Journal: Nanomaterials

Article Title: Engineering of Corneal Tissue through an Aligned PVA/Collagen Composite Nanofibrous Electrospun Scaffold

doi: 10.3390/nano8020124

Figure Lengend Snippet: The proliferation results of the HKs and HCECs: ( a ) proliferation of HKs on the 7% aligned collagen and the 9% PVA-COL (aligned and non-aligned) electrospun scaffolds within seven days; ( b ) proliferation histogram of the HKs in the different electrospun scaffolds cultured for seven days; ( c ) proliferation of HCECs on the 7% aligned collagen and the 9% PVA-COL (aligned and non-aligned) electrospun scaffolds within seven days; and ( d ) proliferation histogram of the HCECs in the different electrospun scaffolds cultured for seven days. The control group was the cells that were seeded on the culture plate, the data represent the means ± SD (* p < 0.05).

Article Snippet: HCECs [ , , , , ] were purchased from the RIKEN BioResource Center (Tsukuba, Japan), and HKs [ , ] were obtained from the He Eye Hospital (Shenyang, China).

Techniques: Cell Culture, Control

Journal: Molecular Vision

Article Title: Enhanced survival in vitro of human corneal endothelial cells using mouse embryonic stem cell conditioned medium

doi:

Figure Lengend Snippet: Cell number in three different mediums. The HCECs were seeded at the density of 9×10 4 cells/well on 12-well plates. The ESC-CM and ESC medium were added to passage 1 cells. Data are expressed as the mean±SEM (n=3). The values with the same letter (a, b, c, d) in each column indicate these values are not significantly different (p>0.05). The same indication is used in all subsequent figures.

Article Snippet: The cell-cycle entrance of HCECs treated with 25%ESC-CM was significantly higher than that of CEM group both in passage 2 (p=0.001) and passage 4 (p=0.000).

Techniques:

Journal: Molecular Vision

Article Title: Enhanced survival in vitro of human corneal endothelial cells using mouse embryonic stem cell conditioned medium

doi:

Figure Lengend Snippet: The morphology and cell size of HCECs cultured in the CEM group and the 25%ESC-CM group. The 25%ESC-CM group maintained the morphology and cell size of HCECs until passage (P6).

Article Snippet: The cell-cycle entrance of HCECs treated with 25%ESC-CM was significantly higher than that of CEM group both in passage 2 (p=0.001) and passage 4 (p=0.000).

Techniques: Cell Culture

Journal: Molecular Vision

Article Title: Enhanced survival in vitro of human corneal endothelial cells using mouse embryonic stem cell conditioned medium

doi:

Figure Lengend Snippet: Increased frequency of proliferating HCECs in conditioned medium from mouse embryonic stem cells. A : Passage 4 HCECs were cultured for 48 h and probed for Ki67 expression. B : Ki67-positive cells were analyzed by flow cytometry. Flow cytometry analysis revealed significantly increased Ki67-positive HCECs in the 25%ESC-CM group (p=0.000). Data are expressed as the mean ± SEM (n=3).

Article Snippet: The cell-cycle entrance of HCECs treated with 25%ESC-CM was significantly higher than that of CEM group both in passage 2 (p=0.001) and passage 4 (p=0.000).

Techniques: Cell Culture, Expressing, Flow Cytometry

Journal: Molecular Vision

Article Title: Enhanced survival in vitro of human corneal endothelial cells using mouse embryonic stem cell conditioned medium

doi:

Figure Lengend Snippet: Conditioned medium from mouse embryonic stem cells stimulated colony formation of HCECs. The colony formation was significantly higher in the 25%ESC-CM group than in the CEM group (p=0.000). Data are expressed as the mean ± SEM (n=5).

Article Snippet: The cell-cycle entrance of HCECs treated with 25%ESC-CM was significantly higher than that of CEM group both in passage 2 (p=0.001) and passage 4 (p=0.000).

Techniques:

Journal: Molecular Vision

Article Title: Enhanced survival in vitro of human corneal endothelial cells using mouse embryonic stem cell conditioned medium

doi:

Figure Lengend Snippet: Conditioned medium from mouse embryonic stem cells promoted cell-cycle entrance of HCECs. The percentage of S phase and G 2 phase cells in 25%ESC-CM group was significantly increased at passage 2 (p = 0.001) and passage 4 (p=0.000). Data are expressed as the mean ± SEM (n=3).

Article Snippet: The cell-cycle entrance of HCECs treated with 25%ESC-CM was significantly higher than that of CEM group both in passage 2 (p=0.001) and passage 4 (p=0.000).

