Application of Single Cell Type-Derived Spheroids Generated by Using a Hanging Drop Culture Technique in Various In Vitro Disease Models: A Narrow Review.

Cell culture methods are indispensable strategies for studies in biological sciences and for drug discovery and testing. Most cell cultures have been developed using two-dimensional (2D) culture methods, but three-dimensional (3D) culture techniques enable the establishment of in vitro models that replicate various pathogenic conditions and they provide valuable insights into the pathophysiology of various diseases as well as more precise results in tests for drug efficacy. However, one difficulty in the use of 3D cultures is selection of the appropriate 3D cell culture technique for the study purpose among the various techniques ranging from the simplest single cell type-derived spheroid culture to the more sophisticated organoid cultures. In the simplest single cell type-derived spheroid cultures, there are also various scaffold-assisted methods such as hydrogel-assisted cultures, biofilm-assisted cultures, particle-assisted cultures, and magnet particle-assisted cultures, as well as non-assisted methods, such as static suspension cultures, floating cultures, and hanging drop cultures. Since each method can be differently influenced by various factors such as gravity force, buoyant force, centrifugal force, and magnetic force, in addition to non-physiological scaffolds, each method has its own advantages and disadvantages, and the methods have different suitable applications. We have been focusing on the use of a hanging drop culture method for modeling various non-cancerous and cancerous diseases because this technique is affected only by gravity force and buoyant force and is thus the simplest method among the various single cell type-derived spheroid culture methods. We have found that the biological natures of spheroids generated even by the simplest method of hanging drop cultures are completely different from those of 2D cultured cells. In this review, we focus on the biological aspects of single cell type-derived spheroid culture and its applications in in vitro models for various diseases.

Modulation of Epithelial-Mesenchymal Transition Is a Possible Underlying Mechanism for Inducing Chemoresistance in MIA PaCa-2 Cells against Gemcitabine and Paclitaxel.

To elucidate the currently unknown molecular mechanisms responsible for the similarity and difference during the acquirement of resistance against gemcitabine (GEM) and paclitaxel (PTX) in patients with pancreatic carcinoma, we examined two-dimensional (2D) and three-dimensional (3D) cultures of parent MIA PaCa-2 cells (MIA PaCa-2-PA) and their GEM resistance cell line (MIA PaCa-2-GR) and PTX resistance (MIA PaCa-2-PR). Using these cells, we examined 3D spheroid configurations and cellular metabolism, including mitochondrial and glycolytic functions, with a Seahorse bio-analyzer and RNA sequencing analysis. Compared to the MIA PaCa-2-PA, (1) the formation of the 3D spheroids of MIA PaCa-2-GR or -PR was much slower, and (2) their mitochondrial and glycolytic functions were greatly modulated in MIA PaCa-2-GR or -PR, and such metabolic changes were also different between their 2D and 3D culture conditions. RNA sequencing and bioinformatic analyses of the differentially expressed genes (DEGs) using an ingenuity pathway analysis (IPA) suggested that various modulatory factors related to epithelial -mesenchymal transition (EMT) including STAT3, GLI1, ZNF367, NKX3-2, ZIC2, IFIT2, HEY1 and FBLX, may be the possible upstream regulators and/or causal network master regulators responsible for the acquirement of drug resistance in MIA PaCa-2-GR and -PR. In addition, among the prominently altered DEGs (Log2 fold changes more than 6 or less than -6), FABP5, IQSEC3, and GASK1B were identified as unique genes associated with their antisense RNA or pseudogenes, and among these, FABP5 and GASK1B are known to function as modulators of cancerous EMT. Therefore, the observations reported herein suggest that modulations of cancerous EMT may be key molecular mechanisms that are responsible for inducing chemoresistance against GEM or PTX in MIA PaCa-2 cells.

Three-Dimensional Spheroid Configurations and Cellular Metabolic Properties of Oral Squamous Carcinomas Are Possible Pharmacological and Pathological Indicators.

