The Rho-Kinase (ROCK) Inhibitor Y-27632 Protects Against Excitotoxicity-Induced Neuronal Death In Vivo and In Vitro

Inhalable nanostructured microparticles of simvastatin, a Nrf2 activator and RhoA/Rho kinase (ROCK) inhibitor, were rationally designed for targeted pulmonary delivery as dry powder inhalers (DPIs) for the treatment of pulmonary hypertension (PH). Advanced particle engineering design technology was employed to develop inhalable dry powders using different dilute feed concentrations and spray drying pump rates. Several analytical techniques were used comprehensively to characterize the physicochemical properties of the resulting powders. Scanning electron microscopy (SEM) was used to visualize particle morphology (shape), surface structure, size, and size distribution. Karl Fischer titration (KFT) was employed to quantify the residual water content in the powders. X-ray powder diffraction (XRPD) was used to determine crystallinity. Hot-stage microscopy (HSM) under cross-polarizing lens was used to observe the presence or absence of birefringence characteristic of crystallinity. Differential scanning calorimetry (DSC) was employed to quantify thermotropic phase behavior. Attenuated total reflectance (ATR)-Fourier-transform infrared (FTIR) spectroscopy and Raman spectroscopy were used to determine the molecular fingerprint of simvastatin powders before and after particle engineering design. In vitro aerosol dispersion performance was performed with three different Food and Drug Administration (FDA)-approved human DPI devices. Cell viability and transepithelial electrical resistance (TEER) were demonstrated using different in vitro human pulmonary cell two and three-dimensional models at the air–liquid interface, and in vivo safety in healthy rats by inhalation. Efficacy was demonstrated in the in vivo lamb model of PH. Four different inhalable powders of simvastatin were successfully produced. They possessed nanostructured surfaces and were in the inhalable size range. Simvastatin retained its crystallinity following particle engineering design. The more dilute feed concentration spray dried at the lower pump rate produced the smallest particles. All powders successfully aerosolized with all three DPI human devices. Inhaled simvastatin as an aerosol restored the endothelial function in the shunt lamb model of PH, as demonstrated by the reduction of pulmonary vascular resistance (PVR) in response to the endothelium-dependent vasodilator acetylcholine. The reviews of this paper are available via the supplemental material section.

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Rho kinase inhibition maintains intestinal and vascular barrier function by upregulation of occludin in experimental necrotizing enterocolitis

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00357.2017 ◽

2018 ◽

Vol 315 (4) ◽

pp. G514-G528 ◽

Cited By ~ 8

Author(s):

Justyna S. Grothaus ◽

Guillermo Ares ◽

Carrie Yuan ◽

Douglas R. Wood ◽

Catherine J. Hunter

Keyword(s):

Tight Junction ◽

Necrotizing Enterocolitis ◽

Therapeutic Target ◽

Rho Kinase ◽

Rock Inhibitor ◽

Cellular Permeability ◽

Rock Inhibition ◽

Rat Pup

Necrotizing enterocolitis (NEC) is a deadly disease that occurs in 5–10% of neonates. Although NEC has been extensively studied, no single therapeutic target has been identified. Rho kinase (ROCK) is a serine/threonine kinase that affects multiple cellular processes, including tight junction (TJ) function, cellular permeability, and apoptosis. We hypothesized that ROCK inhibition would decrease cellular permeability, stabilize TJ proteins (occludin), and decrease the severity of NEC. To test this hypothesis, human colon epithelial cells (Caco-2) and human endothelial cells were studied. Cells were treated with lipopolysaccharide to simulate an in vitro model of NEC. The effect of ROCK inhibition was measured by transepithelial membrane resistance (TEER) and cellular permeability to FITC-dextran. The effects of ROCK inhibition in vivo were analyzed in the rat pup model of NEC. NEC was induced by feeding formula supplemented with Cronobacter sakazakii with or without gavaged ROCK inhibitor. Rat intestines were scored based on histological degree of injury. RNA and protein assays for occludin protein were performed for all models of NEC. Treatment with ROCK inhibitor significantly decreased cellular permeability in Caco-2 cells and increased TEER. Intestinal injury scoring revealed decreased scores in ROCK inhibitor-treated pups compared with NEC only. Both cell and rat pup models demonstrated an upregulation of occludin expression in the ROCK inhibitor-treated groups. Therefore, we conclude that ROCK inhibition protects against experimental NEC by strengthening barrier function via upregulation of occludin. These data suggest that ROCK may be a potential therapeutic target for patients with NEC. NEW & NOTEWORTHY These studies are the first to demonstrate an upregulation of occludin tight junction protein in response to Rho kinase (ROCK) inhibition. Furthermore, we have demonstrated that ROCK inhibition in experimental models of necrotizing enterocolitis (NEC) is protective against NEC in both in vitro and in vivo models of disease.

