Journal: Physiological Reports

Article Title: Pericyte NF- κ B activation enhances endothelial cell proliferation and proangiogenic cytokine secretion in vitro

doi: 10.14814/phy2.12309

Figure Lengend Snippet: Time course of NF- κ B activation in cocultured C 2 C 12 cells and pericytes. p65 DNA binding activity for cocultured C 2 C 12 cells and pericytes in uninjured control (CON) and scratch-injured (INJ) conditions at baseline (BSLN), 3, 6, and 24 h time points. Data are means ± SD. *Significantly increased compared to BSLN for C 2 C 12 cells. † Significantly increased compared to BSLN for pericytes.

Article Snippet: Human primary pericytes isolated from placental tissue were purchased from PromoCell (Heidelberg, Germany).

Techniques: Activation Assay, Binding Assay, Activity Assay, Control

Journal: Physiological Reports

Article Title: Pericyte NF- κ B activation enhances endothelial cell proliferation and proangiogenic cytokine secretion in vitro

doi: 10.14814/phy2.12309

Figure Lengend Snippet: Time course of MCP-1 secretion from cocultured pericytes and C 2 C 12 cells. Monocyte chemoattractant protein-1 (MCP-1) secretion by cocultured C 2 C 12 cells and pericytes in uninjured control (CON) and scratch-injured (INJ) conditions at baseline (BSLN), 3, 6, and 24 h time points. Data are means ± SD. *Significantly increased compared to BSLN for C 2 C 12 cells. † Significantly greater vs. C 2 C 12 cells at 24 h.

Article Snippet: Human primary pericytes isolated from placental tissue were purchased from PromoCell (Heidelberg, Germany).

Techniques: Control

Journal: Physiological Reports

Article Title: Pericyte NF- κ B activation enhances endothelial cell proliferation and proangiogenic cytokine secretion in vitro

doi: 10.14814/phy2.12309

Figure Lengend Snippet: Pericyte transfection efficacy and efficiency in altering NF- κ B activation. (A) Representative images of pericytes transfected with vectors designed to enhance (constitutively active (c.a.) IKK β -EGFP), diminish (dominant negative (d.n.) IKK β -EGFP), or have no effect on (empty vector (e.v.) pEF6/HisB) NF- κ B activity at 24 h post transfection. (B) A luciferase reporter system was used to assess the ability of expression plasmids to alter NF- κ B expression. Data are means ± SD.

Article Snippet: Human primary pericytes isolated from placental tissue were purchased from PromoCell (Heidelberg, Germany).

Techniques: Transfection, Activation Assay, Dominant Negative Mutation, Plasmid Preparation, Activity Assay, Luciferase, Expressing

Journal: Physiological Reports

Article Title: Pericyte NF- κ B activation enhances endothelial cell proliferation and proangiogenic cytokine secretion in vitro

doi: 10.14814/phy2.12309

Figure Lengend Snippet: HMVEC proliferation is affected by pericyte NF- κ B activation in coculture. Human microvascular endothelial cell (HMVEC) number in coculture with pericytes expressing constitutively active IKK β (c.a.), dominant negative IKK β (d.n.), or empty vector control (e.v.). Data are means ± SD. *Significant difference between c.a. and d.n.

Article Snippet: Human primary pericytes isolated from placental tissue were purchased from PromoCell (Heidelberg, Germany).

Techniques: Activation Assay, Expressing, Dominant Negative Mutation, Plasmid Preparation, Control

Journal: Physiological Reports

Article Title: Pericyte NF- κ B activation enhances endothelial cell proliferation and proangiogenic cytokine secretion in vitro

doi: 10.14814/phy2.12309

Figure Lengend Snippet: Pericyte NF- κ B activation affects cytokine secretion in pericyte/HMVEC cocultures. Cytokine secretion of granulocyte-colony stimulating factor (G-CSF), fractalkine, interleukin 6 (IL-6), interleukin 7 (IL-7), interleukin 8 (IL-8), interferon gamma-induced protein-10 (IP-10), monocyte chemoattractant protein-1 (MCP-1), regulated on activation, normal T-cell expressed and secreted (RANTES), and eotaxin in the cocultures of human microvascular endothelial cells (HMVECs) and pericytes that were genetically altered to express constitutively active IKK β (c.a.) or dominant negative IKK β (d.n.) in comparison to empty vector control (e.v.). Significantly greater cytokine concentration was observed in the c.a. IKK β condition compared to both e.v. and d.n. IKK β conditions for all cytokines ( P < 0.05).

