RESUMO
Keratinocyte stem cells play a fundamental role in homeostasis and repair of stratified epithelial tissues. Transplantation of cultured keratinocytes autografts provides a landmark example of successful cellular therapies by restoring durable integrity in stratified epithelia lost to devastating tissue conditions. Despite the overall success of such procedures, failures still occur in case of paucity of cultured stem cells in therapeutic grafts. Strategies aiming at a further amplification of stem cells during keratinocyte ex vivo expansion may thus extend the applicability of these treatments to subjects in which endogenous stem cells pools are depauperated by aging, trauma, or disease. Pharmacological targeting of stem cell signaling pathways is recently emerging as a powerful strategy for improving stem cell maintenance and/or amplification. Recent experimental data indicate that pharmacological inhibition of two prominent keratinocyte signaling pathways governed by apical mTOR and ROCK protein kinases favor stem cell maintenance and/or amplification ex vivo and may improve the effectiveness of stem cell-based therapeutic procedures. In this review, we highlight the pathophysiological roles of mTOR and ROCK in keratinocyte biology and evaluate existing pre-clinical data on the effects of their inhibition in epithelial stem cell expansion for transplantation purposes.
Assuntos
Queratinócitos , Medicina Regenerativa , Diferenciação Celular , Humanos , Queratinócitos/metabolismo , Células-Tronco/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
BACKGROUND: Vitamin B3 (nicotinamide) plays important roles in metabolism as well as in SIRT and PARP pathways. It is also recently reported as a novel kinase inhibitor with multiple targets. Nicotinamide promotes pancreatic cell differentiation from human embryonic stem cells (hESCs). However, its molecular mechanism is still unclear. In order to understand the molecular mechanism involved in pancreatic cell fate determination, we analyzed the downstream pathways of nicotinamide in the derivation of NKX6.1+ pancreatic progenitors from hESCs. METHODS: We applied downstream modulators of nicotinamide during the induction from posterior foregut to pancreatic progenitors, including niacin, PARP inhibitor, SIRT inhibitor, CK1 inhibitor and ROCK inhibitor. The impact of those treatments was evaluated by quantitative real-time PCR, flow cytometry and immunostaining of pancreatic markers. Furthermore, CK1 isoforms were knocked down to validate CK1 function in the induction of pancreatic progenitors. Finally, RNA-seq was used to demonstrate pancreatic induction on the transcriptomic level. RESULTS: First, we demonstrated that nicotinamide promoted pancreatic progenitor differentiation in chemically defined conditions, but it did not act through either niacin-associated metabolism or the inhibition of PARP and SIRT pathways. In contrast, nicotinamide modulated differentiation through CK1 and ROCK inhibition. We demonstrated that CK1 inhibitors promoted the generation of PDX1/NKX6.1 double-positive pancreatic progenitor cells. shRNA knockdown revealed that the inhibition of CK1α and CK1ε promoted pancreatic progenitor differentiation. We then showed that nicotinamide also improved pancreatic progenitor differentiation through ROCK inhibition. Finally, RNA-seq data showed that CK1 and ROCK inhibition led to pancreatic gene expression, similar to nicotinamide treatment. CONCLUSIONS: In this report, we revealed that nicotinamide promotes generation of pancreatic progenitors from hESCs through CK1 and ROCK inhibition. Furthermore, we discovered the novel role of CK1 in pancreatic cell fate determination.
Assuntos
Células-Tronco Embrionárias Humanas , Diferenciação Celular , Endoderma , Humanos , Niacinamida/farmacologia , PâncreasRESUMO
Palmitoylethanolamide (PEA) offers a strong protection against BBB disruption and neurological deficits after cerebral ischaemic/reperfusion (I/R) injury. To date, these BBB protective effects of PEA are mainly attributed to PPARα-mediated actions. However, whether PEA protects against BBB disruption through direct regulation of cytoskeletal microfilaments remains unknown. Here, we identified PEA as a Rho-associated protein kinase (ROCK2) inhibitor (IC50 = 38.4 ± 4.8 µM). In vitro data suggested that PEA reduced the activation of ROCK/MLC signaling and stress fiber formation within microvascular endothelial cells (ECs) after oxygen-glucose deprivation (OGD), and consequently attenuated early (0-4 h) EC barrier disruption. These actions of PEA could not be blocked by the PPARα antagonist GW6471. In summary, the present study described a previously unexplored role of PEA as a ROCK2 inhibitor, and propose a PPARα-independent mechanism for pharmacological effects of PEA.
