primary human umbilical vein ecs  (ATCC)

Journal: Blood Advances

Article Title: Comprehensive analysis of complement activation in a hydroxyurea-treated patient cohort with sickle cell disease

doi: 10.1182/bloodadvances.2025016958

Figure Lengend Snippet: Assessment of complement deposition on the surface of HUVECs treated with heme. (A) Quantification of C3b and C5b-9 deposition on heme- and serum-treated HUVECs (n = 3) as image area (square micrometer) covered with positive signal per cell. (B) Representative immunofluorescence image of HUVECs treated with no heme or 400μM heme followed by 50% NHS stained for C3b (orange) and C5b-9 deposition (green), 20× objective. White arrows indicate complement deposits. Scale bars, 100 μm. Statistics: data passed Shapiro-Wilk test of normality, 1-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test, simple linear regression, data presented as mean ± standard deviation.

Article Snippet: Primary human umbilical vein ECs (HUVECs) from a single donor (Lonza, CC-2517, Lot 0000444770) were maintained on collagenized tissue culture flasks at 37°C, in an environment with 5% CO 2 in Endothelial Cell Growth Medium 2 supplemented with manufacturer’s supplements (PromoCell, C-22111), 10% fetal bovine serum (WISENT, 080-150), and 1% antibiotic-antimycotic (WISENT, 450-115-EL).

Techniques: Immunofluorescence, Staining, Standard Deviation

Journal: Blood Advances

Article Title: Comprehensive analysis of complement activation in a hydroxyurea-treated patient cohort with sickle cell disease

doi: 10.1182/bloodadvances.2025016958

Figure Lengend Snippet: Assessment of complement deposition on HUVECs treated with patient serum. (A) Quantification of C3b deposition as image area (square micrometer) covered with positive signal per cell (crisis n = 23, steady state n = 32, NHS n = 11, ADP crisis n = 20, ADP steady state n = 29). (B) Representative immunofluorescence image of complement deposition on HUVECs treated with 50% NHS and 50% serum from patients with SCD during crisis (20× objective). DAPI (blue), C3b (magenta), C5b-9 (green). White arrows indicate complement deposits. Scale bars, 100 μm. (C) Quantification of C5b-9 deposition as image area (square micrometer) covered with positive signal per cell (crisis n = 23, steady state n = 33, NHS n = 11, ADP crisis n = 21, ADP steady state n = 29). Each NHS data point is an experimental replicate performed with pooled serum from ≥3 healthy adult volunteers. Statistics: data did not pass Shapiro-Wilk normality test, Kruskal-Wallis test with Dunn’s multiple comparisons or Mann-Whitney test, data presented as median with interquartile range and minimum/maximum.

Article Snippet: Primary human umbilical vein ECs (HUVECs) from a single donor (Lonza, CC-2517, Lot 0000444770) were maintained on collagenized tissue culture flasks at 37°C, in an environment with 5% CO 2 in Endothelial Cell Growth Medium 2 supplemented with manufacturer’s supplements (PromoCell, C-22111), 10% fetal bovine serum (WISENT, 080-150), and 1% antibiotic-antimycotic (WISENT, 450-115-EL).

Techniques: Immunofluorescence, MANN-WHITNEY

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: TGF-β1 Promotes Angiogenesis via Endothelial-to-Mesenchymal Transition in Infantile Hemangioma

