Endothelial VEGFR2-PLCγ signaling regulates vascular permeability and antitumor immunity through eNOS/Src
Elin Sjöberg, … , Anna Dimberg, Lena Claesson-Welsh
Elin Sjöberg, … , Anna Dimberg, Lena Claesson-Welsh
Published August 31, 2023
Citation Information: J Clin Invest. 2023;133(20):e161366. https://doi.org/10.1172/JCI161366.
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Research Article Oncology Vascular biology

Endothelial VEGFR2-PLCγ signaling regulates vascular permeability and antitumor immunity through eNOS/Src

Abstract

Endothelial phospholipase Cγ (PLCγ) is essential for vascular development; however, its role in healthy, mature, or pathological vessels is unexplored. Here, we show that PLCγ was prominently expressed in vessels of several human cancer forms, notably in renal cell carcinoma (RCC). High PLCγ expression in clear cell RCC correlated with angiogenic activity and poor prognosis, while low expression correlated with immune cell activation. PLCγ was induced downstream of vascular endothelial growth factor receptor 2 (VEGFR2) phosphosite Y1173 (pY1173). Heterozygous Vegfr2Y1173F/+ mice or mice lacking endothelial PLCγ (Plcg1iECKO) exhibited a stabilized endothelial barrier and diminished vascular leakage. Barrier stabilization was accompanied by decreased expression of immunosuppressive cytokines, reduced infiltration of B cells, helper T cells and regulatory T cells, and improved response to chemo- and immunotherapy. Mechanistically, pY1173/PLCγ signaling induced Ca2+/protein kinase C–dependent activation of endothelial nitric oxide synthase (eNOS), required for tyrosine nitration and activation of Src. Src-induced phosphorylation of VE-cadherin at Y685 was accompanied by disintegration of endothelial junctions. This pY1173/PLCγ/eNOS/Src pathway was detected in both healthy and tumor vessels in Vegfr2Y1173F/+ mice, which displayed decreased activation of PLCγ and eNOS and suppressed vascular leakage. Thus, we believe that we have identified a clinically relevant endothelial PLCγ pathway downstream of VEGFR2 pY1173, which destabilizes the endothelial barrier and results in loss of antitumor immunity.

Authors

Elin Sjöberg, Marit Melssen, Mark Richards, Yindi Ding, Catarina Chanoca, Dongying Chen, Emmanuel Nwadozi, Sagnik Pal, Dominic T. Love, Takeshi Ninchoji, Masabumi Shibuya, Michael Simons, Anna Dimberg, Lena Claesson-Welsh

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Figure 5

PLCγ activates eNOS in a Ca2+-dependent manner to induce VEGF-dependent vascular leakage.

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PLCγ activates eNOS in a Ca2+-dependent manner to induce VEGF-dependent ...
(A) Representative VE-cadherin (VEC) and pVEC Y685 immunostainings of HUVECs pretreated for 15 minutes with DMSO (control) or 5 μM MAPTAM prior to VEGFA stimulation (100 ng/ml, 5 min). Nuclei stained with DAPI (blue). Scale bar: 20 μm. (B and C) Quantification of VEC area (B) and pVEC Y685 levels (C) from A; n = 6 independent experiments, ≥ 3 fields of view/experiment. 1-way ANOVA. (D and E) Representative Western blot (D) and quantification (E) of peNOS S1177 levels in VEGFA-stimulated isolated lung ECs from WT and Vegfr2Y1173F/+ (Y1173F/+) mice; n = 4 mice/genotype. 1-way ANOVA. (F and G) Representative immunostaining (F) and mean fluorescent intensity (MFI) quantification (G) of eNOS and peNOS S1176 in B16F10 melanomas from Vegfr2+/+ (WT) and Vegfr2Y1173F/+ (Y1173F/+) mice; n = 5 (WT) and 3 (Y1173F/+) mice, ≥ 2 fields of view/tumor. Scale bar: 100 μm. Scale bar inset: 20 μm. Unpaired 2-tailed Students’ t test. (H) Immunofluorescent staining with antibodies against VEC and pVEC Y685 in the back skin of NOS3+/+ (WT) and Nos3S1176A/S1167A (S1176A) mice after intradermal injection of PBS or VEGFA. Scale bar: 20 μm. (I) Quantification of MFI values for vascular pVEC Y685 from H; n = 3 mice/genotype, ≥ 2 fields of view/mouse. 1-way ANOVA. (J) Representative time-lapse image of VEGFA-stimulated vascular extravasation of a 2,000 kDa FITC-dextran in the ear dermis of WT and S1176A mice. Scale bar: 50 μm. (K and L) Number of leakage sites/100 μm in WT and S1176A mice in ear dermis veins (K) and capillaries (L); n = 4–5 mice/genotype, ≥ 2 fields of view/mouse. Unpaired 2-tailed Students’ t test. Data represent mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.