Article Snippet: Human primary pericytes isolated from placental tissue were purchased from PromoCell (Heidelberg, Germany).

Techniques: Activation Assay, Dominant Negative Mutation, Comparison, Plasmid Preparation, Control, Concentration Assay

Journal: PLoS ONE

Article Title: A new perfusion culture method with a self-organized capillary network

doi: 10.1371/journal.pone.0240552

Figure Lengend Snippet: (a) Side and top views of the culture dish before setup. The dish was a normal 12-phi glass-bottom dish. A glass separator was set at the center of the dish with a bioinert adhesive. (b) First, we placed 150 μl fibrin gel mixed with HUVECs in the center of the glass plate, so that two sides of the dish were separated by the glass separator and fibrin gel. We also added LF-containing fibrin gel at the edge of the dish. (c) We incubated the dish for 30 min to solidify the fibrin gel. (d) We added 1 ml culture medium to both wells and incubated the dish for 1 week. (e) After 1 week of culture, a vascular network with a perfusable lumen was formed in the glass-bottom region. Then, we cut both edges of the regions to make openings. (f) After the cuts, we increased the amount of culture medium on one side of the dish. This caused a static pressure difference between both openings of the self-organized capillary network, resulting in steady flow inside the apparatus. (g) Low magnification view of the self-organized vascular network. RFP-HUVECs were cultivated in the fibrin gel, and we observed the vascular network formation. (h) High magnification view of (g). Vascular network with a lumen was generated in the fibrin gel region. (i) Low magnification view of the self-organized vascular network after long-term culture with flow. (j) Visualization of the perfusable area by FITC-dextran. Perfusable regions existed near the inlet and outlet, near the glass separator and edge of the well. (k) Flow inside the lumen was visualized using fluorescent beads. (l) Snapshot of the culture system when using whole blood as a tracer. Red blood cells were flowing inside the self-organized vasculature. (m) Projection of multiple frames of (l). Movements of red blood cells were visualized as a stream. Scale bars: 3 mm (g, i, j); 100 μm (h); 500 μm (k); 50 μm (l, m).

Article Snippet: For visualization purposes, we used red fluorescent protein (RFP)-labeled HUVECs and GFP-labeled human placental microvascular pericytes (cAP-0029GFP) from Angio-proteomie Inc. HL60 and NMuMG-Fucci cells were provided by the Riken Bioresource Research Center (RCB2813 and RCB0041, respectively).

Techniques: Adhesive, Incubation, Generated

Journal: PLoS ONE

Article Title: A new perfusion culture method with a self-organized capillary network

doi: 10.1371/journal.pone.0240552

Figure Lengend Snippet: (a) Experimental procedure. Spheroids containing RFP-HUVECs and lung fibroblast were generated and embedded in fibrin gel. After 1 week, sprouts from the spheroids became sufficiently long. Then, we cut the tip of the sprouts from both sides of the well and exerted static pressure to one side of the well. (b) Visualization of the perfusion inside the spheroid using FITC-dextran. (c) High-magnification view of the sprouts connecting two spheroids. FITC-dextran is running through the sprout structure. Scale bar: 1 mm (b); 100 μm (c).

Article Snippet: For visualization purposes, we used red fluorescent protein (RFP)-labeled HUVECs and GFP-labeled human placental microvascular pericytes (cAP-0029GFP) from Angio-proteomie Inc. HL60 and NMuMG-Fucci cells were provided by the Riken Bioresource Research Center (RCB2813 and RCB0041, respectively).

Techniques: Generated

Journal: PLoS ONE

Article Title: A new perfusion culture method with a self-organized capillary network

