Suppressing STAT3 activity protects the endothelial barrier from VEGF-mediated vascular permeability

Vascular permeability triggered by inflammation or ischemia promotes edema, exacerbates disease progression, and impairs tissue recovery. Vascular endothelial growth factor (VEGF) is a potent inducer of vascular permeability. VEGF plays an integral role in regulating vascular barrier function physiologically and in pathologies, such as cancer, ischemic stroke, cardiovascular disease, retinal conditions, and COVID-19-associated pulmonary edema and sepsis, which often leads to acute lung injury, including acute respiratory distress syndrome. However, after initially stimulating permeability, VEGF subsequently mediates angiogenesis to repair damaged tissue. Consequently, understanding temporal molecular regulation of VEG-Finduced vascular permeability will facilitate developing therapeutics that achieve the delicate balance of inhibiting vascular permeability while preserving tissue repair. Here, we demonstrate that VEGF signals through signal transducer and activator of transcription 3 (STAT3) to promote vascular permeability. Specifically, we show that genetic STAT3 ablation reduces vascular permeability in STAT3-deficient endothelium of mice and VEGF-inducible zebrafish crossed with CRISPR/Cas9 generated genomic STAT3 knockout zebrafish. Importantly, STAT3 deficiency does not impair vascular development and function in vivo. We identify intercellular adhesion molecule 1 (ICAM-1) as a STAT3-dependent transcriptional regulator and show VEGF-dependent STAT3 activation is regulated by JAK2. Pyrimethamine, an FDA-approved antimicrobial agent that inhibits STAT3-dependent transcription, substantially reduces VEGF-induced vascular permeability in zebrafish, mouse, and human endothelium. Indeed, pharmacologically targeting STAT3 increases vascular barrier integrity using two additional compounds, atovaquone and C188–9. Collectively, our findings suggest that the VEGF, VEGFR-2, JAK2, and STAT3 signaling cascade regulates vascular barrier integrity, and inhibition of STAT3-dependent activity reduces VEGF-induced vascular permeability in vertebrate models.


Immunoprecipitation
HUVEC were lysed in RIPA buffer (Millipore) supplemented with protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail set V (Sigma). After quantifying protein using the Quick Start Bradford protein assay (Bio-Rad), 500 µg protein lysate was loaded to the supplied spin column and immunoprecipitation was achieved following the manufacturer's protocol (Catch and Release Immunoprecipitation Kit; Catalog No.: 17-500; Millipore).
Zebrafish: Following imaging of vascular permeability, each individual zebrafish was euthanized and genomic DNA was extracted using standard techniques for subsequent STAT3 genotyping using the following pair of primers: 5'-GGTCTTCCACAACCTGCTG-3' and 5'-TAGACGCTGCTCTTCCCAC-3'.

Compounds
Pyrimethamine (PYR; 75 mg/kg; Sigma Aldrich; Catalog No. 46706) or control vehicle was delivered to mice by intraperitoneal injection daily for 15 days. PYR was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 167 mg/ml and then diluted in PBS before injection into mice. For zebrafish studies, 10µM PYR or vehicle (DMSO) was added to zebrafish embryo water for 3 days prior to assessment of vascular permeability and immunoblotting studies. Atovaquone (AQ; Sigma Aldrich; Catalog No. A7986), Catalog No. 573128), and PYR were each dissolved in DMSO and used for in vitro studies at the indicated concentrations. For in vivo studies, C188-9 was dissolved in DMSO at 208mg/ml and diluted in 5% dextrose in water (D5W) before injection. Mice received C188-9 (100 mg/kg) or vehicle (DMSO in D5W) by intraperitoneal injection daily for 1 week. For in vivo studies, Tyrphostin AG 490 (40 mg/kg, Sigma Aldrich; Catalog No.T3434) was dissolved in DMSO at the concentration of 250 mg/ml and diluted in PBS before injection. Mice received AG490 or vehicle (DMSO in PBS) by intraperitoneal injection daily for 1 week.

