Crosslinking-Induced Corneal Endothelium Dysfunction and Its Protection by Topical Ripasudil Treatment

Purpose. To investigate the changes of corneal endothelium under different crosslinking conditions and the protective effect of ripasudil. Methods. Corneal crosslinking groups were infiltrated with riboflavin and subsequently irradiated with 0.54 J/cm2 or 1.08 J/cm2 UVA, while noncrosslinking groups included neither UVA nor riboflavin treatment, only 1.08 J/cm2 UVA and only riboflavin treatment. Corneal opacity, variations in corneal endothelial cells, and corneal thickness of all groups were observed by slit lamp, in vivo confocal microscopy, and optical coherence tomography. Immunofluorescence staining and scanning electron microscopy were performed to evaluate changes in the structure and function of the corneal endothelium. The mice that received a corneal crosslinking dose of 1.08 J/cm2 were instilled with ripasudil to explore its protective effect on the corneal endothelium. Results. Treatment with UVA and riboflavin caused an increase in corneal opacity and corneal thickness and decreased endothelial cell density. Furthermore, treatment with UVA and riboflavin caused endothelial cell DNA damage and destroyed the tight junction and pump function of the endothelium, while riboflavin or the same dose of UVA alone did not affect the endothelium. Ripasudil reduced DNA damage in endothelial cells, increased the density of cells, and protected the endothelium’s integrity and function. Conclusion. Riboflavin combined with UVA can damage the corneal endothelium’s normal functioning. The corneal endothelium’s wound healing is dose-dependent, and the ROCK inhibitor ripasudil maintains the endothelium’s pump and barrier functions.

Advanced Microparticulate/Nanoparticulate Respirable Dry Powders of a Selective RhoA/Rho Kinase (Rock) Inhibitor for Targeted Pulmonary Inhalation Aerosol Delivery

Pulmonary hypertension (PH) is a progressive disease that eventually leads to heart failure and potentially death for some patients. There are many unique advantages to treating pulmonary diseases directly and non-invasively by inhalation aerosols and dry powder inhalers (DPIs) possess additional unique advantages. There continues to be significant unmet medical needs in the effective treatment of PH that target the underlying mechanisms. To date, there is no FDA-approved DPI indicated for the treatment of PH. Fasudil is a novel RhoA/Rho kinase (ROCK) inhibitor that has shown great potential in effectively treating pulmonary hypertension. This systematic study is the first to report on the design and development of DPI formulations comprised of respirable nanoparticles/microparticles using particle engineering design by advanced spray drying. In addition, comprehensive physicochemical characterization, in vitro aerosol aerosol dispersion performance with different types of human DPI devices, in vitro cell-drug dose response cell viability of different human respiratory cells from distinct lung regions, and in vitro transepithelial electrical resistance (TEER) as air-interface culture (AIC) demonstrated that these innovative DPI fasudil formulations are safe on human lung cells and have high aerosol dispersion performance properties.

Lysophosphatidic Acid and Several Neurotransmitters Converge on Rho-Kinase 2 Signaling to Manage Motoneuron Excitability

Intrinsic membrane excitability (IME) sets up neuronal responsiveness to synaptic drive. Several neurotransmitters and neuromodulators, acting through G-protein-coupled receptors (GPCRs), fine-tune motoneuron (MN) IME by modulating background K+ channels TASK1. However, intracellular partners linking GPCRs to TASK1 modulation are not yet well-known. We hypothesized that isoform 2 of rho-kinase (ROCK2), acting as downstream GPCRs, mediates adjustment of MN IME via TASK1. Electrophysiological recordings were performed in hypoglossal MNs (HMNs) obtained from adult and neonatal rats, neonatal knockout mice for TASK1 (task1–/–) and TASK3 (task3–/–, the another highly expressed TASK subunit in MNs), and primary cultures of embryonic spinal cord MNs (SMNs). Small-interfering RNA (siRNA) technology was also used to knockdown either ROCK1 or ROCK2. Furthermore, ROCK activity assays were performed to evaluate the ability of various physiological GPCR ligands to stimulate ROCK. Microiontophoretically applied H1152, a ROCK inhibitor, and siRNA-induced ROCK2 knockdown both depressed AMPAergic, inspiratory-related discharge activity of adult HMNs in vivo, which mainly express the ROCK2 isoform. In brainstem slices, intracellular constitutively active ROCK2 (aROCK2) led to H1152-sensitive HMN hyper-excitability. The aROCK2 inhibited pH-sensitive and TASK1-mediated currents in SMNs. Conclusively, aROCK2 increased IME in task3–/–, but not in task1–/– HMNs. MN IME was also augmented by the physiological neuromodulator lysophosphatidic acid (LPA) through a mechanism entailing Gαi/o-protein stimulation, ROCK2, but not ROCK1, activity and TASK1 inhibition. Finally, two neurotransmitters, TRH, and 5-HT, which are both known to increase MN IME by TASK1 inhibition, stimulated ROCK2, and depressed background resting currents via Gαq/ROCK2 signaling. These outcomes suggest that LPA and several neurotransmitters impact MN IME via Gαi/o/Gαq-protein-coupled receptors, downstream ROCK2 activation, and subsequent inhibition of TASK1 channels.