Assuntos
Amidas/uso terapêutico , Barreira Hematoencefálica/efeitos dos fármacos , Isquemia Encefálica/tratamento farmacológico , Etanolaminas/uso terapêutico , Cadeias Leves de Miosina/metabolismo , Ácidos Palmíticos/uso terapêutico , Traumatismo por Reperfusão/tratamento farmacológico , Quinases Associadas a rho/metabolismo , Amidas/farmacologia , Animais , Barreira Hematoencefálica/metabolismo , Isquemia Encefálica/metabolismo , Linhagem Celular , Etanolaminas/farmacologia , Humanos , Camundongos , Ácidos Palmíticos/farmacologia , Traumatismo por Reperfusão/metabolismo , Transdução de Sinais/efeitos dos fármacos , Quinases Associadas a rho/antagonistas & inibidoresRESUMO
Ripasudil (K-115) is a novel Rho-associated protein kinase (ROCK) inhibitor. The Rho-ROCK pathway regulates key downstream effectors involved in many cellular functions, in particular in the actin cytoskeleton activity. The clinical effects of ripasudil expected on the eye include an intraocular pressure-lowering effect and a wound-healing activity on corneal endothelial cells, but many other functions are currently under investigation. To date, ripasudil has been approved in Japan (2014) for the treatment of glaucoma and ocular hypertension, and several clinical trials are currently investigating its role in the treatment of Fuchs' corneal dystrophy. In this review, we will discuss its pharmacokinetics, pharmacodynamics and clinical efficacy, focusing also on its safety and tolerability profile.
Assuntos
Glaucoma/tratamento farmacológico , Isoquinolinas/uso terapêutico , Hipertensão Ocular/tratamento farmacológico , Sulfonamidas/uso terapêutico , Ensaios Clínicos como Assunto , Células Endoteliais , Humanos , Japão , Quinases Associadas a rho/antagonistas & inibidoresRESUMO
Tumorigenesis involves a complex interplay between genetically modified cancer cells and their adjacent normal tissue, the stroma. We used an established breast cancer mouse model to investigate this inter-relationship. Conditional activation of Rho-associated protein kinase (ROCK) in a model of mammary tumorigenesis enhances tumor growth and progression by educating the stroma and enhancing the production and remodeling of the extracellular matrix. We used peptide matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to quantify the proteomic changes occurring within tumors and their stroma in their regular spatial context. Peptides were ranked according to their ability to discriminate between the two groups, using a receiver operating characteristic tool. Peptides were identified by liquid chromatography tandem mass spectrometry, and protein expression was validated by quantitative immunofluorescence using an independent set of tumor samples. We have identified and validated four key proteins upregulated in ROCK-activated mammary tumors relative to those expressing kinase-dead ROCK, namely, collagen I, α-SMA, Rab14, and tubulin-ß4. Rab14 and tubulin-ß4 are expressed within tumor cells, whereas collagen I is localized within the stroma. α-SMA is predominantly localized within the stroma but is also expressed at higher levels in the epithelia of ROCK-activated tumors. High expression of COL1A, the gene encoding the pro-α 1 chain of collagen, correlates with cancer progression in two human breast cancer genomic data sets, and high expression of COL1A and ACTA2 (the gene encoding α-SMA) are associated with a low survival probability (COLIA, p = 0.00013; ACTA2, p = 0.0076) in estrogen receptor-negative breast cancer patients. To investigate whether ROCK-activated tumor cells cause stromal cancer-associated fibroblasts (CAFs) to upregulate expression of collagen I and α-SMA, we treated CAFs with medium conditioned by primary mammary tumor cells in which ROCK had been activated. This led to abundant production of both proteins in CAFs, clearly highlighting the inter-relationship between tumor cells and CAFs and identifying CAFs as the potential source of high levels of collagen 1 and α-SMA and associated enhancement of tissue stiffness. Our research emphasizes the capacity of MALDI-MSI to quantitatively assess tumor-stroma inter-relationships and to identify potential prognostic factors for cancer progression in human patients, using sophisticated mouse cancer models.
