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1.
Invest Ophthalmol Vis Sci ; 62(13): 12, 2021 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-34661609

RESUMO

Purpose: The purpose of this study was to develop a preclinical compound, ITRI-E-(S)4046, a dual synergistic inhibitor of myosin light chain kinase 4 (MYLK4) and Rho-related protein kinase (ROCK), for reducing intraocular pressure (IOP). Methods: ITRI-E-(S)4046 is an amino-pyrazole derivative with physical and chemical properties suitable for ophthalmic formulation. In vitro kinase inhibition was evaluated using the Kinase-Glo Luminescent Kinase Assays. A comprehensive kinase selectivity analysis of ITRI-E-(S)4046 was performed using the KINOMEscan assay from DiscoverRx. The IOP reduction and tolerability of ITRI-E-(S)4046 were assessed in ocular normotensive rabbits, ocular normotensive non-human primates, and ocular hypertensive rabbits. In vivo studies were conducted to assess drug concentrations in ocular tissue. The adverse ocular effects of rabbit eyes were evaluated following the OECD405 guidelines. Results: ITRI-E-(S)4046 showed highly selective kinase inhibitory activity against ROCK1/2, MYLK4, and mitogen-activated protein kinase kinase kinase 19 (MAP3K19), with high specificity against protein kinase A, G, and C families. In ocular normotensive rabbits and non-human primates, the mean IOP reductions of 0.1% ITRI-E-(S)4046 eye drops were 29.8% and 28.5%, respectively. In hypertonic saline-induced and magnetic beads-induced ocular hypertensive rabbits, the mean IOP reductions of ITRI-E-(S)4046 0.1% eye drops were 46.9% and 22.0%, respectively. ITRI-E-(S)4046 was well tolerated with only temporary and minor signs of hyperemia. Conclusions: ITRI-E-(S)4046 is a novel type of highly specific ROCK1/2 and MYLK4 inhibitor that can reduce IOP in normotensive and hypertensive animal models. It has the potential to become an effective and well-tolerated treatment for glaucoma.


Assuntos
Benzoatos/farmacologia , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Pressão Intraocular/efeitos dos fármacos , Isoquinolinas/farmacologia , Quinase de Cadeia Leve de Miosina/antagonistas & inibidores , Hipertensão Ocular/tratamento farmacológico , Sulfonamidas/farmacologia , beta-Alanina/análogos & derivados , Animais , Modelos Animais de Doenças , Humanos , Macaca , Masculino , Hipertensão Ocular/fisiopatologia , Coelhos , Tonometria Ocular , beta-Alanina/farmacologia , Quinases Associadas a rho/antagonistas & inibidores
2.
Molecules ; 26(16)2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-34443331

RESUMO

KD025, a ROCK2 isoform-specific inhibitor, has an anti-adipogenic activity which is not mediated by ROCK2 inhibition. To identify the target, we searched binding targets of KD025 by using the KINOMEscanTM screening platform, and we identified casein kinase 2 (CK2) as a novel target. KD025 showed comparable binding affinity to CK2α (Kd = 128 nM). By contrast, CK2 inhibitor CX-4945 and ROCK inhibitor fasudil did not show such cross-reactivity. In addition, KD025 effectively inhibited CK2 at a nanomolar concentration (IC50 = 50 nM). We examined if the inhibitory effect of KD025 on adipocyte differentiation is through the inhibition of CK2. Both CX-4945 and KD025 suppressed the generation of lipid droplets and the expression of proadipogenic genes Pparg and Cebpa in 3T3-L1 cells during adipocyte differentiation. Fasudil exerted no significant effect on the quantity of lipid droplets, but another ROCK inhibitor Y-27632 increased the expression of Pparg and Cebpa. Both CX-4945 and KD025 acted specifically in the middle stage (days 1-3) but were ineffective when treated at days 0-1 or the late stages, indicating that CX-4945 and KD025 may regulate the same target, CK2. The mRNA and protein levels of CK2α and CK2ß generally decreased in 3T3-L1 cells at day 2 but recovered thereafter. Other well-known CK2 inhibitors DMAT and quinalizarin inhibited effectively the differentiation of 3T3-L1 cells. Taken together, the results of this study confirmed that KD025 inhibits ROCK2 and CK2, and that the inhibitory effect on adipocyte differentiation is through the inhibition of CK2.


