Journal: Circulation Research

Article Title: Overexpression of VEGF 165 b, an Inhibitory Splice Variant of Vascular Endothelial Growth Factor, Leads to Insufficient Angiogenesis in Patients With Systemic Sclerosis

doi: 10.1161/circresaha.111.242057

Figure Lengend Snippet: Figure 1. VEGF165b mRNA is selectively upregulated in skin of patients with SSc. A, cDNAs from SSc and healthy control skin biopsy samples were ampli- fied with primer pairs specific for VEGF165b (exon 4/exon 8b) or VEGF165 (exon 4/exon 8a) in a semiquantitative RT-PCR assay. Agarose gels from a rep- resentative experiment are shown. Renal cell carcinoma samples were used as a disease control group. -Actin served as the control for normalization. B, Results of SYBR Green quantitative real-time RT-PCR analysis of VEGF165b mRNA expression in skin biopsy samples. The expression level of VEGF165b mRNA in control skin was set to 1; the results in SSc skin are normalized to this value. Data are meanSD. *P0.001 vs control.

Article Snippet: In preliminary experiments, recombinant human VEGF165 and VEGF165b proteins (R&D Systems, cat. no. 293VE and 3045-VE, respectively) were loaded, electrophoresed and blotted to test the specificity of anti-VEGF165b and anti-pan-VEGF antibodies.

Techniques: Control, Reverse Transcription Polymerase Chain Reaction, SYBR Green Assay, Quantitative RT-PCR, Expressing

Journal: Circulation Research

Article Title: Overexpression of VEGF 165 b, an Inhibitory Splice Variant of Vascular Endothelial Growth Factor, Leads to Insufficient Angiogenesis in Patients With Systemic Sclerosis

doi: 10.1161/circresaha.111.242057

Figure Lengend Snippet: Figure 2. Overexpression of VEGF165b in SSc skin determined by immunofluorescence and confocal microscopy. A, Blots of SDS–polyacryl- amide gels loaded with recombinant human (rh) VEGF165b and rh VEGF165 were probed with anti-VEGF165b and anti-pan-VEGF antibodies. Anti- VEGF165b antibody specifically recognizes VEGF165b only. Anti-pan-VEGF antibodies detect both VEGF165b and VEGF165. B–H, Representative micro- photographs of VEGF165b immunostaining in skin sections. In control skin, VEGF165b was constitutively expressed in the epidermis (e) and dermis (B, C). In the majority of control subjects, VEGF165b expres- sion was weak (B). Keratinocytes, fibroblasts (arrow- head), and capillary vessels (arrow) of 6 of 23 con- trol subjects showed an intense VEGF165b immunopositivity (C). In SSc skin, a strong expres- sion of VEGF165b was evident in the epidermis (e), fibroblasts (arrowheads), microvascular endothelial cells (arrows), and perivascular inflammatory cells (*) (D-F). G, In SSc skin, endothelial cells and vascular smooth muscle cells of an arteriole (arrow) strongly expressed VEGF165b. H, Intense immunopositivity for VEGF165b in a large inflammatory infiltrate of SSc hypodermis (*). D, G, and H show early SSc skin, E and F show late SSc skin. I and J, Double immuno- staining for VEGF165b (green) and CD31 (red) in SSc skin. Original magnification, 63. K, Densitometric analysis of VEGF165b immunofluorescent staining. Data are meanSD of optical density in arbitrary units (a.u.). *P0.001 vs control.

Article Snippet: In preliminary experiments, recombinant human VEGF165 and VEGF165b proteins (R&D Systems, cat. no. 293VE and 3045-VE, respectively) were loaded, electrophoresed and blotted to test the specificity of anti-VEGF165b and anti-pan-VEGF antibodies.

Techniques: Over Expression, Confocal Microscopy, Recombinant, Immunostaining, Control, Staining

Journal: Circulation Research

Article Title: Overexpression of VEGF 165 b, an Inhibitory Splice Variant of Vascular Endothelial Growth Factor, Leads to Insufficient Angiogenesis in Patients With Systemic Sclerosis

doi: 10.1161/circresaha.111.242057

Figure Lengend Snippet: Figure 3. VEGF165b and pan- VEGF expression in skin biopsy samples. A, Serial skin sections from SSc patients immunostained with anti- VEGF165b–specific antibodies (upper panels) and anti-pan- VEGF antibodies (lower pan- els). In epidermis (e) and der- mis, VEGF165b-specific and pan-VEGF stains showed simi- lar intensity in the same tissue structures. Asterisks indicate a dermal perivascular inflamma- tory infiltrate. B, Double immu- nostaining for VEGF165b (green) and pan-VEGF (red) in SSc skin. Original magnifica- tion, 63. C, Total protein extracts of SSc and control skin were probed with anti- VEGF165b–specific and anti- pan-VEGF antibodies. Results of a representative experiment are shown. SSc 1 to 3 indi- cates early SSc skin; SSc 4 and 5, late SSc skin. D, Densi- tometric analysis of blot bands. Data are meanSD of optical density in arbitrary units (a.u.). *P0.001 vs VEGF165b in control, **P0.001 vs pan-VEGF in control. E, Different concentra- tions of recombinant human (rh) VEGF165b were probed with anti-VEGF165b–specific and anti-pan-VEGF antibodies.