Assuntos
Fibroblastos Associados a Câncer , Proteômica , Animais , Matriz Extracelular , Fibroblastos , Humanos , Camundongos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Proteínas rab de Ligação ao GTPRESUMO
Since material stiffness controls many cell functions, we reviewed the currently available knowledge on stiffness sensing and elucidated what is known in the context of clinical and experimental articular cartilage (AC) repair. Remarkably, no stiffness information on the various biomaterials for clinical AC repair was accessible. Using mRNA expression profiles and morphology as surrogate markers of stiffness-related effects, we deduced that the various clinically available biomaterials control chondrocyte (CH) phenotype well, but not to equal extents, and only in non-degenerative settings. Ample evidence demonstrates that multiple molecular aspects of CH and mesenchymal stromal cell (MSC) phenotype are susceptible to material stiffness, because proliferation, migration, lineage determination, shape, cytoskeletal properties, expression profiles, cell surface receptor composition, integrin subunit expression, and nuclear shape and composition of CHs and/or MSCs are stiffness-regulated. Moreover, material stiffness modulates MSC immuno-modulatory and angiogenic properties, transforming growth factor beta 1 (TGF-ß1)-induced lineage determination, and CH re-differentiation/de-differentiation, collagen type II fragment production, and TGF-ß1- and interleukin 1 beta (IL-1ß)-induced changes in cell stiffness and traction force. We then integrated the available molecular signaling data into a stiffness-regulated CH phenotype model. Overall, we recommend using material stiffness for controlling cell phenotype, as this would be a promising design cornerstone for novel future-oriented, cell-instructive biomaterials for clinical high-quality AC repair tissue.
Assuntos
Materiais Biocompatíveis/química , Cartilagem Articular/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Mecanotransdução Celular/genética , Osteoartrite/terapia , Regeneração/efeitos dos fármacos , Materiais Biocompatíveis/uso terapêutico , Biomarcadores/metabolismo , Cartilagem Articular/imunologia , Cartilagem Articular/patologia , Cartilagem Articular/cirurgia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Condrócitos/citologia , Condrócitos/metabolismo , Condrogênese/efeitos dos fármacos , Condrogênese/genética , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Regulação da Expressão Gênica , Dureza/fisiologia , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Osteoartrite/genética , Osteoartrite/imunologia , Osteoartrite/cirurgia , Fenótipo , Regeneração/genética , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Rho GTPases play a central role in neuronal survival; however, the antagonistic relationship between Rac and Rho in the regulation of motor neuron survival remains poorly defined. In the current study, we demonstrate that treatment with NSC23766, a selective inhibitor of the Rac-specific guanine nucleotide exchange factors, Tiam1 and Trio, is sufficient to induce the death of embryonic stem cell (ESC)-derived motor neurons. The mode of cell death is primarily apoptotic and is characterized by caspase-3 activation, de-phosphorylation of ERK5 and AKT, and nuclear translocation of the BH3-only protein Bad. As opposed to the inhibition of Rac, motor neuron cell death is also induced by constitutive activation of Rho, via a mechanism that depends on Rho kinase (ROCK) activity. Investigation of Rac and Rho in the G93A mutant, human Cu, Zn-superoxide dismutase (hSOD1) mouse model of amyotrophic lateral sclerosis (ALS), revealed that active Rac1-GTP is markedly decreased in spinal cord motor neurons of transgenic mice at disease onset and end-stage, when compared to age-matched wild type (WT) littermates. Furthermore, although there is no significant change in active RhoA-GTP, total RhoB displays a striking redistribution from motor neuron nuclei in WT mouse spinal cord to motor neuron axons in end-stage G93A mutant hSOD1 mice. Collectively, these data suggest that the intricate balance between pro-survival Rac signaling and pro-apoptotic Rho/ROCK signaling is critical for motor neuron survival and therefore, disruption in the balance of their activities and/or localization may contribute to the death of motor neurons in ALS.