Assuntos
Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Caseína Quinase II/antagonistas & inibidores , Diferenciação Celular/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Células 3T3-L1 , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos
3.
Clin Immunol ; 230: 108823, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34400321

RESUMO

Chronic graft-versus-host disease (cGVHD) is an immune-mediated disorder characterized by chronic inflammation and fibrosis. Rho-associated coiled-coil-containing protein kinases (ROCKs) are key coordinators of tissue response to injury, regulating multiple functions, such as gene expression and cell migration, proliferation and survival. Relevant to cGVHD and autoimmunity, only the ROCK2 isoform drives a pro-inflammatory type 17 helper T (Th17) cell response. Moreover, ROCK2 inhibition shifts the Th17/regulatory T (Treg) cell balance toward Treg cells and restores immune homeostasis in animal models of autoimmunity and cGVHD. Furthermore, the selective inhibition of ROCK2 by belumosudil reduces fibrosis by downregulating both transforming growth factor-ß signaling and profibrotic gene expression, thereby impeding the creation of focal adhesions. Consistent with its anti-inflammatory and antifibrotic activities, belumosudil has demonstrated efficacy in clinical studies, resulting in an overall response rate of >70% in patients with cGVHD who failed 2 to 5 prior lines of systemic therapy. In summary, selective ROCK2 inhibition has emerged as a promising novel therapeutic approach for treating cGVHD and other immunologic diseases with unique mechanisms of action, targeting both immune imbalance and detrimental fibrotic responses.


Assuntos
Doença Enxerto-Hospedeiro/enzimologia , Quinases Associadas a rho/imunologia , Acetamidas/farmacologia , Animais , Doença Crônica , Modelos Animais de Doenças , Fibrose , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/terapia , Humanos , Imunomodulação , Modelos Imunológicos , Inibidores de Proteínas Quinases/farmacologia , Linfócitos T Reguladores/imunologia , Quinases Associadas a rho/antagonistas & inibidores
4.
Cells ; 10(7)2021 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-34359853

RESUMO

Diabetic macular oedema (DMO) is one of the leading causes of vision loss associated with diabetic retinopathy (DR). New insights in managing this condition have changed the paradigm in its treatment, with intravitreal injections of antivascular endothelial growth factor (anti-VEGF) having become the standard therapy for DMO worldwide. However, there is no single standard therapy for all patients DMO refractory to anti-VEGF treatment; thus, further investigation is still needed. The key obstacles in developing suitable therapeutics for refractory DMO lie in its complex pathophysiology; therefore, there is an opportunity for further improvements in the progress and applications of new drugs. Previous studies have indicated that Rho-associated kinase (Rho-kinase/ROCK) is an essential molecule in the pathogenesis of DMO. This is why the Rho/ROCK signalling pathway has been proposed as a possible target for new treatments. The present review focuses on the recent progress on the possible role of ROCK and its therapeutic potential in DMO. A systematic literature search was performed, covering the years 1991 to 2021, using the following keywords: "rho-Associated Kinas-es", "Diabetic Retinopathy", "Macular Edema", "Ripasudil", "Fasudil" and "Netarsudil". Better insight into the pathological role of Rho-kinase/ROCK may lead to the development of new strategies for refractory DMO treatment and prevention.


Assuntos
Retinopatia Diabética/tratamento farmacológico , Edema Macular/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Quinases Associadas a rho/antagonistas & inibidores , Animais , Retinopatia Diabética/complicações , Retinopatia Diabética/fisiopatologia , Humanos , Edema Macular/complicações , Edema Macular/fisiopatologia , Inibidores de Proteínas Quinases/farmacologia , Retina/patologia , Transdução de Sinais/efeitos dos fármacos , Pesquisa Médica Translacional , Quinases Associadas a rho/metabolismo
5.
Int J Mol Sci ; 22(13)2021 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-34202585

RESUMO

Cultured keratinocytes are desirable models for biological and medical studies. However, primary keratinocytes are difficult to maintain, and there has been little research on lingual keratinocyte culture. Here, we investigated the effect of Y-27632, a Rho kinase (ROCK) inhibitor, on the immortalization and characterization of cultured rat lingual keratinocyte (RLKs). Three Y-27632-supplemented media were screened for the cultivation of RLKs isolated from Sprague-Dawley rats. Phalloidin staining and TUNEL assay were applied to visualize cytoskeleton dynamics and cell apoptosis following Y-27632 removal. Label-free proteomics, RT-PCR, calcium imaging, and cytogenetic studies were conducted to characterize the cultured cells. Results showed that RLKs could be conditionally immortalized in a high-calcium medium in the absence of feeder cells, although they did not exhibit normal karyotypes. The removal of Y-27632 from the culture medium led to reversible cytoskeletal reorganization and nuclear enlargement without triggering apoptosis, and a total of 239 differentially expressed proteins were identified by proteomic analysis. Notably, RLKs derived from the non-taste epithelium expressed some molecular markers characteristic of taste bud cells, yet calcium imaging revealed that they rarely responded to tastants. Collectively, we established a high-calcium and feeder-free culture method for the long-term maintenance of RLKs. Our results shed some new light on the immortalization and differentiation of lingual keratinocytes.