doi: 10.1161/ATVBAHA.125.322793

Figure Lengend Snippet: Changes in the expression of endothelial and mesenchymal markers after TGF-β1 (transforming growth factor β1) treatment in hemangioma-derived endothelial cells (HemECs) and human umbilical vein endothelial cells (HUVECs). A , Western blot analysis showing the expression of TGF-β1 in HemECs and HUVECs (n=4 biological replicates). The unpaired 2-tailed Student t tests were used. P =0.001, Cohen d =−7.50 (significant, extremely large effect). B , Immunofluorescence images showing the expression of endothelial and mesenchymal markers before and after TGF-β1 treatment in HemECs and HUVECs (n=4 biological replicates). Scale bar, 50 µm. C , Immunofluorescence analysis showing the mean fluorescence intensity of endothelial and mesenchymal markers before and after TGF-β1 treatment (n=4 biological replicates). The unpaired 2-tailed Student t tests were used. For marker expression in HemECs (TGF-β1 vs untreated), CD31: P =0.001, Cohen d =−7.14 (significant, extremely large effect). VE-cadherin (vascular endothelial cadherin): P =0.030, Cohen d =−2.93 (significant, very large effect). α-SMA (α-smooth muscle actin): P <0.001, Cohen d =13.55 (significant, extremely large effect). COL1A1 (collagen type I alpha 1 chain): P <0.001, Cohen d =11.58 (significant, extremely large effect). For marker expression in HUVECs (TGF-β1 vs untreated), CD31: P =0.72, Cohen d =−1.14 (not significant, large effect). VE-cadherin: P =0.21, Cohen d =1.65 (not significant, very large effect). α-SMA: P =0.92, Cohen d =−0.08 (not significant, negligible effect). COL1A1: P =0.93, Cohen d =−0.11 (not significant, negligible effect).

Article Snippet: HemEC isolation from proliferating IHs was performed as described previously., Primary human umbilical vein ECs (HUVECs) were obtained from the American Type Culture Collection (United States).

Techniques: Expressing, Derivative Assay, Western Blot, Immunofluorescence, Fluorescence, Marker

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: TGF-β1 Promotes Angiogenesis via Endothelial-to-Mesenchymal Transition in Infantile Hemangioma

doi: 10.1161/ATVBAHA.125.322793

Figure Lengend Snippet: TGF-β1 OE (TGF-β1 [transforming growth factor β1] overexpression) promotes hemangioma-derived endothelial cell (HemEC) migration, invasion, and angiogenesis. A , Transwell migration assay results showing the migration ability of HemECs and human umbilical vein endothelial cells (HUVECs) after TGF-β1 OE (n=4 biological replicates). Scale bar, 100 µm. The unpaired 2-tailed Student t tests were used. HUVECs: TGF-β1 OE vs control: P =0.96, Cohen d =0.44 (not significant, small to medium effect). HemECs: TGF-β1 OE vs control: P <0.001, Cohen d =8.85 (significant, extremely large effect). B , Transwell invasion assay results showing the invasion ability of HemECs and HUVECs after TGF-β1 OE (n=4 biological replicates). Scale bar, 100 µm. The unpaired 2-tailed Student t tests were used. HUVECs: P =0.62, Cohen d =−0.58 (not significant, medium effect). HemECs: P <0.001, Cohen d =9.29 (significant, extremely large effect). C , Tube formation assay showing the angiogenic ability of HemECs in vitro after TGF-β1 OE (n=4 biological replicates). Formed vessels are marked with yellow arrows. Scale bar, 100 µm. The unpaired 2-tailed Student t tests were used. For total tube length (TGF-β1 OE vs control), HUVECs: P =0.42, Cohen d =−0.79 (not significant, medium effect). HemECs: P <0.001, Cohen d =8.37 (significant, extremely large effect). For the number of branch sites (Control vs TGF-β1 OE ), HUVECs: P =0.60, Cohen d =−0.47 (not significant, medium effect). HemECs: P =0.017, Cohen d =3.19 (significant, extremely large effect). D , Vessel formation results showing the angiogenic ability of HemECs in vivo after TGF-β1 OE using 4 mice in each group (n=4). Scale bar, 50 µm. The unpaired 2-tailed Student t tests were used. TGF-β1 OE vs control: P =0.001, Cohen d =7.25 (significant, extremely large effect).

Article Snippet: HemEC isolation from proliferating IHs was performed as described previously., Primary human umbilical vein ECs (HUVECs) were obtained from the American Type Culture Collection (United States).