doi: 10.1371/journal.pone.0240552

Figure Lengend Snippet: (a) Initial shape of the vascular network. HUVECs were stained with UEA1 and nuclei were stained with Hoechst 33342. There was a flow-positive region (yellow-dashed line) and non-flow region (green-dashed line). Direction of flow is indicated by a white allow. (b) Kymograph of the yellow-dashed line region. Horizontal direction represents space, and vertical direction represents time. Collective movement toward upstream of the flow was observed. White arrow indicates the flow of cell debris. (c) Kymograph of the green-dashed line region. Cell movement was random. (d–n) Cell shape changes induced by flow: (d, e) Fast flow. When FITC dextran was perfused, the vessel regions near the inlet or outlet showed fast flow. (f) Brightfield view of the fast flow region. Endothelial cells became shaped as spindles aligned parallel to the flow direction. (g) Fluorescence view of the fast flow region. At the floor of the lumen, we observed spindle-shaped cells parallel to the flow direction. (h, i) Slow flow. When FITC dextran was perfused, the vessel regions far from the inlet or outlet showed slow flow. (j) Brightfield view and (k) confocal view of the slow flow region. Endothelial cells did not show any polarity. (l) Low magnification view of the non-flow region. (m) Brightfield view and (n) confocal view of the non-flow region. Vasculatures were disconnected and became thin endothelial cysts with cell debris inside. Scale bars: 1 mm (d, e, h, i, l); 200 μm (f, g, j, k, m, n).

Article Snippet: For visualization purposes, we used red fluorescent protein (RFP)-labeled HUVECs and GFP-labeled human placental microvascular pericytes (cAP-0029GFP) from Angio-proteomie Inc. HL60 and NMuMG-Fucci cells were provided by the Riken Bioresource Research Center (RCB2813 and RCB0041, respectively).

Techniques: Staining, Fluorescence

Journal: PLoS ONE

Article Title: A new perfusion culture method with a self-organized capillary network

doi: 10.1371/journal.pone.0240552

Figure Lengend Snippet: (a) Experiment setup. We prepared two groups of dishes in which a mixture of RFP-HUVECs and NMuMG-Fucci cells were seeded in the fibrin gel. After the perfusable network was formed, we made openings to both groups but transferred medium in only one group of the dishes. (b) Typical appearance of the NMuMG cell colony and its schematic representation. NMuMG-Fucci cells (red and green nuclei) forms colonies outside the vascular lumen. (c) Time course of the cell division monitored by Fucci reporter. (d) Time course of GFP(+) area ratio. 20 NMuMG-Fucci colonies were observed, and the area of GFP(+) areas was obtained using Fiji. (e) GFP(+) area ratio between day 1 and day 0. Statistically significant difference was detected (Student t-test). Scale bars: 50 μm.

Article Snippet: For visualization purposes, we used red fluorescent protein (RFP)-labeled HUVECs and GFP-labeled human placental microvascular pericytes (cAP-0029GFP) from Angio-proteomie Inc. HL60 and NMuMG-Fucci cells were provided by the Riken Bioresource Research Center (RCB2813 and RCB0041, respectively).

Techniques:

Journal: PLoS ONE

Article Title: A new perfusion culture method with a self-organized capillary network

doi: 10.1371/journal.pone.0240552

Figure Lengend Snippet: (a) LM4-GFP cells were introduced into the self-organized capillary network consisting of HUVECs visualized by UEA-1 lectin. (b) High magnification time-lapse view of (a). We observed cancer cells emigrating out of the blood vessels. (c) Detailed morphology of cancer cells on the endothelial cells. Emigrated LM4 cells attached to the blood vessel with highly polarized morphology and multiple protrusions. (c) Max projection image, (c') orthogonal section, and (c'') 3D-reconstructed image. White arrows: cancer cell protrusions. Scale bars: 50 μm (a); 10 μm (b, c).

Article Snippet: For visualization purposes, we used red fluorescent protein (RFP)-labeled HUVECs and GFP-labeled human placental microvascular pericytes (cAP-0029GFP) from Angio-proteomie Inc. HL60 and NMuMG-Fucci cells were provided by the Riken Bioresource Research Center (RCB2813 and RCB0041, respectively).

Techniques:

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Mural Cell-Specific Deletion of Cerebral Cavernous Malformation 3 in the Brain Induces Cerebral Cavernous Malformations.

