ROCK inhibition reduces morphological and functional damage to rod synapses after retinal injury

AbstractRetinal detachment (RD) causes damage, including disjunction, of the rod photoreceptor-bipolar synapse, which disrupts vision and may contribute to the poor visual recovery observed after retinal reattachment surgery. We created a model of iatrogenic RD in adult female pigs to study damage to the rod-bipolar synapse after injury and the ability of a highly specific Rho-kinase (ROCK) inhibitor to preserve synaptic structure and function. This model mimics procedures used in humans when viral vectors or cells are injected subretinally for treatment of retinal disease. Synaptic disjunction by retraction of rod spherules, quantified by image analysis of confocal sections, was present 2 h after detachment and remained 2 days later even though the retina had spontaneously reattached by then. Moreover, spherule retraction occurred in attached retina 1–2 cms from detached retina. Synaptic damage was significantly reduced by ROCK inhibition in detached retina whether injected subretinally or intravitreally. Dark-adapted full-field electroretinograms were recorded in reattached retinas to assess rod-specific function. Reduction in synaptic injury correlated with increases in rod-driven responses in drug-treated eyes. Thus, ROCK inhibition helps prevent synaptic damage and improves functional outcomes after retinal injury and may be a useful adjunctive treatment in iatrogenic RD and other retinal degenerative diseases.

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Rho-Kinase Inhibition Improves Ischemic Perfusion Deficit in Hyperlipidemic Mice

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.2013.195 ◽

2013 ◽

Vol 34 (2) ◽

pp. 284-287 ◽

Cited By ~ 17

Author(s):

Hwa Kyoung Shin ◽

Paul L Huang ◽

Cenk Ayata

Keyword(s):

Single Dose ◽

Vascular Dysfunction ◽

Rho Kinase ◽

Tissue Perfusion ◽

Artery Occlusion ◽

Laser Speckle ◽

Rock Inhibitor ◽

Arterial Blood ◽

Apolipoprotein E Knockout Mice ◽

Rock Inhibition

Hyperlipidemia is a major cardiovascular risk factor associated with progressive cerebrovascular dysfunction and diminished collateral perfusion in stroke. Rho-associated kinase (ROCK) may be an important mediator of hyperlipidemic vascular dysfunction. We tested the efficacy of acute or chronic ROCK inhibition on the size of dynamic perfusion defect using laser speckle flowmetry in hyperlipidemic apolipoprotein E knockout mice fed on a high-fat diet for 8 weeks. Mice were studied at an age before the development of flow-limiting atherosclerotic stenoses in aorta and major cervical arteries. Focal ischemia was induced by distal middle cerebral artery occlusion (dMCAO) during optical imaging. The ROCK inhibitor fasudil (10 mg/kg) was administered either as a single dose 1 hour before ischemia onset, or daily for 4 weeks. Fasudil decreased both baseline arterial blood pressure and cerebrovascular resistance (CVR) by ∼15%, and significantly improved tissue perfusion during dMCAO. Interestingly, peri-infarct depolarizations were also reduced. Chronic treatment did not further enhance these benefits compared with acute treatment with a single dose. These data show that ROCK inhibition improves CVR and ischemic tissue perfusion in hyperlipidemic mice.

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Organized migration of epithelial cells requires control of adhesion and protrusion through Rho kinase effectors

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00333.2006 ◽

2007 ◽

Vol 292 (3) ◽

pp. G806-G817 ◽

Cited By ~ 28

Author(s):

Ann M. Hopkins ◽

A’Drian A. Pineda ◽

L. Matthew Winfree ◽

G. Thomas Brown ◽

Mike G. Laukoetter ◽

...

Keyword(s):

Epithelial Cell ◽

Kinase Inhibitor ◽

Rho Kinase ◽

Leading Edge ◽

Dominant Negative ◽

Actin Binding ◽

Rock Inhibitor ◽

Cell Sheets ◽

Lim Kinase ◽

Rock Inhibition

Migration of epithelial cell sheets, a process involving F-actin restructuring through Rho family GTPases, is both physiologically and pathophysiologically important. Our objective was to clarify the mechanisms whereby the downstream RhoA effector Rho-associated coil-coil-forming kinase (ROCK) influences coordinated epithelial cell motility. Although cells exposed to a pharmacological ROCK inhibitor (Y-27632) exhibited increased spreading in wound closure assays, they failed to migrate in a cohesive manner. Two main phenomena were implicated: the formation of aberrant protrusions at the migrating front and the basal accumulation of F-actin aggregates. Aggregates reflected increased membrane affiliation and detergent insolubility of the actin-binding protein ezrin and enhanced coassociation of ezrin with the membrane protein CD44. While F-actin aggregation following ROCK inhibition was recapitulated by inhibiting myosin light chain (MLC) phosphorylation with the MLC kinase inhibitor ML-7, the latter did not influence protrusiveness and, in fact, significantly decreased cell migration. Our results suggest that excessive protrusiveness downstream of ROCK inhibition reflects an influence of ROCK on F-actin stability via LIM kinase 1 (LIMK-1), which phosphorylates and inactivates cofilin. Y-27632 reduced the levels of both active LIMK-1 and inactive cofilin (phospho forms), and expression of a dominant negative LIMK-1 mutant stimulated leading edge protrusiveness. Furthermore, Y-27632-induced protrusions were partially reversed by overexpression of LIMK-1 to restore cofilin phosphorylation. In summary, our results provide new evidence suggesting that adhesive and protrusive events involved in organized epithelial motility downstream of ROCK are separately coordinated through the phosphorylation of (respectively) MLC and cofilin.

