BACKGROUND: Human mesenchymal stem cells originating from umbilical cord matrix are a promising therapeutic resource, and their differentiated cells are spotlighted as a tissue regeneration treatment. However, there are limitations to the medical use of differentiated cells from human umbilical cord matrix-mesenchymal stem cells (hUCM-MSCs), such as efficient differentiation methods.
METHODS: To effectively differentiate hUCM-MSCs into hepatocyte-like cells (HLCs), we used the ROCK inhibitor, fasudil, which is known to induce endoderm formation, and gelatin, which provides extracellular matrix to the differentiated cells. To estimate a differentiation efficiency of early stage according to combination of gelatin and fasudil, transcription analysis was conducted. Moreover, to demonstrate that organelle states affect differentiation, we performed transcription, tomographic, and mitochondrial function analysis at each stage of hepatic differentiation. Finally, we evaluated hepatocyte function based on the expression of mRNA and protein, secretion of albumin, and activity of CYP3A4 in mature HLCs.
RESULTS: Fasudil induced endoderm-related genes (GATA4, SOX17, and FOXA2) in hUCM-MSCs, and it also induced lipid droplets (LDs) inside the differentiated cells. However, the excessive induction of LDs caused by fasudil inhibited mitochondrial function and prevented differentiation into hepatoblasts. To prevent the excessive LDs formation, we used gelatin as a coating material. When hUCM-MSCs were induced into hepatoblasts with fasudil on high-viscosity (1%) gelatin-coated dishes, hepatoblast-related genes (AFP and HNF4A) showed significant upregulation on high-viscosity gelatin-coated dishes compared to those treated with low-viscosity (0.1%) gelatin. Moreover, other germline cell fates, such as ectoderm and mesoderm, were repressed under these conditions. In addition, LDs abundance was also reduced, whereas mitochondrial function was increased. On the other hand, unlike early stage of the differentiation, low viscosity gelatin was more effective in generating mature HLCs. In this condition, the accumulation of LDs was inhibited in the cells, and mitochondria were activated. Consequently, HLCs originated from hUCM-MSCs were genetically and functionally more matured in low-viscosity gelatin.
CONCLUSIONS: This study demonstrated an effective method for differentiating hUCM-MSCs into hepatic cells using fasudil and gelatin of varying viscosities. Moreover, we suggest that efficient hepatic differentiation and the function of hepatic cells differentiated from hUCM-MSCs depend not only on genetic changes but also on the regulation of organelle states.

Descemet\'s Stripping Only (DSO) is a surgical technique that utilizes the peripheral corneal endothelial cell (CEnC) migration for wound closure. Ripasudil, a Rho-associated protein kinase (ROCK) inhibitor, has shown potential in DSO treatment; however, its mechanism in promoting CEnC migration remains unclear. We observed that ripasudil-treated immortalized normal and Fuchs endothelial corneal dystrophy (FECD) cells exhibited significantly enhanced migration and wound healing, particularly effective in FECD cells. Ripasudil upregulated mRNA expression of Snail Family Transcriptional Repressor (SNAI1/2) and Vimentin (VIM) while decreasing Cadherin (CDH1), indicating endothelial-to-mesenchymal transition (EMT) activation. Ripasudil activated Rac1, driving the actin-related protein complex (ARPC2) to the leading edge, facilitating enhanced migration. Ex vivo studies on cadaveric and FECD Descemet\'s membrane (DM) showed increased migration and proliferation of CEnCs after ripasudil treatment. An ex vivo DSO model demonstrated enhanced migration from the DM to the stroma with ripasudil. Coating small incision lenticule extraction (SMILE) tissues with an FNC coating mix and treating the cells in conjunction with ripasudil further improved migration and resulted in a monolayer formation, as detected by the ZO-1 junctional marker, thereby leading to the reduction in EMT. In conclusion, ripasudil effectively enhanced cellular migration, particularly in a novel ex vivo DSO model, when the stromal microenvironment was modulated. This suggests ripasudil as a promising adjuvant for DSO treatment, highlighting its potential clinical significance.