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Neurônios Motores/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Superóxido Dismutase/fisiologia , Quinases Associadas a rho/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Animais , Morte Celular/fisiologia , Feminino , GTP Fosfo-Hidrolases/genética , Masculino , Camundongos , Camundongos Transgênicos , Neurônios Motores/patologia , Mutação/fisiologia , Proteínas Proto-Oncogênicas c-akt/genética , Quinases Associadas a rho/genéticaRESUMO
Transforming growth factor-ß (TGF-ß)-induced differentiation of orbital fibroblasts into myofibroblasts is an important pathogenesis of Graves' ophthalmopathy (GO) and leads to orbital tissue fibrosis. In the present study, we explored the antifibrotic effects of simvastatin and ROCK inhibitor Y-27632 in primary cultured GO orbital fibroblasts and tried to explain the molecular mechanisms behind these effects. Both simvastatin and Y-27632 inhibited TGF-ß-induced α-smooth muscle actin (α-SMA) expression, which serves as a marker of fibrosis. The inhibitory effect of simvastatin on TGF-ß-induced RhoA, ROCK1, and α-SMA expression could be reversed by geranylgeranyl pyrophosphate, an intermediate in the biosynthesis of cholesterol. This suggested that the mechanism of simvastatin-mediated antifibrotic effects may involve RhoA/ROCK signaling. Furthermore, simvastatin and Y-27632 suppressed TGF-ß-induced phosphorylation of ERK and p38. The TGF-ß-mediated α-SMA expression was suppressed by pharmacological inhibitors of p38 and ERK. These results suggested that simvastatin inhibits TGF-ß-induced myofibroblast differentiation via suppression of the RhoA/ROCK/ERK and p38 MAPK signaling pathways. Thus, our study provides evidence that simvastatin and ROCK inhibitors may be potential therapeutic drugs for the prevention and treatment of orbital fibrosis in GO.
Assuntos
Amidas/farmacologia , Oftalmopatia de Graves/patologia , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Miofibroblastos/efeitos dos fármacos , Piridinas/farmacologia , Sinvastatina/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/efeitos dos fármacos , Quinases Associadas a rho/antagonistas & inibidores , Proteína rhoA de Ligação ao GTP/efeitos dos fármacos , Adulto , Diferenciação Celular/efeitos dos fármacos , Feminino , Fibrose/prevenção & controle , Humanos , Masculino , Pessoa de Meia-Idade , Fosforilação , Cultura Primária de Células , Fator de Crescimento Transformador beta/antagonistas & inibidores , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Salvia przewalskii Maxim is a traditional Chinese herbal medicine and is known to have antibacterial, antiviral, anti-oxidant, anti-thrombotic and anti-depressant properties. However, the major active components of S. przewalskii and its anti-hypoxic effects are still unclear. This study probed the major active component and anti-hypoxic activity of S. przewalskii. The major active components of S. przewalskii were detected by HPLC. The anti-hypoxic effects of S. przewalskii were detected in mice and a rat model of hypoxic preconditioning. The results showed that there are eight active components, including sodium danshensu, rosmarinic acid, lithospermic acid, salvianolic acid B, dihydrotanshinone I, cryptotanshinone, tanshinone I and tanshinone IIA, and each component showed a certain anti-hypoxic effect. Moreover, S. przewalskii enhanced anti-hypoxia in mice, which was manifested as prolonged survival time in acute hypoxic preconditioning and the amelioration of acute hypoxia-induced changes in the activity of superoxide dismutase (SOD) and lactate dehydrogenase (LDH). In addition, S. przewalskii also repaired tissue damage in chronic hypoxia by downregulating hypoxia inducible factor-1α (HIF-1α), proliferating cell nuclear antigen (PCNA), Bcl-2, CDK4, CyclinD1 and P27Kip1 and inhibiting pro-inflammatory cytokines and the RhoA-Rho-associated protein kinase (ROCK) signalling pathway. Our findings provide new insight into the anti-hypoxic effect of S. przewalskii as a promising agent for high-altitude pulmonary hypertension treatment.