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Role of Rho-kinase in reexpansion pulmonary edema in rabbits

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00188.2004 ◽

2005 ◽

Vol 289 (6) ◽

pp. L946-L953 ◽

Cited By ~ 14

Author(s):

Makoto Sawafuji ◽

Akitoshi Ishizaka ◽

Mitsutomo Kohno ◽

Hidefumi Koh ◽

Sadatomo Tasaka ◽

...

Keyword(s):

Pulmonary Edema ◽

Morphological Changes ◽

Rabbit Model ◽

Rho Kinase ◽

Intratracheal Instillation ◽

Epithelial Permeability ◽

Rock Inhibitor ◽

Reexpansion Pulmonary Edema

Reexpansion of a collapsed lung increases the microvascular permeability and causes reexpansion pulmonary edema. Neutrophils and their products have been implicated in the development of this phenomenon. The small GTP-binding proteins Rho and its target Rho-kinase (ROCK) regulate endothelial permeability, although their roles in reexpansion pulmonary edema remain unclear. We studied the contribution of ROCK to pulmonary endothelial and epithelial permeability in a rabbit model of this disorder. Endothelial and epithelial permeability was assessed by measuring the tissue-to-plasma (T/P) and bronchoalveolar lavage (BAL) fluid-to-plasma (B/P) ratios with 125I-labeled albumin. After intratracheal instillation of 125I-albumin, epithelial permeability was also assessed from the plasma leak (PL) index, the ratio of 125I-albumin in plasma/total amount of instilled 125I-albumin. T/P, B/P, and PL index were significantly increased in the reexpanded lung. These increases were attenuated by pretreatment with Y-27632, a specific ROCK inhibitor. However, neutrophil influx, neutrophil elastase activity, and malondialdehyde concentrations in BAL fluid collected from the reexpanded lung were not changed by Y-27632. In endothelial monolayers, Y-27632 significantly attenuated the H2O2-induced increase in permeability and mitigated the morphological changes in the actin microfilament cytoskeleton of endothelial cells. These in vivo and in vitro observations suggest that the Rho/ROCK pathway contributes to the increase in alveolar barrier permeability associated with reexpansion pulmonary edema.

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The effect of Rho Kinase inhibition on corneal nerve regeneration in vitro and in vivo

The Ocular Surface ◽

10.1016/j.jtos.2021.08.011 ◽

2021 ◽

Author(s):

Sonja Mertsch ◽

Inga Neumann ◽

Cosima Rose ◽

Marc Schargus ◽

Gerd Geerling ◽

...

Keyword(s):

Nerve Regeneration ◽

Rho Kinase ◽

Kinase Inhibition ◽

Corneal Nerve ◽

Regeneration In Vitro

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EXTH-64. SMALL GTPASES IN MENINGIOMAS: PROLIFERATION, MIGRATION, SURVIVAL, POTENTIAL TREATMENT AND INTERACTIONS

Neuro-Oncology ◽

10.1093/neuonc/noaa215.418 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii101-ii101

Author(s):

Christoph Kesseler ◽

Julian Kahr ◽

Natalie Waldt ◽

Nele Stroscher ◽

Josephine Liebig ◽

...

