An IP-10 (CXCL10)-derived peptide inhibits angiogenesis

Angiogenesis plays a critical role in processes such as organ development, wound healing, and tumor growth. It requires well-orchestrated integration of soluble and matrix factors and timely recognition of such signals to regulate this process. Previous work has shown that newly forming vessels express the chemokine receptor CXC receptor 3 (CXCR3) and, activation by its ligand IP-10 (CXCL10), both inhibits development of new vasculature and causes regression of newly formed vessels. To identify and develop new therapeutic agents to limit or reverse pathological angiogenesis, we identified a 21 amino acid fragment of IP-10, spanning the α-helical domain residues 77-98, that mimic the actions of the whole IP-10 molecule on endothelial cells. Treatment of the endothelial cells with the 22 amino acid fragment referred to as IP-10p significantly inhibited VEGF-induced endothelial motility and tube formation in vitro, properties critical for angiogenesis. Using a Matrigel plug assay in vivo, we demonstrate that IP-10p both prevented vessel formation and induced involution of nascent vessels. CXCR3 neutralizing antibody was able to block the inhibitory effects of the IP-10p, demonstrating specificity of the peptide. Inhibition of endothelial function by IP-10p was similar to that described for IP-10, secondary to CXCR3-mediated increase in cAMP production, activation of PKA inhibiting cell migration, and inhibition of VEGF-mediated m-calpain activation. IP-10p provides a novel therapeutic agent that inhibits endothelial cell function thus, allowing for the modulation of angiogenesis. © 2012 Yates-Binder et al

IP-10p induces cAMP activation of PKA.

<p>A) HMEC-1 cells were stimulated with 0.1% DMSO (no treatment) and/or with forskolin (25 mM), VEGF (5.2 µM), IP-10 (23.3 µM), IP-10p (10 µM) in combination. Cell lysate was analyzed for total cAMP using cAMP enzyme immunoassay kit. B) HMEC-1 were stimulated with VEGF (5.2 µM) and/or IP-10 (23.3 µM), IP-10p (10 µM) and in combination for 15, 30 and 60 minutes. The cells were lysed in a hypotonic solution containing aprotinin and PMSF. The lysate was then analyzed for PKA activity using a PKA -assay kit (Promega) and absorbance was read at 570 nm with the solubilization buffer serving as a blank. The graph shows the amount of the synthetic substrate phosphorylated by PKA. C) HMEC-1 cells were plated on gelatin-coated glass chamber slides at 1.2×10⁴ cells/well and incubated for 24 hours and then further incubated in 0.5% dialyzed fetal bovine serum for 24 hours. BAPTA AM (5 mM), or Calpain inhibitor I (CI-1, 10 mM) at 37°C for 30 minutes. The BOC-LM-CMAC (Boc) (25 µM,) was added and incubated at 37°C for 30 min, then VEGF (3.9 µM), IP-10 (23.3 µM), and IP-10p (10 µM) was added and incubated for 30 minutes at 37°C. In some experiments the addition of cAMP analogs 8-Br-cAMP (50 µM), activator of PKA, and Rp-8-Br-cAMP (250 µM), inhibitor of PKA for 20 minutes prior to the addition of Boc. Calpain activation was analyzed by fluorescence microscopy. D) Calpain activity was quantified by MetaMorph analysis. Data shown are of at least N = 9 and normalized to no treatment (average ±SEM). *<i>P<</i>0.05. Original magnifications, 10X.</p

Binding of IP-10p to HMEC cells occurs via CXCR3.

<p>A) Biotin tagged IP-10 and IP-10p were incubated with cells and then probed with FITC-conjugated with streptavidin and analyzed on a BD FACSCalbur flow cytometer. Control cells were incubated with FITC-Streptavidin alone. Motility analysis shows the effects of biotinylated IP-10 (23.2 µM), IP-10p (10 µM) C) Binding of IP-10p to endothelial cells is saturable. Endothelial cells were incubated with increasing doses of IP-10p as indicated. Cells were extensively washed in PBS with FITC-Streptavidin analyzed by flow cytometry. Mean fluorescent intensities of labeled cells are plotted against the concentrations of IP-10p. D) IP-10p binds specifically to endothelial cells. Endothelial cell were incubated with 1ug/ml biotin labeled IP-10p and increasing control of unlabeled IP-10p or scrambled control at peptide. Cells were stained with FITC-Streptavidin analyzed by flow cytometry. (µg unlabeled IP-10p ν unlabeled scrambled peptide) E) IP-10 competes with IP-10p for binding. Endothelial cells were incubated with 1ug/ml biotin labeled IP-10p and IP-10. Cells were stained with FITC-Streptavidin analyzed by flow cytometry. Mean fluorescent intensities are plotted as a function of increasing quantities of competitor proteins. Data shown are of N = 6.</p

2D scratch assay of stimulated HMEC-1 cells was used to analyze migration patterns.

