Journal: Circulation Research

Article Title: S100A1 Deficiency Impairs Postischemic Angiogenesis Via Compromised Proangiogenic Endothelial Cell Function and Nitric Oxide Synthase Regulation

doi: 10.1161/circresaha.112.275156

Figure Lengend Snippet: Figure 5. Reduced vascular endothelial growth factor (VEGF)–stimulated Nitric oxide (NO) production in S100A1 knockout (SKO) endothelial cells (ECs) and hindlimbs. A, NO synthase (NOS) activity of skeletal muscle extracts procured at different times after femoral artery resection was assessed by measuring nitrite production (n=6; *P<0.05 SKO vs wild-type [WT]). B, Primary ECs derived from WT and SKO mice or (C) human microvascular ECs (HMVECs) subjected to small interfering RNA (siRNA)–mediated knockdown of S100A1 were treated with 50 ng/mL VEGF for 16 hours. Endothelial NOS (eNOS)–derived nitrite was determined in the cell supernatant as described in the Methods section (*P<0.05 vs untreated WT or VEGF-treated SKO [B] or S100A1 siRNA [C], respectively; experiments were performed in duplicate and repeated 3 times). D, VEGF-dependent phosphorylation status of p-S1177 or p-T495 eNOS was examined in HMVECs subjected to S100A1 knockdown (*P<0.05 vs S100A1 kd pT495 eNOS; #P<0.05 vs scr pS1177 eNOS). E, Protein kinase C (PKC) activity was examined in HMVECs subjected to S100A1 knockdown and treated with 50 ng/mL VEGF for 15 minutes. (n=3 independent assays, performed in triplicate). Calphostin-C (100 ng/mL) was included to inhibit PKC, where indicated.

Article Snippet: Stimulation with human recombinant VEGF (50 ng/ml; R&D Systems 293-VE) was carried out both under normoxic (21%O2) and hypoxic (2%O2) conditions.

Techniques: Knock-Out, Activity Assay, Derivative Assay, Small Interfering RNA, Knockdown, Phospho-proteomics

Journal: Circulation Research

Article Title: S100A1 Deficiency Impairs Postischemic Angiogenesis Via Compromised Proangiogenic Endothelial Cell Function and Nitric Oxide Synthase Regulation

doi: 10.1161/circresaha.112.275156

Figure Lengend Snippet: Figure 7. Augmented activation of hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) in the ischemic hind limb of S100A1 knockout (SKO) mice disturbs VEGF receptor 2 (VEGFR2) signaling. SKO and wild-type (WT) mice were subjected to femoral artery resection, and gastrocnemius muscles of the ischemic and contralateral nonischemic hindlimb were procured and homogenized after 3 days (except for VEGF). Representative samples are shown for all immunoblots. Muscle extracts were immunoblotted for (A) HIF-1α, (B) VEGF (at times indicated), (C) total and p-Y1175 VEGFR2 and VEGFR1, and (D) total and p-S473-Akt. All expression levels were normalized to GAPDH (n=4 for each genotype and condition; *P<0.05 vs ischemic WT).

Article Snippet: Stimulation with human recombinant VEGF (50 ng/ml; R&D Systems 293-VE) was carried out both under normoxic (21%O2) and hypoxic (2%O2) conditions.

Techniques: Activation Assay, Knock-Out, Muscles, Western Blot, Expressing

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Pioglitazone inhibits HIF-1α-dependent angiogenesis in rats by paracrine and direct effects on endothelial cells

doi: 10.1007/s00109-013-1115-0

Figure Lengend Snippet: Pioglitazone is associated with reduced gastrocnemius lectin perfusion in vivo. a Representative confocal images of the gastrocnemius muscle of vehicle- ( left ) and pioglitazone-treated rats perfused with lectin ( green ) immediately prior to sacrifice. Pioglitazone decreases the lectin perfusion ( left ) in both diabetic and non-diabetic models compared to vehicle controls. Values are represented as mean ± SEM of the ischemic reserve (the ratio of the signal in the ligated/non-ligated leg) (* p < 0.05 vs. respective placebo-treated control group). Bar 200 μm. b Western blot showing reduced HIF-1α expression in both non-ligated and ligated limbs exposed to pioglitazone (* p < 0.05 vs. vehicle-treated group). Three different blots, each with samples from three different rats, were quantified. Each lane represents muscle tissue from one animal. c Western blot showing reduced VEGF expression in the gastrocnemius muscles with ligated vessels in pioglitazone-treated animals compared to vehicle-treated animals (* p < 0.05 vs. vehicle-treated group)

