Dataset Information

Notch signalling pathways mediate synovial angiogenesis in response to vascular endothelial growth factor and angiopoietin 2.

ABSTRACT:

Objective

Notch signalling pathways are critical for angiogenesis and endothelial cell (EC) fate; however the mechanisms regulating these processes in the inflamed joint remain to be elucidated. Here, we examine whether Notch signalling mediates vascular endothelial growth factor (VEGF) and angiopoietin 2 (Ang2)-induced vascular function.

Methods

Notch-1 intracellular domain (Notch-1 IC), Notch-4 IC, Delta-like-ligand 4, Hes-related transcriptional repressors-1 and 2 (Hrt-1, Hrt-2) mRNA and/or protein expression was measured by Real-time PCR and/or western blot. VEGF/Ang2 induced EC function was assessed using transwell invasion chambers, matrigel tube formation assays and wound repair scratch assays±Notch-1 siRNA or an ?-secretase inhibitor N-(N-(3,5-Difluorophenacetyl-L-alanly))-S-phenylglycine-t-Butyl Ester (DAPT) in RA synovial explants or human microvascular EC. Interleukin (IL)-6 and IL-8 were measured by ELISA and MMP2 and 9 by gelatine zymography.

Results

Notch-1 IC and Notch-4 IC protein expressions were demonstrated in RA and psoriatic arthritis synovial biopsies, with minimal expression observed in Osteoarthritis (OA). VEGF and Ang2 induced Notch-1 IC/ Notch-4 IC protein expression in synovial explant cultures and human microvascular EC levels were further potentiated by VEGF/Ang2 stimulation in combination. Notch-1, Delta-like-ligand 4, and Hrt-2 mRNA expression were significantly induced by VEGF and Ang2 alone and in combination. Furthermore VEGF/Ang2-induced EC invasion, angiogenesis and migration were inhibited by Notch-1 siRNA or DAPT. Conditioned media from VEGF/Ang2 stimulated RA synovial explants induced EC tube formation, an effect that was inhibited by DAPT. Finally, DAPT significantly decreased VEGF/Ang2 induced IL-6, IL-8, MMP2 and 9 expressions in RA synovial explants.

Conclusions

Notch-1 mediates VEGF/Ang2-induced angiogenesis and EC invasion in inflammatory arthritis.

SUBMITTER: Gao W

PROVIDER: S-EPMC3664379 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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Publications

Notch signalling pathways mediate synovial angiogenesis in response to vascular endothelial growth factor and angiopoietin 2.

Gao Wei W Sweeney Catherine C Walsh Ceara C Rooney Peadar P McCormick Jennifer J Veale Douglas J DJ Fearon Ursula U

Annals of the rheumatic diseases 20121117 6

<h4>Objective</h4>Notch signalling pathways are critical for angiogenesis and endothelial cell (EC) fate; however the mechanisms regulating these processes in the inflamed joint remain to be elucidated. Here, we examine whether Notch signalling mediates vascular endothelial growth factor (VEGF) and angiopoietin 2 (Ang2)-induced vascular function.<h4>Methods</h4>Notch-1 intracellular domain (Notch-1 IC), Notch-4 IC, Delta-like-ligand 4, Hes-related transcriptional repressors-1 and 2 (Hrt-1, Hrt-2 ...[more]

PMID: 23161900

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Project description:Which receptors for prostaglandin E2 (PGE2) and vascular endothelial growth factor A (VEGFA) mediate angiogenesis in the human follicle around the time of ovulation?PGE2 and VEGFA act via multiple PGE2 receptors (PTGERs) and VEGF receptors (VEGFRs) to play complementary roles in follicular angiogenesis.Production of PGE2 and VEGFA by the follicle are prerequisites for ovulation. PGE2 is an emerging regulator of angiogenesis and has not been examined in the context of the human ovulatory follicle. VEGFA is an established regulator of follicular angiogenesis.Ovarian biopsies containing the ovulatory follicle were obtained from 11 women of reproductive age (30-45 years) undergoing surgery for laparoscopic sterilization. In some cases, women received hCG to substitute for the ovulatory LH surge before ovarian biopsy. In addition, aspirates from four women of reproductive age (18-31 years) undergoing gonadotrophin stimulation for oocyte donation were obtained for isolation of human ovarian microvascular endothelial cells (hOMECs).Ovarian biopsies were utilized for immunocytochemical detection of von Willebrand factor to identify endothelial cells. hOMECs were cultured with PGE2, PTGER receptor selective agonists, VEGFA, or VEGFR selective agonists. hOMECs were assessed for proliferation by Ki67 immunocytochemistry. hOMEC migration was determined by counting cells which migrated through a porous membrane in vitro. Sprout formation was quantified by determining sprout number and length from photographs take after culture of hOMECs in a 3-dimensional matrix.Endothelial cells were not observed within the granulosa cell layer of human ovulatory follicles prior to an ovulatory dose of hCG and were first seen amongst granulosa cells 18-34 h after hCG. In vitro, PGE2 enhanced migration and sprout formation but did not alter hOMEC proliferation. Agonists selective for each PTGER increased migration with no change in proliferation. PTGER1 and PTGER2 agonists increased the number of sprouts, while only PTGER1 affected sprout length. VEGFA increased hOMEC proliferation, migration, and formation of structures resembling capillary sprouts. Signaling through VEGFR1 promoted hOMEC migration, proliferation, and the formation of few, long endothelial cell sprouts, while VEGFR2 stimulation promoted hOMEC migration and the formation of many, short sprouts. All effects of treatments in vitro were considered significant at P < 0.05.While primary cultures of hOMECs respond to PGE2 and VEGFA differently than other cultured endothelial cells, hOMECs may not respond to PGE2 and VEGFA in vivo as they do in vitro.Agonists and antagonists selective for PTGER1, PTGER2, VEGFR1, or VEGFR2 may have therapeutic value to promote or prevent ovulation in women.This research was supported by grant funding from the Eunice Kennedy Shriver National Institutes of Child Health and Human Development (HD071875 to D.M.D., T.E.C., M.B.). The authors have no conflicts of interest to disclose.

Dataset Information

Notch signalling pathways mediate synovial angiogenesis in response to vascular endothelial growth factor and angiopoietin 2.

Objective

Methods

Results

Conclusions

Publications

Notch signalling pathways mediate synovial angiogenesis in response to vascular endothelial growth factor and angiopoietin 2.

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