Assuntos
Amidas/farmacologia , Cálcio/metabolismo , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Animais , Técnicas de Cultura de Células , Células Cultivadas , Ratos
6.
Int J Mol Sci ; 22(14)2021 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-34298955

RESUMO

PURPOSE: The effects of Rho-associated coiled-coil containing protein kinase (ROCK) 1 and 2 inhibitor, ripasudil hydrochloride hydrate (Rip), ROCK2 inhibitor, KD025 or rosiglitazone (Rosi) on two-dimension (2D) and three-dimension (3D) cultured human conjunctival fibroblasts (HconF) treated by transforming growth factor (TGFß2) were studied. METHODS: Two-dimension and three-dimension cultured HconF were examined by transendothelial electrical resistance (TEER, 2D), size and stiffness (3D), and the expression of the extracellular matrix (ECM) including collagen1 (COL1), COL4 and COL6, fibronectin (FN), and α-smooth muscle actin (αSMA) by quantitative PCR (2D, 3D) in the presence of Rip, KD025 or Rosi. RESULTS: TGFß2 caused a significant increase in (1) the TEER values (2D) which were greatly reduced by Rosi, (2) the stiffness of the 3D organoids which were substantially reduced by Rip or KD025, and (3) TGFß2 induced a significant up-regulation of all ECMs, except for COL6 (2D) or αSMA (3D), and down-regulation of COL6 (2D). Rosi caused a significant up-regulation of COL1, 4 and 6 (3D), and down-regulation of COL6 (2D) and αSMA (3D). Most of these TGFß2-induced expressions in the 2D and αSMA in the 3D were substantially inhibited by KD025, but COL4 and αSMA in 2D were further enhanced by Rip. CONCLUSION: The findings reported herein indicate that TGFß2 induces an increase in fibrogenetic changes on the plane and in the spatial space, and are inhibited by Rosi and ROCK inhibitors, respectively.


Assuntos
Túnica Conjuntiva/metabolismo , Fibroblastos/metabolismo , Rosiglitazona/farmacologia , Fator de Crescimento Transformador beta2/metabolismo , Quinases Associadas a rho/antagonistas & inibidores , Actinas/biossíntese , Linhagem Celular , Colágeno/biossíntese , Fibronectinas/biossíntese , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Quinases Associadas a rho/metabolismo
7.
Cells ; 10(7)2021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-34209333

RESUMO

Pulmonary hypertension (PH) is a cardiovascular disease caused by extensive vascular remodeling in the lungs, which ultimately leads to death in consequence of right ventricle (RV) failure. While current drugs for PH therapy address the sustained vasoconstriction, no agent effectively targets vascular cell proliferation and tissue inflammation. Rho-associated protein kinases (ROCKs) emerged in the last few decades as promising targets for PH therapy, since ROCK inhibitors demonstrated significant anti-remodeling and anti-inflammatory effects. In this review, current aspects of ROCK inhibition therapy are discussed in relation to the treatment of PH and RV dysfunction, from cell biology to preclinical and clinical studies.


Assuntos
Hipertensão Pulmonar/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Quinases Associadas a rho/antagonistas & inibidores , Animais , Ensaios Clínicos como Assunto , Modelos Animais de Doenças , Aprovação de Drogas , Humanos , Inibidores de Proteínas Quinases/farmacologia , Quinases Associadas a rho/química , Quinases Associadas a rho/metabolismo
8.
Sci Rep ; 11(1): 14475, 2021 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-34262070

RESUMO

Intrauterine growth restriction (IUGR) is one of the most common pathologies of pregnancy. The cardiovascular consequences of IUGR do not disappear in adulthood and can manifest themselves in pathological alterations of vasomotor control. The hypothesis was tested that IUGR weakens anticontractile influence of NO and augments procontractile influence of Rho-kinase in arteries of adult offspring. To model IUGR in the rat, dams were 50% food restricted starting from the gestational day 11 till delivery. Mesenteric and coronary arteries of male offspring were studied at the age of 3 months using wire myography, qPCR, and Western blotting. Contractile responses of mesenteric arteries to α1-adrenoceptor agonist methoxamine as well as influences of NO and Rho-kinase did not differ between control and IUGR rats. However, coronary arteries of IUGR rats demonstrated elevated contraction to thromboxane A2 receptor agonist U46619 due to weakened anticontractile influence of NO and enhanced role of Rho-kinase in the endothelium. This was accompanied by reduced abundance of SODI protein and elevated content of RhoA protein in coronary arteries of IUGR rats. IUGR considerably changes the regulation of coronary vascular tone in adulthood and, therefore, can serve as a risk factor for the development of cardiac disorders.