Article Snippet: Primary antibodies used include VEGF (1:100; Santa Cruz Biotechnologies), HIF-1a (1:100; Abcam, San Francisco, CA), vWF (1:200; Abcam, San Francisco, CA, San Francisco, CA), SMA (1:100; Abcam, San Francisco, CA).

Techniques: In Vivo, Control, Western Blot, Expressing, Muscles

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Pioglitazone inhibits HIF-1α-dependent angiogenesis in rats by paracrine and direct effects on endothelial cells

doi: 10.1007/s00109-013-1115-0

Figure Lengend Snippet: Pioglitazone inhibits HIF-1α in hSkMCs. a Representative immunofluorescence confocal images of hSkMCs in normoxic, hypoxic, or hypoxic plus pioglitazone conditions and stained with HIF-1α ( red ), VEGF ( green ), and the nuclear stain DAPI ( blue ). Pioglitazone reduces nuclear (active) HIF-1α and VEGF expression in hypoxic hSkMCs (* p < 0.05 vs. hypoxia vehicle). Bar 20 μm. b mRNA expression of HIF-1α target genes, HIF-1α and VEGF-R, in hSkMCs exposed to hypoxia plus vehicle or pioglitazone. Pioglitazone reduces HIF-1α and VEGF-R mRNA ( n = 3 experiments. * p < 0.05 vs. hypoxia vehicle)

Article Snippet: Primary antibodies used include VEGF (1:100; Santa Cruz Biotechnologies), HIF-1a (1:100; Abcam, San Francisco, CA), vWF (1:200; Abcam, San Francisco, CA, San Francisco, CA), SMA (1:100; Abcam, San Francisco, CA).

Techniques: Immunofluorescence, Staining, Expressing

Journal: Stem Cells (Dayton, Ohio)

Article Title: Follistatin‐Like 3 Enhances the Function of Endothelial Cells Derived from Pluripotent Stem Cells by Facilitating β‐Catenin Nuclear Translocation Through Inhibition of Glycogen Synthase Kinase‐3β Activity

doi: 10.1002/stem.2820

Figure Lengend Snippet: Induced pluripotent stem cell differentiation toward endothelial cells. Human iPS Cells were differentiated using StemPro serum free media supplemented with BMP4, Activin A, fibroblast growth factor (FGF), and vascular endothelial growth factor (VEGF) for 5 days. The differentiated cells were seeded on collagen IV, while CD144 positive cells were magnetically sorted on day 6 using MicroBeads Kit (Miltenyi BIotec) and culturing in EGM‐2 media (LONZA). (A) : Images show morphology of iPS Cells (left panel) and of their differentiated EC counterparts (right panel) Scale bar: 50 μm. (B) : Real time polymerase chain reaction (PCR) data revealed significantly increased early and (C) late EC marker mRNA expression (Data is means ±SEM [ n = 3], *, p < .05; **, p < .01; ***, p < .001). (D) : Western blots showing protein expression of the EC‐specific marker CD144 in iPS‐ECs and (E) corresponding densitometry. (F) : Immunofluorescent confocal images showing that iPS‐ECs express typical EC markers, CD144, KDR, CD31, and vWF, also costained with DAPI (4′,6‐diamidino‐2‐phenylindole, dihydrochloride). (G) : iPS‐ECs stained positive for CD144, were able to uptake LDL and form tubes in vitro. Scale bar: 50 μm. The data presented are representative or means (±SEM) of three independent experiments. Abbreviations: DAPI, 4′,6‐diamidino‐2‐phenylindole; EC, endothelial cell; GAPDH, Glyceraldehyde 3‐phosphate dehydrogenase; iPS, induced pluripotent stem; LDL, low density lipoprotein.

