Journal: Journal of Cell Science

Article Title: VEGFR2 survival and mitotic signaling depends on joint activation of associated C3ar1/C5ar1 and IL-6R–gp130

doi: 10.1242/jcs.219352

Figure Lengend Snippet: Autocrine C3ar1/C5ar1 signaling is essential for EC viability. (A,B) bEnd.3 (A) or MS-1 (B) cells were incubated for 3 days with medium alone, VEGF-A (VEGF)±C3ar1-A/C5ar1-A (RA) or anti-C3a/anti-C5a mAbs, and growth was assayed daily. (C) bEnd.3 or MS-1 cells were incubated with VEGF-A±RA, after which culture supernatants were assayed for C3a and C5a by ELISAs. (D) Primary murine aortic ECs were incubated with VEGF-A, after which C3, C5, C3ar1 and C5ar1 mRNA levels were quantified by qPCR. (E) Serum-starved HAECs were stimulated with VEGF-A, and intracellular as well as extracellular expression of C5a and C5ar1 was assessed by flow cytometry. (F) HUVECs were incubated with C5a, VEGF-A or VEGF-A±RA, or medium alone, stained with propidium iodide and assayed by flow cytometry (% G2/M+ is indicated). (G) C3a and C5a in culture supernatants from bEnd.3 and MS-1 cells were quantitated by ELISA. (H) C3ar1 and C5ar1 expression in bEnd.3 and MS-1 cells was assayed by flow cytometry (dashed line, isotype control; solid line, anti-C3ar1/anti-C5ar1 mAb-stained cells). Results are representative of four repeat assays. (I) Intracellular and extracellular expression of C5ar1, C5a, C3ar1 and C3a in bEnd.3 cells as assessed by flow cytometry. (J) bEnd.3 and MS-1 cells were incubated for 24 h with RA, after which Fas and FasL expression was assayed by flow cytometry. Red, specific mAb in treated cells; turquoise, specific Ab in untreated cell; purple, Ig control. (K) bEnd.3 cells were incubated for 8 h with RA or anti-C3a/C5a mAbs, after which Bcl-2, Bcl-xL, Bax, and Bim mRNA expression levels were quantified by qPCR. (L) Annexin V staining in bEnd.3 and MS-1 cells incubated with RA, as above. Error bars show s.d. *P<0.05.

Article Snippet: Reagents and antibodies VEGF-A was purchased from ProSpec Bio (Ness Ziona, Israel) or Miltenyi Biotec (San Diego, CA).

Techniques: Incubation, Expressing, Flow Cytometry, Staining, Enzyme-linked Immunosorbent Assay

Journal: Journal of Cell Science

Article Title: VEGFR2 survival and mitotic signaling depends on joint activation of associated C3ar1/C5ar1 and IL-6R–gp130

doi: 10.1242/jcs.219352

Figure Lengend Snippet: Autocrine C3ar1 and C5ar1 signaling participates in VEGF-A-induced angiogenesis in vivo. (A) Equal numbers of primary murine aortic ECs isolated from the aortic rings of Daf1–/–, WT and C3ar1–/–C5ar1–/– mice were cultured for 2 weeks in basal EC growth medium. All panels at same magnification. (B) Equal numbers of primary ECs of each genotype were cultured for 3 days in EC growth medium (consisting of DMEM/F12 medium containing 20% FBS, 2 mM L-glutamine, 1% non-essential amino acid, 0.09 mg/ml EC growth supplement, 1% antibiotic/antimycotic, 100 units/ml penicillin, 100 g/ml streptomycin, 5 ng/ml VEGF-A and 0.09 mg/ml heparin) and cell counts quantified daily. (C) HAECs were transfected with siRNA targeting DAF, C5ar1+C3ar1 or scrambled control. Forty-eight hours after transfection, HAEC growth was monitored daily. (D) C3 and C5 mRNA transcripts harvested from perfused aortas of each genotype relative to WTs. (E) WT or C3ar1–/–C5ar1–/– ECs were incubated for 3 days with media or VEGF-A and growth was quantified daily. (F) VEGF-A production in supernatants and cell lysates of serum-starved primary ECs. (G) Primary aortic ECs from WT and C3ar1–/–C5ar1–/– mice were assayed for VEGFR2 expression by flow cytometry. (H) Primary aortic WT and C3ar1–/–C5ar1–/– ECs were assayed for β3 integrin (CD61) expression by flow cytometry. (I,J) Primary WT and C3ar1–/–C5ar1–/– mouse aortic ECs were assayed for IL-6R (I) and IL-7R (J) expression by flow cytometry. (K) Primary WT and C3ar1–/–C5ar1–/– mouse aortic ECs were incubated with C5a for 24 h and assayed for IL-6 production by ELISA. Error bars show s.d. *P<0.05, **P<0.005.

