Inherent errors of the fixed-frame counting method for corneal endothelial cell density in eye banks

The aim of this work was to analyze the magnitude of inherent errors associated with the fixed-frame counting method for corneal endothelial cell density (ECD) measurements. This technique is common among most eye banks worldwide. Three types of mosaics were used: regular and irregular tessellated mosaics (eight increasing densities ranging from 800 to 3,600 cells/mm² by steps of 400 cells/mm²) generated by a computer, and real mosaics (four specimens) obtained from human corneal endothelium flat mounted and stained with Alizarin red. On the three mosaics, the fixed-frame counting method was applied using a computer program. The ECD was calculated for 3,000 successive random positions from calibrated grids which area ranged from 50 × 50 to 300 × 300 μm² (incremental steps of 25 μm). For each grid, the ECD was expressed either as a single count, a mean of five or a mean of 10 measures. The fixed-frame count was constantly associated with an inherent variability but repeatability increased with larger grid size and ECD. The mean calculated out of 10 measures was the most reliable, but still, we noted ±5 % of residual variability from the real ECD. The 100 × 100 μm² grid manual counts, performed in many eye banks, should be abandoned and upgraded to at least 200 × 200 μm² grid counts. Digital image analysis with a variable frame counting method would be the best alternative.     

Morphological Changes of Human Corneal Endothelial Cells after Rho-Associated Kinase Inhibitor Eye Drop (Ripasudil) Administration: A Prospective Open-Label Clinical Study

Purpose

To investigate the effect and safety of a selective Rho kinase inhibitor, ripasudil 0.4% eye drops, on corneal endothelial cells of healthy subjects.

Design

Prospective, interventional case series.

Methods

In this study, 6 healthy subjects were administered ripasudil 0.4% in the right eye twice daily for 1 week. Morphological changes and corneal endothelial cell density were examined by noncontact and contact specular microscopy. Central corneal thickness and corneal volume of 5 mm-diameter area of center cornea were analyzed by Pentacam Scheimpflug topography. All the above measurements were conducted in both eyes before administration, 1.5 and 6 hours after the initial administration on day 0; and in the same manner after the final administration on day 7.

Results

By noncontact specular microscopy, indistinct cell borders with pseudo guttae were observed, but by contact specular microscopy, morphological changes of corneal endothelial cells were mild and pseudo guttae was not observed after single and repeated administration of ripasudil in all subjects. These changes resolved prior to the next administration, and corneal endothelial cell density, central corneal thickness and corneal volume were not changed throughout the study period.

Conclusion

Transient morphological changes of corneal endothelial cells such as indistinct cell borders with pseudo guttae were observed by noncontact specular microscopy in healthy subjects after ripasudil administration. Corneal edema was not observed and corneal endothelial cell density did not decrease after 1 week repetitive administration. These morphological changes were reversible and corneal endothelial cell morphology returned to normal prior to the next administration.

Trial Registration

JAPIC Clinical Trials Information 142705     

CD39/NTPDase-1 expression and activity in human umbilical vein endothelial cells are differentially regulated by leaf extracts from Rubus caesius and Rubus idaeus

Many experimental studies have demonstrated the favorable biological activities of plants belonging to the genus Rubus, but little is known of the role of Rubus leaf extracts in the modulation of the surface membrane expression and activity of endothelial apyrase. The aim of this study was to assess the influence of 1–15 μg/ml Rubus extracts on CD39 expression and enzymatic activity, and on the activation (ICAM-1 expression) and viability of human umbilical vein endothelial cells (HUVEC). The polyphenolic contents and antioxidative capacities of extracts from dewberry (R. caesius L.) and raspberry (R. idaeus L.) leaves were also investigated. The techniques applied were flow cytometry (endothelial surface membrane expression of ICAM-1 and CD39), malachite green assay (CD39 activity), HPLC-DAD (quantitative analysis of polyphenolic extract), ABTS, DPPH and FRAP spectrometric assays (antioxidant capacity), and the MTT test (cell viability). Significantly increased CD39 expressions and significantly decreased ATPDase activities were found in the cells treated with 15 μg/ml of either extract compared to the results for the controls. Neither of the extracts affected cell proliferation, but both significantly augmented endothelial cell ICAM-1 expression. The overall antioxidant capacities of the examined extracts remained relatively high and corresponded well to the determined total polyphenol contents. Overall, the results indicate that under in vitro conditions dewberry and raspberry leaf extracts have unfavorable impact on endothelial cells.     