Article Snippet: Antibodies against VE‐Cadherin (CD144) (ab33168 and STJ96234), VEGFR (ab9530), GAPDH (ab8245), OCT‐4 (ab19857 and STJ72238), KLF4 (ab72543) eNOS (ab76198), and FSTL3 (STJ112317) were purchased from Abcam, UK or St John's Laboratory, UK.

Techniques: Cell Differentiation, Real-time Polymerase Chain Reaction, Marker, Expressing, Western Blot, Staining, In Vitro

Journal: Stem Cells (Dayton, Ohio)

Article Title: Follistatin‐Like 3 Enhances the Function of Endothelial Cells Derived from Pluripotent Stem Cells by Facilitating β‐Catenin Nuclear Translocation Through Inhibition of Glycogen Synthase Kinase‐3β Activity

doi: 10.1002/stem.2820

Figure Lengend Snippet: FSTL3 is highly expressed in iPS‐ECs. (A) : FSTL3 was progressively induced during EC differentiation in iPS‐ECs at the mRNA level, both preselection (pre‐iPS‐ECs) and postselection with CD144 (iPS‐ECs) (Data is means ± SEM [ n = 3], *, p < .05). (B) : Western Blot showing FSTL3 protein being expressed in parallel with the EC‐specific marker CD144 and (C) corresponding densitometry. (D) : FSTL3 secretion was progressively induced in cell culture media, as shown by ELISA (Data is means ± SEM [ n = 3], *, p < .05). (E) : Immunofluorescent images of FSTL3 in iPS‐ECs, Scale bar: 50 µm. (F) : Immunofluorescent staining showing that FSTL3 is expressed in parallel with the EC‐specific marker CD144 in iPS‐ECs. Scale bar: 50 µm. The data presented are representative or means (±SEM) of three independent experiments. Abbreviations: DAPI, 4′,6‐diamidino‐2‐phenylindole; ECs, endothelial cells; FSTL3, follistatin‐like 3; iPS, induced pluripotent stem.

Article Snippet: Antibodies against VE‐Cadherin (CD144) (ab33168 and STJ96234), VEGFR (ab9530), GAPDH (ab8245), OCT‐4 (ab19857 and STJ72238), KLF4 (ab72543) eNOS (ab76198), and FSTL3 (STJ112317) were purchased from Abcam, UK or St John's Laboratory, UK.

Techniques: Western Blot, Marker, Cell Culture, Enzyme-linked Immunosorbent Assay, Staining

Journal: Stem Cells (Dayton, Ohio)

Article Title: Follistatin‐Like 3 Enhances the Function of Endothelial Cells Derived from Pluripotent Stem Cells by Facilitating β‐Catenin Nuclear Translocation Through Inhibition of Glycogen Synthase Kinase‐3β Activity