Techniques: Over Expression, Derivative Assay, Migration, Transwell Migration Assay, Control, Transwell Invasion Assay, Tube Formation Assay, In Vitro, In Vivo

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: TGF-β1 Promotes Angiogenesis via Endothelial-to-Mesenchymal Transition in Infantile Hemangioma

doi: 10.1161/ATVBAHA.125.322793

Figure Lengend Snippet: Changes in lipid metabolism. A , Oil Red O (ORO) staining showing the number of intracellular lipid droplets (LDs) in hemangioma-derived endothelial cells (HemECs) compared with that in human umbilical vein endothelial cells (HUVECs), with the etomoxir treatment group serving as a positive control (n=4 biological replicates). Scale bar, 20 µm. The Dunnett multiple comparisons test was used. HUVECs: TGF-β1 OE (transforming growth factor β1 overexpression) vs control: P =0.16, Cohen d =1.20 (not significant, large effect). Etomoxir vs control: P <0.001, Cohen d =10.54 (significant, extremely large effect). HemECs: TGF-β1 OE vs control: P =0.001, Cohen d =13.38 (significant, extremely large effect). Etomoxir vs control: P <0.001, Cohen d =24.63 (significant, extremely large effect). B , Western blot analysis showing that CPT1A (carnitine palmitoyltransferase 1A) protein expression decreased in HemECs (n=4 biological replicates). The unpaired 2-tailed Student t test was used. HemEC TGF-β1 OE vs control: P =0.001, Cohen d =−5.84 (significant, extremely large effect). C , Targeted metabolic analysis showing the top 30 differential metabolites (DMs) after TGF-β1 (transforming growth factor β1) treatment in HUVECs (n=6 biological replicates). D , Targeted metabolic analysis showing the top 30 differential metabolites (DMs) after TGF-β1 treatment in HemECs (n=6 biological replicates). E , Quantitative analysis of L-palmitoylcarnitine, with the etomoxir treatment group serving as a positive control (n=6 biological replicates). One-way ANOVA followed by the Dunnett multiple comparisons test was used to compare the TGF-β1 and etomoxir groups to the control group. HUVECs: TGF-β1 vs control: P =0.18, Cohen d =−0.83 (not significant, medium effect). Etomoxir vs control: P <0.001, Cohen d =−2.24 (significant, extremely large effect). HemECs: TGF-β1 vs control: P =0.003, Cohen d =−3.06 (significant, extremely large effect). Etomoxir vs control: P <0.001, Cohen d =−4.76 (significant, extremely large effect). F , Changes in the content of long-chain fatty acids (FAs; chain lengths C 14 –C 18 ; n=4 biological replicates). The paired 2-tailed Student t test with the Welch correction was performed separately for each FA chain length (C14, C16, and C18) to compare the TGF-β1–treated and control groups within HemECs and HUVECs. HemECs: C14: P =0.012, Cohen d =2.50 (significant, extremely large effect). C16: P =0.039, Cohen d =1.73 (significant, extremely large effect). C18: P =0.027, Cohen d =1.89 (significant, extremely large effect). HUVECs: C14: P =0.09, Cohen d =−1.37 (not significant, very large effect). C16: P =0.048, Cohen d =−1.60 (significant, large effect). C18: P =0.049, Cohen d =−1.64 (significant, extremely large effect). G , Palmitate-conjugated BSA (Palm-BSA) stimulated oxygen consumption rate (OCR) in HUVECs (n=4 biological replicates). A paired 2-tailed Student t test was performed. Control: P =0.009, Cohen d =4.11 (significant, extremely large effect). TGF-β1: P =0.010, Cohen d =0.55 (significant, medium effect). Etomoxir: P =0.84, Cohen d = –0.08 (not significant, negligible effect). TGF-β1+etomoxir: P =0.22, Cohen d =1.24 (not significant, large to very large effect). BSA: TGF-β1 vs control: P =0.36, Cohen d =−0.70 (not significant, medium effect). Palm-BSA: TGF-β1 vs control: P =0.08, Cohen d =−4.43 (not significant, extremely large effect). Etomoxir vs control: P <0.001, Cohen d =−4.83 (significant, extremely large effect). TGF-β1+etomoxir vs control: P =0.003, Cohen d =−3.30 (significant, extremely large effect). H , Palm-BSA failed to stimulate the OCR, thus inhibiting the fatty acid oxidation (FAO) effect of TGF-β1 or etomoxir treatment in HemECs (n=4 biological replicates). A paired 2-tailed Student t test was performed. Control: P =0.015, Cohen d =1.73 (significant, extremely large effect). TGF-β1: P =0.23, Cohen d =0.33 (not significant, medium effect). Etomoxir: P =0.64, Cohen d = –0.16 (not significant, small effect). TGF-β1+etomoxir: P =0.76, Cohen d =0.24 (not significant, negligible effect). BSA: TGF-β1 vs control: P =0.006, Cohen d =−2.97 (significant, extremely large effect). Palm-BSA: TGF-β1 vs control: P <0.001, Cohen d =−10.95 (significant, extremely large effect). Etomoxir vs control: P =0.002, Cohen d =−3.93 (significant, extremely large effect). TGF-β1+etomoxir vs control: P =0.003, Cohen d =−3.03 (significant, extremely large effect). I , Rate of FAO after TGF-β1 treatment in HUVECs and HemECs (n=4 biological replicates). The paired 2-tailed Student t test was performed. HemECs: P =0.038, Cohen d =−1.75 (significant, large effect). HUVECs: P =0.89, Cohen d =−0.08 (not significant, negligible effect).