Assuntos
Vasos Coronários/fisiopatologia , Retardo do Crescimento Fetal/etiologia , Artérias Mesentéricas/fisiopatologia , Óxido Nítrico/metabolismo , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Amidas/farmacologia , Animais , Modelos Animais de Doenças , Feminino , Masculino , Artérias Mesentéricas/efeitos dos fármacos , Contração Muscular , Óxido Nítrico Sintase Tipo III/metabolismo , Gravidez , Piridinas/farmacologia , Ratos Wistar , Quinases Associadas a rho/antagonistas & inibidores
9.
Cells ; 10(6)2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-34207965

RESUMO

This study aims to obtain sufficient corneal endothelial cells for regenerative application. We examined the combinatory effects of Rho-associated kinase (ROCK) inhibitor Y-27632 and mesenchymal stem cell-derived conditioned medium (MSC-CM) on the proliferation and senescence of rabbit corneal endothelial cells (rCECs). rCECs were cultured in a control medium, a control medium mixed with either Y-27632 or MSC-CM, and a combinatory medium containing Y-27632 and MSC-CM. Cells were analyzed for morphology, cell size, nuclei/cytoplasmic ratio, proliferation capacity and gene expression. rCECs cultured in a combinatory culture medium showed a higher passage number, cell proliferation, and low senescence. rCECs on collagen type I film showed high expression of tight junction. The cell proliferation marker Ki-67 was positively stained either in Y-27632 or MSC-CM-containing media. Genes related to cell proliferation resulted in negligible changes in MKI67, CIP2A, and PCNA in the combinatory medium, suggesting proliferative capacity was maintained. In contrast, all of these genes were significantly downregulated in the other groups. Senescence marker ß-galactosidase-positive cells significantly decreased in either MSC-CM and/or Y-27632 mixed media. Senescence-related genes downregulated LMNB1 and MAP2K6, and upregulated MMP2. Cell cycle checkpoint genes such as CDC25C, CDCA2, and CIP2A did not vary in the combinatory medium but were significantly downregulated in either ROCK inhibitor or MSC-CM alone. These results imply the synergistic effect of combinatory culture medium on corneal endothelial cell proliferation and high cell number. This study supports high potential for translation to the development of human corneal endothelial tissue regeneration.


Assuntos
Proliferação de Células , Senescência Celular , Meios de Cultivo Condicionados/farmacologia , Endotélio Corneano/citologia , Células-Tronco Mesenquimais/citologia , Inibidores de Proteínas Quinases/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Amidas/farmacologia , Animais , Diferenciação Celular , Movimento Celular , Células Cultivadas , Endotélio Corneano/efeitos dos fármacos , Endotélio Corneano/enzimologia , Inibidores Enzimáticos/farmacologia , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/enzimologia , Piridinas/farmacologia , Coelhos
10.
Sci Rep ; 11(1): 15286, 2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34315994

RESUMO

A pan-ROCK-inhibitor, ripasudil (Rip), and a ROCK2 inhibitor, KD025, were used To study the effects of Rho-associated coiled-coil containing protein kinase (ROCK)1 and 2 on two-dimensional (2D) and three-dimensional (3D) cultures of a TGFß2-treated human trabecular meshwork (HTM) cells. In the presence of 5 ng/mL TGFß2, the effects of these inhibitors were characterized by transendothelial electrical resistance (TEER), FITC-dextran permeability, and the size and stiffness of 3D sphenoids, the expression of extracellular matrix (ECM) including collagen1, 4 and 6, and fibronectin, α-smooth muscle actin, a tissue inhibitor of metalloproteinase (TIMP)1-4, and matrix metalloproteinase (MMP)2, 9 and 14. TGFß2 caused a significant increase in the TEER values, and decrease in FITC-dextran permeability, as well as a decrease in the sizes and stiffness of the 3D sphenoids. In the presence of ROCK inhibitors, the TGFß2-induced effects of the TEER and FITC-dextran permeability were inhibited, especially by KD025. Rip induced a significant increase in sizes and a decrease in the stiffness of the TGFß2-treated 3D sphenoids, although the effects of KD025 were weaker. Gene expressions of most of the ECMs, TIMP2 and MMP9 of 2D and 3D HTM cells were significantly up-regulated by TGFß2. Those were significantly and differently modulated by Rip or KD025.