Article Snippet: In preliminary experiments, recombinant human VEGF165 and VEGF165b proteins (R&D Systems, cat. no. 293VE and 3045-VE, respectively) were loaded, electrophoresed and blotted to test the specificity of anti-VEGF165b and anti-pan-VEGF antibodies.

Techniques: Expressing, Control, Recombinant

Journal: Circulation Research

Article Title: Overexpression of VEGF 165 b, an Inhibitory Splice Variant of Vascular Endothelial Growth Factor, Leads to Insufficient Angiogenesis in Patients With Systemic Sclerosis

doi: 10.1161/circresaha.111.242057

Figure Lengend Snippet: Figure 4. Circulating levels of VEGF165b and pan-VEGF. VEGF165b (A, B) and pan-VEGF (C, D) were mea- sured in SSc and control plasma by ELISA. Data are box plots with median and upper and lower quartiles. Proba- bility values were calculated by the nonparametric Mann-Whitney U test. A, #P0.001, *P0.002 vs control subjects. B, #P0.001, *P0.004 vs control subjects. C, #P0.001, *P0.002 vs control subjects. D, #P0.001, *P0.001 vs control subjects.

Article Snippet: In preliminary experiments, recombinant human VEGF165 and VEGF165b proteins (R&D Systems, cat. no. 293VE and 3045-VE, respectively) were loaded, electrophoresed and blotted to test the specificity of anti-VEGF165b and anti-pan-VEGF antibodies.

Techniques: Control, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Circulation Research

Article Title: Overexpression of VEGF 165 b, an Inhibitory Splice Variant of Vascular Endothelial Growth Factor, Leads to Insufficient Angiogenesis in Patients With Systemic Sclerosis

doi: 10.1161/circresaha.111.242057

Figure Lengend Snippet: Figure 5. In SSc skin, VEGFR-2 is upregulated and coexpressed with VEGF165b in endothelial cells. A–C, Representative micro- photographs of double immunostaining for VEGF165b (green) and VEGFR-2 (red) in SSc and control skin. A, In control skin, weak expression of VEGF165b and very weak or no expression of VEGFR-2 were observed in endothelial cells (arrows). B, C, In the papillary (B) and reticular (C) dermis of SSc skin, VEGF165b and VEGFR-2 were strongly expressed in endothelial cells (arrows). Original magnifi- cation, 63. D, Densitometric analysis of VEGFR-2 immunofluorescent staining. Data are meanSD of optical density in arbitrary units (a.u.). *P0.001 vs control. E, Results of SYBR Green quantitative real-time RT-PCR analysis of VEGFR-2 mRNA expression in skin biopsy samples. VEGFR-2 mRNA expression level in control skin was set to 1; the results in SSc skin are normalized to this value. Data are meanSD. *P0.001 vs control.

Article Snippet: In preliminary experiments, recombinant human VEGF165 and VEGF165b proteins (R&D Systems, cat. no. 293VE and 3045-VE, respectively) were loaded, electrophoresed and blotted to test the specificity of anti-VEGF165b and anti-pan-VEGF antibodies.

Techniques: Double Immunostaining, Control, Expressing, Staining, SYBR Green Assay, Quantitative RT-PCR

Journal: Circulation Research

Article Title: Overexpression of VEGF 165 b, an Inhibitory Splice Variant of Vascular Endothelial Growth Factor, Leads to Insufficient Angiogenesis in Patients With Systemic Sclerosis

doi: 10.1161/circresaha.111.242057

Figure Lengend Snippet: Figure 6. Upregulated VEGF165b correlates with increased expression of TGF-1 in SSc skin. A, B, Representative microphotographs of double immunostaining for VEGF165b (A, green) and TGF-1 (B, red) in SSc skin. VEGF165b and TGF-1 were coexpressed in epidermal keratinocytes (e), dermal fibro- blasts (arrowheads), endothelial cells (arrow), and inflammatory cells. C, Merged image of A and B. Original magnification, 63. D, Scatterplot showing the distribution of sampled pixels plotted as a function of the red (y axis) and green (x axis) emission intensity; colocalized pixels in the image are included in the yellow-orange region.

Article Snippet: In preliminary experiments, recombinant human VEGF165 and VEGF165b proteins (R&D Systems, cat. no. 293VE and 3045-VE, respectively) were loaded, electrophoresed and blotted to test the specificity of anti-VEGF165b and anti-pan-VEGF antibodies.

