Journal: JCI Insight

Article Title: HTATIP2 regulates arteriogenic activity in monocytes from patients with limb ischemia

doi: 10.1172/jci.insight.131419

Figure Lengend Snippet: ( A and B ) Representative bright-field images of tubule formation following coculture of HUVECs with proarteriogenic Mo/MΦs isolated from age-matched controls ( A ) and patients with CLTI ( B , n = 5/group). ( C and D ) The length ( C ) and area ( D ) of EC tubules formed in the coculture assay were quantified using an ImageJ macro. Fold-change in tubule expression is relative to that of assays containing HUVECs only. ( E ) Laser Doppler images of paw perfusion at days 3, 7, and 14 following induction of hindlimb ischemia in nude, athymic mice. ( F ) The ischemic limbs of mice were injected with proarteriogenic Mo/MΦs from controls (left) or patients with CLTI (right) ( n = 7/group). Perfusion ratio calculated by comparison with contralateral limb. ( G – K ) Gastrocnemius muscle from the ischemic leg was analyzed for expression of CD31 (red) and laminin (green, G ) and adductor muscle for α-SMA (red, H ) to quantify capillary/fiber ratio ( I ) and α-SMA + arteriole number ( J ) and diameter ( K ) ( n = 5–7/group). * P < 0.05. Scale bar: 10μm. ( C , D , H , J , and K ) Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01 (Mann-Whitney U test). ( F ) Data were analyzed by 2-way ANOVA and post hoc Bonferroni test. *** P < 0.0001. Mo/MΦ, monocyte/macrophage; CLTI, chronic limb threatening ischemia; EC, endothelial cell; α-SMA, α-smooth muscle actin.

Article Snippet: HUVECs and EA.hy926 cells were expanded in culture using endothelial basal media supplemented with 1 μg/mL hydrocortisone, 100 μg/mL penicillin, 100 μg/mL streptomycin sulphate, 250 ng/mL amphotericin B, 10 ng/mL recombinant human EGF, 3 ng/mL bFGF, 3 μg/mL heparin, and 2% fetal calf serum (Promocell).

Techniques: Isolation, Co-culture Assay, Expressing, Injection, Comparison, MANN-WHITNEY

Journal: JCI Insight

Article Title: HTATIP2 regulates arteriogenic activity in monocytes from patients with limb ischemia

doi: 10.1172/jci.insight.131419

Figure Lengend Snippet: ( A and B ) Representative bright-field images of endothelial cells cocultured with si Control siRNA or si Htatip2 siRNA iBMMs ( n = 6/group). ( C and D ) Quantification of EA.hy926 cell tubule length ( C ) and area ( D ) following coculture. Fold-change in tubule expression is relative to that of assays containing EA.hy926 cells only. ( E ) SMC proliferation was assessed when cells were exposed for 24 hours to conditioned media from si Htatip2 - or si Control -iBMMs ( n = 9/group). ( F and G ) The potential of Htatip2-silenced ( n = 6) or si Control -iBMMs ( n = 7) to promote reperfusion in the ischemic hindlimb of C57BL/6 mice was quantified by laser Doppler imaging over 21 days. P < 0.001 by repeated-measures 2-way ANOVA and * P < 0.05, ** P < 0.01, *** P < 0.001 by post hoc Bonferroni test. ( H – L ) Histological analysis of ischemic limb muscle from si Htatip2- and si Control- iBMM–treated animals for CD31 (red) and laminin (green) staining ( H ) and α-SMA staining ( J , red) to quantify capillary/fiber ratio ( I ), arteriole count ( K ), and arteriole diameter ( L ) respectively. Scale bar: 10 μm. ( C – E , I , K , and L ) Data are presented as mean ± SEM. ( C – E ) Data were analyzed by paired t test. * P < 0.05, ** P < 0.01. ( H – L ) Data were analyzed by unpaired t test. ** P < 0.01, *** P < 0.001. ( C and D ) Data are expressed as fold-change compared with tubule formation of EA.hy926 cells alone. ( G ) Data were analyzed by 2-way ANOVA and post hoc Bonferroni test. * P < 0.05, ** P < 0.01, *** P < 0.0001. Mo/MΦ, monocyte/macrophage; iBMM, immortalized bone marrow macrophage; HTATIP2, HIV-1 Tat interactive protein-2; SMC, smooth muscle cell; α-SMA, α-smooth muscle actin.

Article Snippet: HUVECs and EA.hy926 cells were expanded in culture using endothelial basal media supplemented with 1 μg/mL hydrocortisone, 100 μg/mL penicillin, 100 μg/mL streptomycin sulphate, 250 ng/mL amphotericin B, 10 ng/mL recombinant human EGF, 3 ng/mL bFGF, 3 μg/mL heparin, and 2% fetal calf serum (Promocell).