Article Snippet: HemEC isolation from proliferating IHs was performed as described previously., Primary human umbilical vein ECs (HUVECs) were obtained from the American Type Culture Collection (United States).

Techniques: Staining, Derivative Assay, Positive Control, Over Expression, Control, Western Blot, Expressing

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: TGF-β1 Promotes Angiogenesis via Endothelial-to-Mesenchymal Transition in Infantile Hemangioma

doi: 10.1161/ATVBAHA.125.322793

Figure Lengend Snippet: CPT1A KD (CPT1A [carnitine palmitoyltransferase 1A] knockdown) stimulates hemangioma-derived endothelial cell (HemEC) migration, invasion, and angiogenesis. A , Transwell migration assay results showing the migration ability of HemECs and human umbilical vein endothelial cells (HUVECs) after CPT1A KD , CPT1A KD +L-palmitoylcarnitine, and CPT1A OE +TGF-β1 OE (TGF-β1 [transforming growth factor β1] overexpression; n=4 biological replicates). Scale bar, 100 µm. The unpaired 2-tailed Student t tests were performed. P values were adjusted using the Benjamini-Hochberg correction. HUVECs: CPT1A KD vs control: P =0.10, Cohen d =1.95 (not significant, large effect). CPT1A KD +L-palmitoylcarnitine vs CPT1A KD : P =0.038, Cohen d =−2.75 (significant, extremely large effect). TGF-β1 OE +CPT1A OE vs CPT1A KD +L-palmitoylcarnitine: P =0.10, Cohen d =1.74 (not significant, large effect). HemECs: CPT1A KD vs control: P <0.001, Cohen d =18.11 (significant, extremely large effect). CPT1A KD +L-palmitoylcarnitine vs CPT1A KD : P <0.001, Cohen d =−14.42 (significant, extremely large effect). TGF-β1 OE +CPT1A OE vs CPT1A KD +L-palmitoylcarnitine: P =0.13, Cohen d =−1.53 (not significant, large effect). B , Transwell invasion assay results showing the invasion ability of HemECs and HUVECs after CPT1A KD , CPT1A KD +L-palmitoylcarnitine, or CPT1A OE +TGF-β1 OE (n=4 biological replicates). Scale bar, 100 µm. The unpaired 2-tailed Student t tests were performed. P values were adjusted using the Benjamini-Hochberg correction. HUVECs: CPT1A KD vs control: P =0.19, Cohen d =1.29 (not significant, extremely large effect). CPT1A KD +L-palmitoylcarnitine vs CPT1A KD : P =0.99, Cohen d =0 (not significant, no effect). TGF-β1 OE +CPT1A OE vs CPT1A KD +L-palmitoylcarnitine: P =0.83, Cohen d =0.19 (not significant, small effect). HemECs: CPT1A KD vs control: P =0.005, Cohen d =4.30 (significant, extremely large effect). CPT1A KD +L-palmitoylcarnitine vs CPT1A KD : P =0.005, Cohen d =−4.31 (significant, extremely large effect). TGF-β1 OE +CPT1A OE vs CPT1A KD +L-palmitoylcarnitine: P =0.16, Cohen d =1.33 (not significant, large effect). C , Tube formation assay showing the angiogenic ability of HemECs in vitro after CPT1A KD , CPT1A KD +L-palmitoylcarnitine, and