Techniques: Expressing, Double Immunostaining

Journal: Circulation Research

Article Title: Overexpression of VEGF 165 b, an Inhibitory Splice Variant of Vascular Endothelial Growth Factor, Leads to Insufficient Angiogenesis in Patients With Systemic Sclerosis

doi: 10.1161/circresaha.111.242057

Figure Lengend Snippet: Figure 8. Overexpression of VEGF165b in SSc MVECs results in reduced VEGFR-2 phosphorylation and defec- tive angiogenesis. A, Left panel, Representative blots of VEGF165b and SRp55 expres- sion in healthy MVECs (H-MVEC) and SSc MVECs (SSc-MVEC) at basal condition and after stimulation with TGF- 1. A, Right panel, VEGF165b levels in culture supernatants expressed as meanSD; *P0.001 vs basal H-MVEC, **P0.001 vs basal SSc- MVEC. B, Left panel, Protein extracts from H-MVEC and SSc-MVEC were probed with anti-VEGFR-2 antibodies or immunoprecipitated (IP) with anti-phosphotyrosine antibod- ies and subsequently probed with anti-VEGFR-2; Right panel, VEGFR-2 phosphoryla- tion was analyzed in H-MVEC after stimulation with recombi- nant VEGF165,VEGF165b, com- bination of both, or SSc- MVEC–conditioned medium (c.m.) in absence or presence of anti-VEGF165b blocking anti- bodies. C, Representative images of capillary morpho- genesis on Matrigel after 24 hours. Capillary morphogene- sis was quantified by measur- ing the percent field occu- pancy of capillary projections. Six to 9 photographic fields from 3 plates were scanned for each point. Data are meanSD of triplicate deter- minations. *P0.001 vs basal H-MVECs; **P0.001 vs H-MVECs treated with SSc- MVEC–conditioned medium; #P0.001 vs SSc MVECs in basal conditioned medium (c.m.); ##P0.005 vs SSc MVECs treated with VEGF165 (50 ng/mL). Ab indicates anti- VEGF165b blocking antibodies; IgG, irrelevant isotype- matched immunoglobulin.

Article Snippet: In preliminary experiments, recombinant human VEGF165 and VEGF165b proteins (R&D Systems, cat. no. 293VE and 3045-VE, respectively) were loaded, electrophoresed and blotted to test the specificity of anti-VEGF165b and anti-pan-VEGF antibodies.

Techniques: Over Expression, Phospho-proteomics, Immunoprecipitation, Blocking Assay

Journal: Cancer Research

Article Title: VEGF165b, an Inhibitory Vascular Endothelial Growth Factor Splice Variant

doi: 10.1158/0008-5472.can-04-0934

Figure Lengend Snippet: Fig. 2. A, western blot of recombinant VEGF165b and VEGF165 probed with antibodies to VEGF165b and all iso- forms. The VEGF165b antibody specifically detects the VEGF165b isoform, whereas the pan-VEGF antibody detects both isoforms. B, ELISA for VEGF165b and VEGF165. In- creasing concentrations of recombinant human VEGF165b or VEGF165b were assayed with the ELISA described in Mate- rials and Methods, with either VEGF165b antibody (f) or pan-VEGF antibody (). Both ELISAs detected respective VEGF isoforms at concentrations 100 pgml-1.

Article Snippet: One hundred L of duplicate recombinant human VEGF165 or VEGF165b standards (R&D Systems) diluted in wash buffer (ranging from 15 pg/mL to 4 ng/mL) and blank (wash buffer) or sample were added to each well (Fig. 2B).

Techniques: Western Blot, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: Cancer Research

Article Title: VEGF165b, an Inhibitory Vascular Endothelial Growth Factor Splice Variant

doi: 10.1158/0008-5472.can-04-0934

Figure Lengend Snippet: Fig. 3. Mesentery adjacent to VEGF165b, VEGF165, or control-injected fat pad, fixed, and stained with VEGF165b antibody. Strong fluores- cent staining was seen in mesenteries adjacent to Ad-VEGF165b but not Ad-VEGF165 or control in- jected fat pads. No staining was seen with a no primary control. This shows that Ad-VEGF165b injection resulted in VEGF165b protein secretion into the mesentery.

Article Snippet: One hundred L of duplicate recombinant human VEGF165 or VEGF165b standards (R&D Systems) diluted in wash buffer (ranging from 15 pg/mL to 4 ng/mL) and blank (wash buffer) or sample were added to each well (Fig. 2B).

Techniques: Control, Injection, Staining

Journal: Cancer Research

Article Title: VEGF165b, an Inhibitory Vascular Endothelial Growth Factor Splice Variant

doi: 10.1158/0008-5472.can-04-0934

Figure Lengend Snippet: Fig. 4. VEGFxxxb proteins are found in normal human cells, tissue, and plasma. A, terminally differentiated human podocyte protein extracts were probed with anti-VEGF antibody. This revealed multiple bands corresponding to multiple splice variants of VEGF. When this blot was stripped and reprobed with the anti-VEGF165b antibody, bands appeared consistent with sister splice variants of the common isoforms. VEGF isoforms can be either dimers or monomers, and these bands are consistent with a monomer of VEGF121b (14 kDa, band a), and dimers of VEGF121b (28 kDa, band b), VEGF145b (33 kDa, band c), VEGF165b (38 kDa, band d), and VEGF189b (43.5 kDa, band e) were detected. A series of bands 84 kDa (bands at f) are consistent with large VEGFxxxb isoforms. B, transurethral resection of the prostate tissue probed with VEGFxxxb and pan VEGF antibody also shows expression of multiple isoforms. C, immunohistochemistry of human glomeruli with anti-VEGF165b antibody (i, ii) or nonspecific mouse IgG (iii, iv). This showed that VEGFxxxb was expressed in podocytes on the periphery of the glomerular tuft (arrows). Scale bar is 20 m. D, an ELISA with the VEGF165b antibody was used to detect VEGFxxxb protein in human plasma and tissues. Significant protein expression was seen in normal plasma and benign prostate tissue. A lower level of protein expression was detected in malignant prostate samples. E. mRNA for VEGF165b was expressed in benign (14 of 17) but not malignant (2/9) prostate tissues, whereas VEGF165 isoforms were equally present in both tissue types. Lanes 1, 2, and 3 are benign samples, W is water, b is recombinant VEGF165b cDNA, and a is recombinant VEGF165 cDNA. 7827