Coordinated regulation of autophagy and apoptosis determines endothelial cell fate during Dengue virus type 2 infection

Dengue is the most prevalent mosquito-borne viral disease in tropical regions. Severe cases may progress to Dengue hemorrhagic fever, suggesting vascular endothelial dysfunction in disease pathogenesis. In our previous study, we found that Dengue virus type 2 (DENV2) induced apoptosis of vascular endothelial cells via FasL/Fas- and XIAP-associated factor 1 (XAF1)-dependent pathways. In this paper, we demonstrate that DENV2 can induce autophagy in primary human umbilical vein endothelial cells (HUVECs) and the human umbilical vein endothelial cell line EA.hy926. Inhibition of autophagy with 3-methyl adenine promoted apoptosis, while inhibition of apoptosis with Z-VAD-FMK facilitated autophagy in DENV2-infected HUVECs and EA.hy926 cells. Interferon-alpha-inducible protein 6 (IFI6), a putative apoptosis regulator, inhibited DENV2-induced autophagy in EA.hy926 cells, while XAF1, an inhibitor of anti-apoptotic XIAP, facilitated autophagy. Molecular regulators of apoptosis and autophagy interact at multiple levels to determine cell fate. Our data suggest that XAF1 and IFI6 are involved in regulating the balance between autophagy and apoptosis in DENV2-infected endothelial cells.     

Biological characteristics of rat dorsal root ganglion cell and human vascular endothelial cell in mono- and co-culture

This study aimed to evaluate the biological activity of rat dorsal root ganglion cell (DRGC) and human vascular endothelial cell (HMVEC) in mono- and co-culture. Expression levels of vascular endothelial growth factor (VEGF) and nerve growth factor (NGF) mRNA were measured by quantitative real-time RT-PCR (qRT-PCR). Western blot analysis was used to identify VEGF and NGF protein expressions. Cell injury was assessed by measuring cell viability with methylthiazol tetrazolium (MTT) assay. The results showed that VEGF and NGF mRNA levels in the HMVEC+DRGC group were significantly higher than those in the DRGC and HMVEC groups (all p < 0.05). There were also greater increases in both VEGF and NGF protein expressions in the HMVEC+DRGC group than those in the DRGC and HMVEC groups (all p < 0.05). The results of MTT analysis revealed significant differences in cell viability among the HMVEC+DRGC group and the DRGC and HMVEC groups (all p < 0.05). In summary, our findings provide evidence that DRGC and HMVEC in co-culture may exhibit greater biological activity than DRGC in mono-culture.     

Production of shikonin derivatives by cell suspension cultures of Lithospermum erythrorhizon. III. Comparison of shikonin derivatives of cultured cells and ko-shikon

A method for quantitative analysis of shikonin derivatives using high pressure liquid chromatography (HPLC) was established. With this method the composition of shikonin derivatives in cultured cells and roots of Lithospermum erythrorhizon (ko-shikon) was compared. The composition of shikonin derivatives produced by cell suspension cultures was similar to that of the ko-shikon, and the composition in cultured cells was found to fluctuate less than that of the ko-shikon.     

A new protocol for cultivation of predegenerated adult rat Schwann cells

The purpose of this study was to optimize the methodology of cultivation of predegenerated Schwann cells (SCs). SCs were isolated from 7-day-predegenerated sciatic nerves of adult rats. We applied commercially available culture medium for cultivation of endothelial cells endothelial cell culture medium (EBM-2) instead of Dulbecco’s Modified Eagle’s Medium commonly used to culture adult Schwann cells. Additionally, cell culture medium was supplemented with factors specifically supporting SCs growth as: bovine pituitary extract (5 μg/ml), heregulin (40 ng/ml) and insulin (2.5 ng/ml). Similarly to the reports of others authors, we did not observe any beneficial effects of Forskolin application, so we didn’t supplement our medium with it. Cell culture purity was determined by counting the ratio of GFAP, N-Cadherin and NGFR p75-positive cells to total number of cells. About 94–97 % of cells were confirmed as Schwann cells. As a result, we obtained sufficient number and purity of Schwann cells to be applied in different experimental models in rats. EBM-2 medium coated with fibronectin was the best for cultivation of adult rat Schwann cells.     

