Journal: Lung

Article Title: FLRT3 Overexpression Attenuates Ischemia–Reperfusion Induced Vascular Hyperpermeability and Lung Injury Through RND3

doi: 10.1007/s00408-025-00791-w

Figure Lengend Snippet: Overexpression of FLRT3 promotes cell migration and rescues H/R-induced cell death and inhibition on cell migration. HPMECs were transduced with LV-Control, LV-FLRT3, sh-Control or sh-FLRT3 for 48 h. A Western blot confirmed successful FLRT3 overexpression and knockdown in HPMECs. B CCK-8 assay showed FLRT3 manipulation did not affect baseline cell viability. C Wound healing assay (24 h) demonstrated enhanced migration in LV-FLRT3 group and reduced migration in sh-FLRT3 group. D Transwell migration assay validated the migration changes with FLRT3 manipulation. E Western blot showed LV-FLRT3 restored H/R-induced FLRT3 reduction. F CCK-8 assay revealed FLRT3 overexpression protected against H/R-induced viability decrease. G Wound healing assay demonstrated FLRT3 overexpression preserved migration capacity under H/R conditions. H Transwell assay confirmed the protective effect of FLRT3 on H/R-impaired cell migration. Data are mean ± SD ( n = 3/group). Statistical significance was determined using one-way ANOVA and Tukey’s multiple comparisons test to compare multiple groups. * P < 0.05, ** P < 0.01, *** P < 0.001 compared with the indicated groups

Article Snippet: Human pulmonary microvascular endothelial cells (HPMECs), purchased from ATCC (Manassas, VA, USA), were cultured in endothelial cell medium (ECM, Gibco, USA) containing 10% heat-inactivated fetal bovine serum (FBS, Gibco), 1% endothelial cell growth factor (Beyotime Biotechnology, Jiangsu, China), and 100 IU/ml penicillin and streptomycin (Sigma-Aldrich) at 37 °C in a humidified atmosphere of 5% CO 2 .

Techniques: Over Expression, Migration, Inhibition, Transduction, Control, Western Blot, Knockdown, CCK-8 Assay, Wound Healing Assay, Transwell Migration Assay, Transwell Assay

Journal: Lung

Article Title: FLRT3 Overexpression Attenuates Ischemia–Reperfusion Induced Vascular Hyperpermeability and Lung Injury Through RND3

doi: 10.1007/s00408-025-00791-w

Figure Lengend Snippet: FLRT3 overexpression increases endothelial barrier integrity via cytoskeletal remodeling and restoration of cell–cell junctions after H/R. HPMECs were transduced with LV-Control or LV-FLRT3 for 48 h, then subjected to H/R. A TEER measurements showed FLRT3 overexpression prevented H/R-induced barrier dysfunction. B Evans blue permeability assay demonstrated FLRT3 overexpression reduced H/R-induced barrier leakage. C F-actin staining (FITC-phalloidin, green; DAPI, blue) revealed FLRT3 overexpression maintained cytoskeletal organization under H/R. Scale bar, 50 μm. D Western blot showed FLRT3 overexpression preserved junction protein levels after H/R. E VE-cadherin immunofluorescence demonstrated maintained adherens junctions with FLRT3 overexpression during H/R. Scale bar, 50 μm. F Claudin-5 immunofluorescence showed preserved tight junction organization with FLRT3 overexpression after H/R. Data are mean ± SD ( n = 3/group). Statistical significance was determined using one-way ANOVA and Tukey’s multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001 compared with the indicated groups

Article Snippet: Human pulmonary microvascular endothelial cells (HPMECs), purchased from ATCC (Manassas, VA, USA), were cultured in endothelial cell medium (ECM, Gibco, USA) containing 10% heat-inactivated fetal bovine serum (FBS, Gibco), 1% endothelial cell growth factor (Beyotime Biotechnology, Jiangsu, China), and 100 IU/ml penicillin and streptomycin (Sigma-Aldrich) at 37 °C in a humidified atmosphere of 5% CO 2 .