Article Snippet: One hundred L of duplicate recombinant human VEGF165 or VEGF165b standards (R&D Systems) diluted in wash buffer (ranging from 15 pg/mL to 4 ng/mL) and blank (wash buffer) or sample were added to each well (Fig. 2B).

Techniques: Clinical Proteomics, Expressing, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: Cancer Research

Article Title: VEGF165b, an Inhibitory Vascular Endothelial Growth Factor Splice Variant

doi: 10.1158/0008-5472.can-04-0934

Figure Lengend Snippet: Fig. 5. VEGF165b inhibits VEGF165-mediated binding to VEGFR-2. A, recombinant VEGF165b displaced 125I-VEGF165 from binding to human umbilical vascular endothelial cells (HUVECs) with the same affinity as recombinant VEGF165. B, recombinant VEGF165b also displaced 125I-VEGF165 from binding to human Fc-VEGFR-2 bound to an ELISA plate with the same affinity as recombinant VEGF165. 7828

Article Snippet: One hundred L of duplicate recombinant human VEGF165 or VEGF165b standards (R&D Systems) diluted in wash buffer (ranging from 15 pg/mL to 4 ng/mL) and blank (wash buffer) or sample were added to each well (Fig. 2B).

Techniques: Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: Cancer Research

Article Title: VEGF165b, an Inhibitory Vascular Endothelial Growth Factor Splice Variant

doi: 10.1158/0008-5472.can-04-0934

Figure Lengend Snippet: Fig. 6. VEGF165b does not stimulate signaling by VEGFR-2. A. human microvascular endothelial cells (HMVECs) treated with VEGF165b show reduced level of phosphorylation of VEGFR-2 as detected by immunoprecipitation with anti- phosphotyrosine antibody. Surprisingly, VEGFR-2 phosphorylation by VEGF165 was also inhibited. Bi, phosphotyrosine immunoprecipitation shows that CHO cells transfected with VEGFR-2 display an increase in autophosphorylation of VEGFR-2 upon treatment with VEGF165. This phosphorylation was not induced by VEGF165b, and incubation of cells with both isoforms did not result in VEGF-R2 phosphorylation. Bii. CHO cells transfected with VEGFR-2 display an increase in phosphorylation of p42/p44 MAPK upon treatment with VEGF165. This phosphorylation was not induced by VEGF165b, and incubation of cells with both isoforms did not increase p42-p44 MAPK phosphorylation. Bar chart shows densities of VEGF165b-mediated p42-p44 MAPK phosphorylation relative to total p42-p44 MAPK (n 4, P 0.001 ANOVA, P 0.05, P 0.01 compared with VEGF165, Student Newmann Keuls post hoc test). Biii, these cells also display an increase in phosphorylation of AKT upon treatment with VEGF165. Again, this phosphorylation was not induced by VEGF165b, and incubation of cells with both isoforms did not increase AKT phosphorylation. Bar chart again shows densities of VEGF165b-mediated AKT phosphorylation (n 3, P 0.05 ANOVA, P 0.05, compared with VEGF165, Student Newmann Keuls post hoc test). 7829

Article Snippet: One hundred L of duplicate recombinant human VEGF165 or VEGF165b standards (R&D Systems) diluted in wash buffer (ranging from 15 pg/mL to 4 ng/mL) and blank (wash buffer) or sample were added to each well (Fig. 2B).

Techniques: Phospho-proteomics, Immunoprecipitation, Transfection, Incubation

Journal: Cancer Research

Article Title: VEGF165b, an Inhibitory Vascular Endothelial Growth Factor Splice Variant

doi: 10.1158/0008-5472.can-04-0934

Figure Lengend Snippet: Fig. 7. VEGF165b can result in some signaling in endothelial cells. A, Akt signaling. Human microvascular endothelial cells (HMVECs) treated with VEGF165 display an increase in Akt phosphorylation. This was not seen in cells treated with VEGF165b. However, in contrast to VEGFR-2–transfected CHO cells, VEGF165b did not inhibit VEGF165-mediated Akt phosphorylation. Bar chart shows densities of VEGF165b-mediated Akt phosphorylation (n 3, P 0.05 ANOVA). B, p42-p44 MAPK phosphorylation. Treatment of HMVECs with VEGF165, VEGF165b, or both isoforms resulted in a significant increase in phosphorylation of p42/p44MAPK. (n 4, P 0.05 ANOVA). P 0.05, compared with untreated, Student Newmann Keuls post hoc test). 7830

Article Snippet: One hundred L of duplicate recombinant human VEGF165 or VEGF165b standards (R&D Systems) diluted in wash buffer (ranging from 15 pg/mL to 4 ng/mL) and blank (wash buffer) or sample were added to each well (Fig. 2B).