The objective of the current study was to elucidate the clinicopathological significance and appearance of in vitro three-dimension (3D) spheroid models of oral malignant tumors that were prepared from four pathologically different squamous cell carcinoma (OSCC; low-grade; SSYP and MO-1000, intermediate-grade; LEM2) and oral adenosquamous carcinoma (OASC; high-grade; Mesimo) obtained from patients with different malignant stages. To characterize the biological significance of these cell lines themselves, two-dimensional (2D) cultured cells were subjected to cellular metabolic analysis by a Seahorse bioanalyzer alongside the measurement of the cytotoxicity of cisplatin (CDDP). The appearance of their 3D spheroids was then observed by phase contrast microscopy, and both 2D and 3D cultured cells were subject to trypsin digestion and qPCR analysis of factors related to oncogenic signaling and other related analyses. ATP-linked respiration and proton leaking were significantly different among the four cell lines, and the malignant stages of these cultures were significantly associated with increased ATP-linked respiration and decreased proton leakage. Alternatively, the appearances of these 3D spheroids were also significantly diverse among them, and their differences increased in the order of LEM2, MO-1000, SSYP, and Mesimo. Interestingly, these orders were exactly the same in that the efficacies of CDDP-induced cytotoxicity increased in the same order. qPCR analysis indicated that the levels of expression of oncogenic signaling-related factors varied among these four cell lines, and the values for fibronectin and a key regulator of mitochondrial biogenesis, PGC-1α, were prominently elevated in cultures of the worst malignant Mesimo cells. In addition, although 0.25% trypsin-induced destruction was comparable among all four 2D cultured cells, the values for the 3D spheroids were also substantially varied among these cultures. The findings reported herein indicate that cellular metabolic functions and 3D spheroid architectures may be valuable and useful indicators for estimating the pathological and drug-sensitive aspects of OSCC and OASC malignancies.

3D Spheroid Configurations Are Possible Indictors for Evaluating the Pathophysiology of Melanoma Cell Lines.

To study the molecular mechanisms responsible for inducing the spatial proliferation of malignant melanomas (MM), three-dimension (3D) spheroids were produced from several MM cell lines including SK-mel-24, MM418, A375, WM266-4, and SM2-1, and their 3D architectures and cellular metabolisms were evaluated by phase-contrast microscopy and Seahorse bio-analyzer, respectively. Several transformed horizontal configurations were observed within most of these 3D spheroids, and the degree of their deformity was increased in the order: WM266-4, SM2-1, A375, MM418, and SK-mel-24. An increased maximal respiration and a decreased glycolytic capacity were observed within the lesser deformed two MM cell lines, WM266-4 and SM2-1, as compared with the most deformed ones. Among these MM cell lines, two distinct cell lines, WM266-4 and SK-mel-24, whose 3D appearances were the closest and farthest, respectively, from being horizontally circular-shaped, were subjected to RNA sequence analyses. Bioinformatic analyses of the differentially expressed genes (DEGs) identified KRAS and SOX2 as potential master regulatory genes for inducing these diverse 3D configurations between WM266-4 and SK-mel-24. The knockdown of both factors altered the morphological and functional characteristics of the SK-mel-24 cells, and in fact, their horizontal deformity was significantly reduced. A qPCR analysis indicated that the levels of several oncogenic signaling related factors, including KRAS and SOX2, PCG1α, extracellular matrixes (ECMs), and ZO1 had fluctuated among the five MM cell lines. In addition, and quite interestingly, the dabrafenib and trametinib resistant A375 (A375DT) cells formed globe shaped 3D spheroids and showed different profiles in cellular metabolism while the mRNA expression of these molecules that were tested as above were different compared with A375 cells. These current findings suggest that 3D spheroid configuration has the potential for serving as an indicator of the pathophysiological activities associated with MM.

G-Protein-Coupled Receptors Mediate Modulations of Cell Viability and Drug Sensitivity by Aberrantly Expressed Recoverin 3 within A549 Cells.

To elucidate the currently unknown molecular mechanisms responsible for the aberrant expression of recoverin (Rec) within cancerous cells, we examined two-dimensional (2D) and three-dimensional (3D) cultures of Rec-negative lung adenocarcinoma A549 cells which had been transfected with a plasmid containing human recoverin cDNA (A549 Rec) or an empty plasmid as a mock control (A549 MOCK). Using these cells, we measured cytotoxicity by several anti-tumor agents (2D), cellular metabolism including mitochondrial and glycolytic functions by a Seahorse bio-analyzer (2D), the physical properties, size and stiffness of the 3D spheroids, trypsin sensitivities (2D and 3D), and RNA sequencing analysis (2D). Compared with the A549 MOCK, the A549 Rec cells showed (1) more sensitivity toward anti-tumor agents (2D) and a 0.25% solution of trypsin (3D); (2) a metabolic shift from glycolysis to oxidative phosphorylation; and (3) the formation of larger and stiffer 3D spheroids. RNA sequencing analysis and bioinformatic analyses of the differentially expressed genes (DEGs) using Gene Ontology (GO) enrichment analysis suggested that aberrantly expressed Rec is most likely associated with several canonical pathways including G-protein-coupled receptor (GPCR)-mediated signaling and signaling by the cAMP response element binding protein (CREB). The findings reported here indicate that the aberrantly expressed Rec-induced modulation of the cell viability and drug sensitivity may be GPCR mediated.