Techniques: Over Expression, Transduction, Control, Permeability, Staining, Western Blot, Immunofluorescence

Journal: Lung

Article Title: FLRT3 Overexpression Attenuates Ischemia–Reperfusion Induced Vascular Hyperpermeability and Lung Injury Through RND3

doi: 10.1007/s00408-025-00791-w

Figure Lengend Snippet: The FLRT3 protein is destabilized through the autophagic–lysosomal pathway after H/R. A qRT-PCR revealed unchanged FLRT3 mRNA levels in HPMECs after H/R ( n = 3/group). B qRT-PCR showed stable FLRT3 mRNA levels in lung tissue after I/R ( n = 5/group). C , D Protein stability assay using CHX (20 μg/ml) demonstrated accelerated FLRT3 degradation in H/R-treated HPMECs, with significant reduction at 2 h post-CHX ( n = 3/group). E Western blot showed proteasome inhibitor MG132 (5 μM) failed to prevent H/R-induced FLRT3 degradation ( n = 3/group). F Lysosomal inhibitors NH 4 Cl (10 µM) and CQ (10 μM) restored FLRT3 levels in H/R-treated HPMECs. Western blot quantification showed significant restoration of FLRT3 levels with both inhibitors ( n = 3/group). G Autophagy inhibitor Baf-A1 (100 nM) increased both LC3-II/LC3-I ratio and FLRT3 levels under H/R conditions ( n = 3/group). Data are presented as mean ± SD. Statistical significance was determined using unpaired Student’s t test to compare two groups and one-way ANOVA and Tukey’s multiple comparisons test to compare multiple groups. Two‐way ANOVA with Tukey’s post hoc test was used to compare multiple groups with two categorical variables. ** P < 0.01 compared with the indicated groups. Baf-A1 Bafilomycin-A1, CHX cycloheximide, CQ chloroquine, NH 4 Cl ammonium chloride

Article Snippet: Human pulmonary microvascular endothelial cells (HPMECs), purchased from ATCC (Manassas, VA, USA), were cultured in endothelial cell medium (ECM, Gibco, USA) containing 10% heat-inactivated fetal bovine serum (FBS, Gibco), 1% endothelial cell growth factor (Beyotime Biotechnology, Jiangsu, China), and 100 IU/ml penicillin and streptomycin (Sigma-Aldrich) at 37 °C in a humidified atmosphere of 5% CO 2 .

Techniques: Quantitative RT-PCR, Stability Assay, Western Blot

Journal: Lung

Article Title: FLRT3 Overexpression Attenuates Ischemia–Reperfusion Induced Vascular Hyperpermeability and Lung Injury Through RND3

doi: 10.1007/s00408-025-00791-w

Figure Lengend Snippet: RND3 is a target of FLRT3. A HumanBase Gene Networks analysis predicted functional correlation between FLRT3 and RND3 in lung tissue. B Co-IP assay demonstrated endogenous FLRT3–RND3 protein interaction in HPMECs, with input and IgG controls confirming specificity. C GST pulldown assay validated direct FLRT3-RND3 binding. D Double immunofluorescence (FLRT3-red, RND3-green) showed protein co-localization in HPMECs. Scale bar, 50 μm. E Western blot revealed FLRT3 overexpression increased RND3 levels while FLRT3 knockdown reduced RND3 expression. F Western blot showed FLRT3 overexpression restored H/R-induced RND3 reduction ( P < 0.01). Data are mean ± SD ( n = 3/group). Statistical significance was determined using one-way ANOVA and Tukey’s multiple comparisons test. * P < 0.05, ** P < 0.01 compared with the indicated groups

Article Snippet: Human pulmonary microvascular endothelial cells (HPMECs), purchased from ATCC (Manassas, VA, USA), were cultured in endothelial cell medium (ECM, Gibco, USA) containing 10% heat-inactivated fetal bovine serum (FBS, Gibco), 1% endothelial cell growth factor (Beyotime Biotechnology, Jiangsu, China), and 100 IU/ml penicillin and streptomycin (Sigma-Aldrich) at 37 °C in a humidified atmosphere of 5% CO 2 .