Assuntos
Inibidores de Proteínas Quinases/farmacologia , Fator de Crescimento Transformador beta2/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Linhagem Celular Transformada , Células Cultivadas , Humanos
11.
Lab Chip ; 21(16): 3128-3136, 2021 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-34180491

RESUMO

Integrins are key players in platelet adhesion and aggregation. Integrin molecular tensions, the forces transmitted by integrin molecules, are regulated by both mechanical and biochemical cues, and the outside-in and inside-out signaling has been extensively studied. While the mechanical properties of platelets at static status have been studied by atomic force microscopy, traction force microscopy and tension sensors, the biomechanical properties of flowing platelets remain elusive. Herein, we report microfluidic chips grafted with integrin tension sensors for microfluidic-force mapping in platelets. Specifically, the process of integrin αIIbß3 mediating tension transmission and platelet adhesion under low flow rates has been obtained, and the process of platelet clustering at post-stenotic regions has been demonstrated. We found that flowing shear force can postpone the integrin-mediated tension transmission and platelet adhesion. We further evaluated the effect of Y-27632, a ROCK inhibitor that has been proven to reduce integrin-mediated platelet adhesion, at a series of concentrations and demonstrated that microfluidic chips with integrin tension sensors are sensitive to the concentration-dependent effects of Y-27632. Given their low cost and scalable throughput, these chips are ideal technical platforms for biological studies of platelets at flowing status and for platelet inhibitor or potential antiplatelet drug screening.


Assuntos
Plaquetas , Integrinas , Microfluídica , Quinases Associadas a rho/antagonistas & inibidores , Animais , Cães , Adesividade Plaquetária , Estresse Mecânico
12.
Int J Mol Sci ; 22(9)2021 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-34066490

RESUMO

There is an unmet need for simplified in vitro models of malignancy and metastasis that facilitate fast, affordable and scalable gene and compound analysis. "Adherent" cancer cell lines frequently release "free-floating" cells into suspension that are viable and can reattach. This, in a simplistic way, mimics the metastatic process. We compared the gene expression profiles of naturally co-existing populations of floating and adherent cells in SW620 (colon), C33a (cervix) and HeLa (cervix) cancer cells. We found that 1227, 1367 and 1333 genes were at least 2-fold differentially expressed in the respective cell lines, of which 122 were shared among the three cell lines. As proof of principle, we focused on the anti-metastatic gene NM23-H1, which was downregulated both at the RNA and protein level in the floating cell populations of all three cell lines. Knockdown of NM23-H1 significantly increased the number of floating (and viable) cells, whereas overexpression of NM23-H1 significantly reduced the proportion of floating cells. Other potential regulators of these cellular states were identified through pathway analysis, including hypoxia, mTOR (mechanistic target of rapamycin), cell adhesion and cell polarity signal transduction pathways. Hypoxia, a condition linked to malignancy and metastasis, reduced NM23-H1 expression and significantly increased the number of free-floating cells. Inhibition of mTOR or Rho-associated protein kinase (ROCK) significantly increased cell death specifically in the floating and not the adherent cell population. In conclusion, our study suggests that dynamic subpopulations of free-floating and adherent cells is a useful model to screen and identify genes, drugs and pathways that regulate the process of cancer metastasis, such as cell detachment and anoikis.


Assuntos
Modelos Biológicos , Neoplasias/patologia , Animais , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/genética , Linhagem Celular Tumoral , Forma Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Metástase Neoplásica , Neoplasias/genética , Inibidores de Proteínas Quinases/farmacologia , Reprodutibilidade dos Testes , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/metabolismo
13.
Molecules ; 26(9)2021 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-34062818

RESUMO

Salivary gland stem cells (SGSCs) are potential cell sources for the treatment of salivary gland diseases. The control of cell survival is an essential factor for applying stem cells to regenerative medicine or stem cell-based research. The purpose of this study was to investigate the effects of the ROCK inhibitor Y-27632 on the survival of SGSCs and its underlying mechanisms. SGSCs were isolated from mouse submandibular glands and cultured in suspension. Treatment with Y-27632 restored the viability of SGSCs that was significantly decreased during isolation and the subsequent culture. Y-27632 upregulated the expression of anti-apoptotic protein BCL-2 in SGSCs and, in the apoptosis assay, significantly reduced apoptotic and necrotic cell populations. Matrigel was used to mimic the extracellular environment of an intact salivary gland. The expression of genes regulating apoptosis and the ROCK signaling pathway was significantly reduced when SGSCs were embedded in Matrigel. SGSCs cultured in Matrigel and treated with Y-27632 showed no difference in the total numbers of spheroids and expression levels of apoptosis-regulating genes. Matrigel-embedded SGSCs treated with Y-27632 increased the number of spheroids with budding structures and the expression of acinar cell-specific marker AQP5. We demonstrate the protective effects of Y-27632 against dissociation-induced apoptosis of SGSCs during their culture in vitro.