BACKGROUND: Orthodontic pain affects the physical and mental health of patients. The spinal trigeminal subnucleus caudalis (SPVC) contributes to the transmission of pain information and serves as a relay station for integrating orofacial damage information. Recently, glial cells have been found to be crucial for both acute and maintenance phases of pain. It has also been demonstrated that rho kinase (ROCK) inhibitors can manage different pain models by inhibiting glial cell activation. Here, we hypothesized that orthodontic pain is related to glial cells in the SPVC, and Fasudil, a representative rho/rock kinase inhibitor, can relieve orthodontic pain by regulating the function of glial cells and the related inflammatory factors. In this study, we constructed a rat model of tooth movement pain and used immunofluorescence staining to evaluate the activation of microglia and astrocytes. Quantitative real-time PCR was used to detect the release of related cytokines and the expression of pain-related genes in the SPVC. Simultaneously, we investigated the effect of Fasudil on the aforementioned indicators.
RESULTS: In the SPVC, the expression of c-Fos peaked on day 1 along with the expression of OX42 (related to microglial activation), CD16 (a pro-inflammatory factor), and CD206 (an anti-inflammatory factor) on day 3 after tooth movement, followed by a gradual decrease. GFAP-staining showed that the number of activated astrocytes was the highest on day 5 and that cell morphology became complex. After Fasudil treatment, the expression of these proteins showed a downward trend. The mRNA levels of pro-inflammatory factors (IL-1β and TNF-α) peaked on day 3, and the mRNA expression of the anti-inflammatory factor TGF-β was the lowest 3 days after tooth movement. Fasudil inhibited the mRNA expression of pain-related genes encoding CSF-1, t-PA, CTSS, and BDNF.
CONCLUSIONS: This study shows that tooth movement can cause the activation of glial cells in SPVC, and ROCK inhibitor Fasudil can inhibit the activation of glial cells and reduce the expression of the related inflammatory factors. This study presents for the first time the potential application of Fasudil in othodontic pain.

Cerebral cavernous malformation (CCM) is a hemorrhagic cerebrovascular disease where lesions develop in the setting of endothelial mutations of CCM genes, with many cases also harboring somatic PIK3CA gain of function (GOF) mutations. Rapamycin, an mTORC1 inhibitor, inhibited progression of murine CCM lesions driven by Ccm gene loss and Pik3ca GOF, but it remains unknown if rapamycin is beneficial in the absence of induction of Pik3ca GOF. We investigated the effect of rapamycin at three clinically relevant doses on lesion development in the Ccm3-/-PDGFb-icreERPositive murine model of familial CCM disease, without induction of Pik3ca GOF. Lesion burden, attrition, and acute and chronic hemorrhaging were compared between placebo and rapamycin-treated mice. Plasma miRNome was compared to identify potential biomarkers of rapamycin response. Outlier, exceptionally large CCM lesions (> 2 SD above the mean lesion burden) were exclusively observed in the placebo group. Rapamycin, across all dosages, may have prevented the emergence of large outlier lesions. Yet rapamycin also appeared to exacerbate mean lesion burden of surviving mice when outliers were excluded, increased attrition, and did not alter hemorrhage. miR-30c-2-3p, decreased in rapamycin-treated mouse plasma, has gene targets in PI3K/AKT and mTOR signaling. Progression of outlier lesions in a familial CCM model may have been halted by rapamycin treatment, at the potential expense of increased mean lesion burden and increased attrition. If confirmed, this can have implications for potential rapamycin treatment of familial CCM disease, where lesion development may not be driven by PIK3CA GOF. Further studies are necessary to determine specific pathways that mediate potential beneficial and detrimental effects of rapamycin treatment, and whether somatic PIK3CA mutations drive particularly aggressive lesions.

Cisplatin is an effective chemotherapy agent against various solid malignancies; however, it is associated with irreversible bilateral sensorineural hearing loss, emphasizing the need for drug development to prevent this complication, with the current options being very limited. Rho-associated coiled-coil-containing protein kinase (ROCK) is a serine-threonine protein kinase involved in various cellular processes, including apoptosis regulation. In this study, we used a transgenic zebrafish model (Brn3C: EGFP) in which hair cells within neuromasts are observed in green under fluorescent microscopy without the need for staining. Zebrafish larvae were exposed to cisplatin alone or in combination with various concentrations of Y-27632, a potent ROCK inhibitor. Hair cell counts, apoptosis assessments using the terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling assay, FM1-43FX labeling assay and behavioral analyses (startle response and rheotaxis) were performed to evaluate the protective effects of Y-27632 against cisplatin-induced ototoxicity. Cisplatin treatment reduced the number of hair cells in neuromasts, induced apoptosis, and impaired zebrafish larval behaviors. Y-27632 demonstrated a dose-dependent protective effect against cisplatin-induced hair cell loss and apoptosis. These findings suggest that Y-27632, as a ROCK inhibitor, mitigates cisplatin-induced hair cell loss and associated ototoxicity in zebrafish.