doi: 10.1002/stem.2820

Figure Lengend Snippet: FSTL3 regulates endothelial cell (EC) marker expression in induced pluripotent stem (iPS)‐ECs. iPS‐ECs were transfected with the EX‐FSTL3 plasmid carrying the red mCherry fluorophore, or an empty control vector plasmid (EX‐mCherry). Forty‐eight hours later, FSTL3 overexpression induced (A) mRNA and (B, C) protein expression of EC markers CD144, eNOS, and KDR (Data is means ± SEM [ n = 3], **, p < .01; ***, p < .001). FSTL3 knockdown by short hairpin RNA (shRNA) in iPS‐ECs resulted in suppression of EC markers at the (D) mRNA and (E, F) protein levels when the cells were harvested 72 hours later (Data is means ± SEM [ n = 3], ***, p < .001). (G) : Fluorescent microscope images of iPS‐ECs transfected with EX‐FSTL3 plasmid carrying the red mCherry fluorophore, or an empty control vector plasmid (EX‐mCherry). Scale bar: 50 µm. (H) : Treatment of the cells with 25–50 ng/ml of recombinant FSTL3 (R FSTL3) increased EC marker expression such as CD144, eNOS, and KDR compared to phosphate buffered saline (PBS) control (R CTL) (Data is means ± SEM [ n = 3], ***, p < .001). ELISA showing FSTL3 concentration in cell culture media after FSTL3 overexpression compared to control (Data is means ± SEM [ n = 3], **, p < .01). The data presented are representative or means (±SEM) of three independent experiments. Abbreviations: FSTL3, follistatin‐like 3; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase. (I) Enzyme‐linked immunosorbent assay (ELISA) showed increased concentration of FSTL3 in the media after overexpression with EX‐FSTL3 plasmid (Data is means ± SEM [n < 3], **, p < .01

Article Snippet: Antibodies against VE‐Cadherin (CD144) (ab33168 and STJ96234), VEGFR (ab9530), GAPDH (ab8245), OCT‐4 (ab19857 and STJ72238), KLF4 (ab72543) eNOS (ab76198), and FSTL3 (STJ112317) were purchased from Abcam, UK or St John's Laboratory, UK.

Techniques: Marker, Expressing, Transfection, Plasmid Preparation, Control, Over Expression, Knockdown, shRNA, Microscopy, Recombinant, Saline, Enzyme-linked Immunosorbent Assay, Concentration Assay, Cell Culture

Journal: Stem Cells (Dayton, Ohio)

Article Title: Follistatin‐Like 3 Enhances the Function of Endothelial Cells Derived from Pluripotent Stem Cells by Facilitating β‐Catenin Nuclear Translocation Through Inhibition of Glycogen Synthase Kinase‐3β Activity

doi: 10.1002/stem.2820

Figure Lengend Snippet: FSTL3 induced vascular tube formation in vivo and significantly improved angiogenesis, neovascularization, and blood flow recovery in the hind limb ischemic model. Human induced pluripotent stem (iPS)‐ECs overexpressing FSTL3 enhanced the formation of vascular‐like tubes in vivo. (A) : iPS‐ECs overexpressing FSTL3 (EX‐FSTL3), significantly induced angiogenesis, after subcutaneous injection in severe combined immunodeficiency (SCID) mice, in comparison to the control iPS‐ECs (PBS CTL) or control iPS‐ECs expressing mCherry (EX‐mCherry), as shown by H&E staining and (B) capillary density quantification (Data is means ± SEM [ n = 3], **, p < .01). Scale bar: 50 µm. (C) : phosphate buffered saline (PBS)‐CTL and iPS‐ECs overexpressing mCherry (EX‐mCherry) or FSTL3 (EX‐FSTL3) were injected intramuscularly in NOD.CB17‐Prkdcscid/NcrCrl SCID mice immediately after induction of hind limb ischemia. Laser Doppler images show blood flow (BF) in the lower limbs of mice in prone position on day 14. (D) : BF recovery in the ischemic foot (calculated as a percentage ratio between ischemic foot BF and the contralateral foot) for each of the conditions. Statistical analysis shows significantly higher BF recovery in the EX‐FSTL3 treated mice at 14 days in comparison to controls; (Data is means ± SEM [ n = 3], *, p < .05). (E) : Sections of adductor muscles were stained with CD144 antibody for each condition. (F) : Quantified capillary density of CD144‐stained cells, expressed as capillary number per mm 2 (Data is means ± SEM [ n = 3], *, p < .05). (G) Quantified capillary density of CD144‐ and mCherry‐stained cells (Data is means ± SEM; **, p < .01; quantification from 10 random microscopic fields at ×40, scale: 50 μm). The data presented are representative or means (±SEM) of three independent experiments. Abbreviations: DAPI, 4′,6‐diamidino‐2‐phenylindole; ECs, endothelial cells; FSTL3, follistatin‐like 3.