Assuntos
Amidas/farmacologia , Piridinas/farmacologia , Glândulas Salivares/efeitos dos fármacos , Quinases Associadas a rho/antagonistas & inibidores , Animais , Apoptose , Morte Celular , Sobrevivência Celular , Células Cultivadas , Colágeno/química , Combinação de Medicamentos , Matriz Extracelular/metabolismo , Feminino , Laminina/química , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Necrose , Proteoglicanas/química , Esferoides Celulares , Células-Tronco/citologia , Glândula Submandibular/efeitos dos fármacos
14.
Biochem Biophys Res Commun ; 566: 164-169, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34126347

RESUMO

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 & inibidores
15.
Exp Neurol ; 343: 113794, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34166685

RESUMO

Regeneration is bungled following CNS injuries, including spinal cord injury (SCI). Inherent decay of permissive conditions restricts the regrowth of the mature CNS after an injury. Hypertrophic scarring, insignificant intrinsic axon-growth activity, and axon-growth inhibitory molecules such as myelin inhibitors and scar inhibitors constitute a significant hindrance to spinal cord repair. Besides these molecules, a combined absence of various mechanisms responsible for axonal regeneration is the main reason behind the dereliction of the adult CNS to regenerate. The neutralization of specific inhibitors/proteins by stymieing antibodies or encouraging enzymatic degradation results in improved axon regeneration. Previous efforts to induce regeneration after SCI have stimulated axonal development in or near lesion sites, but not beyond them. Several pathways are responsible for the axonal growth obstruction after a CNS injury, including SCI. Herein, we summarize the axonal, glial, and intrinsic factor which impedes the regeneration. We have also discussed the methods to stabilize microtubules and through this to maintain the proper cytoskeletal dynamics of growth cone as disorganized microtubules lead to the failure of axonal regeneration. Moreover, we primarily focus on diverse inhibitors of axonal growth and molecular approaches to counteract them and their downstream intracellular signaling through the RhoA/ROCK pathway.


Assuntos
Transdução de Sinais/fisiologia , Traumatismos da Medula Espinal/metabolismo , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Humanos , Glicoproteína Associada a Mielina/metabolismo , Regeneração Nervosa/efeitos dos fármacos , Regeneração Nervosa/fisiologia , Proteínas Nogo/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Traumatismos da Medula Espinal/tratamento farmacológico , Quinases Associadas a rho/antagonistas & inibidores , Proteína rhoA de Ligação ao GTP/antagonistas & inibidores
16.
PLoS One ; 16(5): e0251411, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33974655

RESUMO

Cells exert traction forces on the extracellular matrix to which they are adhered through the formation of focal adhesions. Spatial-temporal regulation of traction forces is crucial in cell adhesion, migration, cellular division, and remodeling of the extracellular matrix. By cultivating cells on polyacrylamide hydrogels of different stiffness we were able to investigate the effects of substrate stiffness on the generation of cellular traction forces by Traction Force Microscopy (TFM), and characterize the molecular dynamics of the focal adhesion protein zyxin by Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Recovery After Photobleaching (FRAP). As the rigidity of the substrate increases, we observed an increment of both, cellular traction generation and zyxin residence time at the focal adhesions, while its diffusion would not be altered. Moreover, we found a positive correlation between the traction forces exerted by cells and the residence time of zyxin at the substrate elasticities studied. We found that this correlation persists at the subcellular level, even if there is no variation in substrate stiffness, revealing that focal adhesions that exert greater traction present longer residence time for zyxin, i.e., zyxin protein has less probability to dissociate from the focal adhesion.