Laurdan Monitors Different Lipids Content in Eukaryotic Membrane During Embryonic Neural Development

We describe a method based on fluorescence-lifetime imaging microscopy (FLIM) to assess the fluidity of various membranes in neuronal cells at different stages of development [day 12 (E12) and day 16 (E16) of gestation]. For the FLIM measurements, we use the Laurdan probe which is commonly used to assess membrane water penetration in model and in biological membranes using spectral information. Using the FLIM approach, we build a fluidity scale based on calibration with model systems of different lipid compositions. In neuronal cells, we found a marked difference in fluidity between the internal membranes and the plasma membrane, being the plasma membrane the less fluid. However, we found no significant differences between the two cell groups, E12 and E16. Comparison with NIH3T3 cells shows that the plasma membranes of E12 and E16 cells are significantly more fluid than the plasma membrane of the cancer cells.     

Detection of cytokines in supernatant from hematopoietic stem/progenitor cells co-cultured with mesenchymal stem cells and endothelial progenitor cells

This study aimed to investigate the significance of cytokine expression in supernatant from hematopoietic stem/progenitor cells (HSCs/HPCs) co-cultured with mesenchymal stem cells (MSCs) or endothelial progenitor cells (EPCs). Mononuclear cells (MNCs) were isolated from normal human umbilical cord blood and then cultured solely or co-cultured with MSCs or EPCs. Changes in the number of MNCs and HSCs/HPCs were observed, and MNC proliferation was tested by carboxyfluorescein diacetate succinimidyl ester. The cultured supernatants of the treated MSCs and EPCs were collected at 24 h after co-culture and used to determine the concentrations of IL-3, IL-6, stem cell factor (SCF), TPO, Flt3l, and VEGF. The total number and proliferation of MNCs increased significantly when co-cultured with MSCs or EPCs than when cultured alone, particularly when MNCs were co-cultured with EPCs. The differences in IL-3 and Flt3l concentrations between groups were not significant. However, IL-6 in the MSC group was significantly higher than that in the two other groups. The SCF and TPO concentrations were highly expressed in the EPC group. The VEGF concentrations in the MSC group and the EPC group were higher than those in the control group. These results indicated that MSCs and EPCs possibly favor the proliferation of MNCs and HSCs/HPCs. IL-6 and VEGF may be related to hematopoietic reconstitution and homing ability of HSCs/HPCs. TPO may have a specific relationship with the promotion of HSCs/HPCs differentiation.     

Increased Expression of Angiogenic Factors in Cultured Human Brain Arteriovenous Malformation Endothelial Cells

To compare the mRNA level of angiogenic factor vascular endothelial growth factor (VEGF), matrix metalloproteinases (MMP)-2, and MMP-9 in cultured human brain arteriovenous malformation (AVM) endothelial cells (ECs) and normal brain endothelial cells (BECs). Tissue explants both from deformed vessels of AVM and normal microvessel were put into culture for endothelial cells. After the monolayer adherent ECs reached confluence, they were tested with endothelial specific marker CD34 and von Willebrand factor (vWF) by immunochemical assay. mRNA levels of VEGF-A, MMP-2, and MMP-9 in AVM endothelial cells (AVMECs) and BECs were measured by PCR. Immunostaining confirmed that more than 95 % of the cultured cells were CD34 (Fig. 1b) and/or vWF positive. Expression levels of VEGF-A and MMP-2 mRNAs were significantly higher in AVMECs than in BECs. The MMP-9 level was also increased in AVMECs, but the difference was not statistically significant. Vascular tissue explants adherent method is a better approach for isolation and culture of AVMECs. Cultured AVMECs expressed higher angiogenic factors (VEGF, MMP-2) than the controlled BECs, implicating angiogenesis plays an important role in the pathogenesis of AVM.     

β-adrenergic receptor antagonists inhibit vasculogenesis of embryonic stem cells by downregulation of nitric oxide generation and interference with VEGF signalling

