHR and Wistar-Kyoto (WKY) rats were mated and served as controls. At 8-10 months of age, heart weight and left ventricular weight/body weight ratios were increased in SHR compared to WKY, SHRC, and SHROC. Aortic medial areas of SHR and SHROC were similar and were larger than WKY and SHRC. Nuclear density in SHR and SHROC was less than WKY and SHRC suggesting hypertrophy of the medial wall. In coronary vessels, medial thickness was greatest in SHR, while there was no difference among WKY, SHRC, SHROC. These data suggest that early, short-term treatment of SHR with captopril permanently attenuated the structural alterations in the heart and coronary vessels that are commonly seen in adult SHR.
Asunto(s)
Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Antihipertensivos/uso terapéutico , Captopril/uso terapéutico , Enfermedades Cardiovasculares/tratamiento farmacológico , Hipertensión/tratamiento farmacológico , Análisis de Varianza , Animales , Aorta Torácica/efectos de los fármacos , Presión Sanguínea/efectos de los fármacos , Vasos Coronarios/efectos de los fármacos , Corazón/efectos de los fármacos , Tamaño de los Órganos/efectos de los fármacos , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Factores de TiempoRESUMEN
To determine the role of the renin-angiotensin system (RAS) on cardiovascular remodeling in a pressure overload model of cardiac hypertrophy, a subdiaphragmatic aortic band was placed in adult male, Sprague-Dawley rats. Rats were left untreated (AB) or given captopril (Cap, 400 mg/l) (AB-Cap). Sham-operated controls were either left untreated (S) or given Cap (S-Cap). After 4 wk, rats were catheterized, and carotid and femoral mean arterial pressures (CMAP and FMAP in mmHg, respectively) were recorded. Hearts were isolated, and minimal coronary resistance (MCR) was determined. Hearts were then perfusion fixed, total and regional heart weights were recorded, and sections were processed for vessel morphology. Changes in coronary artery medical thickness and perivascular fibrosis were assessed by quantitative image analysis. CMAP was significantly higher in AB and AB-Cap than S or S-Cap rats (P < 0.05). There was no difference in FMAP in AB vs. S rats, but AB-Cap and S-Cap had lower FMAP values than S rats. Total heart weight and left ventricular weight-to-body weight ratios were increased in AB and AB-Cap rats compared with S and S-Cap rats (P < 0.05). MCR of AB was greater than S and S-Cap rats. MCR of AB-Cap rats was significantly greater than S and S-Cap rats but was significantly less than AB rats. In coronary vessels, medial thickness was greatest in AB, whereas there was no difference among AB-Cap, S, and S-Cap rats. Similarly, the increase in perivascular fibrosis was greatest in AB rats, and there was no difference among AB-Cap, S, and S-Cap rats. These data suggest that the RAS, independent of increased arterial pressure, is critical for the development of the vascular and fibrotic changes that occur in this model of pressure overload hypertrophy.
Asunto(s)
Captopril/farmacología , Cardiomegalia/patología , Vasos Coronarios/efectos de los fármacos , Vasos Coronarios/patología , Animales , Aorta , Presión Sanguínea/efectos de los fármacos , Constricción , Circulación Coronaria/efectos de los fármacos , Fibrosis , Técnicas In Vitro , Masculino , Miocardio/patología , Tamaño de los Órganos/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Resistencia Vascular/efectos de los fármacosRESUMEN
Lifetime treatment with oral captopril prevents the development of hypertension in spontaneously hypertensive rats (SHR). We tested the hypothesis that this treatment also prevents the hypertensive response that occurs when untreated NaCl-sensitive SHR are placed on a high NaCl diet. Female SHR were continuously treated with oral captopril before conception and throughout lactation, and the offspring were similarly treated with oral captopril throughout life. At 6 weeks of age, treated male SHR were placed on an 8% (or remained on a 1%) NaCl diet, and systolic arterial pressure, heart rate, and body weight were monitored for 2 weeks. The 8% NaCl diet caused a rapid increase in arterial pressure in the lifetime captopril-treated rats, and 18 days after the initiation of the diet, the mean arterial pressure of this group was 136 +/- 7 mm Hg compared with 100 +/- 2 mm Hg in the 1% NaCl diet rats. The results of a second experiment confirmed the hypertensive effect of the high NaCl diet in lifetime captopril-treated SHR and demonstrated that after 18 days on the diet the dietary NaCl-induced hypertensive response was greater in magnitude in lifetime captopril-treated compared with untreated SHR. The results also demonstrated that lifetime captopril-treated Wistar-Kyoto rats, which are normotensive irrespective of captopril treatment, display no significant increase in arterial pressure when given a high NaCl diet. A third experiment demonstrated that rapidly progressing NaCl sensitivity is also present in female lifetime captopril-treated SHR.(ABSTRACT TRUNCATED AT 250 WORDS)