Assuntos
Hipóxia/tratamento farmacológico , Extratos Vegetais/farmacologia , Salvia/química , Quinases Associadas a rho/efeitos dos fármacos , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Hipóxia Celular/efeitos dos fármacos , Citocinas/efeitos dos fármacos , Coração/efeitos dos fármacos , Hipertensão Pulmonar/tratamento farmacológico , Hipóxia/induzido quimicamente , Camundongos , Modelos Animais , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
Ocular hypertension is believed to be involved in the etiology of primary open-angle glaucoma. Although many pharmaceutical agents have been shown to be effective for the reduction of intraocular pressure (IOP), a significant opportunity to improve glaucoma treatments remains. Thus, the aims of the present study were: (1) to evaluate the IOP-lowering effect of four compounds RU-551, RU-555, RU-839 (pyrimido[1,2-a]benzimidazole), and RU-615 (imidazo[1,2-a]benzimidazole) on steroid-induced ocular hypertension in rats after single drop and chronic applications; and (2) to test in silico and in vitro conventional rho-associated kinase (ROCK) inhibitory activity of the selected compound. This study demonstrated that RU-551, RU-555, RU-839, and RU-615 significantly reduced IOP in Sprague Dawley rats with dexamethasone (DEXA) induced ocular hypertension after single drop administration (0.1%), however RU-615 showed the best IOP lowering effect as indicated by maximum IOP reduction of 22.32% from baseline. Repeated dose topical application of RU-615 caused sustained reduction of IOP from baseline throughout the 3 weeks of treatment with maximum IOP reduction of 30.31% on day 15. This study also showed that the steroid-induced increase in IOP is associated with increased retinal oxidative stress and significant retinal ganglion cells (RGCs) loss. Prolonged treatment with RU-615 over 3 weeks results in normalization of IOP in DEXA-treated rats with partial restoration of retinal antioxidant status (catalase, glutathione and superoxide dismutase) and subsequent protective effect against RGC loss. Thus, IOP lowering activity of RU-615 together with antioxidant properties might be the factors that contribute to prevention of further RGC loss. In vitro part of this study explored the ROCK inhibitory activity of RU-615 using dexamethasone-treated human trabecular meshwork cells as a possible mechanism of action of its IOP lowering activity. However, this study didn't show conventional ROCK inhibition by RU-615 which was later confirmed by in silico consensus prediction. Therefore, in the future studies it is important to identify the upstream target receptors for RU-615 and then delineate the involved intracellular signalling pathways which are likely to be other than ROCK inhibition.
Assuntos
Benzimidazóis/química , Benzimidazóis/farmacologia , Dexametasona/farmacologia , Pressão Intraocular/efeitos dos fármacos , Hipertensão Ocular/tratamento farmacológico , Hipertensão Ocular/fisiopatologia , Animais , Benzimidazóis/uso terapêutico , Domínio Catalítico , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Humanos , Modelos Moleculares , Hipertensão Ocular/induzido quimicamente , Hipertensão Ocular/patologia , Estresse Oxidativo/efeitos dos fármacos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Ratos , Ratos Sprague-Dawley , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/patologia , Fatores de Tempo , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/químicaRESUMO
Alterations in mechanical properties in the extracellular matrix are modulated by myofibroblasts and are required for progressive fibrotic diseases. Recently, we reported that fibroblasts depleted of SPIN90 showed enhanced differentiation into myofibroblasts via increased acetylation of microtubules in the soft matrix; the mechanisms of the underlying signaling network, however, remain unclear. In this study, we determine the effect of depletion of SPIN90 on FAK/ROCK signaling modules. Transcriptome analysis of Spin90 KO mouse embryonic fibroblasts (MEF) and fibroblasts activated by TGF-ß revealed that Postn is the most significantly upregulated gene. Knockdown of Postn by small interfering RNA suppressed cell adhesion and myofibroblastic differentiation and downregulated FAK activity in Spin90 KO MEF. Our results indicate that SPIN90 depletion activates FAK/ROCK signaling, induced by Postn expression, which is critical for myofibroblastic differentiation on soft matrices mimicking the mechanical environment of a normal tissue.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Moléculas de Adesão Celular/metabolismo , Regulação para Baixo/genética , Fibroblastos/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais , Quinases Associadas a rho/metabolismo , Animais , Diferenciação Celular , Adesões Focais/metabolismo , Camundongos Knockout , Miofibroblastos/metabolismoRESUMO
Rho-associated protein kinase (ROCK), a ubiquitously expressed signaling messenger and downstream effector of Rho, is activated by several bioactive factors in the aqueous humor (AH). Rho-ROCK signaling regulates a wide spectrum of fundamental cellular events, including cell adhesion, motility, proliferation, differentiation, and apoptosis. Previous studies, including our own, found that ROCK inhibitor lowers intraocular pressure (IOP) via a direct effect on the conventional AH outflow pathway, by regulation of contractile properties, fibrotic activity, and permeability of the trabecular meshwork (TM) and Schlemm's canal (SC) tissues, influencing extracellular matrix (ECM) production. Recently, a novel ROCK inhibitor, ripasudil, has been introduced in Japan. Other ROCK inhibitors are now in clinical trials as new IOP-lowering drugs for glaucoma patients. To date, ripasudil, administered together with other glaucoma medications, has proved safe and efficient in lowering IOP as well as additional effects such as prostaglandin analogs, beta-blockers, and carbonic anhydrase inhibitors, all of which help lower IOP by different mechanisms. In addition, we found that long-term treatment with ripasudil exerted an additional IOP-lowering effect, especially in eyes with high IOP, suggesting that late-onset remodeling of the ECM in glaucomatous eyes may elicit mild and delayed changes in IOP levels. ROCK inhibitors have also shown several additional effects, including increased retinal blood flow, direct protection of neurons against various types of stress, and regulation of wound healing; these benefits may potentially be useful in glaucoma treatment.