CPT1A OE +TGF-β1 OE (n=4 biological replicates). Scale bar, 100 µm. D , Vessel formation results showing the angiogenic ability of HemECs in vivo after CPT1A KD , CPT1A KD +L-palmitoylcarnitine, and CPT1A OE +TGF-β1 OE (n=4 biological replicates). Scale bar, 50 µm. The unpaired 2-tailed Student t tests were performed. P values were adjusted using the Benjamini-Hochberg correction. Total length: HUVECs: CPT1A KD vs control: P =0.90, Cohen d =0.11 (not significant, small effect). CPT1A KD +L-palmitoylcarnitine vs CPT1A KD : P =0.18, Cohen d =1.31 (not significant, large effect). TGF-β1 OE +CPT1A OE vs CPT1A KD +L-palmitoylcarnitine: P =0.32, Cohen d =−0.93 (not significant, large effect). HemECs: CPT1A KD vs control: P =0.003, Cohen d =2.73 (significant, extremely large effect). CPT1A KD +L-palmitoylcarnitine vs CPT1A KD : P =0.013, Cohen d =−3.53 (significant, extremely large effect). TGF-β1 OE +CPT1A OE vs CPT1A KD +L-palmitoylcarnitine: P =0.84, Cohen d =0.17 (not significant, small effect). Number of branch sites. HUVECs: CPT1A KD vs control: P =0.91, Cohen d =0.10 (not significant, small effect). CPT1A KD +L-palmitoylcarnitine vs CPT1A KD : P =0.71, Cohen d= −0.33 (not significant, medium effect). TGF-β1 OE +CPT1A OE vs CPT1A KD +L-palmitoylcarnitine: P =0.85, Cohen d =0.16 (not significant, small effect). HemECs: CPT1A KD vs control: P =0.001, Cohen d =3.35 (significant, extremely large effect). CPT1A KD +L-palmitoylcarnitine vs CPT1A KD : P =0.013, Cohen d =−6.40 (significant, extremely large effect). TGF-β1 OE +CPT1A OE vs CPT1A KD +L-palmitoylcarnitine: P =0.66, Cohen d =−0.38 (not significant, small effect). D , Vessel formation results showing the angiogenic ability of HemECs in vivo after CPT1A KD , CPT1A KD +L-palmitoylcarnitine, and CPT1A OE +TGF-β1 OE (n=4 biological replicates). Scale bar, 50 µm. The unpaired 2-tailed Student t tests with the Welch correction were performed. CPT1A KD vs control: P <0.001, Cohen d =8.90 (significant, extremely large effect). CPT1A KD +L-palmitoylcarnitine vs CPT1A KD : P =0.014, Cohen d =−3.81 (significant, extremely large effect). TGF-β1 OE +CPT1A OE vs CPT1A KD +L-palmitoylcarnitine: P =0.72, Cohen d =0.25 (not significant, negligible to small effect).

Article Snippet: HemEC isolation from proliferating IHs was performed as described previously., Primary human umbilical vein ECs (HUVECs) were obtained from the American Type Culture Collection (United States).

Techniques: Knockdown, Derivative Assay, Migration, Transwell Migration Assay, Over Expression, Control, Transwell Invasion Assay, Tube Formation Assay, In Vitro, In Vivo

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: TGF-β1 Promotes Angiogenesis via Endothelial-to-Mesenchymal Transition in Infantile Hemangioma