All-trans Retinoic Acids Synergistically and Beneficially Affect In Vitro Glaucomatous Trabecular Meshwork (TM) Models Using 2D and 3D Cell Cultures of Human TM Cells.

We report herein on the effects of all-trans retinoic acid (ATRA) on two-dimensional (2D) and three-dimensional (3D) cultures of human trabecular meshwork (HTM) cells that were treated with transforming growth factor ß2 (TGF-ß2). In the presence of 5 ng/mL TGF-ß2, the effects of ATRA on the following were observed: (1) the barrier function of the 2D HTM monolayers, as determined by trans-endothelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC) dextran permeability measurements; (2) a Seahorse cellular bio-metabolism analysis; (3) physical properties, including the size and stiffness, of 3D spheroids; (4) the gene expression of extracellular matrix (ECM) molecules, ECM modulators including tissue inhibitor of metalloproteinases (TIMPs), matrix metalloproteinases (MMPs), tight junction (TJ)-related molecules, and endoplasmic reticulum (ER)-stress-related factors. ATRA significantly inhibited the TGF-ß2-induced increase in the TEER values and FITC dextran permeability of the 2D monolayers, while an ATRA monotreatment induced similar effects as TGF-ß2. A real-time metabolic analysis revealed that ATRA significantly inhibited the TGF-ß2-induced shift in metabolic reserve from mitochondrial oxidative phosphorylation to glycolysis in 2D HTM cells, whereas ATRA alone did not induce significant metabolic changes. In contrast, ATRA induced the formation of substantially downsized and softer 3D spheroids in the absence and presence of TGF-ß2. The different effects induced by ATRA toward 2D and 3D HTM cells were also supported by the qPCR analysis of several proteins as above. The findings reported here indicate that ATRA may induce synergistic and beneficial effects on TGF-ß2-treated 2D- and 3D-cultured HTM cells; those effects varied significantly between the 2D and 3D cultures.

FGF-2 enhances fibrogenetic changes in TGF-ß2 treated human conjunctival fibroblasts.

The objective of the current study was to examine the effects of fibroblast growth factor-2 (FGF-2) on conjunctival fibrogenesis that was induced by the presence of transforming growth factor-ß2 (TGF-ß2). Two-dimension (2D) and three-dimension (3D) cultured human conjunctival fibroblasts (HconF) were used for this purpose. The 2D and 3D cultured HconF were characterized by transendothelial electrical resistance (TEER) and FITC dextran permeability measurements (2D), real-time metabolic analyses (2D), size and stiffness measurements (3D), and the mRNA expression of extracellular matrix molecules, their modulators, Tissue inhibitor of metalloproteinases and matrix metalloproteinases and ER-stress related genes (2D and 3D). FGF-2 significantly increased planar proliferation, as evidenced by TEER values and FITC dextran permeability, and shifted glucose metabolism to the energetic phenotype of 2D HconF cells, and the stiffness of the 3D spheroids, and these effects were further enhanced in the presence of TGF-ß2. Analyses of the expression of possible candidate molecules involved in cell architecture and stress indicated that some additive effects caused by both factors were also recognized in some of these molecules. The findings reported herein indicate that the FGF-2, either along or additively with TGF- ß2 increased the fibrogenetic changes on the plane as well as in the spatial space of HconF cells.

Fatty acid metabolism is involved in both retinal physiology and the pathology of retinal vascular diseases.

