VEGFR3 Modulates Vascular Permeability by Controlling VEGF/VEGFR2 Signaling

Abstract

Rationale: Vascular endothelial growth factor (VEGF) is the main driver of angiogenesis and vascular permeability via VEGF receptor 2 (VEGFR2), whereas lymphangiogenesis signals are transduced by VEGFC/D via VEGFR3. VEGFR3 also regulates sprouting angiogenesis and blood vessel growth, but to what extent VEGFR3 signaling controls blood vessel permeability remains unknown.

Objective: To investigate the role of VEGFR3 in the regulation of VEGF-induced vascular permeability.

Methods and results: Long-term global Vegfr3 gene deletion in adult mice resulted in increased fibrinogen deposition in lungs and kidneys, indicating enhanced vascular leakage at the steady state. Short-term deletion of Vegfr3 in blood vascular endothelial cells increased baseline leakage in various tissues, as well as in tumors, and exacerbated vascular permeability in response to VEGF, administered via intradermal adenoviral delivery or through systemic injection of recombinant protein. VEGFR3 gene silencing upregulated VEGFR2 protein levels and phosphorylation in cultured endothelial cells. Consistent with elevated VEGFR2 activity, vascular endothelial cadherin showed reduced localization at endothelial cell-cell junctions in postnatal retinas after Vegfr3 deletion, or after VEGFR3 silencing in cultured endothelial cells. Furthermore, concurrent deletion of Vegfr2 prevented VEGF-induced excessive vascular leakage in mice lacking Vegfr3.

Conclusions: VEGFR3 limits VEGFR2 expression and VEGF/VEGFR2 pathway activity in quiescent and angiogenic blood vascular endothelial cells, thereby preventing excessive vascular permeability.

Keywords: VE-Cadherin; VEGF receptor regulation; blood vessels; vascular biology; vascular leakage.

Figure 1.. Global long-term deletion of Vegfr3 results in vascular leakage.

A–D, Immunostaining and quantification of % fibrinogen area in Vegfr3^iΔ (R3^iΔ) and control (CNTR; Vegfr3^flox/flox) adult mouse lungs (A and B) and kidneys (C and D). Mice were euthanized 12 wk after tamoxifen induction. *P<0.05. Error bars, SEM. Numbers (N) are indicated on the bars.

Figure 2.. PdgfbiCreER^T2 activation does not lead to Vegfr3 deletion in lymphatic vasculature.

A, LYVE1 (gray), VEGFR3 (red), and PECAM1 (green) staining of ear skin of Vegfr3^iΔEC adult mice. The mice were treated with tamoxifen via gavage on postnatal days (P) 3 to 5 and were analyzed at 8 mo of age. B and C, No significant fluid retention in organs of adult Vegfr3^iΔ mice. Weights of lung, spleen, and thyroid tissues normalized to body weight of Vegfr3^iΔ and control mice 4 mo after tamoxifen administration. The mice were treated with tamoxifen via gavage for 3 consecutive days when they were 12 wk old and analyzed 16 wk later. P>0.05. Error bars, SEM. Numbers (N) are indicated below the bars.

Figure 3.. Short-term Vegfr3 deletion in the blood vessel endothelium results in increased vascular permeability in retina, trachea, ear skin, and in a tumor isograft.

A–C, Eight-week-old Vegfr3^iΔEC (R3^iΔEC) and control (CNTR) mice were analyzed 7 d after tamoxifen administration. Representative images of 70-kDa Texas red dextran extravasation in baseline conditions, in retinal (A) and ear skin (C) blood vessels (stained for podocalyxin in gray), 12 min after dextran injection. B, Quantification of 70-kDa Texas red dextran colocalization with retinal blood vessels 30 min after intravenous administration. D, Representative images of 70-kDa Texas red dextran and 50-nm dragon green nanoparticle extravasation from tracheal blood vessels (podocalyxin, gray) 12 min after administration. E, Sections from Lewis lung carcinoma (LLC) tumors grown in Vegfr3^iΔEC or control mice stained for fibrinogen (green) and PECAM1 (red). F, Quantification of fibrinogen signal intensity normalized to total PECAM1 pixel density. ***P<0.001. Error bars, SEM. Numbers (N) are indicated on the bars.