Techniques: Functional Assay, Co-Immunoprecipitation Assay, GST Pulldown Assay, Binding Assay, Immunofluorescence, Western Blot, Over Expression, Knockdown, Expressing

Journal: Lung

Article Title: FLRT3 Overexpression Attenuates Ischemia–Reperfusion Induced Vascular Hyperpermeability and Lung Injury Through RND3

doi: 10.1007/s00408-025-00791-w

Figure Lengend Snippet: RND3 is required for FLRT3 overexpression related cytoprotective effects after H/R in HPMEC cells. H/R-induced HPMECs were transduced with LV-FLRT3 and/or sh-RND3. A Western blot showed that overexpression of FLRT3 led to an increase in RND3 protein expression, while knockdown of RND3 in FLRT3-overexpressing HPMECs resulted in a reduction in RND3 protein expression levels. B , C Western blot showed RND3 knockdown reversed FLRT3-mediated protein expression under H/R. D TEER measurements revealed RND3 knockdown compromised FLRT3’s barrier-protective effect. E Evans blue assay validated RND3’s requirement in FLRT3-mediated barrier protection. F F-actin staining (phalloidin, green; DAPI, blue) showed RND3 knockdown prevented FLRT3’s cytoskeletal preservation. Scale bar, 50 μm. G Western blot demonstrated RND3 knockdown reversed FLRT3’s protection of junction proteins (VE-cadherin, ZO-1, claudin-5, and occludin). H , I Immunofluorescence of VE-cadherin ( H ) and claudin-5 ( I ) confirmed RND3’s role in FLRT3-mediated junction maintenance. Scale bar, 50 μm. Data are mean ± SD ( n = 3/group). Statistical significance was determined using one-way ANOVA and Tukey’s multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001 compared with the indicated groups

Article Snippet: Human pulmonary microvascular endothelial cells (HPMECs), purchased from ATCC (Manassas, VA, USA), were cultured in endothelial cell medium (ECM, Gibco, USA) containing 10% heat-inactivated fetal bovine serum (FBS, Gibco), 1% endothelial cell growth factor (Beyotime Biotechnology, Jiangsu, China), and 100 IU/ml penicillin and streptomycin (Sigma-Aldrich) at 37 °C in a humidified atmosphere of 5% CO 2 .

Techniques: Over Expression, Transduction, Western Blot, Expressing, Knockdown, Evans Blue Assay, Staining, Preserving, Immunofluorescence

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: GSK-3Beta-Dependent Activation of GEF-H1/ROCK Signaling Promotes LPS-Induced Lung Vascular Endothelial Barrier Dysfunction and Acute Lung Injury

doi: 10.3389/fcimb.2017.00357

Figure Lengend Snippet: LPS induces GSK-3beta activation in dose- and time-dependent manners in HPMECs. Expression of P-GSK-3beta and GSK-3beta was detected after incubation with different concentrations of LPS for 1 h (A) . The expression of P-GSK-3beta was represented as a histogram according to band intensities (B) . Expression of P-GSK-3beta and GSK-3beta was examined at indicated time points after stimulation with LPS (0.1 μg/ml) in HPMECs (C) . The Western blotting results are presented as a histogram showing the band intensity values (D) . * P < 0.05 vs. LPS un-treatment group.

Article Snippet: Primary human pulmonary micro-vascular endothelial cells (HPMECs) were obtained ScienCell Research Laboratories and maintained in ScienCell Endothelial Cell Medium in a humidified 37°C, 5% CO 2 incubator.

Techniques: Activation Assay, Expressing, Incubation, Western Blot

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: GSK-3Beta-Dependent Activation of GEF-H1/ROCK Signaling Promotes LPS-Induced Lung Vascular Endothelial Barrier Dysfunction and Acute Lung Injury