To study the pathophysiological roles of the fatty acid-binding proteins (FABPs) within the retina, we performed; (1) immunolabeling of human retinas, wild type (WT) rat and mouse retinas, rat models for diabetic retinopathy (DR) and retinitis pigmentosa (RP) with anti-FABP3, FABP4, FABP5, FABP7, FABP8 and FABP12, (2) electroretinogram (ERG) measurements of WT and FABP4-deficient (Fabp4-/-) mice, (3) ELISA or gas chromatography measurements of plasma (P-) and vitreous (V-) levels of FABP4 and vascular endothelial growth factor A (VEGFA), and fatty acids (FAs) from patients with retinal vascular disease (RVD) including proliferative DR (PDR, n = 30) and retinal vein occlusion (RVO, n = 18) and non-RVD (n = 18). Within the human retina, diverse expressions of FABP3, FABP4, FABP7 and FABP8 were identified. In contrast, positive immunoreactivities toward only FABP4 and FABP12 were detected in the cases of rat and mouse retinas, and interestingly, the FABP4 labeling patterns for the WT, DR and RP rat retinas were different. The ERG amplitudes of Fabp4-/- mice were enhanced compared with those of WT mice. The concentrations of V-FABP4, V-VEGFA and total FAs were significantly higher in RVD patients than in non-PDR patients (P < 0.05). The V-FAs levels of each were significantly and positively correlated with V-FABP4 and V-VEGFA, although no significant correlation between vitreous (V-) and plasma (P-) FABP4, VEGFA and FAs were detected. The current study reveals that V-FAs appear to have significant roles in both retinal physiology as well as the pathogenesis of RVD with FABP4, which is commonly expressed within the retina in most species.

Hypoxia Differently Affects TGF-ß2-Induced Epithelial Mesenchymal Transitions in the 2D and 3D Culture of the Human Retinal Pigment Epithelium Cells.

The hypoxia associated with the transforming growth factor-ß2 (TGF-ß2)-induced epithelial mesenchymal transition (EMT) of human retinal pigment epithelium (HRPE) cells is well recognized as the essential underlying mechanism responsible for the development of proliferative retinal diseases. In vitro, three-dimensional (3D) models associated with spontaneous O2 gradients can be used to recapitulate the pathological levels of hypoxia to study the effect of hypoxia on the TGF-ß2-induced EMT of HRPE cells in detail, we used two-dimensional-(2D) and 3D-cultured HRPE cells. TGF-ß2 and hypoxia significantly and synergistically increased the barrier function of the 2D HRPE monolayers, as evidenced by TEER measurements, the downsizing and stiffening of the 3D HRPE spheroids and the mRNA expression of most of the ECM proteins. A real-time metabolic analysis indicated that TGF-ß2 caused a decrease in the maximal capacity of mitochondrial oxidative phosphorylation in the 2D HRPE cells, whereas, in the case of 3D HRPE spheroids, TGF-ß2 increased proton leakage. The findings reported herein indicate that the TGF-ß2-induced EMT of both the 2D and 3D cultured HRPE cells were greatly modified by hypoxia, but during these EMT processes, the metabolic plasticity was different between 2D and 3D HRPE cells, suggesting that the mechanisms responsible for the EMT of the HRPE cells may be variable during their spatial spreading.

Establishment of appropriate glaucoma models using dexamethasone or TGFß2 treated three-dimension (3D) cultured human trabecular meshwork (HTM) cells.

To establish appropriate ex vivo models for a glaucomatous trabecular meshwork (TM), two-dimensional (2D) and three-dimensional (3D) cultures of human trabecular meshwork cells (HTM) were prepared in the presence of 250 nM dexamethasone (DEX) or 5 ng/mL TGFß2, and characterized by the following analyses; transendothelial electrical resistance (TEER) measurements, FITC dextran permeability, scanning electron microscopy and the expression of the extracellular matrix (ECM) including collagen (COL)1, 4 and 6, and fibronectin (FN), α-smooth muscle actin (α-SMA), tissue inhibitor of metalloproteinase (TIMP)1-4, and matrix metalloproteinase (MMP)2, 9 and 14. DEX and TGFß2 both caused a significant increase or decrease in the TEER values and FITC dextran permeability. During the 3D spheroid culture, DEX or TGFß2 induced a mild and significant down-sizing and an increase in stiffness, respectively. TGFß2 induced a significant up-regulation of COL1 and 4, FN, α-SMA, and MMP 2 and 14 (2D) or COL1 and 6, and TIMP2 and 3 (3D), and DEX induced a significant up-regulation of FN (3D) and TIMP4 (2D and 3D). The findings presented herein indicate that DEX or TGFß2 resulted in mild and severe down-sized and stiff 3D HTM spheroids, respectively, thus making them viable in vitro HTM models for steroid-induced and primary open angle glaucoma.