Figure 4.. Vegfr3 deletion increases VE-Cadherin internalization in endothelial cells.

A and B, VE-Cadherin (green) and Claudin-5 (red) staining of P5 retinas of Vegfr3^iΔEC mice. Note the nonjunctional green staining indicating VE-Cadherin internalization (asterisks).C, Quantification of VE-Cadherin/Claudin-5 colocalization. *P<0.05. Error bars, SEM. Numbers (N) are indicated on the bars (N pooled together from 2 independent experiments). D and E, Immunofluorescence staining of VEGFR3 (green), VE-Cadherin (red), and 4′,6-diamidino-2-phenylindole (DAPI; blue) in cultured lung endothelial cells (ECs) from Vegfr3^iΔEC mice treated with 4-hydroxytamoxifen in vitro (D), and in human umbilical vein endothelial cells (HUVECs) after transduction with lentivirus expressing shFLT4 or scrambled control vector (E). mLEC indicates mouse lung EC.

Figure 5.. Vegfr3 deletion increases VEGFR2 levels.

A, Western blotting of VEGFR2, VEGFR3 and heat shock protein 70 in the lungs of Vegfr3^iΔ and control mice. B, Validation of vascular endothelial growth factor receptor (VEGFR) 3 silencing in human umbilical vein endothelial cells (HUVECs) after transduction with lentivirus expressing shFLT4 or scrambled control, using 2 different primary antibodies against VEGFR3. C–F, Quantification of VEGFR3 and VEGFR2 protein (C and D) and mRNA levels (E and F) in HUVECs transduced with lentivirus expressing shFLT4 or scrambled control. G, mRNA levels of NOTCH1 target genes HEY1, HES1 and DLL4 in VEGFR3-silenced HUVECs (4 independent experiments). H, Western blotting of VEGFR2 and pVEGFR2 from transduced HUVECs, stimulated with VEGF₁₆₅ for 10 min before lysis. *P<0.05, **P<0.01, ***P<0.001, Error bars, SEM. Numbers (N) are indicated on the bars.

Figure 6.. VEGFR3 restricts vascular permeability by suppressing VEGF/VEGFR2 signaling.

A and B, Representative whole-mount images and quantification of nanoparticle (FluoSpheres 0.1 μm) leakage in tracheal blood vessels of adult Vegfr3^iΔ (R3^iΔ) and control (CNTR) mice 2 wk after tamoxifen administration. The mice received vascular endothelial growth factor (VEGF) intravenously 15 min before termination. C and D, Vegfr3 loss of function does not affect bradykinin-induced permeability. Representative images and quantification of nanoparticle (0.1 μm) leakage in the tracheas of adult Vegfr3^iΔ and CNTR mice 2 wk after tamoxifen administration. The mice received 1 mg/kg bradykinin intravenously, 4 min before termination. E–G, Miles assay of VEGF or VEGFC-induced vascular permeability in Vegfr3^iΔEC (R3^iΔEC) vs CNTR mice and in compound Vegfr2;Vegfr3^iΔEC (R2;R3^iΔEC) vs littermate CNTR (Vegfr2^flox/flox;Vegfr3^flox/flox) 48 h after transduction of the ear skin with the indicated adenoviral vectors. For C–G, the mice were induced at the age of 9 wk with tamoxifen via gavage and analyzed 1 wk thereafter. *P<0.05, ***P<0.001. Error bars, SEM. Numbers (N) are indicated on the bars.

Figure 7.. Schematic summarizing the effects of vascular endothelial growth factor receptor (VEGFR) 3 in VEGFR2-regulated vascular permeability in steady state and after VEGF stimulation.

VEGFR3 suppresses VEGFR2 mRNA and protein levels, thus limiting VEGF-induced vascular permeability. On VEGFR3 loss of function, VEGFR2 expression levels increase, resulting in increased sensitivity to VEGF signals, enhanced VE-Cadherin internalization and excessive vascular leakage.