La inhibición de Rho quinasa post infarto mejora el remodelado y la función ventricular: mecanismos involucrados a nivel preclínico / Rho-kinase inhibition post myocardial infarction improves remodeling and systolic function: a preclinical study of intervening factors in a pre-clinical study

Resumen: Objetivo: Determinar algunos mecanismos moleculares por los cuales la activación de ROCK cardíaca post infarto del miocardio (IAM) participa en el remodelado y en deterioro de la función sistólica. Métodos: Determinación simultánea de niveles de proteínas blanco de ROCK cardíaca, de función sistólica in vivo del ventrículo izquierdo (VI) y de fibrosis e hipertrofia cardíaca en ratas con IAM en condiciones de inhibición de ROCK con fasudil. Resultados : Siete días post IAM la masa ventricular relativa aumentó significativamente en un 30% en el grupo MI y se redujo con fasudil. La disfunción sistólica VI mejoró significativamente con fasudil mientras que la activación de ROCK cardíaca se redujo a niveles del grupo control. El inhibidor de ROCK también redujo significativamente los niveles cardíacos elevados de las isoformas ROCK1 y ROCK2, de MHC-β y del colágeno miocárdico. En el grupo con IAM aumentaron significativamente los niveles de fosforilación de ERK 42 y ERK 44 (en 2 veces y en 63%, respectivamente), mientras que en el grupo IAM tratado con fasudil estos niveles fueron similares a los del grupo control. El IAM aumentó significativamente los niveles fosforilados del factor de transcripción GATA-4, que se normalizaron con el inhibidor de ROCK. Conclusiones: La disfunción sistólica post IAM se asoció fuertemente con la activación del ROCK cardíaca y con la fosforilación de proteínas río abajo de ROCK que promueven remodelado cardíaco como β-MHC y la vía ERK / GATA-4.

Abstracts: Objective: to determine some molecular mechanisms by which cardiac ROCK activation after myocardial infarction (MI) intervene in cardiac systolic function decline and remodeling. Methods: simultaneous measurement of different cardiac ROCK target proteins levels, in vivo left ventricular (LV) systolic function, myocardial fibrosis, and hypertrophy in rats with MI under ROCK inhibition with fasudil were performed. Results: seven days after MI the relative ventricular mass increased significantly by 30% in the MI groupand was reduced with fasudil. LV systolic dysfunction improved significantly with fasudil whereas at the same time cardiac ROCK activation was reduced to sham levels. The ROCK inhibitor also reduced increased cardiac levels of both ROCK1 and ROCK2 isoforms, β-MHC levels and myocardial collagen volume fraction decline. MI significantly increased phosphorylation levels of ERK 42 and ERK 44 by 2-fold and 63% respectively whereas in the fasudil-treated MI group these levels were similar to those in the sham group. MI significantly increased phosphorylated levels of the transcription factor GATA-4 which were normalyzed by the ROCK inhibitor. Conclusion: LV systolic dysfunction after MI was strongly associated to cardiac ROCK activation and subsequent phosphorylation of ROCK target proteins that promote ventricular remodeling, such as β-MHC and the ERK/GATA-4 pathway. ROCK inhibition with fasudil significantly improved systolic function, diminished myocardial fibrosis, and normalized β-MHC and ERK/GATA-4 phosphorylation levels.

Rho kinase inhibitors for glaucoma treatment - Review / Inibidores da Rho-Quinase para o tratamento do glaucoma - Revisão

ABSTRACT Glaucoma is a progressive optic neuropathy characterized by the loss of ganglion cells and their axons. A major risk factor for glaucomatous visual field loss is elevated intraocular pressure (IOP), and several studies have shown that lowering IOP reduces the risk of glaucomatous progression. Currently, an increasing number of researches involve Rho kinase inhibitors, which are a new pharmacological class of hypotensive agents specifically targeting the diseased trabecular outflow pathway. Rho kinase inhibitors reduce IOP by increasing aqueous humor drainage through the primary outflow pathway in the eye, which is known as the trabecular meshwork. In addition to improving the outflow facility of the trabecular meshwork, Rho kinase inhibitors also enhance retinal ganglion cell survival after ischemic injury and increase ocular blood flow.