<p>A) The dose response used to determine the optimal concentration IP-10p (10 µM) used to compare to IP-10 (34.9 µM) B) HMEC-1 cells were grown to 80 to 85% confluence in a 12-well plate and quiesced in 0.5% dialyzed fetal bovine serum for 24 hours. A 1-mm scratch was made to the confluent of monolayer using a rubber policeman. The cells were then incubated in 0.5% dialyzed with/without IP-10 (23.2 µM), IP-10p (10 µM), VEGF (3.9 µM) or and/or scrambled control (10 µM) for 24 hours. As expected, IP-10p inhibited motility of the HMEC-1 as wells as inhibited VEGF induced motility. The results are N = 6 (average ±SEM). *P<0.05.</p

Structure model of IP-10 and IP-10p.

<p>Full Length Sequence of IP-10 with C-terminal 22 amino acids highlighted comprising IP-10p.</p

CXCR3 neutralizing antibody blocks IP-10p inhibition affects.

<p>A) HMEC-1 cells were grown, detached and resuspended in serum-free medium and pretreated with a neutralizing antibody to CXCR3 (0.5 µg/ml) 30 minutes prior to addition to VEGF (3.9 µM), IP-10 (34.9 µM), IP-10p (10 µM) and/or IgG (control). Treated cells (1 X10⁴ cells/well) were added to 24-well culture plates coated with growth factor reduce Matrigel and incubated for 24 hours. Endothelial tubes were allowed to form. B) Quantification of the endothelial tube was done using MetaMorph C) CXCR3 siRNA down regulation of CXCR3 was used on the HMEC-1 cells and incubated on GFR-Matrigel in the presence of IP-10 VEGF (3.9 µM), IP-10 (34.9 µM) and/or IP-10p (10 µM). Quantification of the endothelial cell tube density was shown using MetaMorph analysis. D) To demonstrate that the IP-10p inhibition of motility is mediated via CXCR3, a siRNA down regulation of CXCR3 was used on the HMEC-1 cells. The 2-D scratch assay was performed on the CXCR3 knockdown cells under the same condition above. Both IP-10 and IP-10p were unable to block VEGF induced motility. E) Immunofluorescence staining to verify siRNA knockdown of CXCR3. Data shown are of N = 6 and normalized to no treatment (average ±SEM). *P<0.05. Original magnifications, 4X.</p

IP-10p is able to inhibit angiogenesis.

<p>A) C57BL/6J mice were subcutaneously implanted with 750 µl of Matrigel containing VEGF (23.3 µM), IP-10 (1.2 µM) and/or, of IP-10p (41 µM). Ten days post inoculation; the Matrigel plug was removed and stained with Masson Trichrome to visualize endothelial infiltration. Vessels were quantified by counting vessel infiltration in to the plug on day 10. B) To verify that IP-10p is able to inhibit regression of newly formed vessel <i>in vivo</i> GFR- Matrigel supplemented with VEGF (10.4 µM) was injected into both the left and right side groin of mice. After 10 days the left side Matrigel plug was removed (control, <i>n</i> = 6) to show vessel invasion. The remaining right side Matrigel plug in the mice was injected with either IP-10 (1.2 µM) and/or, IP-10p (41 µM) at days 10 and 12. Day 17, the Matrigel plugs were removed and stained with Masson’s Trichrome. C) To validate the invasion of endothelial cells plugs were stained with CD31. IP-10p treated plugs showed a significant regression of vessels compared to the saline controls. D) Vasculature is a marker of vessel maturation Desmin to validate mature vessels. Images shown represent N = 8 Original magnifications, 10X and/or 40X.</p

IP-10p is able to inhibit tube formation.

<p>A) The dose response used to determine the optimal concentration IP-10p (10 µM) used to compare to IP-10 (34.9 µM). B) HMEC-1 cells were grown, detached and resuspended in serum-free medium either with or without VEGF (3.9 µM), IP-10 (34.9 µM), IP-10p (10 µM) and/or scrambled control (10 µM) for 24 hours. Treated cells (1 x10⁴ cells/well) were added to 24-well culture plates coated with growth factor reduced Matrigel and incubated for 24 hours. C) Newly formed endothelial tubes in A were analyzed and quantified using MetaMorph image programming. Data shown are of N = 6 and normalized to no treatment (average ±SEM). *P<0.05. Original magnifications, 4X.</p

IP-10p induces dissociation of newly formed tubes.

<p>A) The dose response used to determine the optimal concentration IP-10p (10 µM) used to compare to IP-10 (34.9 µM). B) HMEC-1 cells were treated with VEGF (3.9 µM) and plated on GFR-Matrigel to form endothelial tubes. The newly formed tubes were incubated in 0.5% dialyzed FBS medium for 24 hours with VEGF (48 hours) in the presence of IP-10 (34.9 µM) (24 hrs+IP-10), or IP-10p (10 µM) (24 hrs+IP-10). C) Quantification of the endothelial tube area was determined, using MetaMorph. Data shown are of at least N = 6 and normalized to no treatment (average ±SEM). *<i>P<</i>0.05. Original magnifications, 4X.</p