Diverse effects of pan-ROCK and ROCK2 inhibitors on 2 D and 3D cultured human trabecular meshwork (HTM) cells treated with TGFß2.

A pan-ROCK-inhibitor, ripasudil (Rip), and a ROCK2 inhibitor, KD025, were used To study the effects of Rho-associated coiled-coil containing protein kinase (ROCK)1 and 2 on two-dimensional (2D) and three-dimensional (3D) cultures of a TGFß2-treated human trabecular meshwork (HTM) cells. In the presence of 5 ng/mL TGFß2, the effects of these inhibitors were characterized by transendothelial electrical resistance (TEER), FITC-dextran permeability, and the size and stiffness of 3D sphenoids, the expression of extracellular matrix (ECM) including collagen1, 4 and 6, and fibronectin, α-smooth muscle actin, a tissue inhibitor of metalloproteinase (TIMP)1-4, and matrix metalloproteinase (MMP)2, 9 and 14. TGFß2 caused a significant increase in the TEER values, and decrease in FITC-dextran permeability, as well as a decrease in the sizes and stiffness of the 3D sphenoids. In the presence of ROCK inhibitors, the TGFß2-induced effects of the TEER and FITC-dextran permeability were inhibited, especially by KD025. Rip induced a significant increase in sizes and a decrease in the stiffness of the TGFß2-treated 3D sphenoids, although the effects of KD025 were weaker. Gene expressions of most of the ECMs, TIMP2 and MMP9 of 2D and 3D HTM cells were significantly up-regulated by TGFß2. Those were significantly and differently modulated by Rip or KD025.

Rosiglitasone and ROCK Inhibitors Modulate Fibrogenetic Changes in TGF-ß2 Treated Human Conjunctival Fibroblasts (HconF) in Different Manners.

PURPOSE: The effects of Rho-associated coiled-coil containing protein kinase (ROCK) 1 and 2 inhibitor, ripasudil hydrochloride hydrate (Rip), ROCK2 inhibitor, KD025 or rosiglitazone (Rosi) on two-dimension (2D) and three-dimension (3D) cultured human conjunctival fibroblasts (HconF) treated by transforming growth factor (TGFß2) were studied. METHODS: Two-dimension and three-dimension cultured HconF were examined by transendothelial electrical resistance (TEER, 2D), size and stiffness (3D), and the expression of the extracellular matrix (ECM) including collagen1 (COL1), COL4 and COL6, fibronectin (FN), and α-smooth muscle actin (αSMA) by quantitative PCR (2D, 3D) in the presence of Rip, KD025 or Rosi. RESULTS: TGFß2 caused a significant increase in (1) the TEER values (2D) which were greatly reduced by Rosi, (2) the stiffness of the 3D organoids which were substantially reduced by Rip or KD025, and (3) TGFß2 induced a significant up-regulation of all ECMs, except for COL6 (2D) or αSMA (3D), and down-regulation of COL6 (2D). Rosi caused a significant up-regulation of COL1, 4 and 6 (3D), and down-regulation of COL6 (2D) and αSMA (3D). Most of these TGFß2-induced expressions in the 2D and αSMA in the 3D were substantially inhibited by KD025, but COL4 and αSMA in 2D were further enhanced by Rip. CONCLUSION: The findings reported herein indicate that TGFß2 induces an increase in fibrogenetic changes on the plane and in the spatial space, and are inhibited by Rosi and ROCK inhibitors, respectively.

Detection of significantly high vitreous concentrations of fatty acid-binding protein 4 in patients with proliferative diabetic retinopathy.

