Non-muscle myosin II (NMII)[1]

Using bovine corneal endothelial cells, the therapeutic potential of targeting NMII to improve CEC migration was examined (BCEC). By decreasing retrograde actin flow and boosting lamellar foot process persistence, blebbistatin, a direct inhibitor of myosin motility, stimulates migration and directional persistence in CECs and speeds up in vitro wound healing [1].

In a rabbit corneal endothelial scratch model, blebbistatin (0.05 mL, 20 μM; intracameral injection; once daily; for 6 days; New Zealand white rabbit) therapy improves wound healing [1].

Protein permeability assay on BCEC monolayer[1]
BCECs were cultured in control medium or media containing 10 μM Y27632 or 10 μM blebbistatin until cellular confluence on Transwell filters (0.4 lm pore-size polycarbonate filters). After washing and equilibrating in fresh media for 30 min, FITC-conjugated 4 K, 40 K, and 500 K dextran at a concentration of 3 mg/mL were added to the upper compartment. After further incubation for 4 h, the medium from the lower compartment was assessed for diffused FITC-dextran measured by fluorometry (excitation, 492 nm; emission, 520 nm).

Immunofluorescence confocal microscopy[1]
BCECs were cultured on cover slides, fixed at the indicated time points in 4% paraformaldehyde (pH 7.4) for 30 min at room temperature, permeabilized with 0.5% Triton X-100 for 5 min, and blocked with 10% bovine serum albumin for 30 min. The cells then were incubated with the indicated primary antibodies overnight at 4 °C. After washing twice with PBS for 15 min, samples were incubated with Alexa Fluor-conjugated secondary antibody at room temperature for 1 h. For actin labeling, samples were stained with Alexa 546 phalloidin at room temperature for 10 min. After several washes, all samples were mounted in fluorescent mounting solution. Immunofluorescent images were obtained using laser scanning confocal microscope and analyzed by Zen software. For Y27632 or blebbistatin washout experiment, cells were plated and incubated with 10 μM Y27632 or 10 μM blebbistatin simultaneously at the beginning. Later, Y27632 or blebbistatin was removed from the replete culture media for the time spans as indicated in Fig. 5f before cells were harvested and processed for confocal microscopic examination.

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Live-cell imaging[1]
For single cell migration study, BCECs were plated into a 12-well plate and incubated with control medium or media containing 10 μM Y27632, 10 μM blebbistatin, and 10 μM ML-7 for 2 days. The plate was then placed in a humidified, CO2-equilibrated chamber equipped with a Zeiss Axiovert 200 M inverted microscope and a motorized stage. Five fields per condition were captured every 30 min for 16 h using a 10×/0.3 EC Plan-Neofluar objective lens. The nuclear positions of 10 randomly selected cells per field (50 cells per condition) were tracked by MetaMorph software. The migration speed, directionality ratio, and mean square displacement were analyzed using DiPer program generously provided by Gorelik et al. For in vitro wound healing study, cells were seeded in a 6-well plate and incubated with control medium until confluence. Prior to imaging, the wound was generated in each well by scratching with a 200 μl pipette tip, and the media were changed to serum free media containing vehicle control, 10 μM Y27632, 10 μM blebbistatin, or 10 μM ML-7, separately. Images were taken with a 10×/0.3 EC Plan-Neofluar objective lens every 30 min for 16 h. The change in wound areas was analyzed by ImageJ software.

Animal/Disease Models: New Zealand white rabbits (16-20 weeks; 3-3.5 kg)[1]
Doses: 0.05 mL; 20 μM
Route of Administration: Intracameral injection; daily; for 6 days
Experimental Results: Resulted in significant improvement of corneal clarity and corneal edema resolution, implying the restoration of an intact corneal endothelial monolayer.