Article Snippet: Antibodies against VE‐Cadherin (CD144) (ab33168 and STJ96234), VEGFR (ab9530), GAPDH (ab8245), OCT‐4 (ab19857 and STJ72238), KLF4 (ab72543) eNOS (ab76198), and FSTL3 (STJ112317) were purchased from Abcam, UK or St John's Laboratory, UK.

Techniques: In Vivo, Injection, Comparison, Control, Expressing, Staining, Saline, Muscles

Journal: Stem cell research & therapy

Article Title: Acidic preconditioning of endothelial colony-forming cells (ECFC) promote vasculogenesis under proinflammatory and high glucose conditions in vitro and in vivo.

doi: 10.1186/s13287-018-0872-7

Figure Lengend Snippet: Fig. 1 Acidic preconditioning increased endothelial colony-forming cell (EFCF) adhesion and pro-angiogenic factor release. a Nonpreconditioned (npECFC) or preconditioned ECFC (pECFC) (grey and black bars, respectively) were seeded onto fibronectin, collagen type I, or TNFα-activated human umbilical vein endothelial cells (HUVEC) and, after 30 min or 2 h, the number of adherent cells was counted (n = 5). b ECFC were perfused onto collagen or TNFα-activated HUVEC for 15 min at shear force 1 dyn/cm2 and the number of adherent cells was counted (n = 4). c ECFC were seeded onto fibronectin for 30 min and focal adhesion kinase (FAK) phosphorylation was determined by Western blot. Each membrane was reprobed with anti-actin antibody to calculate the relative integrated optical density (IOD) (n = 4). d ECFC were stained with antibodies against ICAM, E-selectin, α5, β1, or αVβ3 and then analyzed by flow cytometry (n = 3). e Interleukin (IL)-8, transforming growth factor (TGF)β1, basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), and IL-4 or IL-10, vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) cytokines were measured in the supernatants of npECFC or pECFC after 24 or 48 h, respectively (n = 5). *p < 0.05 vs npECFC. AU, arbitrary units; MFI, mean fluorescence intensity

Article Snippet: Quantification of pro-angiogenic factor levels The concentration of basic fibroblast growth factor (bFGF), IL-10 (Biolegend, San Diego, CA, USA), transforming growth factor (TGF)β1, IL-8, IL-4 (eBioscience), vascular endothelial growth factor (VEGF; R&D Systems, Inc., Minneapolis, MN, USA), platelet-derived growth factor (PDGF; Abcam), and epidermal growth factor (EGF; Life Technologies, Carlsbad, CA, USA) in ECFC culture supernatants were measured by enzyme-linked immunosorbent assay (ELISA) at the indicated time points following the manufacturer’s instructions.

Techniques: Shear, Phospho-proteomics, Western Blot, Membrane, Staining, Flow Cytometry, Derivative Assay, Fluorescence

Journal: Stem cell research & therapy

Article Title: Acidic preconditioning of endothelial colony-forming cells (ECFC) promote vasculogenesis under proinflammatory and high glucose conditions in vitro and in vivo.

doi: 10.1186/s13287-018-0872-7

Figure Lengend Snippet: Fig. 2 Monosodium urate (MSU) induced endothelial colony-forming cell (EFCF) necrosis. a ECFC were treated with MSU at the indicated concentration and the cell death was analyzed by fluoresce microscopy after 24 h (n = 5). b Kinetics of ECFC death were studied by fluorescence microscopy (n = 4). c Representative images of viable (V) and necrotic (N) cells are shown. Original magnification, 1200×. Scale bar = 10 μm. d The percentage of cell death was measured by fluorescence microscopy in the presence or absence of Z-VAD-fmk (30 μM). The percentage of positive cells for active caspase-3 (e), interleukin (IL)-1β levels (f), and reactive oxygen species (ROS) formation (g) were analyzed by flow cytometry after 24 h (n = 3). *p < 0.05 vs untreated. NS, not significant