Nicotinamide promotes cardiomyocyte derivation and survival through kinase inhibition in human pluripotent stem cells

Nicotinamide, the amide form of Vitamin B3, is a common nutrient supplement that plays important role in human fetal development. Nicotinamide has been widely used in clinical treatments, including the treatment of diseases during pregnancy. However, its impacts during embryogenesis have not been fully understood. In this study, we show that nicotinamide plays multiplex roles in mesoderm differentiation of human embryonic stem cells (hESCs). Nicotinamide promotes cardiomyocyte fate from mesoderm progenitor cells, and suppresses the emergence of other cell types. Independent of its functions in PARP and Sirtuin pathways, nicotinamide modulates differentiation through kinase inhibition. A KINOMEscan assay identifies 14 novel nicotinamide targets among 468 kinase candidates. We demonstrate that nicotinamide promotes cardiomyocyte differentiation through p38 MAP kinase inhibition. Furthermore, we show that nicotinamide enhances cardiomyocyte survival as a Rho-associated protein kinase (ROCK) inhibitor. This study reveals nicotinamide as a pleiotropic molecule that promotes the derivation and survival of cardiomyocytes, and it could become a useful tool for cardiomyocyte production for regenerative medicine. It also provides a theoretical foundation for physicians when nicotinamide is considered for treatments for pregnant women.

Intravitreal Fasudil Monotherapy for Treatment of Refractory Diabetic Macular Edema: a Prospective, Interventional Case-series

Purpose: To evaluate the effect of three monthly intravitreal injections of a Rho-associated protein kinase (ROCK) inhibitor (Fasudil, Asahi Kasei Pharma Corporation, Tokyo, Japan) on refractory diabetic macular edema (DME).Study design: Prospective – clinical Methods: This interventional case series included 10 eyes of 10 patients with DME unresponsive to at least six previous intravitreal bevacizumab (IVB) injections. Eligible eyes underwent intravitreal injection of 0.025 mg/0.05 ml Fasudil. Best-corrected visual acuity (BCVA) and central macular thickness (CMT) were evaluated as functional and anatomical response indicators, respectively. Results: Mean age was 60.1±5.1 (range, 53-68) years. Five cases responded to treatment; two with both anatomical and functional response (reduction of CMT from 521 to 395 and from 390 to 301microns and improvement of BCVA from 0.3 to 0.1 LogMAR and 0.6 to 0.4 LogMAR, respectively) and three with only functional improvement (0.7 to 0.4; 0.7 to 0.4; and 0.3 to 0.1 LogMAR). Of note, cases with no significant CMT change had morphologic improvement of retinal microstructure to some extent. No adverse event was observed during the study period.Conclusion: Monotherapy with intravitreal injection of ROCK inhibitors seems to have moderate visual benefits in eyes with DME refractory to IVB. Such effects may be functionally significant without obvious anatomical improvement.

Substrate stiffness controls the cell cycle of human mesenchymal stem cells via cellular traction

The microenvironment of human mesenchymal stem cells (hMSCs) regulates their self-renewal and differentiation properties. Previously it was shown that hMSCs remained quiescent on soft (0.25 kPa) polyacrylamide (PA) gels but re-entered into cell cycle on a stiff (7.5 kPa) gel. However, how cells behave on intermediate stiffness and what intracellular factors transmit mechanical changes to cell interior thereby regulating cell cycle remained unknown. In this work we demonstrated that PA gels between 1 and 5 kPa act as a mechanical switch in regulating cell cycle of hMSCs. By experiments on cell-cycle exit and re-entry, we found that hMSCs demonstrated a sharp transition from quiescence to proliferation between 1 and 5 kPa. Further studies with ROCK inhibitor Y-27632 revealed that contractile proteins, but not cell spread area, accounts for the sensitivity of hMSCs towards substrate stiffness and hence correlates with their changes in cell cycle. These observations therefore suggest that substrate stiffness regulates hMSC proliferation through contractile forces as generated by cellular contractile proteins in a unique pattern which is distinct from other cell types as studied.