Article Snippet: Reagents and antibodies VEGF-A was purchased from ProSpec Bio (Ness Ziona, Israel) or Miltenyi Biotec (San Diego, CA).

Techniques: In Vivo, Isolation, Cell Culture, Transfection, Incubation, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Journal of Cell Science

Article Title: VEGFR2 survival and mitotic signaling depends on joint activation of associated C3ar1/C5ar1 and IL-6R–gp130

doi: 10.1242/jcs.219352

Figure Lengend Snippet: C3ar1/C5ar1 signaling induces VEGFR2 phosphorylation. (A) HUVECs were incubated for 24 h with EBM-2 medium, VEGF-A or VEGF-A+RA. (B–D) Male mice (n=8) were inoculated subcutaneously with RM1 prostate cancer cells. (B) Representative images of tumor sections of WT, Daf1–/– and C3ar1–/–C5ar1–/– mice, following CD31 staining to identify blood vessels (brown). Scale bars: 150 μm. (C) The numbers of CD31-positive areas were quantified in 5–10 independent fields per tumor implant. (D) Tumors were collected 10 days after injection and weighed. (E) Corneal neovascularization assessed by fluorescence microscopy using fluorescein-dextran in WT, Daf1–/–, C3ar1–/–C5ar1–/– and Daf1–/–C3ar1–/–C5ar1–/– (n=3 for each), induced by placing a non-penetrating suture in one cornea of each mouse. Representative data are shown. (F–I) Retinas from day 5 neonatal mice (n=6 WT, 7 Daf1–/– and 5 C3ar1–/–C5ar1–/– mice) were isolated and the retinal vasculature was visualized with isolectin B4. (F–H) Whole mounts of the retinas (F) were analyzed for retinal vessel length (distance to angiogenic from optic nerve) (G) and number of retinal branch points (H). ON, optical nerve. AF, angiogenic front (distance from ON). (I) Representative images depicting the angiogenic front of retinal growth. Scale bars: 50 μm. Error bars show s.d. *P<0.02; NS, non-significant.

Article Snippet: Reagents and antibodies VEGF-A was purchased from ProSpec Bio (Ness Ziona, Israel) or Miltenyi Biotec (San Diego, CA).

Techniques: Incubation, Staining, Injection, Fluorescence, Microscopy, Isolation

Journal: Journal of Cell Science

Article Title: VEGFR2 survival and mitotic signaling depends on joint activation of associated C3ar1/C5ar1 and IL-6R–gp130

doi: 10.1242/jcs.219352

Figure Lengend Snippet: VEGF-A induces C3ar1/C5ar1 signaling in ECs via IL-6 and p-STAT3. (A) Serum-starved primary ECs were incubated for 5 min with VEGF-A alone, VEGF-A+RA, RA alone or C5a alone, after which the p-Y1094/Y1059 VEGFR2 auto-phosphorylation sites were assessed by immunoblotting. PP2 was used as a control. (B) Densitometry of bands from three separate experiments from A showed that all changes were significant compared to untreated cells (*P<0.05, **P<0.005). (C) Serum-starved MS-1 cells were cultured for 3 days with medium alone or with VEGF-A±anti-IL-6 neutralizing mAb and growth was quantified daily. (D) Serum-starved MS-1 cells were cultured with IL-6±RA and growth was quantified daily. (E) Serum-starved MS-1 cells were incubated with VEGF-A, VEGF-A±RA, anti-IL-6 mAb or IL-6±RA, after which pY705-STAT3 was assayed by flow cytometry. (F) Following incubation of serum-starved MS-1 cells with medium alone, the cells were alternatively incubated for increasing times with VEGF-A, C5a or VEGF-A, after which endogenously produced IL-6, endogenously produced VEGF-A or endogenously produced C5a in the culture supernatant was assayed by ELISA. (G,H) Serum-starved bEnd.3 cells were incubated for increasing times with VEGF-A±RA or anti-IL-6 mAb, and assayed for pY743-STAT3 and p-Tyk2 expression (G) or VEGFR2, C5ar1 and C3ar1 intracellular expression (H) by flow cytometry. Error bars show s.d.

Article Snippet: Reagents and antibodies VEGF-A was purchased from ProSpec Bio (Ness Ziona, Israel) or Miltenyi Biotec (San Diego, CA).