Techniques: Control, Expressing, Imaging, Staining

Journal: Cancers

Article Title: Protein Disulphide Isomerase A1 Is Involved in the Regulation of Breast Cancer Cell Adhesion and Transmigration via Lung Microvascular Endothelial Cells

doi: 10.3390/cancers12102850

Figure Lengend Snippet: Effects of PDIA1 inhibition on wound-healing and migration of breast cancer cells and endothelial cells. ECIS Wound-Healing Assay of hLMVEC, MCF-7 and MDA-MB-231 cells and breast cancer sublines with silencing of PDIA1 (shN, shPDIA1-1 and shPDIA1-3). Real time tracings in hLMVECs ( A ), MCF-7 ( C ) and MDA-MB-231 ( E ) cell lines after addition of bepristat 2a at various concentrations (1, 10, 30 or 50 µM) and after PDIA1 silencing in MCF-7 ( G ) and MDA-MB-231 ( I ) cell lines. Area under the curve boxplots represent AUC quantitation of changes in migration rate of bepristat 2a-treated hLMVECs ( B ), MCF-7 ( D ) and MDA-MB-231 ( F ) cell lines versus non-treated controls as well as MCF-7 ( H ) or MDA-MB-231 ( J ) sublines transduced against PDIA1 (shPDIA1-1, shPDIA1-3) or wild type cells regarding to negative sequence (shN). The line graphs and AUC boxplots represent mean ± SD of three independent experiments. Statistical analysis was calculated using parametric one-way ANOVA followed by Dunnett’s multiple comparisons test (* p = 0.05, ** p = 0.01, *** p = 0.001).

Article Snippet: Human lung microvascular endothelial cell line (hLMVECs) was obtained from Cell Applications (San Diego, CA, USA).

Techniques: Inhibition, Migration, Wound Healing Assay, Quantitation Assay, Sequencing

Journal: Cancers

Article Title: Protein Disulphide Isomerase A1 Is Involved in the Regulation of Breast Cancer Cell Adhesion and Transmigration via Lung Microvascular Endothelial Cells

doi: 10.3390/cancers12102850

Figure Lengend Snippet: Effects of exogenous PDIA1 and PDIA3 on adhesive interaction between breast cancer cells and different substrates. Effect of exogenous proteins PDIA1 and PDIA3 on adhesion of MCF-7 ( A – F ) and MDA-MB-231 ( G – L ) cells to collagen type I, fibronectin and lung microvascular hLMVEC cells, respectively. Data represent mean ± SD of three independent experiments. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparisons test (* p = 0.05, ** p = 0.01, *** p = 0.001).

Article Snippet: Human lung microvascular endothelial cell line (hLMVECs) was obtained from Cell Applications (San Diego, CA, USA).

Techniques: Adhesive

Journal: PLoS ONE

Article Title: Prox1 and FOXC2 Act as Regulators of Lymphangiogenesis and Angiogenesis in Oral Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0092534

Figure Lengend Snippet: Prox1 (a) and FOXC2 (b) expression were not observed in non-tumor oral mucosa. The lymphoendothelial cells (arrow) showed immunoreactivity to Prox1 (c) and expression of FOXC2 was found in lymphoendothelial cells (arrow) and vascular endothelial cells (arrow head) (d). Expression of Prox1 (e) and FOXC2 (f) were observed in cytoplasm of the cancer cells. LYVE1 positive lymphovessels (g) and CD34 positive blood vessels were counted for MVD and LVD, respectively. Original magnification was 200-fold. Bar, 100 μm. LEC; lymphoendothelial cells, VEC; vascular endothelial cells.

Article Snippet: Primary human umbilical vein endothelial cells (HUVECs) and primary human dermal lymphatic microvascular endothelial cells (HDLMVECs) were purchased from Cell Applications (San Diego, CA, USA) and maintained in Endothelial growth media (Cell Applications) and Microvacular endothelial growth media (Cell Applications) under the conditions of 5% CO 2 in air at 37°C, respectively.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Prox1 and FOXC2 Act as Regulators of Lymphangiogenesis and Angiogenesis in Oral Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0092534

Figure Lengend Snippet: a , Cell growth of endothelial cells treated with conditioned medium from OSCC cells. The growth of HUVECs and HDLMVECs were significantly enhanced by the addition of negative siRNA treated KON cell culture supernatant. Further, HUVECs or HDLMVECs proliferation was inhibited when added to FOXC2 or Prox1 siRNA treated KON cell culture supernatant, respectively. b , Migration of endothelial cells co-cultured with OSCC cells. The migration of HUVECs and HDLMVECs were enhanced by co-culture with negative siRNA treated KON cells. Moreover, co-cultivation with FOXC2 or Prox1 siRNA treated KON cells suppressed HUVECs or HDLMVECs migration, respectively.

Article Snippet: Primary human umbilical vein endothelial cells (HUVECs) and primary human dermal lymphatic microvascular endothelial cells (HDLMVECs) were purchased from Cell Applications (San Diego, CA, USA) and maintained in Endothelial growth media (Cell Applications) and Microvacular endothelial growth media (Cell Applications) under the conditions of 5% CO 2 in air at 37°C, respectively.