doi: 10.1161/ATVBAHA.120.314586

Figure Lengend Snippet: Figure 1. The SM22α-Cre specificity in the mouse brain vasculature. mT/mG reporter mice were bred with SM22α-Cre deleter mice (mT/mG:SM22α-Cre), and brain tissues were harvested at postnatal day 6 (P6). A, Sections of cerebellum and cerebrum were detected for mG expression by fluorescence microscopy. SM22α-Cre-driven mG was specifically detected in microvessels of both cerebrum and cerebellum (arrowheads) but not in control mT/mG mice (arrows). High power images of arrowhead-indicated regions are shown on the right. n=3 mice per group. B, Cerebral sections were immunostained with anti- PDGFR-β followed by an allophycocyanin (APC)-conjugated secondary antibody with an IgG isotype as a control. mG expression was colocalized with the pericyte (PC) marker PDGFR-β in the brain microvasculature of mT/mG:SM22α-Cre (arrowhead), but not in the IgG staining or in the control mT/mG mice (arrow). n=3 mice per group. C, mG+ and mG− cell populations were isolated from P6 mT/mG:SM22α- Cre brain tissues, and gene expression was determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) with specific PC and endothelial cell (EC) markers as indicated. mG+ cells expressed PC marker genes PDGFRB (PDGFR-β, SCPG4 [NG-2], and ANPEP [CD13], but not EC marker genes PECAM1 [CD31], VEGFR2 [VEGFR2] and CDH5 [VE-cadherin]) with normalization by GAPDH. Data are mean±SEM; n=3; ***P<0.001 by unpaired 2-tailed Student t test. D and E, Mouse brain microvascular pericytes (mBMVPCs) and ECs (mBMVECs) were isolated from wild-type (WT) mice at P6, and immunostained with PC marker PDGFR-β and EC marker VE-cadherin. Phase images (D) and immunofluorescence images (E) are presented. F–I, Ccm3 deletion was specifically in mouse brain PCs but not in mouse brain ECs. mBMVPCs and mBMVECs were isolated from P6 WT and Ccm3smKO brain tissues. F, Cells were immunostained with PC marker PDGFR-β and EC marker VE-cadherin. G, Ccm3 gene expression was determined by qRT-PCR. n=3; ***P<0.001 by unpaired 2-tailed Student t test. H, CCM3 protein was determined by Western blotting. Representative blot form 3 experiments. I, CCM3 protein was determined by immunostaining using an anti-CCM3 antibody with costaining of antipaxillin antibody. n=3. Scale bar: 50 μm (A, B, and D); 25 μm (E and F); 10 μm (I).

Article Snippet: Human brain microvascular ECs (cAP0002) and human brain microvascular pericytes (hBMVPCs; cAP-0030) were purchased from Angio-Proteomie (Boston).

Techniques: Expressing, Fluorescence, Microscopy, Control, Marker, Staining, Isolation, Gene Expression, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Immunofluorescence, Western Blot, Immunostaining

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Mural Cell-Specific Deletion of Cerebral Cavernous Malformation 3 in the Brain Induces Cerebral Cavernous Malformations.

doi: 10.1161/ATVBAHA.120.314586

Figure Lengend Snippet: Figure 3 Continued. Representative images are shown in E. Quantification of % GFAP coverage on CD31+-vessel was quantified by Image J (F). G–I, Mouse brain microvascular pericytes (mBMVPCs) were isolated from neonatal WT and CCM3 smKO brains. 4×105 WT and CCM3- knockout (KO) mBMVPCs were seeded on fibronectin-coated culture slides for indicated times (0–16 h), and unattached cells were washed away. Cells were fixed with 4% paraformaldehyde (PFA) and stained with phalloidin (red) and 4′,6-diamidino-2-phenylindole (DAPI; blue). Representative images for each time point are shown (G). Cell area (H) and cell length (I) were measured by Image J software. Ten fields were counted and n=3 repeated experiments. J and K, RNA-seq analyses. The endogenous CCM3 was knocked out by CRISPR/Cas9. mRNA from confluent WT and CCM3-KO human brain microvascular pericytes (hBMVPCs) were subjected to RNA-seq analyses. J, Gene expression value was estimated by Cufflinks (v1.2.0) and genes with >2-fold change between WT and KO were defined as differential expression. K, Gene Ontology analysis using GOstats was performed and the significant pathways (muscle cell migration and extracellular matrix [ECM] organization) are presented. n=2. Data are means±SEM. Scale bars: 25 μm (A, C, G, and I); 500 nm (E).

Article Snippet: Human brain microvascular ECs (cAP0002) and human brain microvascular pericytes (hBMVPCs; cAP-0030) were purchased from Angio-Proteomie (Boston).