Techniques:

Journal: Molecular Vision

Article Title: Enhanced survival in vitro of human corneal endothelial cells using mouse embryonic stem cell conditioned medium

doi:

Figure Lengend Snippet: Conditioned medium from mouse embryonic stem cells inhibited the apoptosis of HCECs. The apoptosis/necrosis rate of passage 4 HCECs in 25%ESC-CM group was significantly lower than in CEM group (p=0.001). Data are expressed as the mean ± SEM (n=3).

Article Snippet: The cell-cycle entrance of HCECs treated with 25%ESC-CM was significantly higher than that of CEM group both in passage 2 (p=0.001) and passage 4 (p=0.000).

Techniques:

Journal: Investigative Ophthalmology & Visual Science

Article Title: Varicella Zoster Virus Induces Differential Cell-Type Specific Responses in Human Corneal Epithelial Cells and Keratocytes

doi: 10.1167/iovs.18-25801

Figure Lengend Snippet: Phase-contrast imaging of VZV-infected primary HCECs and HKs. HCEC and HK cell types were verified by IFA. All DAPI-positive HCECs expressed the epithelial cell marker cytokeratin 18 (A1, red) and all DAPI-positive HKs expressed the fibroblast cell marker fibronectin (A2, green). HCECs and HKs were mock- or VZV-infected and analyzed at 7 days postinfection by phase microscopy and IFA using mouse anti-VZV glycoprotein E (gB) antibody. In mock-infected HCECs, phase images showed a cell monolayer without a CPE (A3) and no VZV gB (A4), whereas VZV-infected HCECs showed a CPE with areas of cell accumulation on phase-contrast (A5) that contained VZV gB by IFA (A6, red). In mock-infected HKs, phase images showed a monolayer of cells without CPE (A7) and no VZV gB (A8), whereas VZV-infected HKs showed a CPE on phase-contrast (A9) that corresponded to cells expressing VZV gB (A10, red). Blue color indicates cell nuclei. Mag 400X, A1 and A2; 100X, A3-A10. At 3, 5, and 7 days postinfection, infectious virus transmission from VZV-infected HCECs and HKs was measured by serially diluting cells onto uninfected HFLs. After 3 days of co-culture, HFLs were stained with crystal violet and the number of PFU/mL was determined. VZV-infected HCECs significantly increased the amount of PFU/mL at each time point: 3 DPI (367 ± 219), 5 DPI (2300 ± 82), 7 DPI (5250 ± 204; mean PFU/mL ± SEM; n = 3 [B]). In contrast, VZV-infected HKs significantly decreased PFU/mL at each time point: 3 DPI (14,666 ± 1171), 5 DPI (8333 ± 1353), 7 DPI (5400 ± 493; mean PFU/mL ± SEM; n = 3; [C]). Dashed lines represent a 1-fold (no) change relative to control groups (*P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: The VZV Gilden strain (GenBank #MH379685) and primary HCECs and HKs from adult human cornea (ScienCell, Carlsbad, CA, USA) were used.

Techniques: Imaging, Infection, Marker, Microscopy, Expressing, Virus, Transmission Assay, Co-Culture Assay, Staining, Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Varicella Zoster Virus Induces Differential Cell-Type Specific Responses in Human Corneal Epithelial Cells and Keratocytes

doi: 10.1167/iovs.18-25801

Figure Lengend Snippet: Differential morphology of VZV-infected primary HCECs and HKs. HCECs and HKs were mock- or VZV-infected and at 7 days postinfection, analyzed by IFA using mouse anti-VZV glycoprotein B (gB, red) and rabbit anti-GAPdH (green). Mock-infected HCECs expressed GAPdH but not VZV gB (A1); a corresponding surface plot showed HCECs in a monolayer (A2). VZV-infected HCECs contained infected cells expressing VZV gB (A3, red-yellow) and the corresponding surface plot showed that these cells were piling up over the monolayer (A4, red), similar to the elevated VZV-antigen-positive pseudodendrites seen in patients with VZV epithelial keratitis. Mock-infected HKs expressed GAPdH but not VZV gB (B1) and were present as a monolayer on a corresponding surface plot (B2). VZV-infected HKs contained classic plaques with areas of cell clearing/lysis and peripheral cells expressing VZV gB (B3) as confirmed on a surface plot (B4). Blue color indicates cell nuclei. Mag 400X. Cell counts of VZV-infected HCECs and HKs at 1, 3, 5 and 7 days postinfection were counted using a hemocytometer. Cell counts were normalized to 1 DPI and reported as a fold-difference. VZV-infected HCECs significantly increased cell counts at each time point: 3 DPI (2.09 ± 0.40), 5 DPI (2.00 ± 0.18), 7 DPI (2.45 ± 0.16; fold difference relative to 1 DPI ± SEM; n = 3 [C]), consistent with the lack of cell death seen on IFA images. VZV-infected HKs significantly decreased cell counts at 5 and 7 DPI compared to 1 DPI: 3 DPI (0.99 ± 0.07), 5 DPI (0.64 ± 0.07), 7 DPI (0.13 ± 0.01; fold difference relative to 1 DPI ± SEM; n = 3 [D]), consistent with cell death seen on IFA images. Dashed lines represent a 1-fold (no) change relative to control groups (*P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: The VZV Gilden strain (GenBank #MH379685) and primary HCECs and HKs from adult human cornea (ScienCell, Carlsbad, CA, USA) were used.