RESUMO Glaucoma é uma neuropatia óptica progressiva, caracterizada pela perda de células ganglionares e seus axônios. O principal fator de risco que leva à perda de campo visual relacionada ao glaucoma é a elevação da pressão intraocular (PIO) e vários estudos mostraram que a redução da pressão intraocular diminui o risco de progressão do glaucoma. Atualmente, uma nova classe de drogas hipotensoras foi desenvolvida e tem sido cada vez mais estudada, os inibidores da Rho-Kinase. Essas drogas reduzem a pressão intraocular aumentando a drenagem de humor aquoso através da via de drenagem primária do humor aquoso no olho, a malha trabecular. Além de aumentar o escoamento pela malha trabecular, inibidores da Rho-kinase também aumentam a sobrevivência das células ganglionares retinianas após isquemia e aumentam o fluxo ocular sanguíneo.

Inhibition of Rho-associated kinases disturbs the collective cell migration of stratified TE-10 cells

BACKGROUND: The collective cell migration of stratified epithelial cells is considered to be an important phenomenon in wound healing, development, and cancer invasion; however, little is known about the mechanisms involved. Furthermore, whereas Rho family proteins, including RhoA, play important roles in cell migration, the exact role of Rho-associated coiled coil-containing protein kinases (ROCKs) in cell migration is controversial and might be cell-type dependent. Here, we report the development of a novel modified scratch assay that was used to observe the collective cell migration of stratified TE-10 cells derived from a human esophageal cancer specimen. RESULTS: Desmosomes were found between the TE-10 cells and microvilli of the surface of the cell sheet. The leading edge of cells in the cell sheet formed a simple layer and moved forward regularly; these rows were followed by the stratified epithelium. ROCK inhibitors and ROCK small interfering RNAs (siRNAs) disturbed not only the collective migration of the leading edge of this cell sheet, but also the stratified layer in the rear. In contrast, RhoA siRNA treatment resulted in more rapid migration of the leading rows and disturbed movement of the stratified portion. CONCLUSIONS: The data presented in this study suggest that ROCKs play an important role in mediating the collective migration of TE-10 cell sheets. In addition, differences between the effects of siRNAs targeting either RhoA or ROCKs suggested that distinct mechanisms regulate the collective cell migration in the simple epithelium of the wound edge versus the stratified layer of the epithelium.

Cinnamyl alcohol attenuates vasoconstriction by activation of K+ channels via NO-cGMP-protein kinase G pathway and inhibition of Rho-kinase

Cinnamyl alcohol (CAL) is known as an antipyretic, and a recent study showed its vasodilatory activity without explaining the mechanism. Here we demonstrate the vasodilatory effect and the mechanism of action of CAL in rat thoracic aorta. The change of tension in aortic strips treated with CAL was measured in an organ bath system. In addition, vascular strips or human umbilical vein endothelial cells (HUVECs) were used for biochemical experiments such as Western blot and nitrite and cyclic guanosine monophosphate (cGMP) measurements. CAL attenuated the vasoconstriction of phenylephrine (PE, 1 microM)-precontracted aortic strips in an endothelium-dependent manner. CAL-induced vasorelaxation was inhibited by pretreatment with NG-nitro-L-arginine methyl ester (L-NAME; 10(-4) M), methylene blue (MB; 10(-5) M) and 1 H-[1,2,4]-oxadiazolole-[4,3-a] quinoxalin-10one, (ODQ; 10(-6) or 10(-7) M) in the endothelium-intact aortic strips. Atrial natriuretic peptide (ANP; 10(-8) or 10(-9) M) did not affect the vasodilatory effect of CAL. The phosphorylation of endothelial nitric oxide synthase (eNOS) and generation of nitric oxide (NO) were stimulated by CAL treatment in HUVECs and inhibited by treatment with L-NAME. In addition, cGMP and PKG1 activation in aortic strips treated with CAL were also significantly inhibited by L-NAME. Furthermore, CAL relaxed Rho-kinase activator calpeptin-precontracted aortic strips, and the vasodilatory effect of CAL was inhibited by the ATP-sensitive K+ channel inhibitor glibenclamide (Gli; 10(-5) M) and the voltage-dependent K+ channel inhibitor 4-aminopyridine (4-AP; 2 x 10(-4) M). These results suggest that CAL induces vasorelaxation by activating K+ channels via the NO-cGMP-PKG pathway and the inhibition of Rho-kinase.