Techniques: Isolation, Knock-Out, Staining, Software, RNA Sequencing, CRISPR, Gene Expression, Quantitative Proteomics, Migration

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Mural Cell-Specific Deletion of Cerebral Cavernous Malformation 3 in the Brain Induces Cerebral Cavernous Malformations.

doi: 10.1161/ATVBAHA.120.314586

Figure Lengend Snippet: Figure 5. Cerebral cavernous malformation (CCM)3-knockout (KO) pericytes (PCs) attenuates PC migration and endothelial cell (EC)-PC interactions. CCM3-KO human brain microvascular pericytes (hBMVPCs) were re-expressed with vector (VC), CCM3-wild type (WT), or CCM3-4KE by lentivirus infection. A, WT, KO/VC, KO/CCM3-WT, and KO/CCM3-4KE hBMVPCs were harvested and subjected to Western blotting to test for adhesion complexes and RhoA-pMLC signaling. Relative protein levels were quantified and fold changes are presented by keeping WT as 1.0. B and C, 4×105 hBMVPCs were seeded on fibronectin-coated culture slides for 16 h. Cells were fixed with 4% paraformaldehyde (PFA) followed costaining with phosphor-paxillin (green) and phalloidin (red) with DAPI counterstaining (blue) (B). Number of FA per cell was measured by Image J software. Ten fields were counted and n=3 repeated experiments. D and E, Rescue PC migration by CCM3-WT but not by paxillin-defective CCM3-4KE mutant. WT, KO/VC, KO/CCM3-WT, and KO/CCM3-4KE hBMVPCs were subjected to wound injury followed by incubation for 24 h. D, Representative images of cell migration are shown. Dashed lines indicate the remaining gaps. E, Quantitation of EC migration. The percentage of unhealed wound was quantified, n=3. F and G, EC-PC interactions in 3-dimensional spheroid sprouting assay. Human brain microvascular ECs (hBMVECs) were infected with EGFP (enhanced green fluorescent protein)-expressing retroviruses, whereas WT, KO/VC, KO/CCM3-WT, and KO/CCM3-4KE hBMVPCs were infected with mCherry-expressing lentiviruses. ECs and PCs (2:1 ratio) were seeded to beads and coated with microbeads, embedded in fibrin gels and grown in EGM-2 endothelial growth medium for 4 d. A representative image of 10 beads for each sample is shown in F and percentage of PC coverage of sprouts is quantified in G. n=10, *P<0.05; **P<0.01 (1- way ANOVA). Additional 2 independent experiments were performed. Error bars indicate SEM. Scale bar: 10 μm (B); 100 μm (D and F).

Article Snippet: Human brain microvascular ECs (cAP0002) and human brain microvascular pericytes (hBMVPCs; cAP-0030) were purchased from Angio-Proteomie (Boston).

Techniques: Knock-Out, Migration, Plasmid Preparation, Infection, Western Blot, Software, Mutagenesis, Incubation, Quantitation Assay, Expressing

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Mural Cell-Specific Deletion of Cerebral Cavernous Malformation 3 in the Brain Induces Cerebral Cavernous Malformations.

doi: 10.1161/ATVBAHA.120.314586

Figure Lengend Snippet: Figure 6. Cerebral cavernous malformation (CCM)3 loss in pericyte (PC) induces extracellular matrix (ECM) deposition in CCM. A and B, Increased ECM deposition in CCM3-deficient human brain microvascular pericytes (hBMVPCs). Wild-type (WT) and CCM3-KO hBMVPCs were cultured confluently on fibronectin-coated culture slides for 16 h. Cells were subjected to costaining with fibronectin (green) and Col IV (red) with DAPI counterstaining (blue). Mean fluorescence intensity (MFI)/per cell were measured by Image J software. Ten fields were counted and n=3 repeated experiments. C and D, P6 WT and Ccm3smKO mouse brain sections for staining of CD31 with fibronectin or NG-2. IgG isotype was used as a control. Representative images are shown (C). Normalized fibronectin MFI were measure by Image J (by taking WT as 1.0). Scale bar: 50 μm (A and C). E, A model for CCM3-depleted PC in promoting CCM lesion progression. The brain microvessels have an extraordinarily high PC to endothelial cell (EC) ratio, and PCs have multiple slender processes extending longitudinally to cover capillary EC for vascular integrity. Integrin-mediated matrix interactions and PC migration are critical in this process. Focal adhesion- mediated cell adhesion is important for cell migration, but the extent of adhesion can govern migration speed. We observed that CCM3- deficient PCs exhibit excess adhesion due to enhanced ECM deposition, ITG-β1 (integrin β1) activation and paxillin-mediated focal adhesion. We propose that CCM3 loss in PCs enhances PC adhesion while reducing PC protrusion and migration along EC, leading to the disruption of PC-EC interactions and resulting in CCM lesion formation.