doi: 10.3389/fcimb.2017.00357

Figure Lengend Snippet: Involvement of GSK-3beta in LPS-induced GEF-H1/ROCK signaling activation. HPMECs were incubated with LPS (0.1 μg/ml) at different indicated times, and the GEF-H1 and myosin-associated phosphatase type 1 (P-MYPT 1: the substrate of ROCK) were detected by Western blot assay (A) . The expression of GEF-H1 and P-MYPT 1 were represented as a histogram according to band intensities (B) . * < 0.05 vs. LPS un-treatment group. Inhibition effect of GSK-3beta activity in HPMECs was analyzed by Western blot (C,D) . * P < 0.05 vs. the negative control group, # P < 0.05 vs. the corresponding LPS treatment group. HPMECs were pretreated with SB-216763 (20 μM) for 1 h and then were exposed to LPS (0.1 μg/ml) for 1 h. The expression of GEF-H1 and P-MYPT 1 were determined by Western blot (E) . The Western blotting results are presented as a histogram showing the band intensity values (F) . * P < 0.05 vs. the negative control group, # P < 0.05 vs. the corresponding LPS treatment group.

Article Snippet: Primary human pulmonary micro-vascular endothelial cells (HPMECs) were obtained ScienCell Research Laboratories and maintained in ScienCell Endothelial Cell Medium in a humidified 37°C, 5% CO 2 incubator.

Techniques: Activation Assay, Incubation, Western Blot, Expressing, Inhibition, Activity Assay, Negative Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: GSK-3Beta-Dependent Activation of GEF-H1/ROCK Signaling Promotes LPS-Induced Lung Vascular Endothelial Barrier Dysfunction and Acute Lung Injury

doi: 10.3389/fcimb.2017.00357

Figure Lengend Snippet: GSK-3beta signaling is involved in LPS-induced HPMECs barrier disruption. The HPMECs were plated on the gold microelectrodes. When HPMECs formed monolayers and reached stable TER values, the SB-216763 (20 μM) was added. After 1 h, the medium or LPS (0.1 μg/ml) was added for another 6 h. The HPMEC monolayers permeability was determined by real-time TER measurement (A) . The results of the 3 h LPS stimulation were represented as a histogram in (B) according to the TER curves. * P < 0.05 vs. negative control. # P < 0.05 vs. corresponding LPS-stimulated group.

Article Snippet: Primary human pulmonary micro-vascular endothelial cells (HPMECs) were obtained ScienCell Research Laboratories and maintained in ScienCell Endothelial Cell Medium in a humidified 37°C, 5% CO 2 incubator.

Techniques: Disruption, Permeability, Negative Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: GSK-3Beta-Dependent Activation of GEF-H1/ROCK Signaling Promotes LPS-Induced Lung Vascular Endothelial Barrier Dysfunction and Acute Lung Injury

doi: 10.3389/fcimb.2017.00357

Figure Lengend Snippet: LPS induces degradation of beta-catenin and ZO-1 in HPMECs monolayer. LPS (0.1 μg/ml) induced down-regulation of ZO-1 expression and increase of phosphorylated degradation of beta-catenin in a time-dependent manner (A) . The Western blotting results are presented as a histogram showing the band intensity values (B) . * P < 0.05 vs. LPS un-treatment group.

Article Snippet: Primary human pulmonary micro-vascular endothelial cells (HPMECs) were obtained ScienCell Research Laboratories and maintained in ScienCell Endothelial Cell Medium in a humidified 37°C, 5% CO 2 incubator.

Techniques: Expressing, Western Blot

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: GSK-3Beta-Dependent Activation of GEF-H1/ROCK Signaling Promotes LPS-Induced Lung Vascular Endothelial Barrier Dysfunction and Acute Lung Injury

doi: 10.3389/fcimb.2017.00357

Figure Lengend Snippet: GSK-3beta/GEF-H1/ROCK signaling is required for LPS-induced degradation of beta-catenin and ZO-1. After transfection with GEF-H1 siRNA and Control siRNA for 48 h, HPMECs were treated with SB-216763 (20 μM) and/or Y-27632 (10 μM) for another 1 h prior to LPS stimulation (0.1 μg/ml) for 3 h. The expression of ZO-1 was determined by immunoblotting, and GAPDH protein was used as loading control (A) . The Western blotting results are presented as a histogram showing the band intensity values (B) . The expression of P-beta-catenin was determined by immunoblotting, and GSK-3beta and GAPDH proteins were used as control (C) . The Western blotting results are presented as a histogram showing the band intensity values (D) . * P < 0.05 vs. negative control. # P < 0.05 vs. corresponding LPS-stimulated group. NS, no significance.