Techniques: Phospho-proteomics, Transfection

Journal: Cancer Research

Article Title: VEGF165b, an Inhibitory Vascular Endothelial Growth Factor Splice Variant

doi: 10.1158/0008-5472.can-04-0934

Figure Lengend Snippet: Fig. 8. VEGF165b inhibits VEGF165 mediated angiogenesis in rabbit cornea in vivo. A, MCF-7 cells transfected with either pcDNA3, VEGF165b, or VEGF165 were implanted into the rabbit cornea (arrows). Angiogenesis (arrowheads) was seen in eyes of rabbits implanted with VEGF165 expressing cells, but not when implanted with cells transfected with pcDNA3 alone or with VEGF165b alone. B, VEGF165b inhibits angiogenesis in the rabbit cornea. When cells transfected with VEGF165 were mixed with cells expressing VEGF165b, no angiogenesis was seen, compared with significant angiogenesis when VEGF165 expressing cells were mixed with pcDNA3 transfected cells, indicating that VEGF-mediated angiogenesis in the cornea could be blocked by VEGF165b expression. C, angiogenic score after implantation with cells expressing control vector, VEGF165 or VEGF165b. ††, P 0.01 two way ANOVA, Buonferroni post hoc. D, quantification of angiogenesis showing that VEGF165b inhibits vessel growth in the eye. †††, P 0.001 for treatment, two way ANOVA, Buonferroni post hoc. 7831

Article Snippet: One hundred L of duplicate recombinant human VEGF165 or VEGF165b standards (R&D Systems) diluted in wash buffer (ranging from 15 pg/mL to 4 ng/mL) and blank (wash buffer) or sample were added to each well (Fig. 2B).

Techniques: In Vivo, Transfection, Expressing, Control, Plasmid Preparation

Journal: Cancer Research

Article Title: VEGF165b, an Inhibitory Vascular Endothelial Growth Factor Splice Variant

doi: 10.1158/0008-5472.can-04-0934

Figure Lengend Snippet: Fig. 9. VEGF165b does not stimulate increased vascularity of mesenteric tissue and inhibits VEGF165-mediated increased vascularity. A, injection of adenovirus-expressing enhanced GFP (Ad-EGFP) resulted in a background level of angiogenesis on day 7 (ii) compared with day 1 (i). Ad-VEGF165 injection resulted in florid angiogenesis 7 days (iv) after adenovirus injection (iii). Ad-VEGF165b injection did not result in any more angiogenesis on day 7 (vi) compared with day 1 (v) than EGFP (i to ii). Injection of both Ad-VEGF165b and Ad-VEGF165 resulted in no greater angiogenesis on day 7 (viii) compared with day 1 (vii) than Ad-EGFP (i to ii). B, quantification of the increase in vessel area in six mesenteries showed that there was a significant increase in fractional vessel area in Ad-VEGF165 compared with Ad-EGFP–injected mesenteries, which was not the case with either Ad-VEGF165b or combined Ad-VEGF165 and Ad-VEGF165b injection (P 0.01, ANOVA). 7832

Article Snippet: One hundred L of duplicate recombinant human VEGF165 or VEGF165b standards (R&D Systems) diluted in wash buffer (ranging from 15 pg/mL to 4 ng/mL) and blank (wash buffer) or sample were added to each well (Fig. 2B).

Techniques: Injection, Expressing

Journal: Cancer Research

Article Title: VEGF165b, an Inhibitory Vascular Endothelial Growth Factor Splice Variant

doi: 10.1158/0008-5472.can-04-0934

Figure Lengend Snippet: Fig. 10. VEGF165b is not angiogenic and inhibits VEGF165-mediated angiogenesis. A, fluorescent staining of mesenteries after injection of Ad-VEGF165b into the fat pad of rat mesenteries. Lectin staining shows extensive sprouting (arrows), branching (arrowheads), and increased density by Ad-VEGF165 injection (i) but not Ad-VEGF165b (ii) or combined injection (iii). Proliferating endothelial cells (arrows) were also markedly more common in Ad-VEGF165 (iv) than in VEGF165b (v) or combined (vi) injection. There was a significant increase in branch point density (B), sprout density (C), proliferating endothelial cell density (D), and vessel density (E) and a decrease in vessel length (F) in Ad-VEGF165-treated compared with Ad-EGFP but not in Ad-VEGF165b or Ad-VEGF165b combined with Ad-VEGF165b, indicating that VEGF165b did not cause angiogenesis and specifically inhibited angiogenesis in this in vivo model. † P 0.05, P 0.001, P 0.01, P 0.05 compared with Ad-EGFP, P 0.01, P 0.05 compared with Ad-VEGF165 Student Newmann Keuls post hoc test. 7833

Article Snippet: One hundred L of duplicate recombinant human VEGF165 or VEGF165b standards (R&D Systems) diluted in wash buffer (ranging from 15 pg/mL to 4 ng/mL) and blank (wash buffer) or sample were added to each well (Fig. 2B).