Techniques: Infection, Expressing, Lysis, Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Varicella Zoster Virus Induces Differential Cell-Type Specific Responses in Human Corneal Epithelial Cells and Keratocytes

doi: 10.1167/iovs.18-25801

Figure Lengend Snippet: Proinflammatory cytokines and immune cell migration in conditioned supernatant from mock- and VZV-infected primary HCECs and HKs. At 7 days postinfection, conditioned supernatant from mock- and VZV-infected cells were analyzed for proinflammatory cytokines IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, IFN-γ, and TNF-α by multiplex assays (Meso Scale Discovery). Compared to the respective mock-infected cells, supernatant from VZV-infected HCECs contained significantly increased levels of IL-2, IL-6, IL-8, IL-10, IL-12p70 and IFNγ; IL-1β and IL-13 were unchanged and; IL-4 and TNF-α were not detected (ND; A, black bars; n = 5). Supernatant from VZV-infected HKs contained significantly decreased levels of IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12p70, IL-13 and IFNγ; IL-8 was increased and; TNF-α was not detected (A, gray bars; n = 5). Results are reported as fold difference of VZV-infected compared to mock-infected cells. In chemotaxis assays, supernatants from mock- and VZV-infected cells at 7 days postinfection were placed in the bottom chamber, peripheral blood mononuclear cells (PBMCs) or neutrophils were placed in the top chamber separated from the bottom chamber by a 5-μm filter and the number of immune cells migrating through the filter and toward conditioned supernatant was quantitated 4 hours later. Compared to mock-infected supernatant, VZV-infected HCEC supernatant significantly increased PBMC infiltration (1.73 ± 0.15, mean fold difference ± SEM; n = 3), media only control had less migrating PBMCs than CCL2 (20 pg/mL, positive control) diluted in HCEC medium (0.03 ± 0.01 versus 2.68 ± 0.48, respectively; mean fold difference ± SEM; n = 3 [B]). Compared to mock-infected cells, VZV-infected HK supernatant did not increase PBMC migration (0.99 ± 0.16, mean fold difference ± SEM; n = 3), media only control had less migrating PBMCs than CCL2 diluted in HK medium (0.27 ± 0.27 versus 2.19 ± 0.40, respectively, mean fold difference ± SEM; n = 3 [C]). Compared to mock-infected HCEC supernatant, neutrophils significantly increased migration toward VZV-infected HCEC supernatant (1.29 ± 0.04, mean fold difference ± SEM; n = 3), anti-IL-8 antibody (αIL-8) in VZV-infected HCEC supernatant and an αIL-8 with IL-8 cytokine did not significantly increase neutrophil migration (0.89 ± 0.05 and 1.02 ± 0.07, respectively, mean fold difference relative to mock ± SEM; n = 3) and IL-8 diluted in HCEC medium significantly increased neutrophil infiltration (2.90 ± 0.45, mean fold difference ± SEM; n = 3 [D]). Compared to mock-infected HK supernatant, neutrophils significantly increased migration toward VZV-infected HK supernatant (3.01 ± 0.68, mean fold difference ± SEM; n = 3), αIL-8 in VZV-infected HK supernatant and a αIL-8 with IL-8 cytokine was not significantly increased (1.00 ± 0.10 versus 1.37 ± 0.11, respectively, mean fold difference relative to mock ± SEM; n = 3), IL-8 diluted in HK medium significantly increased PBMC infiltration (43.31 ± 12.22, mean fold difference ± SEM; n = 3 [E]). Dashed lines represent a 1-fold difference relative to control group (*P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: The VZV Gilden strain (GenBank #MH379685) and primary HCECs and HKs from adult human cornea (ScienCell, Carlsbad, CA, USA) were used.