Proper hydration and the clarity of the cornea are maintained through the crucial function of the corneal endothelium. Inflammation of the corneal endothelium, known as endotheliitis, can disrupt endothelial function, resulting in alterations to vision. Corneal endotheliitis is characterised by corneal oedema, the presence of keratic precipitates, inflammation within the anterior chamber, and occasionally, limbal injection, neovascularisation, and the concurrent or overlapping presence of uveitis. The aetiology of this condition is diverse, predominantly viral, but it may also be drug-induced, result from bacterial or fungal infections, be associated with systemic diseases and procedures, or remain idiopathic with no identifiable cause. To date, no standardised protocol for the treatment of this ocular disease exists, and in severe cases, corneal transplantation may be required. A 31-year-old male was transferred to our hospital for the management of corneal endothelial decompensation resulting from corneal endotheliitis. Hormonal therapy and antiviral medications proved ineffective, rendering the patient a candidate for corneal transplantation. As a final measure, treatment with the ROCK inhibitor netarsudil was initiated. The patient demonstrated significant improvement in symptoms, and the inflammation was successfully managed after nine months. In this study, a novel approach employing ROCK inhibitor therapy was utilised for the treatment of corneal endotheliitis, leading to marked recovery during patient follow-up. This case report represents the inaugural application of the ROCK inhibitor netarsudil in managing corneal endothelial decompensation attributed to corneal endotheliitis. These findings suggest that this method warrants consideration as a potential novel treatment option for similar conditions.

Successful engineering of functional salivary glands necessitates the creation of cell-instructive environments for ex vivo expansion and lineage specification of primary human salivary gland stem cells (hS/PCs). Herein, basement membrane mimetic hydrogels were prepared using hyaluronic acid, cell adhesive peptides, and hyperbranched polyglycerol (HPG), with or without sulfate groups, to produce \"hyperGel+\" or \"hyperGel\", respectively. Differential scanning fluorescence experiments confirmed the ability of the sulphated HPG precursor to stabilize fibroblast growth factor 10. The hydrogels were nanoporous, cytocompatibile and cell-permissive, enabling the development of multicellular hS/PC spheroids in 14 days. Incorporation of sulfated HPG species in the hydrogel enhanced cell proliferation. Culture of hS/PCs in hyperGel+ in the presence of a Rho kinase inhibitor, Y-27632 (Y-27), led to the development of spheroids with a central lumen, increased the expression of acinar marker aquaporin-3 at the transcript level (AQP3), and decreased the expression of ductal marker keratin 7 at both the transcript (KRT7) and the protein levels (K7). Reduced expression of transforming growth factor beta (TGF-β) targets SMAD2/3 was also observed in Y27-treated cultures, suggesting attenuation of TGF-β signaling. Thus, hyperGel+ cooperates with the ROCK inhibitor to promote the development of lumened spheroids with enhanced expression of acinar markers.

To better understand molecular aspects of equine endometrial function, there is a need for advanced in vitro culture systems that more closely imitate the intricate 3-dimensional (3D) in vivo endometrial structure than current techniques. However, development of a 3D in vitro model of this complex tissue is challenging. This study aimed to develop an in vitro 3D endometrial tissue (3D-ET) with an epithelial cell phenotype optimized by treatment with a Rho-associated protein kinase (ROCK) inhibitor. Equine endometrial epithelial (eECs) and mesenchymal stromal (eMSCs) cells were isolated separately, and eECs cultured in various concentrations of Rock inhibitor (0, 5, 10 µmol) in epithelial medium (EC-medium) containing 10% knock-out serum replacement (KSR). The optimal concentration of Rock inhibitor for enhancing eEC proliferation and viability was 10 µM. However, 10 µM Rock inhibitor in the 10% KSR EC-medium was able to maintain mucin1 (Muc1) gene expression for only a short period. In contrast, fetal bovine serum (FBS) was able to maintain Muc1 gene expression for longer culture durations. An in vitro 3D-ET was successfully constructed using a collagen-based scaffold to support the eECs and eMSCs. The 3D-ET closely mimicked in vivo endometrium by displaying gland-like eEC-derived structures positive for the endometrial gland marker, Fork headbox A2 (FOXA2), and by mimicking the 3D morphology of the stromal compartment. In addition, the 3D-ET expressed the secretory protein MUC1 on its glandular epithelial surface and responded to LPS challenge by upregulating the expression of the interleukin-6 (IL6) and prostaglandin F synthase (PGFS) genes (P < 0.01), along with an increase in their secretory products, IL-6 (P < 0.01) and prostaglandin F2alpha (PGF2α) (P < 0.001) respectively. In the future, this culture system can be used to study both normal physiology and pathological processes of the equine endometrium.