The fatty acid-binding protein4 (FABP4) and vascular endothelial growth factor A (VEGFA) play key roles in the metabolic and cardiovascular diseases, and proliferative diabetic retinopathy (PDR), respectively. To identify FABP4 in vitreous fluid in PDR, vitreous concentrations of FABP4 (V-FABP4) and VEGFA (V-VEGFA) from PDR (n = 20) and non-PDR (n = 20) patients were determined by Enzyme-Linked ImmunoSorbent Assays. The data, which included height and weight, systemic blood pressures, several blood biochemical parameters and blood flow at the optic nerve head (ONH) by laser speckle flowgraphy (LSFG) were collected. The levels of V-FABP4 and V-VEGFA were significantly higher in PDR patients than in non-PDR patients (P < 0.001) with a high positive correlation (r = 0.72, P < 0.001) between them. The findings were not affected by body mass index values and the presence of vitreous hemorrhaging. Among the clinical parameters, V-FABP4 correlated positively with creatinine and negatively with age and aspartate transaminase (AST) levels, while V-VEGFA correlated positively with fasting plasma glucose and hemoglobin A1c (HbA1c) levels but negatively with AST. Multiple regression analyses indicated that V-VEGFA, or V-FABP4, AST and HbA1c were independent predictors of V-FABP4 or V-VEGFA, respectively. Both were negatively correlated, but more evident in V-FABP4, with the ONH ocular blood flow.

NF-κB activation in retinal microglia is involved in the inflammatory and neovascularization signaling in laser-induced choroidal neovascularization in mice.

PURPOSE: To evaluate Nuclear Factor NF-κB (NF-κB) signaling on microglia activation, migration, and angiogenesis in laser-induced choroidal neovascularization (CNV). METHODS: Nine-week-old C57BL/6 male mice were randomly assigned to IMD-0354 treated or untreated groups (5 mice, 10 eyes per group). CNV was induced with a 532-nm laser. Laser spots (power 250 mW, spot size 100 µm, time of exposure 50 ms) were created in each eye using a slit-lamp delivery system. Selective inhibitor of nuclear factor kappa-B kinase subunit beta (IKK2) inhibitor IMD-0354 (10 µg) was delivered subconjunctivally; vehicle-treated mice were the control. The treatment effect on CNV development was assessed at five days post-CNV induction in vivo in C57BL/6 and Cx3cr1gfp/wt mice by fluorescent angiography, fundus imaging, and ex vivo by retinal flatmounts immunostaining and Western blot analysis of RPE/Choroidal/Scleral complexes (RCSC) lysates. In vitro evaluations of IMD-0354 effects were performed in the BV-2 microglial cell line using lipopolysaccharide (LPS) stimulation. RESULTS: IMD-0354 caused a significant reduction in the fluorescein leakage and size of the laser spot, as well as a reduction in microglial cell migration and suppression of phospho-IκBα, Vascular endothelial growth factor (VEGF-A), and Prostaglandin-endoperoxide synthase 2 (COX-2). In vivo and ex vivo observations demonstrated reduced lesion size in mice, CD68, and Allograft inflammatory factor 1 (IBA-1) positive microglia cells migration to the laser injury site in IMD-0354 treated eyes. The data further corroborate with GFP-positive cells infiltration of the CNV site in Cx3cr1wt/gfp mice. In vitro IMD-0354 (10-25 ng/ml) treatment reduced NF-κB activation, expression of COX-2, caused decreased Actin-F presence and organization, resulting in reduced BV-2 cells migration capacity. CONCLUSION: The present data indicate that NF-κB activation in microglia and it's migration capacity is involved in the development of laser CNV in mice. Its suppression by NF-κB inhibition might be a promising therapeutic strategy for wet AMD.

Fatty acid-binding protein 4 is an independent factor in the pathogenesis of retinal vein occlusion.