The β-adrenoceptor antagonist Propranolol has been successfully used to treat infantile hemangioma. However, its mechanism of action is so far unknown. The hypothesis of this research was that β-adrenoceptor antagonists may interfere with endothelial cell differentiation of stem cells. Specifically, the effects of the non-specific β-adrenergic receptor (β-adrenoceptor) antagonist Propranolol, the β₁-adrenoceptor-specific antagonist Atenolol and the β₂-adrenoceptor-specific antagonist ICI118,551 on vasculogenesis of mouse embryonic stem (ES) cells were investigated. All three β-blockers dose-dependently downregulated formation of capillary structures in ES cell-derived embryoid bodies and decreased the expression of the vascular cell markers CD31 and VE-cadherin. Furthermore, β-blockers downregulated the expression of fibroblast growth factor-2 (FGF-2), hypoxia inducible factor-1α (HIF-1α), vascular endothelial growth factor 165 (VEGF₁₆₅), VEGF receptor 2 (VEGF-R2) and phospho VEGF-R2, as well as neuropilin 1 (NRP1) and plexin-B1 which are essential modulators of embryonic angiogenesis with additional roles in vessel remodelling and arteriogenesis. Under conditions of β-adrenoceptor inhibition, the endogenous generation of nitric oxide (NO) as well as the phosphorylation of endothelial nitric oxide synthase (eNOS) was decreased in embryoid bodies, whereas an increase in NO generation was observed with the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP). Consequently, vasculogenesis of ES cells was restored upon treatment of differentiating ES cells with β-adrenoceptor antagonists in the presence of NO donor. In summary, our data suggest that β-blockers impair vasculogenesis of ES cells by interfering with NO generation which could be the explanation for their anti-angiogenic effects in infantile hemangioma.     

Isolation and characterization of mesenchymal stem cells derived from fetal bovine liver

Bovine liver-derived mesenchymal stem cells (bLMSCs) were isolated from the liver tissue of 4–6 months old fetal calf, and then characterized by immunofluorescence and RT-PCR. We found that primary bLMSCs could be subcultured to 44 passages, the total culture time in vitro was 192 days. The results of surface antigen detection showed that bBMSCs expressed CD29, CD44, CD73, CD90, CD106 and CD166 but not expressed endothelial cells and hematopoietic cells specific marker CD34, CD45 and BLA-DR. The results of growth kinetics, colony-forming cell assay and cell cycle analysis indicated that the fetal bovine LMSCs had good proliferation ability in vitro. The cells from passages 7 were successfully induced to differentiate into osteoblasts, adipocytes and chondrocytes. The results indicate the potential for multi-lineage differentiation of bLMSCs that may represent an ideal candidate for cellular transplantation therapy.     

The Interruption of PKC-ι Signaling and TRAIL Combination Therapy Against Glioblastoma Cells

Glioblastoma is a highly aggressive type of brain cancer which currently has limited options for treatment. It is imperative to develop combination therapies that could cause apoptosis in glioblastoma. The aim of this study was to characterize the affect of modified ICA-1, a PKC-iota inhibitor, on the growth pattern of various glioblastoma cell lines. T98G and U87 glioblastoma cells were treated with ICA-1 alone and the absolute cell numbers of each group were determined for cell growth expansion analysis, cell viability analysis, and cell death analysis. Low dose ICA-1 treatment alone significantly inhibited cell growth expansion of high density glioblastoma cells without inducing cell death. However, the high dose ICA-1 treatment regimen provided significant apoptosis for glioblastoma cells. Furthermore, this study was conducted to use a two layer molecular level approach for treating glioblastoma cells with ICA-1 plus an apoptosis agent, tumor-necrosis factor-related apoptosis-inducing ligand (TRAIL), to induce apoptosis in such chemo-refractory cancer cells. Following ICA-1 plus TRAIL treatment, apoptosis was detected in glioblastoma cells via the TUNEL assay and via flow cytometric analysis using Annexin-V FITC/PI. This study offers the first evidence for ICA-1 alone to inhibit glioblastoma cell proliferation as well as the novel combination of ICA-1 with TRAIL to cause robust apoptosis in a caspase-3 mediated mechanism. Furthermore, ICA-1 plus TRAIL simultaneously modulates down-regulation of PKC-iota and c-Jun.     

P53 induction accompanying G2/M arrest upon knockdown of tumor suppressor HIC1 in U87MG glioma cells

Hypermethylated in cancer 1 (HIC1) is a novel tumor suppressor gene (tsg) frequently silenced by epigenetic modification, predominantly by methylation in different tumors. HIC1 functionally co-operates with p53 in cultured cells as well as in transgenic animals to suppress tumors and has binding site on its promoter. Its over expression often leads to cell cycle arrests. Although HIC1 proven to have role as tsg, its regulation to cell cycle and dependency upon p53 is grossly unknown. In this study, we investigated the role of HIC1 in cell cycle and proliferation of glioma cell line U87MG which has wild type p53, in both serum-containing and serum-deprived medium. Microscopic analysis and MTT assay showed reduced cell number and rate of proliferation upon HIC1 knock down compared to control siRNA (p = 0.025) and untreated cells (p = 0.03) in serum-containing medium and serum-free medium (p = 0.014 vs control siRNA; p = 0.018 vs untreated cells). Cell cycle analysis revealed an arrest at G2/M phase of cell cycle with no demonstrable increase in apoptosis with both medium. An increased expression of p53 concomitant with HIC1 knockdown was observed. Furthermore P21, a p53 responsive gene, along with p27 was significantly increased in comparison with controls. Our results demonstrated an important role of HIC1 for the normal progression of cell cycle, and at molecular level, it could affect the homeostasis of p53 as well as number of cell cycle-related genes, which may or may not be directly linked to p53.     