Article Snippet: Primary human pulmonary micro-vascular endothelial cells (HPMECs) were obtained ScienCell Research Laboratories and maintained in ScienCell Endothelial Cell Medium in a humidified 37°C, 5% CO 2 incubator.

Techniques: Transfection, Control, Expressing, Western Blot, Negative Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: GSK-3Beta-Dependent Activation of GEF-H1/ROCK Signaling Promotes LPS-Induced Lung Vascular Endothelial Barrier Dysfunction and Acute Lung Injury

doi: 10.3389/fcimb.2017.00357

Figure Lengend Snippet: GSK-3beta/GEF-H1/ROCK pathway is involved in LPS-induced HPMECs barrier disruption by beta-catenin and ZO-1. HPMECs monolayer was pretreated with SB-216763 (20 μM) (A) , GEF-H1 siRNA (B) , or Y-27632 (10 μM) (C) , for indicated times and then was exposed to LPS (0.1 μg/ml) for 3 h before fixation and staining with anti-beta-catenin and anti-ZO-1 antibody as described in Materials and Methods. Beta-catenin (green) and ZO-1 (green) were visualized by immunofluorescence microscopy. Red arrows not only represent the expression of beta-catenin and ZO-1 in the membrane of HPMECs but also represent the cell-cell gaps formation in the ECs monolayer.

Article Snippet: Primary human pulmonary micro-vascular endothelial cells (HPMECs) were obtained ScienCell Research Laboratories and maintained in ScienCell Endothelial Cell Medium in a humidified 37°C, 5% CO 2 incubator.

Techniques: Disruption, Staining, Immunofluorescence, Microscopy, Expressing, Membrane

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: MicroRNA-30a as a candidate underlying sex-specific differences in neonatal hyperoxic lung injury: implications for BPD

doi: 10.1152/ajplung.00372.2018

Figure Lengend Snippet: Differential sex-specific expression on miR-30a in vitro. A and B: miR-30a-3p and miR-30a-5p expression in human pulmonary microvascular endothelial cells (HPMECs) exposed to room air (RA) (RA-5% CO2) and 72 h of hyperoxia (95% O2-5% CO2) (n = 3/group). Values are means ± SE. Significant differences between RA and hyperoxia within each sex are indicated by ***P < 0.001. Significant differences between male and female mice in normoxia or hyperoxia are indicated by #P < 0.05 and ##P < 0.01.

Article Snippet: Neonatal human pulmonary microvascular endothelial cells (HPMECs) were purchased from ScienCell (female lot nos: 5016, 10169, 10160, 15900 and 17799; male lot nos.

Techniques: Expressing, In Vitro

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: MicroRNA-30a as a candidate underlying sex-specific differences in neonatal hyperoxic lung injury: implications for BPD

doi: 10.1152/ajplung.00372.2018

Figure Lengend Snippet: Delta-like ligand 4 (Dll4) expression in male and female human neonatal pulmonary microvascular endothelial cells upon exposure to hyperoxia in vitro: DLL4 mRNA (A) and protein (B) expression in male and female neonatal human pulmonary microvascular endothelial cells (HPMECs) exposed to room air (RA) (RA-5% CO2) and 72 h of hyperoxia (95% O2-5% CO2) (n = 3/group). Values are means ± SE. Significant differences between RA and hyperoxia within each sex are indicated by *P < 0.05 and **P < 0.01. Significant differences between male and female mice in normoxia or hyperoxia are indicated by ##P < 0.01. NS, not significant.

Article Snippet: Neonatal human pulmonary microvascular endothelial cells (HPMECs) were purchased from ScienCell (female lot nos: 5016, 10169, 10160, 15900 and 17799; male lot nos.