Article Snippet: Human brain microvascular ECs (cAP0002) and human brain microvascular pericytes (hBMVPCs; cAP-0030) were purchased from Angio-Proteomie (Boston).

Techniques: Cell Culture, Fluorescence, Software, Staining, Control, Migration, Activation Assay, Disruption

Journal: Investigative ophthalmology & visual science

Article Title: Apratoxin S4 Inspired by a Marine Natural Product, a New Treatment Option for Ocular Angiogenic Diseases.

doi: 10.1167/iovs.19-26936

Figure Lengend Snippet: FIGURE 2. Apratoxin S4 inhibits HRPC activation. (a) HRPCs were treated with 25 nM Apratoxin S4 or 10 lg/mL aflibercept for 24 hours. Representative Western blot demonstrated a reduced level of PDGFRb in 10 nM Apratoxin S4–treated HRPCs (n ¼ 3). (b) AlamarBlue assay demonstrated reduced HRPC proliferation after treatment with 1 and 10 nM Apratoxin S4 for 24 hours (n ¼ 3). (c) DAPI staining (left) and quantitative analysis (right) of the motility of Apratoxin S4–treated HRPCs (n ¼ 3). After 4 hours of treatment of Apratoxin S4, HRPCs motility was reduced in a dose-dependent manner. Scale bar: 50 lM. (d) HRECs and HRPCs were co-cultured and treated with Apratoxin S4 for 16 hours. Representative images (left) and quantification of Apratoxin S4’s impact on HRPC (red) to HREC (green) ratio and total tube length (right) in Matrigel (n ¼ 3). Scale bar, 200 lM. All images shown are representative and data are represented as means 6 SEM; 1-way ANOVA followed by Tukey’s multiple comparisons test, *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Primary human retinal endothelial cells (HRECs) and primary human retinal pericytes (HRPCs) were purchased from AngioProteomie (Boston, MA, USA) and maintained in endothelial growth media (EGM) or pericyte growth media (PGM) according to supplier’s instruction.

Techniques: Activation Assay, Western Blot, Alamar Blue Assay, Staining, Cell Culture

Journal: Investigative ophthalmology & visual science

Article Title: Apratoxin S4 Inspired by a Marine Natural Product, a New Treatment Option for Ocular Angiogenic Diseases.

doi: 10.1167/iovs.19-26936

Figure Lengend Snippet: FIGURE 5. Apratoxin S4 specifically inhibits pathological neovascularization in the eye. (a) In OIR, Apratoxin S4 significantly decreased the formation of pathological neovascular tufts (delineated in higher power by white boundary line) without affecting the organized normal revascularization at P17. Scale bar: 500 lM (retina tile images) and 100 lM (higher magnification images). Vehicle, n ¼ 6; Apratoxin S4, n ¼ 6 per dosage. (b) Representative image and volume-rendered examples of NG2 (red) and CD31 (green) stained abnormal neovascular tufts as well as quantitative analysis of pericyte versus EC ratio in P17 OIR retina flatmount prepared from mice subjected to 0.0625 mg/kg Apratoxin S4 or vehicle treatment. Scale bar: 10 lM. (c) Representative Western blot demonstrating a reduced level of pSmad1, 5, 8 in P17 OIR retinal tissue prepared from Apratoxin S4–treated mice (n ¼ 3). (d) Combination of Apratoxin S4 and aflibercept in OIR in C57BL/6J mice resulted in enhanced reduction of neovascular tufts compared with single treatment, whereas organized revascuarization is not affect. Scale bar: 500 lM (retina tile images) and 100 lM (higher magnification images), n ¼ 6 per group. All images shown are representative and data are represented as means 6 SEM; 1-way ANOVA followed by Tukey’s multiple comparisons test, *P < 0.5, **P < 0.01, ***P < 0.001.

Article Snippet: Primary human retinal endothelial cells (HRECs) and primary human retinal pericytes (HRPCs) were purchased from AngioProteomie (Boston, MA, USA) and maintained in endothelial growth media (EGM) or pericyte growth media (PGM) according to supplier’s instruction.

Techniques: Staining, Western Blot