Techniques: Cell Culture, Migration, Co-Culture Assay

Journal: Stem Cells Translational Medicine

Article Title: Spontaneous In Vivo Chondrogenesis of Bone Marrow-Derived Mesenchymal Progenitor Cells by Blocking Vascular Endothelial Growth Factor Signaling

doi: 10.5966/sctm.2015-0321

Figure Lengend Snippet: Inhibitory effect of sFlk-1 expressed by bone marrow-derived mesenchymal stromal/stem cells (MSCs) on in vitro endothelial organization and proliferation. Human umbilical vein endothelial cell proliferation assay performed by using supernatants collected by naïve (control) or sFlk-1-expressing MSCs. (A): Cell metabolic activity was measured on the basis of the colorimetric MTS assay and represented by using optical density units. (B): Immunofluorescence staining for CD31 in green showing the capillary-like structure formation in the direct coculture of either control or sFlk-1-expressing MSCs with human dermal microvascular endothelial cells. (C): Representative images of tubular structure formation of endothelial cells (stained with calcein assay medium in green after 48 hours) cultured in medium conditioned by control or sFlk-1 expressing MSCs supplemented with 0, 10, or 50 ng/ml of VEGF. Scale bars = 300 µm. (D): Total tube number, length, branching points, and loops were quantified by image analysis. Ten images per sample were analyzed. Data are presented as mean ± SD (n = 4 samples/group from 2 independent experiments). ∗, p < .05; ∗∗, p < .01. Abbreviations: OD, optical density; VEGF, vascular endothelial growth factor.

Article Snippet: In vitro tube formation assay was performed with human dermal microvascular endothelial cells (HDMECs) as previously described [ 29 ], either by using control or sFlk-1 MSC conditioned media (Endothelial Cell Basal Medium MV [PromoCell, Heidelberg, Germany, http://www.promocell.com/ ] with 5% FBS and 1% penicillin-streptomycin solution) extracted after 48 hours or by direct coculture of control or sFlk-1 MSC.

Techniques: Derivative Assay, In Vitro, Proliferation Assay, Expressing, Activity Assay, MTS Assay, Immunofluorescence, Staining, Cell Culture

Journal: Stem Cells Translational Medicine

Article Title: Spontaneous In Vivo Chondrogenesis of Bone Marrow-Derived Mesenchymal Progenitor Cells by Blocking Vascular Endothelial Growth Factor Signaling

doi: 10.5966/sctm.2015-0321

Figure Lengend Snippet: In vivo blocking of angiogenesis. (A): Representative macroscopic picture of the explants of engineered tissue generated by naïve (left) or sFlk-1-expressing (right) MSCs after 12 weeks in vivo. (B): Vessel density quantified within the total area of the implant generated by control (naïve) or sFlk-1 MSCs at different time points by histomorphometric analysis (n = 3–4). ∗, p < .05. (C): Immunohistochemistry for mouse CD31. Representative images at ×20 magnification include the central implant area generated by naive (top row) or sFlk-1 (bottom row) MSCs at 1, 4, 8, or 12 weeks. Black arrowheads indicate the blood vessels. Scale bar = 100 µm. Abbreviation: MSC, bone marrow-derived mesenchymal stromal/stem cell.

Article Snippet: In vitro tube formation assay was performed with human dermal microvascular endothelial cells (HDMECs) as previously described [ 29 ], either by using control or sFlk-1 MSC conditioned media (Endothelial Cell Basal Medium MV [PromoCell, Heidelberg, Germany, http://www.promocell.com/ ] with 5% FBS and 1% penicillin-streptomycin solution) extracted after 48 hours or by direct coculture of control or sFlk-1 MSC.

Techniques: In Vivo, Blocking Assay, Generated, Expressing, Immunohistochemistry, Derivative Assay

Journal: Stem Cells Translational Medicine

Article Title: Spontaneous In Vivo Chondrogenesis of Bone Marrow-Derived Mesenchymal Progenitor Cells by Blocking Vascular Endothelial Growth Factor Signaling

doi: 10.5966/sctm.2015-0321

Figure Lengend Snippet: In vivo chondrogenesis. Histological staining with Safranin-O for glycosaminoglycans and immunohistochemistry for type II collagen of engineered tissue generated by naïve (control) or sFlk-1 MSCs after 4 (A) or 12 (B) weeks in vivo. Fluorescence staining with DAPI (in blue) and a specific anti-human nuclei antibody (in red) of constructs generated by control or sFlk-1 MSCs after 4 (A) or 12 (B) weeks in vivo. Scale bar = 100 µm. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; MSC, bone marrow-derived mesenchymal stromal/stem cell.

Article Snippet: In vitro tube formation assay was performed with human dermal microvascular endothelial cells (HDMECs) as previously described [ 29 ], either by using control or sFlk-1 MSC conditioned media (Endothelial Cell Basal Medium MV [PromoCell, Heidelberg, Germany, http://www.promocell.com/ ] with 5% FBS and 1% penicillin-streptomycin solution) extracted after 48 hours or by direct coculture of control or sFlk-1 MSC.

Techniques: In Vivo, Staining, Immunohistochemistry, Generated, Fluorescence, Construct, Derivative Assay