Assuntos
Estresse Mecânico , Zixina/química , Citoesqueleto de Actina/efeitos dos fármacos , Amidas/farmacologia , Animais , Bovinos , Adesão Celular , Citocalasina D/farmacologia , Células Endoteliais , Recuperação de Fluorescência Após Fotodegradação , Adesões Focais , Proteínas de Fluorescência Verde , Microscopia Intravital , Cinética , Lasers , Camundongos , Camundongos Endogâmicos BALB C , Piridinas/farmacologia , Proteínas Recombinantes de Fusão/química , Vinculina/química , Quinases Associadas a rho/antagonistas & inibidores
17.
Invest Ophthalmol Vis Sci ; 62(6): 21, 2021 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-34015079

RESUMO

Purpose: This study investigated the neuroprotective effects of administration of ROCK inhibitor E212 on ischemic optic neuropathy. Methods: Rats received an intravitreal injection of either E212 or PBS immediately after optic nerve infarct. The oxidative stress in the retina was detected by performing superoxide dismutase activity and CellROX assays. The integrity of retinal pigment epithelium was determined by staining of zona occludens 1. The visual function, retinal ganglion cell (RGC) density, and RGC apoptosis were determined by using flash visual-evoked potential analysis, retrograde FluoroGold labeling, and TdT-dUTP nick end-labeling assay. Macrophage infiltration was detected by staining for ED1. The protein levels of TNF-α, p-CRMP, p-AKT1, p-STAT3, and CD206 were evaluated using Western blotting. Results: Administration of E212 resulted in a 1.23-fold increase in the superoxide dismutase activity of the retina and 2.28-fold decrease in RGC-produced reactive oxygen species as compared to the levels observed upon treatment with PBS (P < 0.05). Moreover, E212 prevented the disruption of the blood-retinal barrier (BRB) in contrast to PBS. The P1-N2 amplitude and RGC density in the E212-treated group were 1.75- and 2.05-fold higher, respectively, than those in the PBS-treated group (P < 0.05). The numbers of apoptotic RGCs and macrophages were reduced by 2.93- and 2.54-fold, respectively, in the E212-treated group compared with those in the PBS-treated group (P < 0.05). The levels of p-AKT1, p-STAT3, and CD206 were increased, whereas those of p-PTEN, p-CRMP2, and TNF-α were decreased after treatment with E212 (P < 0.05). Conclusions: Treatment with E212 suppresses oxidative stress, BRB disruption, and neuroinflammation to protect the visual function in ischemic optic neuropathy.


Assuntos
Neuropatia Óptica Isquêmica/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Células Ganglionares da Retina/efeitos dos fármacos , Quinases Associadas a rho/antagonistas & inibidores , Animais , Anti-Inflamatórios/uso terapêutico , Antioxidantes/uso terapêutico , Apoptose/efeitos dos fármacos , Barreira Hematorretiniana/efeitos dos fármacos , Western Blotting , Contagem de Células , Modelos Animais de Doenças , Potenciais Evocados Visuais/fisiologia , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Injeções Intravítreas , Masculino , Neuropatia Óptica Isquêmica/metabolismo , Neuropatia Óptica Isquêmica/fisiopatologia , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Células Ganglionares da Retina/patologia , Superóxido Dismutase/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo
18.
Adv Ther ; 38(7): 3760-3770, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34028695

RESUMO

INTRODUCTION: We aimed to evaluate the additional effects and safety of a Rho-associated protein kinase (ROCK) inhibitor, ripasudil hydrochloride hydrate, in Japanese patients with primary open-angle glaucoma (POAG) with an intraocular pressure (IOP) of at most 15 mmHg undergoing prostaglandin F2α (FP) receptor agonist monotherapy (FP monotherapy). METHODS: In this prospective observational study, 30 Japanese patients with POAG and IOP of at most 15 mmHg (mean age 59.4 years; 10 men) who were undergoing FP monotherapy in both eyes were administered an additional dose of ripasudil hydrochloride hydrate (GLANATEC ophthalmic solution 0.4%: ripasudil) in one eye. The following factors were investigated at 1 and 3 months after the initiation of ripasudil treatment: (1) magnitude of change in IOP between the treated and contralateral untreated eyes, (2) number of treated eyes showing 20% and 30% IOP reduction, (3) IOP difference between treated and contralateral untreated eyes, and (4) safety during the treatment period. Both (1) and (3) were analyzed using the mixed-effect model for repeated measurements. RESULTS: The treated eyes showed significant reduction in IOP at 1 month (- 1.92 mmHg, P < 0.001) and 3 months (- 1.81 mmHg, P < 0.001). In contrast, contralateral untreated eyes did not show IOP reduction at 1 month (0.53 mmHg, P = 0.016) and 3 months (0.38 mmHg, P = 0.15). IOP reduction of - 20% and - 30% was achieved in 9 (30%) and 3 (10%) treated eyes, respectively. There were significant differences in IOP between the treated and contralateral untreated eyes at 1 month (- 2.46 mmHg, P < 0.001) and 3 months (- 2.20 mmHg, P < 0.001). Two patients experienced local adverse events (facial edema, one patient at week 1; blepharitis, one patient at 1 month); they recovered quickly after stopping ripasudil administration. CONCLUSION: In patients with POAG with an IOP of at most 15 mmHg undergoing FP monotherapy, the addition of ripasudil resulted in significant IOP lowering at 1 and 3 months. Ripasudil could be used to enhance the outcome of FP monotherapy. TRIAL REGISTRATION: Registered UMIN ID: UMIN000030742.