Assuntos
Humor Aquoso/enzimologia , Glaucoma , Pressão Intraocular/efeitos dos fármacos , Isoquinolinas/uso terapêutico , Sulfonamidas/uso terapêutico , Quinases Associadas a rho/antagonistas & inibidores , Glaucoma/tratamento farmacológico , Glaucoma/enzimologia , Glaucoma/fisiopatologia , Humanos , Quinases Associadas a rho/biossínteseRESUMO
OBJECTIVES: To evaluate the expression of the Rho/Rho-associated protein kinase (ROCK) pathway in the corpus cavernosum of patients with severe erectile dysfunction (ED) compared with healthy human corpus cavernosum, and to test the functional effects of two Rho kinase inhibitors (RKIs) on erectile tissue of patients with severe ED, which did not respond to phosphodiesterase type 5 inhibitors (PDE5Is). PATIENTS AND METHODS: Human corpus cavernosum samples were obtained after consent from men undergoing penile prosthesis implantation (n = 7 for organ bath experiments, n = 17 for quantitative PCR [qPCR]). Potent control subjects (n = 5) underwent penile needle biopsy. qPCR was performed for the expression of RhoA and ROCK subtypes 1 and 2. Immunohistochemistry staining against ROCK and α smooth muscle actin (αSMA) was performed on the corpus cavernosum of patients with ED. Tissue strips were precontracted with phenylephrine and incubated with 1 µm of the PDE5I vardenafil or with DMSO (control). Subsequently, increasing concentrations of the RKIs azaindole or Y-27632 were added, and relaxation of tissue was quantified. RESULTS: The expression of ROCK1 was unchanged (P > 0.05), while ROCK2 (P < 0.05) was significantly upregulated in patients with ED compared with controls. ROCK1 and ROCK2 protein colocalized with αSMA, confirming the presence of this kinase in cavernous smooth muscle cells and/or myofibroblasts. After incubation with DMSO, 10 µm azaindole and 10 µm Y-27632 relaxed precontracted tissues with 49.5 ± 7.42% (P = 0.1470 when compared with vehicle) and 85.9 ± 10.3% (P = 0.0016 when compared with vehicle), respectively. Additive effects on relaxation of human corpus cavernosum were seen after preincubation with 1 µm vardenafil. CONCLUSION: The RKI Y-27632 causes a significant relaxation of corpus cavernosum in tissue strips of patients with severe ED. The additive effect of vardenafil and Y-27632 shows that a combined inhibition of Rho-kinase and phosphodiesterase type 5 could be a promising orally administered treatment for severe ED.
Assuntos
Amidas/farmacologia , Inibidores Enzimáticos/farmacologia , Disfunção Erétil/tratamento farmacológico , Pênis/efeitos dos fármacos , Inibidores da Fosfodiesterase 5/uso terapêutico , Piridinas/farmacologia , Dicloridrato de Vardenafila/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Sinergismo Farmacológico , Humanos , Masculino , Pessoa de Meia-Idade , Falha de TratamentoRESUMO
The differential activity of the Hippo-signalling pathway between the outer- and inner-cell populations of the developing preimplantation mouse embryo directs appropriate formation of trophectoderm and inner cell mass (ICM) lineages. Such distinct signalling activity is under control of intracellular polarization, whereby Hippo-signalling is either supressed in polarized outer cells or activated in apolar inner cells. The central role of apical-basolateral polarization to such differential Hippo-signalling regulation prompted us to reinvestigate the role of potential upstream molecular regulators affecting apical-basolateral polarity. This study reports that the chemical inhibition of Rho-associated kinase (Rock) is associated with failure to form morphologically distinct blastocysts, indicative of compromised trophectoderm differentiation, and defects in the localization of both apical and basolateral polarity factors associated with malformation of tight junctions. Moreover, Rock-inhibition mediates mislocalization of the Hippo-signalling activator Angiomotin (Amot), to the basolateral regions of outer cells and is concomitant with aberrant activation of the pathway. The Rock-inhibition phenotype is mediated by Amot, as RNAi-based Amot knockdown totally rescues the normal suppression of Hippo-signalling in outer cells. In conclusion, Rock, via regulating appropriate apical-basolateral polarization in outer cells, regulates the appropriate activity of the Hippo-signalling pathway, by ensuring correct subcellular localization of Amot protein in outer cells.