The epiretinal membrane is a fibrocontractile tissue that forms on the inner surface of the retina, causing visual impairment ranging from mild to severe, and even retinal detachment. Müller glial cells actively participate in the formation of this membrane. Current research is constantly seeking for new therapeutic approaches that aim to prevent or treat cellular dysfunctions involved in the progression of this common fibrosis condition. The Rho GTPases signaling pathway regulates several processes associated with the epiretinal membrane, such as cell proliferation, migration, and contraction. Rho kinase (ROCK), an effector of the RhoA GTPase, is an interesting potential therapeutic target. This study aimed to evaluate the effects of a ROCK inhibitor (Y27632) on human Müller cells viability, growth, cytoskeletal organization, expression of extracellular matrix components, myofibroblast differentiation, migration, and contractility. Müller cells of the MIO-M1 lineage were cultured and treated for different periods with the inhibitor. Viability was evaluated by MTT assay and trypan blue exclusion method, and growth was evaluated by growth curve and BrdU incorporation assay. The actin cytoskeleton was stained with fluorescent phalloidin, intermediate filaments and microtubules were analyzed with immunofluorescence for vimentin and α-tubulin. Gene and protein expression of collagens I and V, laminin and fibronectin were evaluated by rt-PCR and immunofluorescence. Chemotactic and spontaneous cell migration were studied by transwell assay and time-lapse observation of live cells, respectively. Cell contractility was assessed by collagen gel contraction assay. The results showed that ROCK inhibition by Y27632 did not affect cell viability, but decreased cell growth and proliferation after 72 h. There was a change in cell morphology and organization of F-actin, with a reduction in the cell body, disappearance of stress fibers and formation of long, branched cell extensions. Microtubules and vimentin filaments were also affected, possibly because of F-actin alterations. The inhibitor also reduced gene expression and immunoreactivity of smooth muscle α-actin, a marker of myofibroblasts. The expression of extracellular matrix components was not affected by the inhibitor. Chemotactic cell migration showed no significant changes, while cell contractility was substantially reduced. No spontaneous migration of MIO-M1 cells was observed. In conclusion, pharmacological inhibition of ROCK in Müller cells could be a potentially promising approach to treat epiretinal membranes by preventing cell proliferation, contractility and transdifferentiation, without affecting cell viability.

The impairment in microvascular network formation could delay the restoration of blood flow after acute limb ischemia. A high-content screen of a GSK-published kinase inhibitor library identified a set of ROCK inhibitor hits enhancing endothelial network formation. Subsequent kinase activity profiling against a panel of 224 protein kinases showed that two indazole-based ROCK inhibitor hits exhibited high selectivity for ROCK1 and ROCK2 isoforms compared to other ROCK inhibitors. One of the chemical entities, GSK429286, was selected for follow-up studies. We found that GSK429286 was ten times more potent in enhancing endothelial tube formation than Fasudil, a classic ROCK inhibitor. ROCK1 inhibition by RNAi phenocopied the angiogenic phenotype of the GSK429286 compound. Using an organotypic angiogenesis co-culture assay, we showed that GSK429286 formed a dense vascular network with thicker endothelial tubes. Next, mice received either vehicle or GSK429286 (10 mg/kg i.p.) for seven days after hindlimb ischemia induction. As assessed by laser speckle contrast imaging, GSK429286 potentiated blood flow recovery after ischemia induction. At the histological level, we found that GSK429286 significantly increased the size of new microvessels in the regenerating areas of ischemic muscles compared with vehicle-treated ones. Our findings reveal that selective ROCK inhibitors have in vitro pro-angiogenic properties and therapeutic potential to restore blood flow in limb ischemia.