Assuntos
Anti-Hipertensivos , Glaucoma de Ângulo Aberto , Glaucoma , Quinases Associadas a rho/antagonistas & inibidores , Anti-Hipertensivos/uso terapêutico , Glaucoma/tratamento farmacológico , Glaucoma de Ângulo Aberto/tratamento farmacológico , Humanos , Pressão Intraocular , Japão , Masculino , Pessoa de Meia-Idade , Prostaglandinas/uso terapêutico , Resultado do Tratamento
19.
Nat Methods ; 18(5): 528-541, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33941937

RESUMO

Human pluripotent stem cells (hPSCs) are capable of extensive self-renewal yet remain highly sensitive to environmental perturbations in vitro, posing challenges to their therapeutic use. There is an urgent need to advance strategies that ensure safe and robust long-term growth and functional differentiation of these cells. Here, we deployed high-throughput screening strategies to identify a small-molecule cocktail that improves viability of hPSCs and their differentiated progeny. The combination of chroman 1, emricasan, polyamines, and trans-ISRIB (CEPT) enhanced cell survival of genetically stable hPSCs by simultaneously blocking several stress mechanisms that otherwise compromise cell structure and function. CEPT provided strong improvements for several key applications in stem-cell research, including routine cell passaging, cryopreservation of pluripotent and differentiated cells, embryoid body (EB) and organoid formation, single-cell cloning, and genome editing. Thus, CEPT represents a unique poly-pharmacological strategy for comprehensive cytoprotection, providing a rationale for efficient and safe utilization of hPSCs.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Crioprotetores/farmacologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Polifarmacologia , Técnicas de Cultura de Células , Criopreservação/métodos , Crioprotetores/química , Regulação da Expressão Gênica/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Humanos , Células-Tronco Pluripotentes/fisiologia , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismo
20.
J Control Release ; 334: 237-247, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-33915222

RESUMO

ROCK, one of the downstream regulators of Rho, controls actomyosin cytoskeleton organization, stress fiber formation, smooth muscle contraction, and cell migration. ROCK plays an important role in the pathologies of cerebral and coronary vasospasm, hypertension, cancer, and arteriosclerosis. Pharmacological-induced systemic inhibition of ROCK affects both the pathological and physiological functions of Rho-kinase, resulting in hypotension, increased heart rate, decreased lymphocyte count, and eventually cardiovascular collapse. To overcome the adverse effects of systemic ROCK inhibition, we developed a bioreductive prodrug of a ROCK inhibitor, fasudil, that functions selectively under hypoxic conditions. By masking fasudil's active site with a bioreductive 4-nitrobenzyl group, we synthesized a prodrug of fasudil that is inactive in normoxia. Reduction of the protecting group initiated by hypoxia reveals an electron-donating substituent that leads to fragmentation of the parent molecule. Under normoxia the fasudil prodrug displayed significantly reduced activity against ROCK compared to its parent compound, but under severe hypoxia the prodrug was highly effective in suppressing ROCK activity. Under hypoxia the prodrug elicited an antiproliferative effect on disease-afflicted pulmonary arterial smooth muscle cells and pulmonary arterial endothelial cells. The prodrug displayed a long plasma half-life, remained inactive in the blood, and produced no drop in systemic blood pressure when compared with fasudil-treated controls. Due to its selective nature, our hypoxia-activated fasudil prodrug could be used to treat diseases where tissue-hypoxia or hypoxic cells are the pathological basis of the disease.


Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina , Hipóxia , Pró-Fármacos , Inibidores de Proteínas Quinases , Quinases Associadas a rho , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/efeitos adversos , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Células Endoteliais , Humanos , Hipóxia/tratamento farmacológico , Pró-Fármacos/efeitos adversos , Pró-Fármacos/farmacologia , Inibidores de Proteínas Quinases/efeitos adversos , Inibidores de Proteínas Quinases/farmacologia , Quinases Associadas a rho/antagonistas & inibidores
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