Techniques: Incubation, Western Blot, Cell Culture, Flow Cytometry, Produced, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Journal of Cell Science

Article Title: VEGFR2 survival and mitotic signaling depends on joint activation of associated C3ar1/C5ar1 and IL-6R–gp130

doi: 10.1242/jcs.219352

Figure Lengend Snippet: Signaling studies. (A) bEnd.3 cells were incubated for 5 min with VEGF-A±RA or anti-IL-6 mAb, and cell extracts were assayed for p-Src, p-AKT and p-ERK by Luminex assay. (B) bEnd.3 cells were incubated for 5 min with medium alone, C5a or IL-6, and cell extracts were assayed for p-Src, p-AKT and p-ERK by Luminex assay. (C,D) bEnd.3 cells were incubated with C5a (C) or IL-6±SU5416 (30 μM) (D), after which cell extracts were assayed for Src, AKT and ERK phosphorylation by Luminex assay. (E) HeLa cells were incubated with VEGF-A±RA, after which cell extracts were assayed for p-AKT, p-ERK and p-Src by Luminex assay. (F) bEnd.3 cells were incubated for 1 h with C5a, C3a or VEGF-A, after which cells were lysed and assayed for PLC activity by EnzCheck Direct Phospholipase C Assay Kit. (G–I) bEnd.3 cells were incubated with C5a or IL-6±SU5416, after which cells were assayed for Src (G), AKT (H) and ERK (I) phosphorylation by Luminex assay. (J) bEnd.3 cells were incubated with VEGF-A or VEGF+SU5416, after which culture supernatants were assayed for C5a and IL-6 by ELISA. (K) bEnd.3 cells were incubated with C5a or IL-6±SU5416 and assayed for Bcl-2, Bcl-xL, Bim and Bax mRNA expression. Error bars show s.d. *P<0.05.

Article Snippet: Reagents and antibodies VEGF-A was purchased from ProSpec Bio (Ness Ziona, Israel) or Miltenyi Biotec (San Diego, CA).

Techniques: Incubation, Luminex, Activity Assay, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Journal of Cell Science

Article Title: VEGFR2 survival and mitotic signaling depends on joint activation of associated C3ar1/C5ar1 and IL-6R–gp130

doi: 10.1242/jcs.219352

Figure Lengend Snippet: VEGFR2, gp130, C3ar1 and C5ar1 are co-associated. (A) Anti-C3ar1, anti-C5ar1, anti-VEGFR2, anti-gp130 and anti-CD31 IPs were prepared from detergent extracts of serum-starved bEnd.3 cells and immunoblots of the IPd proteins were probed for C5ar1, C3ar1, VEGFR2, gp130 and CD31. (B–D) Following 3 h pre-incubation with cytochalasin D (to prevent receptor internalization), serum-starved bEnd.3 cells were incubated for 30 min with VEGF-A. Biotin-labeled C5a or biotin-labeled-C3a was then added for 5 min, after which the cells were extracted and anti-C5ar1, anti-VEGFR2, anti-gp130 and anti-CD31 IPs prepared. Immunoblots of the proteins IPd by the different antibodies were probed for biotin-C3a (B) or iotin-C5a (C) with streptavidin peroxidase (representative of two repeat experiments). (D) Serum-starved bEnd.3 cells were incubated with VEGF-A, after which biotin-labeled C5a was added for 5 min. The cells were extracted, anti-C5ar1, anti-VEGFR2, anti-gp130 and anti-CD31 IPs prepared, and immunoblots of the IPd proteins were probed for biotin-C5a with streptavidin peroxidase. Densitometry of band intensity for each IP was graphed over time. (E) HEK293 cells were transfected with VEGFR2-Luc donor plasmid and increasing (0-5 µg) amounts of acceptor C5ar1-GFP plasmid. A second set of cells was transfected with C5ar1-Luc donor plasmid and increasing (0-5 µg) amounts of acceptor VEGFR2-GFP or C5ar1-GFP plasmid. Immediately after the addition of 5 μM luciferase substrate BRET signal was collected; no interaction between C5ar1 or VEGFR2 and a different GPCR, the protease-activated receptor 1 (PAR-1) was observed (not shown). Error bars show s.d. (F) bEnd.3 cells were plated on slides and, following serum starvation for 24 h, the cells were stained with differentially tagged antibodies against VEGFR2, C5ar1 and IL-6R, and examined by confocal microscopy (cells were visualized at 63×; 5× digital enlargements of region marked by yellow arrow are below each image). Scale bar: 200 μm. (G) bEnd.3 cells were plated on slides and, following serum starvation for 24 h, the cells were fixed and permeabilized, then stained for VEGFR2 and EEA1, or C5ar1 and EEA1, and examined by confocal microscopy. Arrows indicate colocalization (cells were visualized at 63×). Scale bars: 200 μm. (H) Diagram of the VEGFR2–IL-6R–C5ar1 signaling complex and integrated signaling cascade.