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Rabbit corneal endothelium wounding model[1]
The rabbit corneal endothelium wounding procedure was performed as described previously with modifications. New Zealand white rabbits aged 16 to 20 weeks and weighing 3 to 3.5 kg were anesthetized with an intramuscular injection of xylazine 5 mg/kg and ketamine 40 mg/kg. Alcaine Ophthalmic Solution (0.5%) was instilled to further minimize ocular pain. The area of endothelial cells to be removed was marked with an 8.0-mm marker. After creating the corneal incision with a 2.75-mm blade, CECs within the 8.0-mm mark were gently removed by using posterior capsule polisher, and the areas of denuded Descemet membrane were confirmed by 0.06% trypan blue staining. Missed areas were debrided again to achieve complete cell removal. After wounding, intracameral injection of 0.05 mL of vehicle control, 20 μM Y27632, or 20 μM blebbistatin was administered immediately after wounding and once daily thereafter in different groups of animals. Topical 0.3% gentamicin sulfate was instilled after the procedure for infection control. During the follow-up evaluation, the corneas were observed and photographed with a surgical microscope. The corneal thickness was measured by ultrasound biomicroscopy. Briefly, after intramuscular and topical anesthesia, the ultrasound gel was applied on the cornea. A 50-MHz transducer-probe, aligned vertically to the corneal surface, was used to acquire the image of the anterior chamber. Only the image of the central cornea was acquired, in which the lens was clearly visible through un-dilated pupil. After acquiring the images, the corneal thickness was measured by drawing a line perpendicular to the corneal apex. The whole procedure was performed by an independent technician without prior knowledge related to the experimental condition, and the corneal thickness derived from the images was further analyzed by the author (W.T.H). The animals were euthanized at the indicated time point, and the corneas were harvested. To quantify the wounding extent, the corneas were stained for denuded Descemet’s membrane with 0.2% trypan blue for 1 min. After capturing the image with operating microscope, the wounding extent was quantified by ImageJ software. For immunofluorescent staining, the corneas were fixed in 4% paraformaldehyde (pH 7.4) for 5 min at room temperature, radially incised to allow flat mounting, and were subjected to immunostaining and observation as described above.

[1]. Targeting non-muscle myosin II promotes corneal endothelial migration through regulating lamellipodial dynamics. J Mol Med (Berl). 2019 Sep;97(9):1345-1357.

[2]. Discovery of the migrasome, an organelle mediating release of cytoplasmic contents during cell migration. Cell Res. 2015 Jan;25(1):24-38.

Blebbistatin is a pyrroloquinoline that is 1,2,3,3a-tetrahydro-H-pyrrolo[2,3-b]quinolin-4-one substituted by a hydroxy group at position 3a, a methyl group at position 6 and a phenyl group at position 1. It acts as an inhibitor of ATPase activity of non-muscle myosin II. It has a role as an inhibitor. It is a pyrroloquinoline, a cyclic ketone, a tertiary alcohol and a tertiary alpha-hydroxy ketone.

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Corneal endothelial cell (CEC) dysfunction causes corneal edema that may lead to blindness. In addition to corneal transplantation, simple descemetorhexis has been proposed to treat centrally located disease with adequate peripheral cell reserve, but promoting the centripetal migration of CECs is pivotal to this strategy. Here, we show that targeting non-muscle myosin II (NMII) activity by Y27632, a ROCK inhibitor, or blebbistatin, a selective NMII inhibitor, promotes directional migration of CECs and accelerates in vitro wound healing. The lamellipodial protrusion persistence is increased, and actin retrograde flow is decreased after NMII inhibition. Counteracting lamellipodial protrusion by actin-related protein 2/3 (ARP2/3) inhibitor abolishes this migration-promoting effect. Although both Y27632 and blebbistatin accelerate wound healing, cell junctional integrity and barrier function are better preserved after blebbistatin treatment, leading to more rapid corneal deturgescence in rabbit corneal endothelial wounding model. Our findings indicate that NMII is a promising therapeutic target in the treatment of CEC dysfunction. KEY MESSAGES: NMII inhibition promotes directional migration and wound healing of CECs in vitro. Lamellipodial protrusion persistence is increased after NMII inhibition. Selective NMII inhibitor preserves junctional integrity better than ROCK inhibitor. Selective NMII inhibitor accelerates corneal deturgescence after wounding in vivo.[1]

C18H16N2O2

292.33

292.121

C, 73.95; H, 5.52; N, 9.58; O, 10.95

674289-55-5

(-)-Blebbistatin;856925-71-8

3476986

Light yellow to yellow solid powder

1.3±0.1 g/cm3

507.3±60.0 °C at 760 mmHg

210-212ºC

260.6±32.9 °C

0.0±1.4 mmHg at 25°C

1.681

1.62

1

3

1

22

497

0

LZAXPYOBKSJSEX-UHFFFAOYSA-N

InChI=1S/C18H16N2O2/c1-12-7-8-15-14(11-12)16(21)18(22)9-10-20(17(18)19-15)13-5-3-2-4-6-13/h2-8,11,22H,9-10H2,1H3

3a-hydroxy-6-methyl-1-phenyl-2,3-dihydropyrrolo[2,3-b]quinolin-4-one

Blebbistatin; (+/-)-Blebbistatin; 674289-55-5; (+-)-Blebbistatin; CHEBI:75379; UNII-20WC4J7CQ6; 3a-hydroxy-6-methyl-1-phenyl-2,3-dihydropyrrolo[2,3-b]quinolin-4-one; 20WC4J7CQ6;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : 25 mg/mL (85.52 mM)

Solubility in Formulation 1: 2.5 mg/mL (8.55 mM) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 + to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)