The main objective of current study was to identify the fatty acid-binding protein 4 (FABP4) expressed in both adipocytes and macrophages in vitreous fluid from patients with retinal vein occlusion (RVO). Patients with RVO (n = 14, CRVO; central RVO n = 5, BRVO; branch RVO n = 9) and non-RVO (macular hole or epiretinal membrane, n = 18) were surgically treated by a 25 or 27G vitrectomy. Undiluted vitreous fluid samples obtained as the result of surgery were subjected to enzyme-linked immunosorbent assays to measure the levels of FABP4 and vascular endothelial growth factor A (VEGFA).Data including ocular blood flow by laser speckle flow graphy (LSFG), height and weight, systemic blood pressures and several blood biochemistry values were collected. Among the LSFG mean blur rate (MBR) values of the optic nerve head (ONH) at baseline, MA (MBR of all area), MV (MBR of the vascular area), and MV-MT (MBR of the tissue area) were significantly decreased in patients with CRVO. The levels of V-FABP4 and V-VEGFA were relatively or significantly (P< 0.05) higher in the BRVO or CRVO patients compared to the non-RVO patients, respectively. A positive correlation (r = 0.36, P = 0.045) or a negative correlation (r = -0.51, P = 0.006) was observed between Log V-FABP4 and Log V-VEGF, or Log V-FABP4 and MV-MT at post-operative 1-week, respectively. Furthermore, neither of these factors were affected with respect to sex, body mass index and several clinical parameters that were collected, except that a positive correlation was observed for Log V-FABP4 with blood urea nitrogen. Stepwise multivariable regression analyses indicated that MV-MT at post-operative 1week was independently associated with Log V-FABP4 after adjustment for age and gender, and gender and Log V-FABP4 were independently associated with Log V-VEGFA after adjustment for age. The findings reported herein suggest that an independent factor, FABP4 may be synergistically involved in the pathogenesis of RVO with VEGFA.

Unique and progressive retinal degeneration in a patient with cancer associated retinopathy.

PURPOSE: To report clinical course of a patient with cancer-associated retinopathy (CAR) medicated by steroid therapy, focusing on retinal degeneration progression. OBSERVATIONS: A 67 years-old female patient, who had a surgical history of endometrial carcinoma with adjuvant chemotherapy, was referred to our hospitals for the complaints of sudden reduced visual acuity and visual field constriction in the right eye. Best corrected visual acuity (BCVA) was 0.4 and 1.0 in right and left eyes, respectively. Funduscopy showed almost normal appearance in both eyes. Fluorescein angiography showed slight fluorescein leakage from the optic disc in both eyes and an inferior arcade vessel in the right eye. Optical coherence tomography (OCT) images showed loss of ellipsoid zone (EZ) and thinning of outer retinal layers at the nasal area of the fovea in both eyes. Goldmann perimetry (GP) demonstrated several paracentral absolute scotomas with peripheral visual field constriction in the right eye, and a paracentral relative scotoma with preserved peripheral visual field in the left eye. Ten months after the first visit, retinopathy progressed in both eyes. Funduscopy indicated mild retinal degeneration along with arcade veins with white sheathing of retinal arteries. Slightly visible EZ at the fovea and loss of EZ and interdigitation zone and thinning of outer retinal layers at other areas were observed in OCT images from both eyes. GP showed no response in both eyes. Oral prednisolone therapy was started and gradually tapered over a 3-month period. Twelve and fifteen months after the first visit, BCVA, EZ at the fovea in OCT images, and visual field gradually improved, whereas retinal degeneration along arcade veins became apparent. CONCLUSIONS AND IMPORTANCE: We reported a patient with CAR who exhibited progressive retinal degeneration and good response to oral prednisolone therapy. This case expands the clinical spectrum of CAR.

ROCK inhibitors beneficially alter the spatial configuration of TGFß2-treated 3D organoids from a human trabecular meshwork (HTM).

To elucidate molecular pharmacology of Rho-associated coiled-coil containing protein kinase inhibitors (ROCK-i, Ripasudil and Y27632) on their efficiency for aqueous outflow, 2D or 3D cultures of a human trabecular meshwork (HTM) were prepared in the presence of TGFß2. Those were examined by transendothelial electrical resistance (TEER, 2D), electronic microscopy (EM, 2D and 3D), expression of the extracellular matrix (ECM) including collagen1 (COL1), COL4 and COL6, and fibronectin (FN) by immunolabeling and/or quantitative PCR (3D), and solidity of 3D organoids by a micro-squeezer. TGFß2 significantly increased the TEER values in 2D cultures, and the ECM expression indicated that the 3D organoids assumed a more densely packed shape. ROCK-i greatly reduced the TGFß2-induced enhancement of TEER and the immunolabeled ECM expression of the 3D organoids. In contrast, the mRNA expression of COL1 was increased, and those of COL4 and FN were unchanged. EM revealed that TGFß2 caused the HTM cells to become more compact and abundant ECM deposits within the 3D organoids were observed. These were significantly inhibited by ROCK-i. The dense solids caused by the presence of TGFß2 were significantly suppressed by ROCK-i. Current study indicates that ROCK-i cause beneficial effects toward the spatial configuration of TGFß2-induced HTM 3D organoids.