Article Snippet: Reagents and antibodies VEGF-A was purchased from ProSpec Bio (Ness Ziona, Israel) or Miltenyi Biotec (San Diego, CA).

Techniques: Western Blot, Incubation, Labeling, Transfection, Plasmid Preparation, Luciferase, Staining, Confocal Microscopy

Journal: International Journal of Retina and Vitreous

Article Title: Safety and tolerability evaluation after repeated intravitreal injections of a humanized anti-VEGF-A monoclonal antibody (PRO-169) versus ranibizumab in New Zealand white rabbits

doi: 10.1186/s40942-020-00235-y

Figure Lengend Snippet: ERG analysis of NZW rabbits as a function of time after intravitreal injections. a The effects of the intravitreal PRO-169 (white bars) and ranibizumab (dark bars) on the dark-adapted ERG responses after 30 days of each injection. The dark-adapted retinal response is represented by the mean ± SEM V Max ratio of the ERG (b-wave). b Time dependent effects of repeated injections of PRO-169 and ranibizumab on retinal function of rabbits. The difference of the log semi saturation constant (experimental-control) is represented by the mean ± SEM. V Max ratios are around 1 and log σ differences are around 0, indicating no damage to the road system

Article Snippet: PRO-169 is a recombinant, humanized anti-VEGF-A mAb with a molecular mass of 149 kDa, structurally similar and with a target specificity like bevacizumab (Avastin, Genentech, South San Francisco, CA) [ , ].

Techniques: Injection

Journal: International Journal of Retina and Vitreous

Article Title: Safety and tolerability evaluation after repeated intravitreal injections of a humanized anti-VEGF-A monoclonal antibody (PRO-169) versus ranibizumab in New Zealand white rabbits

doi: 10.1186/s40942-020-00235-y

Figure Lengend Snippet: Histologic examination of the central retina for PRO-169 ( a ) vs ranibizumab ( b ) after the 1st Ivt injection (D32), for PRO-169 ( c ) vs ranibizumab ( d ) after the 2nd Ivt injection (D63), and for PRO-169 ( e ) vs ranibizumab ( f ) after the 3rd Ivt injection (D94). No retinal toxicity was found in any eyes (x40)

Article Snippet: PRO-169 is a recombinant, humanized anti-VEGF-A mAb with a molecular mass of 149 kDa, structurally similar and with a target specificity like bevacizumab (Avastin, Genentech, South San Francisco, CA) [ , ].

Techniques: Injection

Journal: International Journal of Molecular Sciences

Article Title: Müller Glia Co-Regulate Barrier Permeability with Endothelial Cells in an Vitro Model of Hyperglycemia

doi: 10.3390/ijms252212271

Figure Lengend Snippet: Barrier recovery response to TNF-α and ARVA via trans-endothelial/epithelial resistance (TEER). ( A ) TEER quantification of cell barriers in presence of TNF-α (5 ng/mL) in ( A1 ) normoglycemic groups (solid lines) and ( A2 ) hyperglycemic groups (dashed lines). ECM: Transwell membrane coated with fibronectin and collagen IV at a 1:1 ratio, 1 mg/mL, ECs: endothelial cells monolayers on coated membranes with ECM. MG: Müller glia monolayers on coated membranes with ECM. COMBO: ECs monolayer on top of CM-coated membrane and MG monolayer on bottom of same membrane. ( B ) TEER quantification of cell barriers in presence of Anti-Rat VEGF-A (ARVA) (1 μg/mL) in normoglycemic groups (solid lines) and hyperglycemic groups (dashed lines). COMBO: ECs monolayer on top of ECM-coated membrane and MG monolayer on bottom of same membrane. Hyperglycemic COMBO: COMBO in hyperglycemic conditions. COMBO + ARVA: COMBOs treated with ARVA (1 μg/mL). Hyperglycemic COMBOs + ARVA: hyperglycemic COMBOs treated with ARVA (1 μg/mL). ΔΩ is percent TEER recovery, measured by TEER change between day 4 and day 6. ΔΩ = COMBOs, ΔΩ C + A = COMBOs +ARVA, ΔΩ D = Hyperglycemic COMBOs, and ΔΩ DC + A = hyperglycemic COMBOs +ARVA. **** ( p < 0.0001) correspond to the statistical difference between hyperglycemic COMBOs and hyperglycemic COMBOs + ARVA.

Article Snippet: This study used a rat anti-VEGF-A (ARVA) molecule (Leinco Technologies, V142, Fenton, MO, USA) that operates in a similar fashion as bevacizumab, an anti-VEGF-A agent used to treat aberrant angiogenesis in humans [ ].

Techniques: Membrane