doi: 10.1161/ATVBAHA.125.322793

Figure Lengend Snippet: CPT1A KD (CPT1A [carnitine palmitoyltransferase 1A] knockdown) suppresses hemangioma-derived endothelial cell (HemEC) autophagy. A , Transmission electron microscopy (TEM) results showing changes in autophagy after CPT1A KD , CPT1A KD +L-palmitoylcarnitine, and CPT1A OE +TGF-β1 OE (TGF-β1 [transforming growth factor β1] overexpression) in HemECs and human umbilical vein endothelial cells (HUVECs; n=4 biological replicates). Scale bar, 1 µm. B , Autophagic vesicles per field (n=4 biological replicates). The unpaired 2-tailed Student t tests were performed. P values were adjusted using the Benjamini-Hochberg correction. HUVECs: CPT1A KD vs control: P =0.33, Cohen d =0.83 (not significant, large effect). CPT1A KD +L-palmitoylcarnitine vs CPT1A KD : P =0.11, Cohen d =−1.54 (not significant, large effect). TGF-β1 OE +CPT1A OE vs CPT1A KD +L-palmitoylcarnitine: P =0.06, Cohen d =1.89 (not significant, large effect). HemECs: CPT1A KD vs control: P =0.004, Cohen d =−3.24 (significant, extremely large effect). CPT1A KD +L-palmitoylcarnitine vs CPT1A KD : P <0.001, Cohen d =9.71 (significant, extremely large effect). TGF-β1 OE +CPT1A OE vs CPT1A KD +L-palmitoylcarnitine: P =0.017, Cohen d =−2.55 (significant, large effect). C , Immunoblot analysis of TGF-β1, CPT1A, and AMPK (5’-monophosphate [AMP]–activated protein kinase; n=3 biological replicates).

Article Snippet: HemEC isolation from proliferating IHs was performed as described previously., Primary human umbilical vein ECs (HUVECs) were obtained from the American Type Culture Collection (United States).

Techniques: Knockdown, Derivative Assay, Transmission Assay, Electron Microscopy, Over Expression, Control, Western Blot

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: TGF-β1 Promotes Angiogenesis via Endothelial-to-Mesenchymal Transition in Infantile Hemangioma

doi: 10.1161/ATVBAHA.125.322793

Figure Lengend Snippet: Changes in the expression of endothelial and mesenchymal markers after R(+) propranolol or S(−) propranolol treatment in hemangioma-derived endothelial cells (HemECs) and human umbilical vein endothelial cells (HUVECs). A , Immunofluorescence images showing the expression of endothelial and mesenchymal markers before and after R(+) propranolol or S(−) propranolol treatment in HemECs and HUVECs. Scale bar, 100 µm (n=4 biological replicates). B , Immunofluorescence analysis showing the mean fluorescence intensity of endothelial and mesenchymal markers before and after R(+) propranolol or S(−) propranolol treatment in HemECs (n=4 biological replicates). The Dunnett multiple comparisons test was used. R(+) propranolol vs control: CD31: P <0.001, Cohen d =16.23 (significant, extremely large effect). VE-cadherin (vascular endothelial cadherin): P <0.001, Cohen d =11.58 (significant, extremely large effect). α-SMA (α-smooth muscle actin): P <0.001, Cohen d =−20.50 (significant, extremely large effect). COL1A1 (collagen type I alpha 1 chain): P =0.001, Cohen d =−11.23 (significant, extremely large effect). S(−) propranolol vs control: CD31: P =0.20, Cohen d =0.99 (not significant, large effect). VE-cadherin: P =0.07, Cohen d =2.62 (not significant, very large effect). α-SMA: P =0.75, Cohen d =0.21 (not significant, small effect). COL1A1: P =0.88, Cohen d =0.11 (not significant, negligible effect). C , Immunofluorescence analysis showing the mean fluorescence intensity of endothelial and mesenchymal markers before and after R(+) propranolol or S(−) propranolol treatment in HUVECs (n=4 biological replicates). The Dunnett multiple comparisons test was used. R(+) propranolol vs control: CD31: P =0.46, Cohen d =0.54 (not significant, medium effect). VE-cadherin: P =0.24, Cohen d =−0.94 (not significant, large effect). α-SMA: P =0.65, Cohen d =0.33 (not significant, small effect). COL1A1: P =0.52, Cohen d =0.48 (not significant, small effect). S(−) propranolol vs control: CD31: P =0.10, Cohen d =−1.36 (not significant, very large effect). VE-cadherin: P =0.79, Cohen d =1.90 (not significant, very large effect). α-SMA: P =0.93, Cohen d =−0.07 (not significant, negligible effect). COL1A1: P =0.87, Cohen d =0.1 (not significant, negligible effect).

Article Snippet: HemEC isolation from proliferating IHs was performed as described previously., Primary human umbilical vein ECs (HUVECs) were obtained from the American Type Culture Collection (United States).

Techniques: Expressing, Derivative Assay, Immunofluorescence, Fluorescence, Control