Article Snippet: Quantification of pro-angiogenic factor levels The concentration of basic fibroblast growth factor (bFGF), IL-10 (Biolegend, San Diego, CA, USA), transforming growth factor (TGF)β1, IL-8, IL-4 (eBioscience), vascular endothelial growth factor (VEGF; R&D Systems, Inc., Minneapolis, MN, USA), platelet-derived growth factor (PDGF; Abcam), and epidermal growth factor (EGF; Life Technologies, Carlsbad, CA, USA) in ECFC culture supernatants were measured by enzyme-linked immunosorbent assay (ELISA) at the indicated time points following the manufacturer’s instructions.

Techniques: Concentration Assay, Microscopy, Fluorescence, Flow Cytometry

Journal: Stem cell research & therapy

Article Title: Acidic preconditioning of endothelial colony-forming cells (ECFC) promote vasculogenesis under proinflammatory and high glucose conditions in vitro and in vivo.

doi: 10.1186/s13287-018-0872-7

Figure Lengend Snippet: Fig. 4 Acidic preconditioning improved endothelial colony-forming cell (EFCF) proliferation and tubule formation under adverse conditions. Nonpreconditioned or preconditioned ECFC (npECFC or pECFC, respectively) were treated with high glucose, tumor necrosis factor (TNF)α, or their combination, and the different angiogenic responses were measured. a Cell counts were performed after 48 h using a Neubauer chamber (n = 4). b Cells were seeded on Geltrex™matrix and the number of branch points per field was measured after 18 h. c Representative images of six different experiments. Original magnification, 40×. *p < 0.05 vs untreated npECFC; #p < 0.05 vs npECFC with the same treatment; &p < 0.05 vs TNFα or glucose alone

Article Snippet: Quantification of pro-angiogenic factor levels The concentration of basic fibroblast growth factor (bFGF), IL-10 (Biolegend, San Diego, CA, USA), transforming growth factor (TGF)β1, IL-8, IL-4 (eBioscience), vascular endothelial growth factor (VEGF; R&D Systems, Inc., Minneapolis, MN, USA), platelet-derived growth factor (PDGF; Abcam), and epidermal growth factor (EGF; Life Technologies, Carlsbad, CA, USA) in ECFC culture supernatants were measured by enzyme-linked immunosorbent assay (ELISA) at the indicated time points following the manufacturer’s instructions.

Techniques:

Journal: Stem cell research & therapy

Article Title: Acidic preconditioning of endothelial colony-forming cells (ECFC) promote vasculogenesis under proinflammatory and high glucose conditions in vitro and in vivo.

doi: 10.1186/s13287-018-0872-7

Figure Lengend Snippet: Fig. 3 Preconditioned endothelial colony-forming cells (EFCF) are more resistant to DAMP- or TNFα-induced cell death. Nonpreconditioned or preconditioned ECFC (npECFC or pECFC, respectively) were incubated with monosodium urate (MSU), tumor necrosis factor (TNF)α, or histones at the indicated concentrations. a The nuclear morphology was analyzed by fluorescence microscopy and (b) the percentage of annexin V and propidium iodide (PI) double-positive cells was measured by flow cytometry after 24 h (n = 6). c ECFC were incubated with high glucose, TNFα, or their combination and cell death was analyzed after 24 h (n = 6). *p < 0.05 vs untreated npECFC; #p < 0.05 vs npECFC with the same treatment

Article Snippet: Quantification of pro-angiogenic factor levels The concentration of basic fibroblast growth factor (bFGF), IL-10 (Biolegend, San Diego, CA, USA), transforming growth factor (TGF)β1, IL-8, IL-4 (eBioscience), vascular endothelial growth factor (VEGF; R&D Systems, Inc., Minneapolis, MN, USA), platelet-derived growth factor (PDGF; Abcam), and epidermal growth factor (EGF; Life Technologies, Carlsbad, CA, USA) in ECFC culture supernatants were measured by enzyme-linked immunosorbent assay (ELISA) at the indicated time points following the manufacturer’s instructions.