Batimastat Nanoparticles Associated with Transcatheter Arterial Chemoembolization Decrease Hepatocellular Carcinoma Recurrence

Hepatocellular carcinoma (HCC) is a malignant tumor characterized by easy metastasis and frequent recurrence. Transarterial chemoembolization (TACE) remains the routine treatment for patients with HCC who are not eligible for surgical resection or percutaneous tumor ablation; however, 5-year survival rates following interventional therapy are only 17–38.8 %, with liver recurrence due to incomplete embolization and tumor angiogenesis being a significant reason for treatment failure. Ischemia and hypoxia induced by TACE is correlated with an increased expression of angiogenic factor and stimulates an increase in angiogenesis, including endothelial cells (ECs) proliferation. Matrix metalloproteinases (MMPs) are zinc-dependent proteolytic endopeptidases involved in tumor angiogenesis. In addition, MMPs stimulate tumor cell growth, migration and invasion, and metastasis. Hypoxia enhanced EC migration in a MMP-2-dependent manner while MMP inhibitors (MMPIs) significantly decreased the number of migrating cells in hypoxic cultures. We hypothesize batimastat (synthetic MMPI) nanoparticles associated with TACE could decrease HCC recurrence and metastasis. At first, batimastat nanoparticles were made from batimastat and poly(lactic-co-glycolic acid). Then, nanoparticles were mixed with lipiodol and chemotherapeutic drugs solution. The mixture was infused super-selectively into supplied artery of HCC through catheter. The disseminated area of batimastat might be same with TACE-induced hypoxia area. In the hypoxia area, batimastat inhibited the activity of MMPs, weakened the angiogenesis of tumor vascular system and migration of HCC cells. HCC cells could not escape from hypoxia area and tumor angiogenesis inhibited could not supply sufficient nutrients and O₂ to residual HCC cells. With the help of batimastat, the killing effect of chemotherapeutic drugs might be enhanced. The rate of complete necrosis of HCC lesion might be increased and local recurrence and metastasis of HCC might be reduced. The hypothesis might increase the clinical efficacy of TACE and improve the prognosis of HCC patients.     

Gene expression profiling of endometrium versus bone marrow-derived mesenchymal stem cells: upregulation of cytokine genes

Postulated Stem/progenitor cells involved in endometrium regeneration are epithelial, mesenchymal, and endothelial. Bone marrow (BM) has been implicated in endometrial stem cells. We aimed at studying gene expression profiling of endometrial mesenchymal stem cells compared to BM MSCS to better understand their nature and functional phenotype. Endometrial tissues were obtained from premenopausal hysterectomies (n = 3), minced and enzymatically digested as well as Normal BM aspirates (n=3). Immunophenotyping, differentiation to mesoderm, and proliferation were studied. The expression profile of 84 genes relevant to mesenchymal stem cells was performed. Fold change calculations were determined with SA Biosciences data analysis software. VEGF, G-CSF, and GM-CSF in cultures supernatants of MSCs were assayed by Luminex immunoassay. Endo MSCs possess properties similar to BM MSCs. Cumulative population doubling was significantly higher in Endo MSCs compared to BM MSCs (p < 0.001). 52 core genes were shared between both generated MSCs including stemness, self-renewal, members of the Notch, TGFB, FGF, and WNT.16 downregulated genes (VCAM, IGF1)and 16 upregulated in Endo MSCs compared to BM (p < 0.05 → fourfolds). They included mostly cytokine and growth factor genes G-CSF, GM-CSF, VWF, IL1b, GDF15, and KDR. VEGF and G-CSF levels were higher in Endo MSCs supernatants (p < 0.0001). Cells sharing MSC and endothelial cell characteristics could be isolated from the human endometrium. Endo MSCs share a core genetic profile with BM MSCs including stemness. They show upregulation of genes involved in vasculogenesis, angiogenesis, cell adhesion, growth proliferation, migration, and differentiation of endothelial cells, all contributing to endometrial function.