Techniques: Migration, Infection, Multiplex Assay, Chemotaxis Assay, Control, Positive Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Varicella Zoster Virus Induces Differential Cell-Type Specific Responses in Human Corneal Epithelial Cells and Keratocytes

doi: 10.1167/iovs.18-25801

Figure Lengend Snippet: Matrix metalloproteinases (MMPs) and activity in conditioned supernatant from mock- and VZV-infected primary HCECs and keratocytes (HKs). At 7 days postinfection, mock- and VZV-infected conditioned supernatant from HCECs and HKs were analyzed for MMPs-1, -2, -3, -9 and -10 using Meso Scale Discovery multiplex assays. Compared to the respective mock-infected cells, conditioned supernatant from VZV-infected HCECs significantly increased levels of MMP-1 and -9, but not MMP-3 or -10; MMP-2 was not detected (ND; A, black bars), whereas supernatant from VZV-infected HKs contained significantly decreased levels of MMP-1, -2, -3 and -9; MMP-10 was ND (A, gray bars). Overall MMP activity was measured as substrate cleavage, reported as relative fluorescence unit (RFU, excitation 485 nm and emission 520 nm). Compared to the respective mock-infected cells, supernatant from VZV-infected HCECs had significantly increased MMP activity (1.44 ± 0.06, mean fold difference ± SEM; n = 3), while supernatant from VZV-infected HKs had significantly decreased MMP activity (0.84 ± 0.00, mean fold difference ± SEM; n = 3 [B]). MMP-1 enzyme (500 pg/mL) served as the positive control (1.78 ± 0.03, mean fold difference ± SEM; n = 3), while MMP-1 enzyme with the trypsin deactivating enzyme at 100 μg/mL (0.01 ± 0.00, mean fold difference ± SEM; n = 3) was used as the negative control. Dashed lines represent a 1-fold difference relative to control group (*P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: The VZV Gilden strain (GenBank #MH379685) and primary HCECs and HKs from adult human cornea (ScienCell, Carlsbad, CA, USA) were used.

Techniques: Activity Assay, Infection, Multiplex Assay, Fluorescence, Positive Control, Negative Control, Control

Journal: British Journal of Pharmacology

Article Title: Smooth muscle‐generated methylglyoxal impairs endothelial cell‐mediated vasodilatation of cerebral microvessels in type 1 diabetic rats

doi: 10.1111/bph.13617

Figure Lengend Snippet: Panel A shows methylglyoxal levels in culture media from hcSMCs grown in 5.5 mM glucose and 25 mM glucose at 0.6, 12 and 24 h. Values shown in graph are for five independent experiments carried out using three separate preparations of hcSMCs. * P < 0.05, significantly different from 5.5 mM glucose. Panels B and C show viability of hcECs and hcSMCs 24 h after addition of a single addition of varying concentrations of methylglyoxal to the culture chambers. Values shown in graph are for eight independent experiments carried out using four separate preparations of hcECs and hcSMCs. * P < 0.05, significantly different from 0 μM methylglyoxal. Inset shows Western blots for Glo‐I and actin in hbECs and hbSMCs. Panels D shows representative images of changes in mitochondrial ROS in hbECs before and after exposure to 30 μM methylglyoxal. The third row also shows mitochondria ROS levels in hbECs that were pretreated for 30 min with 20 μM of MnTBAP, a cell permeable synthetic metalloporphyrin that exhibits SOD activity. For these studies, cells were loaded with the mitochondria‐localizing probe MitoTracker Green (100 nM), followed by the fluorogenic, mitochondria‐targeted, superoxide/ROS probe MitoSOX Red (2 μM), for 15 min each. Values shown in the graph below are for n = 5 independent experiments carried out using three separate preparations of hbECs. * P < 0.05, significantly different from 0 μM methylglyoxal. # P < 0.05, significantly different from 30 μM methylglyoxal. Panels E shows representative autoradiograms for claudin‐5 and occludin in hbECs, 24 h after adding a single addition of varying concentrations of methylglyoxal to the culture chamber. Values shown in the graph below are for n = 5 independent experiments carried out using three separate preparations of hcECs and hcSMCs.

Article Snippet: Human cECs (hcECs) and human cerebral microvascular SMCs (hcSMCs; 1 × 10 6 , ScienCell, Carlsbad, CA) were incubated with varying concentrations of methylglyoxal (1–1000 μM) for 24 h at 37°C.

Techniques: Western Blot, Activity Assay