Assuntos
Desenvolvimento Embrionário , Peptídeos e Proteínas de Sinalização Intercelular/análise , Proteínas dos Microfilamentos/análise , Proteínas Serina-Treonina Quinases/metabolismo , Quinases Associadas a rho/fisiologia , Angiomotinas , Animais , Blastocisto/metabolismo , Via de Sinalização Hippo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Proteínas dos Microfilamentos/metabolismo , Transdução de Sinais , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismoRESUMO
PURPOSE: Lines of evidence suggest that Rho-associated protein kinase (ROCK)-mediated myosin phosphatase-targeting subunit 1 (MYPT1) phosphorylation plays a central role in smooth muscle contraction. However, the physiological significance of MYPT1 phosphorylation at Thr696 catalyzed by ROCK in bladder smooth muscle remains controversial. We attempt to directly observe the quantitative protein expression of Rho A/ROCK and phosphorylation of MYPT1 at Thr696 after carbachol administration in rat bladder smooth muscle cells (RBMSCs). MATERIALS AND METHODS: Primary cultured smooth muscle cells were obtained from rat bladders. The effects of both concentration and time-course induced by the muscarinic agonist carbachol were investigated by assessing the expression of Rho A/ROCK and MYPT1 phosphorylation at Thr696 using Western blot. RESULTS: In the dose-course studies, carbachol showed significant increase in phosphorylation of MYPT1 at Thr696 (p-MYPT1) from concentrations of 15-100 µM based on Western blot results (p < 0.05, ANOVA test). In the time-course studies, treatment of cells with 15 µM of carbachol significantly enhanced the expression of p-MYPT1 from 3 to 15 h (p < 0.05, ANOVA test) and induced the expression of Rho A from 10 to 120 min (p < 0.05, ANOVA test). CONCLUSIONS: Carbachol can induce the expression of ROCK pathway, leading to MYPT1 phosphorylation at Thr696 and thereby sustained RBSMCs contraction.
Assuntos
Carbacol/farmacologia , Músculo Liso/efeitos dos fármacos , Fosfatase de Miosina-de-Cadeia-Leve/efeitos dos fármacos , Quinases Associadas a rho/metabolismo , Análise de Variância , Animais , Western Blotting , Células Cultivadas , Relação Dose-Resposta a Droga , Músculo Liso/citologia , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Fosforilação/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Sensibilidade e Especificidade , Transdução de Sinais/efeitos dos fármacos , Bexiga Urinária/citologia , Quinases Associadas a rho/genéticaRESUMO
The number of patients with diabetes is on an increasing trend, thus leading to the belief that diabetes will be the largest medical problem of the 21st century. Islet transplantation can improve glycometabolic control in patients with type 1 diabetes. We studied the viability of Rho-associated protein kinase (ROCK) inhibitor Y-27632 in a culture system in vitro on freshly isolated rat islets. Islet isolation was conducted on a Lewis rat, and studies of culture solutions were split into two groups, one group using ROCK inhibitor Y-27632, and another without. On the seventh day of culture, we evaluated the differences for the cell morphology, viability, and insulin secretion. The Y-27632 group maintained form better than the group without Y-27632. With strong expression of Bcl-2 observed with the Y-27632 group, and expression suppressed with Bax, inhibition of apoptosis by Y-27632 was confirmed. The Y-27632 group predominantly secreted insulin. For islet transplantation, Y-27632 inhibited cell apoptosis in a graft and was also effective in promoting insulin secretion. We were able to confirm effective morphological and functional culture maintenance by separating islets from a rat and adding ROCK inhibitor Y-27632 to the medium.