Techniques: Staining, Injection, In Vivo

Journal: Cancer Research

Article Title: VEGF165b, an Inhibitory Vascular Endothelial Growth Factor Splice Variant

doi: 10.1158/0008-5472.can-04-0934

Figure Lengend Snippet: Fig. 11. Mimicking a switch in splicing proan- giogenic to antiangiogenic isoforms inhibits tumor growth in vivo. A, mice that received injections of A375 melanoma cells transfected with VEGF165 grew larger tumors more quickly than VEGF165b (B). Mice that received injections of equal amounts of both transfected cell types grew at an interme- diate rate (C). D, VEGF165b expressing cells grew significantly smaller tumors than VEGF165-ex- pressing cells or a mixture of the two types and were not significantly different from control tu- mors. (P 0.001, two-way ANOVA). E, doubling time was significantly greater in VEGF165b ex- pressing tumor cells than in VEGF165 (P 0.01 ANOVA, P 0.01, compared with VEGF165).

Article Snippet: One hundred L of duplicate recombinant human VEGF165 or VEGF165b standards (R&D Systems) diluted in wash buffer (ranging from 15 pg/mL to 4 ng/mL) and blank (wash buffer) or sample were added to each well (Fig. 2B).

Techniques: In Vivo, Transfection, Expressing, Control

Journal: Circulation Research

Article Title: Overexpression of VEGF 165 b, an Inhibitory Splice Variant of Vascular Endothelial Growth Factor, Leads to Insufficient Angiogenesis in Patients With Systemic Sclerosis

doi: 10.1161/circresaha.111.242057

Figure Lengend Snippet: Figure 1. VEGF165b mRNA is selectively upregulated in skin of patients with SSc. A, cDNAs from SSc and healthy control skin biopsy samples were ampli- fied with primer pairs specific for VEGF165b (exon 4/exon 8b) or VEGF165 (exon 4/exon 8a) in a semiquantitative RT-PCR assay. Agarose gels from a rep- resentative experiment are shown. Renal cell carcinoma samples were used as a disease control group. -Actin served as the control for normalization. B, Results of SYBR Green quantitative real-time RT-PCR analysis of VEGF165b mRNA expression in skin biopsy samples. The expression level of VEGF165b mRNA in control skin was set to 1; the results in SSc skin are normalized to this value. Data are meanSD. *P0.001 vs control.

Article Snippet: Matrigel (50 μl; 10–12 mg/ml) was pipetted into culture wells and polymerized for 30 minutes to 1 hour at 37°C, as described elsewhere.14 Healthy-MVECs (30 x 103 cells/well) were incubated in MCDB medium containing 2% FBS, or in 2% FBS-MCDB medium supplemented with recombinant human VEGF165 (10 ng/ml; R&D Systems), VEGF165b (10 ng/ml; R&D Systems), or combination of VEGF165 and VEGF165b (both 10 ng/ml).

Techniques: Control, Reverse Transcription Polymerase Chain Reaction, SYBR Green Assay, Quantitative RT-PCR, Expressing

Journal: Circulation Research

Article Title: Overexpression of VEGF 165 b, an Inhibitory Splice Variant of Vascular Endothelial Growth Factor, Leads to Insufficient Angiogenesis in Patients With Systemic Sclerosis

doi: 10.1161/circresaha.111.242057

Figure Lengend Snippet: Figure 2. Overexpression of VEGF165b in SSc skin determined by immunofluorescence and confocal microscopy. A, Blots of SDS–polyacryl- amide gels loaded with recombinant human (rh) VEGF165b and rh VEGF165 were probed with anti-VEGF165b and anti-pan-VEGF antibodies. Anti- VEGF165b antibody specifically recognizes VEGF165b only. Anti-pan-VEGF antibodies detect both VEGF165b and VEGF165. B–H, Representative micro- photographs of VEGF165b immunostaining in skin sections. In control skin, VEGF165b was constitutively expressed in the epidermis (e) and dermis (B, C). In the majority of control subjects, VEGF165b expres- sion was weak (B). Keratinocytes, fibroblasts (arrow- head), and capillary vessels (arrow) of 6 of 23 con- trol subjects showed an intense VEGF165b immunopositivity (C). In SSc skin, a strong expres- sion of VEGF165b was evident in the epidermis (e), fibroblasts (arrowheads), microvascular endothelial cells (arrows), and perivascular inflammatory cells (*) (D-F). G, In SSc skin, endothelial cells and vascular smooth muscle cells of an arteriole (arrow) strongly expressed VEGF165b. H, Intense immunopositivity for VEGF165b in a large inflammatory infiltrate of SSc hypodermis (*). D, G, and H show early SSc skin, E and F show late SSc skin. I and J, Double immuno- staining for VEGF165b (green) and CD31 (red) in SSc skin. Original magnification, 63. K, Densitometric analysis of VEGF165b immunofluorescent staining. Data are meanSD of optical density in arbitrary units (a.u.). *P0.001 vs control.