Techniques: Incubation, Fluorescence, Microscopy, Flow Cytometry

Journal: Stem cell research & therapy

Article Title: Acidic preconditioning of endothelial colony-forming cells (ECFC) promote vasculogenesis under proinflammatory and high glucose conditions in vitro and in vivo.

doi: 10.1186/s13287-018-0872-7

Figure Lengend Snippet: Fig. 5 Acidic preconditioning improves endothelial colony-forming cell (EFCF) migration under stressful conditions. Nonpreconditioned or preconditioned ECFC (npECFC or pECFC, respectively) were treated with high glucose, tumor necrosis factor (TNF)α, or their combination, and the different angiogenic responses were measured. a The extent of cell migration into the scratched area was measured after 0, 6, and 18 h (n = 5). b Images show the kinetic of gap closure in npECFC or pECFC treated with both high glucose and TNFα. Original magnification, 40×. c Chemotaxis in response to SDF1 (20 ng/ml) was determined using transwell inserts. The number of migrated cells was counted after 6 h (n = 4). *p < 0.05 vs untreated npECFC; #p < 0.05 vs npECFC with the same treatment; &p < 0.05 vs TNFα or glucose alone

Article Snippet: Quantification of pro-angiogenic factor levels The concentration of basic fibroblast growth factor (bFGF), IL-10 (Biolegend, San Diego, CA, USA), transforming growth factor (TGF)β1, IL-8, IL-4 (eBioscience), vascular endothelial growth factor (VEGF; R&D Systems, Inc., Minneapolis, MN, USA), platelet-derived growth factor (PDGF; Abcam), and epidermal growth factor (EGF; Life Technologies, Carlsbad, CA, USA) in ECFC culture supernatants were measured by enzyme-linked immunosorbent assay (ELISA) at the indicated time points following the manufacturer’s instructions.

Techniques: Migration, Chemotaxis Assay

Journal: Stem cell research & therapy

Article Title: Acidic preconditioning of endothelial colony-forming cells (ECFC) promote vasculogenesis under proinflammatory and high glucose conditions in vitro and in vivo.

doi: 10.1186/s13287-018-0872-7

Figure Lengend Snippet: Fig. 6 Acidic preconditioning enhanced endothelial colony-forming cell (EFCF)-mediated neovascularization in ischemic hind limbs of normoglycemic and diabetic mice. Phosphate-buffered saline (PBS), and nonpreconditioned or preconditioned ECFC (npECFC or pECFC, respectively) were infused intravenously in normoglycemic and type 2 diabetic (T2D) mice 5 h after ischemia-inducing surgery. a Perfusion index was calculated by Doppler analysis after 7 and 14 days postischemia. Representative images of T2D animals are shown. b Histologic analysis of gastrocnemius muscles, stained with hematoxylin and eosin (H/E) or Masson’s trichrome, was performed after 14 days postischemia. Original magnification, 400×. Scale bar = 80 μm c Inflammation score was calculated in H/E stained sections. d Vascular density was measured in Masson’s trichrome stained sections. n = 6 per group. *p < 0.05 vs PBS; #p < 0.05 vs npECFC; &p < 0.05 vs the same treatment in the normoglycemic group; ¥p < 0.05 vs no ischemia. AU, arbitrary units

Article Snippet: Quantification of pro-angiogenic factor levels The concentration of basic fibroblast growth factor (bFGF), IL-10 (Biolegend, San Diego, CA, USA), transforming growth factor (TGF)β1, IL-8, IL-4 (eBioscience), vascular endothelial growth factor (VEGF; R&D Systems, Inc., Minneapolis, MN, USA), platelet-derived growth factor (PDGF; Abcam), and epidermal growth factor (EGF; Life Technologies, Carlsbad, CA, USA) in ECFC culture supernatants were measured by enzyme-linked immunosorbent assay (ELISA) at the indicated time points following the manufacturer’s instructions.

Techniques: Saline, Muscles, Staining