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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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Effects of a Rho kinase inhibitor on pressure overload induced cardiac hypertrophy and associated diastolic dysfunction

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.01078.2007 ◽

2008 ◽

Vol 294 (4) ◽

pp. H1804-H1814 ◽

Cited By ~ 83

Author(s):

Arintaya Phrommintikul ◽

Lavinia Tran ◽

Andrew Kompa ◽

Bing Wang ◽

Anastasia Adrahtas ◽

...

Keyword(s):

Cardiac Hypertrophy ◽

Diastolic Dysfunction ◽

Kinase Inhibitor ◽

Diastolic Pressure ◽

Rho Kinase ◽

Pressure Overload ◽

Rock Inhibitor ◽

Abdominal Aortic ◽

Vehicle Treatment ◽

Rock Inhibition

The RhoA-Rho kinase (ROCK) signaling pathway has an important role in cardiovascular diseases. However, the effect of Rho kinase inhibition on pressure overload-induced cardiac hypertrophy (POH) and associated diastolic dysfunction has not been evaluated. This study examined the effect of a selective ROCK inhibitor (GSK-576371) in a POH model, induced by suprarenal abdominal aortic constriction. POH rats were divided into the following four groups: 1 (GSK 1, n = 9) or 3 (GSK 3, n = 10) mg/kg bid GSK-576371, 1 mg·kg−1·day−1 ramipril ( n = 10) or vehicle ( n = 11) treatment for 4 wk. Sham animals ( n = 11) underwent surgery without banding. Echocardiograms were performed before surgery and posttreatment, and hemodynamic data were obtained at completion of the study. Echocardiography showed an increase in relative wall thickness of the left ventricle (LV) following POH + vehicle treatment compared with sham animals. This was attenuated by both doses of GSK-576371 and ramipril. Vehicle treatment demonstrated abnormal diastolic parameters, including mitral valve (MV) inflow E wave deceleration time, isovolumic relaxation time, and MV annular velocity, which were dose dependently restored toward sham values by GSK-576371. LV end diastolic pressure was increased following POH + vehicle treatment compared with sham (6.9 ± 0.7 vs. 3.2 ± 0.7 mmHg, P = 0.008) and was reduced with GSK 3 and ramipril treatment (1.7 ± 0.7, P < 0.01 and 2.9 ± 0.6 mmHg, P < 0.01, respectively). Collagen I deposition in the LV was increased following POH + vehicle treatment (32.2%; P < 0.01) compared with sham animals and was significantly attenuated with GSK 1 (21.7%; P < 0.05), GSK 3 (23.8%; P < 0.01), and ramipril (35.5%; P < 0.01) treatment. These results suggest that ROCK inhibition improves LV geometry and reduces collagen deposition accompanied by improved diastolic function in POH.

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Clinical Phenotype of PDE6B-Associated Retinitis Pigmentosa

International Journal of Molecular Sciences ◽

10.3390/ijms22052374 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2374

Author(s):

Laura Kuehlewein ◽

Ditta Zobor ◽

Katarina Stingl ◽

Melanie Kempf ◽

Fadi Nasser ◽

...

Keyword(s):

Visual Acuity ◽

Genetic Testing ◽

Retinitis Pigmentosa ◽

Fundus Autofluorescence ◽

Retinal Disease ◽

New Drugs ◽

Autofluorescence Imaging ◽

Full Field ◽

Tertiary Referral Center ◽

The Right

In this retrospective, longitudinal, observational cohort study, we investigated the phenotypic and genotypic features of retinitis pigmentosa associated with variants in the PDE6B gene. Patients underwent clinical examination and genetic testing at a single tertiary referral center, including best-corrected visual acuity (BCVA), kinetic visual field (VF), full-field electroretinography, full-field stimulus threshold, spectral domain optical coherence tomography, and fundus autofluorescence imaging. The genetic testing comprised candidate gene sequencing, inherited retinal disease gene panel sequencing, whole-genome sequencing, and testing for familial variants by Sanger sequencing. Twenty-four patients with mutations in PDE6B from 21 families were included in the study (mean age at the first visit: 32.1 ± 13.5 years). The majority of variants were putative splicing defects (8/23) and missense (7/23) mutations. Seventy-nine percent (38/48) of eyes had no visual acuity impairment at the first visit. Visual acuity impairment was mild in 4% (2/48), moderate in 13% (6/48), and severe in 4% (2/48). BCVA was symmetrical in the right and left eyes. The kinetic VF measurements were highly symmetrical in the right and left eyes, as was the horizontal ellipsoid zone (EZ) width. Regarding the genetic findings, 43% of the PDE6B variants found in our patients were novel. Thus, this study contributed substantially to the PDE6B mutation spectrum. The visual acuity impairment was mild in 83% of eyes, providing a window of opportunity for investigational new drugs. The EZ width was reduced in all patients and was highly symmetric between the eyes, making it a promising outcome measure. We expect these findings to have implications on the design of future PDE6B-related retinitis pigmentosa (RP) clinical trials.