Keyword(s):

Overall Survival ◽

Cell Lines ◽

Small Gtpases ◽

Potential Treatment ◽

Rock Inhibitor ◽

Meningioma Cell ◽

And Migration ◽

Proliferation And Migration

Abstract PURPOSE To evaluate the role of the small GTPases RhoA, Rac1 and Cdc42 in meningiomas as therapeutic targets and their interactions in meningiomas. EXPERIMENTAL DESIGN We analyzed expression of GTPases in human meningioma samples and meningioma cell lines of various WHO grades. Malignant IOMM-Lee meningioma cells were used to generate shRNA mediated knockdowns of GTPases RhoA, Rac1 or Cdc42 and to study knockdown effects on proliferation and migration, as well as analysis of cell morphology by confocal microscopy. The same tests were used to investigate effects of the two inhibitors Fasudil and EHT-1864 of malignant IOMM-Lee, KT21 and benign Ben-Men cells and the effects of these drugs on IOMM-Lee knockdown cells. The effects of GTPase knockdowns and Fasudil treatment were studied in terms of overall survival by intracranial xenografts of mice. Potential interactions of GTPases regarding NF2, mTOR and FAK-Paxillin were examined. RESULTS Small GTPases were upregulated in meningiomas of higher tumor grades. Reduced proliferation and migration could be achieved by GTPase knockdown in IOMM-Lee cells. Additionally, the ROCK-inhibitor Fasudil and Rac1-inhibitor EHT-1864 reduced proliferation in different meningioma cell lines and reduced proliferation and migration independent of GTPase knockdowns/status. Moreover, overall survival in vivo could also be increased by knockdowns of RhoA and Rac1 as well as Fasudil treatment. GTPase expression was affected dependent on the NF2 status but effects were not very distinct, indicating that NF2 is not strongly involved in GTPase regulation in meningiomas. In terms of mTOR and FAK-Paxillin signaling, each GTPase changes those pathways in a different manner. CONCLUSION Small GTPases are important effectors in meningioma proliferation and migration in vitro as well as survival in vivo and their inhibition should be considered as potential treatment option.

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Hydrogel Tissue Construct-Based High-Content Compound Screening

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057110388269 ◽

2010 ◽

Vol 16 (1) ◽

pp. 120-128 ◽

Cited By ~ 8

Author(s):

Vy Lam ◽

Tetsuro Wakatsuki

Keyword(s):

Kinase Inhibitor ◽

Physiological Mechanism ◽

Rho Kinase ◽

Assay System ◽

Physiological Status ◽

Multiple Parameters ◽

Compound Screening ◽

Tissue Construct

Current pharmaceutical compound screening systems rely on cell-based assays to identify therapeutic candidates and potential toxicities. However, cells grown on 2D substrata or in suspension do not exhibit the mechanical or physiological properties of cells in vivo. To address this limitation, the authors developed an in vitro, high-throughput, 3D hydrogel tissue construct (HTC)–based assay system to quantify cell and tissue mechanical properties and multiple parameters of physiology. HTC mechanics was quantified using an automated device, and physiological status was assessed using spectroscopy-based indicators that were read on microplate readers. To demonstrate the application of this system, the authors screened 4 test compounds—rotenone (ROT), cytochalasin D (CD), 2,4-dinitrophenol (DNP), and Rho kinase inhibitor (H-1152)—for their ability to modulate HTC contractility without affecting actin integrity, mitochondrial membrane potential (MMP), or viability. All 4 compounds dose-dependently reduced HTC contractility. However, ROT was toxic, DNP dissipated MMP, and CD reduced both intracellular F-actin and viability. H-1152 was found to be the best candidate compound since it reduced HTC contractility with minimal side effects. The authors propose that their HTC-based assay system can be used to screen for compounds that modulate HTC contractility and assess the underlying physiological mechanism(s) of compound activity and toxicity.

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In Vitro and In Vivo Assays to Analyze the Contribution of Rho Kinase in Angiogenesis

Methods in Enzymology - Small GTPases in Disease, Part B ◽

10.1016/s0076-6879(07)00428-4 ◽

2008 ◽

pp. 395-412 ◽

Cited By ~ 5

Author(s):

Kenjiro Sawada ◽

Ken‐ichirou Morishige ◽

Seiji Mabuchi ◽

Seiji Ogata ◽

Chiaki Kawase ◽

...

Keyword(s):

Rho Kinase ◽

In Vivo Assays

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Effects of Short-Term Inhibition of Rho Kinase on Dromedary Camel Oocyte In Vitro Maturation

Animals ◽

10.3390/ani10050750 ◽

2020 ◽

Vol 10 (5) ◽

pp. 750

Author(s):

Hammed A. Tukur ◽

Riyadh S. Aljumaah ◽

Ayman Abdel-Aziz Swelum ◽

Abdullah N. Alowaimer ◽

Mutassim Abdelrahman ◽

...