Techniques: Expressing, In Vitro

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: MicroRNA-30a as a candidate underlying sex-specific differences in neonatal hyperoxic lung injury: implications for BPD

doi: 10.1152/ajplung.00372.2018

Figure Lengend Snippet: Delta-like ligand 4 (Dll4) is an miR-30a-5p target in neonatal human pulmonary microvascular endothelial cells. TargetScan reported miR-30a binding sites 59–66bp in the 3′UTR of DLL4 (A). Increased mir30a-5p expression after mir30a-5p mimic transfection in HPMECs (B). DLL4 mRNA (C) and protein (D) expression after mir30a-5p mimic transfection in HPMECs. DLL4 mRNA (E) and protein (F) expression after mir30a-5p inhibitor transfection in HPMECs. DLL4 protein expression (G) after mir30a-5p mimic transfection with and without target protector in HPMECs. Values are means ± SE from 3 independent experiments (n = 3/group). Significant differences between indicated groups are indicated by *P < 0.05 and **P < 0.01. HPMEC, human pulmonary microvascular endothelial cell; UTR, untranslated region; NC, normal control.

Article Snippet: Neonatal human pulmonary microvascular endothelial cells (HPMECs) were purchased from ScienCell (female lot nos: 5016, 10169, 10160, 15900 and 17799; male lot nos.

Techniques: Binding Assay, Expressing, Transfection, Control

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: MicroRNA-30a as a candidate underlying sex-specific differences in neonatal hyperoxic lung injury: implications for BPD

doi: 10.1152/ajplung.00372.2018

Figure Lengend Snippet: Effect of miR30a-3p overexpression and inhibition on DLL4 expression in HPMECs. Increased mir30a-3p expression after mir30a-3p mimic transfection in HPMECs (A). DLL4 mRNA (B) and protein (C) expression after mir30a-3p mimic transfection in HPMECs. DLL4 mRNA (D) and protein (E) expression after mir30a-3p inhibitor transfection in HPMECs. Values are means ± SE from three independent experiments (n = 3/group). Significant differences between indicated groups are indicated by *P < 0.05 and **P < 0.01. DLL4, delta-like ligand 4; HPMEC, human pulmonary microvascular endothelial cell. NC, normal control.

Article Snippet: Neonatal human pulmonary microvascular endothelial cells (HPMECs) were purchased from ScienCell (female lot nos: 5016, 10169, 10160, 15900 and 17799; male lot nos.

Techniques: Over Expression, Inhibition, Expressing, Transfection, Control

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: MicroRNA-30a as a candidate underlying sex-specific differences in neonatal hyperoxic lung injury: implications for BPD

doi: 10.1152/ajplung.00372.2018

Figure Lengend Snippet: miR30a-5p inhibition under hyperoxic conditions decreases increases Dll4 expression in female HPMECs. DLL4 mRNA (A) and protein (B) expression after inhibition of mir30a-5p in HPMECs upon exposure to hyperoxia. Values are means ± SE from three independent experiments (n = 3/group). Significant differences between indicated groups are indicated by **P < 0.01. DLL4, delta-like ligand 4; HPMEC, human pulmonary microvascular endothelial cell. NC, normal control.

Article Snippet: Neonatal human pulmonary microvascular endothelial cells (HPMECs) were purchased from ScienCell (female lot nos: 5016, 10169, 10160, 15900 and 17799; male lot nos.

Techniques: Inhibition, Expressing, Control

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: MicroRNA-30a as a candidate underlying sex-specific differences in neonatal hyperoxic lung injury: implications for BPD

doi: 10.1152/ajplung.00372.2018

Figure Lengend Snippet: miR30a-5p mimic increases sprouting angiogenesis in female HPMECs. Angiogenic potential was quantified based on the maximum length of sprouts protruding from HPMEC-coated cytodex-3 microcarrier beads suspended in a fibrin gel (n = 3/group). A: representative images from male and female HPMECs subjected to sprouting angiogenesis assay treated with miR30a-5p mimic or negative control. Scale bar represents 200 μm. B: maximum sprouting distance in male and female HPMECs. Values are means ± SE. Significant differences between treatment and control groups is indicated by *P < 0.05. HPMEC, human pulmonary microvascular endothelial cell.

Article Snippet: Neonatal human pulmonary microvascular endothelial cells (HPMECs) were purchased from ScienCell (female lot nos: 5016, 10169, 10160, 15900 and 17799; male lot nos.

Techniques: Angiogenesis Assay, Negative Control, Control