Efectividad del uso combinado de un inhibidor de Rho Kinasa y de un antagonista del receptor de angiotensina II en la prevención de hipertrofia ventricular en ratas hipertensas / Effectiveness of the combined use of a Rho-kinase inhibitor and an Angiotensin II receptor antagonist in the prevention of left ventricular hypertrophy in hypertensive rats

La actividad de Rho kinasa (ROCK) cardíaca en la hipertensión arterial (HTA) y el efecto del tratamiento antihipertensivo conjunto han sido poco estudiados. Hemos planteado que la adición de un inhibidor de ROCK al tratamiento antihipertensivo convencional podría tener efectos preventivos adicionales al uso aislado del antihipertensivo. Objetivo: Determinar la actividad de ROCK ventricular y parámetros de remodelamiento cardíaco en ratas hipertensas con y sin tratamiento antihipertensivo, adicionando un inhibidor directo de ROCK. Métodos. Se usaron ratas Sprague Dawley de 150 grs. ( n = 12 - 13/grupo) unifrectomizadas tratadas con desoxicorticosterona (DOCA, 100 mg/Kg/sem sbc) durante 6 semanas. Como controles se usaron ratas unifrectomizadas. Otros 3 grupos recibieron DOCA y además el antagonista del receptor de angiotensina n, candesartán (10 mg/kg/día) o el inhibidor de la vía ROCK fasudil (50 mg/Kg/dia), o la combinación de ambos (5 y 25 mg/Kg/dia, respectivamente), vía gavage desde la tercera semana post cirugía, durante 3 semanas. Al finalizar los tratamientos se determinó la masa corporal (MC), presión arterial sistólica (PAS) y la masa cardíaca relativa (MCR). Además se midió en el ventrículo izquierdo la fosforilación de la fosfatasa de la miosina (MYPT-1) como índice de activación de ROCK, la infiltración de macrófagos/ monocitos (células ED1 positivas), la expresión proteica de colágeno I (por Western blot) y la expresión génica de la subunidad gp91 de NADPH oxidasa y eNOS por RTPCR. Resultados: Con respecto de las ratas sham, en las ratas hipertensas se observó hipertrofia cardiaca de 63 por ciento (p < 0,05), junto a aumento significativo (p < 0,05) de MYPT-1 fosforilado/total (300 por ciento), de colágeno miocárdico I (en 14 veces), de células ED1 en 270 por ciento y de la expresión génica de la subunidad gp91 de NADPH...

Background: The effect of cardiac Rho-kinase (ROCK) on hypertension (HT) and cardiac hypertrophy prevention and also the combined anti-hypertensive treatment have been scarcely studied. We hypothesized that the addition of a ROCK inhibitor to conventional anti-hypertensive treatment may have additional beneficial effects. Ainv to determine ventricular ROCK activity and ventricular remodeling in hypertensive rats treated with Angiotensin II inhibition with the addition of a ROCK inhibitor. Methods: Sprague-Dawley rats weighing 150 grams had one kidney removed and received deoxycortisterone acétate (DOCA, 100 mg/kg/week, during 6 weeks). Unilaterally nephrectomized rats were used as controls. The other 3 groups received DOCA along with the Angiotensin II receptor blocker candesartan (10 mg/kg/day) or the combination of both agents (5 and 25 mg/kg/day, respectively) and ROCK inhibitor fasudil (50 mg/kg/day) for 3 weeks starting 3 weeks after surgery. Body mass (BM), systolic blood pressure (SBP) and relative cardiac mass (RCM) were measured. In addition, myosin phosphatase (MYPT-1) phosphorylation was measured as an indicator of ROCK activation. Cardiac infiltration of macrophages/monocytes (ED1 positive cells), collagen I protein contení (by Western Blot) and also cardiac gene expression of NADPH oxydase GP91 subunit and eNOS were determined by RT-PCR. Results: In hypertensive rats we observed cardiac hypertrophy by 63 percent (p < 0.05), a 300 percent increase in cardiac MYPT-1 phosphorylation (p< 0.05), 14 times increase in myocardial collagen type 1,270 percent increase in ED1 cells, a 75 percent increased gene expression of NADPH oxydase GP91 subunit and a 37 percent reduction (p< 0.05) in the gene expression of cardiac eNOS. In hypertensive DOCA rats treated during 3 week with candesartan, fasudil or the combination of both, we observed a significant reduction in cardiac hypertrophy and normalization of SBP, MYPT-1 phosphorylation, ...