Article Snippet: Matrigel (50 μl; 10–12 mg/ml) was pipetted into culture wells and polymerized for 30 minutes to 1 hour at 37°C, as described elsewhere.14 Healthy-MVECs (30 x 103 cells/well) were incubated in MCDB medium containing 2% FBS, or in 2% FBS-MCDB medium supplemented with recombinant human VEGF165 (10 ng/ml; R&D Systems), VEGF165b (10 ng/ml; R&D Systems), or combination of VEGF165 and VEGF165b (both 10 ng/ml).

Techniques: Over Expression, Confocal Microscopy, Recombinant, Immunostaining, Control, Staining

Journal: Circulation Research

Article Title: Overexpression of VEGF 165 b, an Inhibitory Splice Variant of Vascular Endothelial Growth Factor, Leads to Insufficient Angiogenesis in Patients With Systemic Sclerosis

doi: 10.1161/circresaha.111.242057

Figure Lengend Snippet: Figure 3. VEGF165b and pan- VEGF expression in skin biopsy samples. A, Serial skin sections from SSc patients immunostained with anti- VEGF165b–specific antibodies (upper panels) and anti-pan- VEGF antibodies (lower pan- els). In epidermis (e) and der- mis, VEGF165b-specific and pan-VEGF stains showed simi- lar intensity in the same tissue structures. Asterisks indicate a dermal perivascular inflamma- tory infiltrate. B, Double immu- nostaining for VEGF165b (green) and pan-VEGF (red) in SSc skin. Original magnifica- tion, 63. C, Total protein extracts of SSc and control skin were probed with anti- VEGF165b–specific and anti- pan-VEGF antibodies. Results of a representative experiment are shown. SSc 1 to 3 indi- cates early SSc skin; SSc 4 and 5, late SSc skin. D, Densi- tometric analysis of blot bands. Data are meanSD of optical density in arbitrary units (a.u.). *P0.001 vs VEGF165b in control, **P0.001 vs pan-VEGF in control. E, Different concentra- tions of recombinant human (rh) VEGF165b were probed with anti-VEGF165b–specific and anti-pan-VEGF antibodies.

Article Snippet: Matrigel (50 μl; 10–12 mg/ml) was pipetted into culture wells and polymerized for 30 minutes to 1 hour at 37°C, as described elsewhere.14 Healthy-MVECs (30 x 103 cells/well) were incubated in MCDB medium containing 2% FBS, or in 2% FBS-MCDB medium supplemented with recombinant human VEGF165 (10 ng/ml; R&D Systems), VEGF165b (10 ng/ml; R&D Systems), or combination of VEGF165 and VEGF165b (both 10 ng/ml).

Techniques: Expressing, Control, Recombinant

Journal: Circulation Research

Article Title: Overexpression of VEGF 165 b, an Inhibitory Splice Variant of Vascular Endothelial Growth Factor, Leads to Insufficient Angiogenesis in Patients With Systemic Sclerosis

doi: 10.1161/circresaha.111.242057

Figure Lengend Snippet: Figure 4. Circulating levels of VEGF165b and pan-VEGF. VEGF165b (A, B) and pan-VEGF (C, D) were mea- sured in SSc and control plasma by ELISA. Data are box plots with median and upper and lower quartiles. Proba- bility values were calculated by the nonparametric Mann-Whitney U test. A, #P0.001, *P0.002 vs control subjects. B, #P0.001, *P0.004 vs control subjects. C, #P0.001, *P0.002 vs control subjects. D, #P0.001, *P0.001 vs control subjects.

Article Snippet: Matrigel (50 μl; 10–12 mg/ml) was pipetted into culture wells and polymerized for 30 minutes to 1 hour at 37°C, as described elsewhere.14 Healthy-MVECs (30 x 103 cells/well) were incubated in MCDB medium containing 2% FBS, or in 2% FBS-MCDB medium supplemented with recombinant human VEGF165 (10 ng/ml; R&D Systems), VEGF165b (10 ng/ml; R&D Systems), or combination of VEGF165 and VEGF165b (both 10 ng/ml).

Techniques: Control, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Circulation Research

Article Title: Overexpression of VEGF 165 b, an Inhibitory Splice Variant of Vascular Endothelial Growth Factor, Leads to Insufficient Angiogenesis in Patients With Systemic Sclerosis

doi: 10.1161/circresaha.111.242057

Figure Lengend Snippet: Figure 5. In SSc skin, VEGFR-2 is upregulated and coexpressed with VEGF165b in endothelial cells. A–C, Representative micro- photographs of double immunostaining for VEGF165b (green) and VEGFR-2 (red) in SSc and control skin. A, In control skin, weak expression of VEGF165b and very weak or no expression of VEGFR-2 were observed in endothelial cells (arrows). B, C, In the papillary (B) and reticular (C) dermis of SSc skin, VEGF165b and VEGFR-2 were strongly expressed in endothelial cells (arrows). Original magnifi- cation, 63. D, Densitometric analysis of VEGFR-2 immunofluorescent staining. Data are meanSD of optical density in arbitrary units (a.u.). *P0.001 vs control. E, Results of SYBR Green quantitative real-time RT-PCR analysis of VEGFR-2 mRNA expression in skin biopsy samples. VEGFR-2 mRNA expression level in control skin was set to 1; the results in SSc skin are normalized to this value. Data are meanSD. *P0.001 vs control.