Keyword(s):

In Vitro Maturation ◽

Rho Kinase ◽

Positive Outcome ◽

Dromedary Camel ◽

Second Phase ◽

Short Term ◽

Rock Inhibitor ◽

Nuclear Maturation ◽

Significant Difference

This is the first report on a biphasic in vitro maturation (IVM) approach with a meiotic inhibitor to improve dromedary camel IVM. Spontaneous meiotic resumption poses a major setback for in vitro matured oocytes. The overall objective of this study was to improve in vitro maturation of dromedary camel oocytes using ROCK inhibitor (Y-27632) in a biphasic IVM to prevent spontaneous meiotic resumption. In the first experiment, we cultured immature cumulus–oocyte complexes (COCs, n = 375) in a prematuration medium supplemented with ROCK inhibitor (RI) for 2 h, 4 h, 6 h, and 24 h before submission to normal in vitro maturation to complete 28 h. The control was cultured for 28 h in the absence of RI. In the first phase of experiment two, we cultured COCs (n = 480) in the presence or absence (control) of RI for 2 h, 4 h, 6 h, and 24 h, and conducted real-time relative quantitative PCR (qPCR) on selected mRNA transcripts. The same was done in the second phase, but qPCR was done after completion of normal IVM. Assessment of nuclear maturation showed that pre-IVM for 4 h yielded an increase in MII oocyte (54.67% vs. 26.6% of control; p < 0.05). As expected, the same group showed the highest degree (2) of cumulus expansion. In experiment 2, qPCR results showed significantly higher expression of ACTB and BCL2 in the RI group treated for 4 h when compared with the other groups. However, their relative quantification after biphasic IVM did not reveal any significant difference, except for the positive response of BCL2 and BAX/BCL2 ratio after 4 and 6 h biphasic IVM. In conclusion, RI prevents premature oocyte maturation and gave a significantly positive outcome during the 4 h treatment. This finding is a paradigm for future investigation on dromedary camel biphasic IVM and for improving the outcome of IVM in this species.

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The Vasopressin Receptor 2 Mutant R137L Linked to the Nephrogenic Syndrome of Inappropriate Antidiuresis (NSIAD) Signals through an Alternative Pathway that Increases AQP2 Membrane Targeting Independently of S256 Phosphorylation

Cells ◽

10.3390/cells9061354 ◽

2020 ◽

Vol 9 (6) ◽

pp. 1354

Author(s):

Marianna Ranieri ◽

Maria Venneri ◽

Tommaso Pellegrino ◽

Mariangela Centrone ◽

Annarita Di Mise ◽

...

Keyword(s):

Water Permeability ◽

Collecting Duct ◽

Alternative Pathway ◽

Water Channel ◽

Rho Kinase ◽

Membrane Targeting ◽

Rock Inhibitor ◽

Activating Mutations ◽

Osmotic Water

NSIAD is a rare X-linked condition, caused by activating mutations in the AVPR2 gene coding for the vasopressin V2 receptor (V2R) associated with hyponatremia, despite undetectable plasma vasopressin levels. We have recently provided in vitro evidence that, compared to V2R-wt, expression of activating V2R mutations R137L, R137C and F229V cause a constitutive redistribution of the AQP2 water channel to the plasma membrane, higher basal water permeability and significantly higher basal levels of p256-AQP2 in the F229V mutant but not in R137L or R137C. In this study, V2R mutations were expressed in collecting duct principal cells and the associated signalling was dissected. V2R-R137L and R137C mutants had significantly higher basal pT269-AQP2 levels -independently of S256 and PKA-which were reduced to control by treatment with Rho kinase (ROCK) inhibitor. Interestingly, ROCK activity was found significantly higher in V2R-R137L along with activation of the Gα12/13–Rho–ROCK pathway. Of note, inhibition of ROCK reduced the basal elevated osmotic water permeability to control. To conclude, our data demonstrate for the first time that the gain-of-function mutation of the V2R, R137L causing NSIAD, signals through an alternative PKA-independent pathway that increases AQP2 membrane targeting through ROCK-induced phosphorylation at S/T269 independently of S256 of AQP2.

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