La sobrexpresión génica vascular de NADPH oxidasa en la hipertensión arterial experimental se reduce solamente con inhibición directa de Rho kinasa / Vascular gene overexpression of NADPH oxydase in experimental hypertension is only reduced by direct Rho kinase inhibition

Uno de los objetivos del tratamiento antihipertensivo, más allá de normalizar las cifras tensionales, es disminuir/regresar el remodelado patológico hipertensivo cardiovascular y renal, de manera de reducir eficientemente el riesgo dado por esta patología. Al respecto, la vía de señalización intracelular de la Rho kinasa (ROCK) es un mecanismo de vasoconstricción y de promoción de remodelado que podría ser un blanco atractivo en el tratamiento antihipertensivo. Objetivo: Evaluar a nivel vascular los niveles de TGF beta, la expresión génica vascular de NADPH oxidasa (fuente de stress oxidativo vascular), y el grado de inflamación parietal y su dependencia de ROCK en la hipertensión arterial (HTA) experimental. Métodos: Se compararon 5 grupos experimentales. Se usó el modelo de HTA por administración de deoxicorticosterona (DOCA, 100mg/Kg, sc una vez/semana) + sal en ratas Sprague Dawley uninefrectomizadas. Como controles, se usaron ratas uninefrectomizadas (Sham). Las ratas DOCA se randomizaron para recibir el inhibidor específico de ROCK fasudil (Fas, 50 mg/kg/d, por gavage, durante 3 semanas), desde la semana 3 post cirugía, o candesartán (Cand, 10 mg/kg/d, por gavage, durante 3 semanas), o fasudil (25 mg/kg/d) + candesartan (5 mg/ kg/d por gavage, 3 semanas ). Al finalizar los experimentos se midieron en la aorta los niveles de MYPT1 fosforilada/total, blanco de ROCK y estimador de su activación (por Western blot), de TGF beta (por Western blot), los niveles de mRNA de las subunidades p22 Phox y gp 91 de la NADPH oxidasa (por RT PCR) y el número de células infamatorias ED1 en anillos aórticos (por inmunohistoquímica). Resultados: La presión arterial sistólica aumentó en las ratas DOCA a 172 +/- 7 mm Hg (p < 0,05) y fue normalizada después de 3 semanas de tratamiento con candesartán, fasudil y de candesartán + fasudil. La actividad de ROCK en aorta aumentó en 4 veces en las ratas hipertensas (p < 0,05)...

Background: Rho kinase (ROCK) activity promotes vasoconstriction and pathological vascular remodeling in experimental hypertension. Our working hypothesis is that ROCK inhibition could be an attractive target to prevent vascular remodeling in hypertension. Objectives: We evaluated vascular TGF beta, the genic expression of NADPH oxydase (a vascular oxidative stress source) and its dependency from ROCK activation in experimental hypertension in the rat. Methods: Five experimental groups were compared. Hypertension was induced by the administration of salt and deoxycorticosterone acetate (DOCA, 100 mg/Kg, weekly) to unilaterally nephrectomized rats. Unilaterally nephrectomized rats were used as controls (controls). DOCA rats were randomized to receive either Fasudil (a ROCK inhibitor, 50 mg/Kg/day) or candesartan (CAND, 10 mg/Kg/day for 3 weeks), starting 3 weeks after surgery. The other group received fasudil (25 mg/Kg/day) plus CAND (5 mg/Kg/day) for 3 weeks. After treatment, phosphorilated MYPT1 (a ROCK target expressing ROCK activation) was measured in aortic wall rings by Western Blot. We also determined TGF-beta (Western Blot), p22 Phox and gp 91subnits of NADPH oxydase mRNA (RT-PCR) and the number of ED1 infammatory cells. Results: In DOCA rats, SBP increased to 172 +/- 7 mm Hg (p < 0,05), and returned to normal values after 3 weeks with candesartan, fasudil or both combined. In these rats, ROCK activity in aorta was increased 4 times (p < 0,05) and returned to control values in the 3 groups receiving treatment. p22 Phox and gp 91subnits of NADPH oxydase mRNA were increased by 80 and 90 percent, respectively (p<0,05). These changes were reduced to control values in rats receiving fasudil and candesartan + fasudil. Gene expression of TGF-beta increased 4 times, and the number of ED1...