Article Snippet: Matrigel (50 μl; 10–12 mg/ml) was pipetted into culture wells and polymerized for 30 minutes to 1 hour at 37°C, as described elsewhere.14 Healthy-MVECs (30 x 103 cells/well) were incubated in MCDB medium containing 2% FBS, or in 2% FBS-MCDB medium supplemented with recombinant human VEGF165 (10 ng/ml; R&D Systems), VEGF165b (10 ng/ml; R&D Systems), or combination of VEGF165 and VEGF165b (both 10 ng/ml).

Techniques: Double Immunostaining, Control, Expressing, Staining, SYBR Green Assay, Quantitative RT-PCR

Journal: Circulation Research

Article Title: Overexpression of VEGF 165 b, an Inhibitory Splice Variant of Vascular Endothelial Growth Factor, Leads to Insufficient Angiogenesis in Patients With Systemic Sclerosis

doi: 10.1161/circresaha.111.242057

Figure Lengend Snippet: Figure 6. Upregulated VEGF165b correlates with increased expression of TGF-1 in SSc skin. A, B, Representative microphotographs of double immunostaining for VEGF165b (A, green) and TGF-1 (B, red) in SSc skin. VEGF165b and TGF-1 were coexpressed in epidermal keratinocytes (e), dermal fibro- blasts (arrowheads), endothelial cells (arrow), and inflammatory cells. C, Merged image of A and B. Original magnification, 63. D, Scatterplot showing the distribution of sampled pixels plotted as a function of the red (y axis) and green (x axis) emission intensity; colocalized pixels in the image are included in the yellow-orange region.

Article Snippet: Matrigel (50 μl; 10–12 mg/ml) was pipetted into culture wells and polymerized for 30 minutes to 1 hour at 37°C, as described elsewhere.14 Healthy-MVECs (30 x 103 cells/well) were incubated in MCDB medium containing 2% FBS, or in 2% FBS-MCDB medium supplemented with recombinant human VEGF165 (10 ng/ml; R&D Systems), VEGF165b (10 ng/ml; R&D Systems), or combination of VEGF165 and VEGF165b (both 10 ng/ml).

Techniques: Expressing, Double Immunostaining

Journal: Circulation Research

Article Title: Overexpression of VEGF 165 b, an Inhibitory Splice Variant of Vascular Endothelial Growth Factor, Leads to Insufficient Angiogenesis in Patients With Systemic Sclerosis

doi: 10.1161/circresaha.111.242057

Figure Lengend Snippet: Figure 8. Overexpression of VEGF165b in SSc MVECs results in reduced VEGFR-2 phosphorylation and defec- tive angiogenesis. A, Left panel, Representative blots of VEGF165b and SRp55 expres- sion in healthy MVECs (H-MVEC) and SSc MVECs (SSc-MVEC) at basal condition and after stimulation with TGF- 1. A, Right panel, VEGF165b levels in culture supernatants expressed as meanSD; *P0.001 vs basal H-MVEC, **P0.001 vs basal SSc- MVEC. B, Left panel, Protein extracts from H-MVEC and SSc-MVEC were probed with anti-VEGFR-2 antibodies or immunoprecipitated (IP) with anti-phosphotyrosine antibod- ies and subsequently probed with anti-VEGFR-2; Right panel, VEGFR-2 phosphoryla- tion was analyzed in H-MVEC after stimulation with recombi- nant VEGF165,VEGF165b, com- bination of both, or SSc- MVEC–conditioned medium (c.m.) in absence or presence of anti-VEGF165b blocking anti- bodies. C, Representative images of capillary morpho- genesis on Matrigel after 24 hours. Capillary morphogene- sis was quantified by measur- ing the percent field occu- pancy of capillary projections. Six to 9 photographic fields from 3 plates were scanned for each point. Data are meanSD of triplicate deter- minations. *P0.001 vs basal H-MVECs; **P0.001 vs H-MVECs treated with SSc- MVEC–conditioned medium; #P0.001 vs SSc MVECs in basal conditioned medium (c.m.); ##P0.005 vs SSc MVECs treated with VEGF165 (50 ng/mL). Ab indicates anti- VEGF165b blocking antibodies; IgG, irrelevant isotype- matched immunoglobulin.

Article Snippet: Matrigel (50 μl; 10–12 mg/ml) was pipetted into culture wells and polymerized for 30 minutes to 1 hour at 37°C, as described elsewhere.14 Healthy-MVECs (30 x 103 cells/well) were incubated in MCDB medium containing 2% FBS, or in 2% FBS-MCDB medium supplemented with recombinant human VEGF165 (10 ng/ml; R&D Systems), VEGF165b (10 ng/ml; R&D Systems), or combination of VEGF165 and VEGF165b (both 10 ng/ml).

Techniques: Over Expression, Phospho-proteomics, Immunoprecipitation, Blocking Assay