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Immortalization and Characterization of Rat Lingual Keratinocytes in a High-Calcium and Feeder-Free Culture System Using ROCK Inhibitor Y-27632

International Journal of Molecular Sciences ◽

10.3390/ijms22136782 ◽

2021 ◽

Vol 22 (13) ◽

pp. 6782

Author(s):

Zixing Chen ◽

Wenmeng He ◽

Thomas Chun Ning Leung ◽

Hau Yin Chung

Keyword(s):

Calcium Imaging ◽

Cultured Cells ◽

Rho Kinase ◽

Culture Method ◽

Taste Bud ◽

Label Free ◽

Sprague Dawley ◽

Rock Inhibitor ◽

High Calcium

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.

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Pathogenic STX3 variants affecting the retinal and intestinal transcripts cause an early-onset severe retinal dystrophy in microvillus inclusion disease subjects

Human Genetics ◽

10.1007/s00439-021-02284-1 ◽

2021 ◽

Author(s):

Andreas R. Janecke ◽

Xiaoqin Liu ◽

Rüdiger Adam ◽

Sumanth Punuru ◽

Arne Viestenz ◽

...

Keyword(s):

Early Onset ◽

Single Nucleotide Polymorphism Array ◽

Outer Nuclear Layer ◽

Retinal Dystrophy ◽

Geographic Origin ◽

Retinal Disease ◽

Homozygosity Mapping ◽

Rod Photoreceptor ◽

Loss Of Function ◽

Knockout Mouse Model

AbstractBiallelic STX3 variants were previously reported in five individuals with the severe congenital enteropathy, microvillus inclusion disease (MVID). Here, we provide a significant extension of the phenotypic spectrum caused by STX3 variants. We report ten individuals of diverse geographic origin with biallelic STX3 loss-of-function variants, identified through exome sequencing, single-nucleotide polymorphism array-based homozygosity mapping, and international collaboration. The evaluated individuals all presented with MVID. Eight individuals also displayed early-onset severe retinal dystrophy, i.e., syndromic—intestinal and retinal—disease. These individuals harbored STX3 variants that affected both the retinal and intestinal STX3 transcripts, whereas STX3 variants affected only the intestinal transcript in individuals with solitary MVID. That STX3 is essential for retinal photoreceptor survival was confirmed by the creation of a rod photoreceptor-specific STX3 knockout mouse model which revealed a time-dependent reduction in the number of rod photoreceptors, thinning of the outer nuclear layer, and the eventual loss of both rod and cone photoreceptors. Together, our results provide a link between STX3 loss-of-function variants and a human retinal dystrophy. Depending on the genomic site of a human loss-of-function STX3 variant, it can cause MVID, the novel intestinal-retinal syndrome reported here or, hypothetically, an isolated retinal dystrophy.

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Full-field stimulus testing: Role in the clinic and as an outcome measure in clinical trials of severe childhood retinal disease

Progress in Retinal and Eye Research ◽

10.1016/j.preteyeres.2021.101000 ◽

2021 ◽

pp. 101000

Author(s):

Alejandro J. Roman ◽

Artur V. Cideciyan ◽

Vivian Wu ◽

Alexandra V. Garafalo ◽

Samuel G. Jacobson

Keyword(s):

Clinical Trials ◽

Outcome Measure ◽

Retinal Disease ◽

Full Field

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A visible light-controllable Rho kinase inhibitor based on a photochromic phenylazothiazole

Chemical Communications ◽

10.1039/d1cc04905d ◽

2021 ◽

Author(s):

Kazuya Matsuo ◽

Sampreeth Thayyil ◽

Mitsuyasu Kawaguchi ◽

Hidehiko Nakagawa ◽

Nobuyuki Tamaoki

Keyword(s):

Protein Kinase ◽

Visible Light ◽

Kinase Inhibitor ◽

Coiled Coil ◽

Rho Kinase ◽

Rock Inhibitor ◽

Serine Threonine Kinase ◽

Threonine Kinase ◽

Rho Kinase Inhibitor

Rho-associated coiled-coil-containing protein kinase (ROCK) is a serine-threonine kinase, whose inhibitors are useful for the regulation of actomyosin system. Here, we developed a photoswitchable ROCK inhibitor based on a phenylazothiazole...

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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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