Glutathione S-transferase (GST) pull-down assays
The cDNA fragment encoding full-length human STAT3 was subcloned from pLEGFP-WT-STAT3 1 into the mammalian expression vector pEBG 2 using KpnI and NotI restriction enzyme sites and the following subcloning primer pairs: forward: 5'-GGGGTACCTCGAGCTCAAGCTTCAGGATGG-3' and reverse: 5'-ATAAGAATGCGGCCGCTCACTTGTAGTCCATGGGGGAGGTA-3'. pLEGFP-WT-STAT3 was a gift from Dr. George Stark (Addgene plasmid # 71450; http://n2t.net/addgene:71450; RRID:Addgene_71450) 1 . pEBG was generously shared by Dr. David Baltimore (Addgene plasmid # 22227; http://n2t.net/addgene:22227; RRID:Addgene_22227) 2 . Freestyle 293F cells (Thermo Fisher Scientific) grown in FreeStyle™ 293 Expression Medium (Thermo Fisher Scientific;12338018) in an incubator at 37°C with 8% CO2 were transfected with pEBG-STAT3 plasmid according to the instructions of the Freestyle 293 Expression System (Thermo Fisher Scientific) and then harvested by centrifugation at 1500 x g for 10 minutes. Cell pellets were suspended in PBS containing protease inhibitor cocktail (Roche) and lysed on ice using 15 s pulses of sonication repeated 7 times with a Sonic Dismembrator, Model 100 (Fisher Scientific). Lysates in 1% Triton, 5 mM DTT were centrifuged at 12000g for 10 minutes at 4°C, and Glutathione Sepharose 4B (GE Healthcare) was used to bind the GST fusion proteins from the supernatant. GST protein-coated beads were incubated with pre-cleared HUVEC cell lysates at 4°C overnight. The bead-protein complexes were then washed 5 times with pre-chilled PBS, and the proteins were eluted using 10mM Glutathione elution buffer at room temperature. The proteins were boiled for 5 minutes in the Laemmli sample buffer and then analyzed by immunoblotting.

Quantitation of zebrafish vascular permeability
ImageJ software was used to quantitate the extent of Texas Red-dextran in the extravascular space. Mean gray value was measured after converting the image type to 8-bit gray, setting scale to pixels, inverting the image and identifying 4 areas of extravascular space in the middle of the zebrafish trunk region using drawing/selection tools to avoid intersegmental vessels evident by green fluorescent signal originating from FITC-dextran. The background gray value minus the average gray value of the four regions is used for statistical analysis. . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2020. . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2020. ;https://doi.org/10.1101https://doi.org/10. /2020 Supplemental Figure 2: Densitometry analysis for immunoblotting of VEGF-stimulated HUVEC upon pretreatment with STAT3 inhibitors, atovaquone (AQ) and pyrimethamine (PYR). Densitometry was performed on the immunoblotting image depicted in Figure 4B by quantitating phosphorylated protein relative to total protein levels as indicated.
. CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2020. ;https://doi.org/10.1101https://doi.org/10. /2020 Supplemental Figure 5: Pharmacological inhibition of STAT3 stabilizes endothelial barrier integrity following VEGF stimulation in HUVEC and mice. A) Serum-starved HUVECs were pretreated with 10 µM C188-9 for 5 minutes prior to human VEGF-165 protein (25ng/ml) stimulation for 0, 2 and 5 minutes. VEGF stimulation promotes disorganization of ZO-1 (green) at endothelial cell junctions. ZO-1 organization is maintained when HUVEC were pretreated with C188-9. p-STAT3 (Y705; red) was reduced upon treatment with C188-9. Nuclei were stained with DAPI (blue). B) Depiction of selected ZO-1 staining to help visualize its organization or disorganization upon VEGF treatment in the absence of STAT3 inhibitor, C188-9. C) Serumstarved HPAEC were pretreated with 10 µM C188-9 for 5 minutes prior to human VEGF-165 protein (25ng/ml) stimulation for 0 and 5 minutes. After stimulation with VEGF protein for 5 minutes, the structure of tight junction marked with ZO-1 was disrupted (i.e. jagged-like ZO-1 green staining). ZO-1 organization is maintained when HPAEC were pretreated with C188-9. Nuclei were stained with DAPI (blue). D) Depiction of selected ZO-1 staining. (E) Mice were administered C188-9 or vehicle prior to tail vein injection with 1% Evans blue and VEGF (2.5 µg/ml) or PBS vehicle being injected into the root of the footpad. Quantitation of Evans blue leakage in C57BL/6 wildtype mice. n=5 mice per group. Each mouse was injected with PBS on right anterior and posterior footpads and VEGF on left anterior and posterior footpads. Multiple biological replicates were performed and depicted findings are representative. *P<0.05, paired t-test.
. CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2020. ; https://doi.org/10.1101/2020.10.27.358374 doi: bioRxiv preprint Supplemental Figure 6: Pyrimethamine (PYR) inhibits STAT3 activity in zebrafish. Zebrafish were exposed to embryo water containing DMSO or 25 μM PYR for 3 days at starting 6 hours post-fertilization. Protein lysates were harvested from 3 days post-fertilization embryos by sonication in RIPA buffer after removing yolk sac and immunoblotting was performed using antibodies against zebrafish p-STAT3 (Y708) and cofilin.
. CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 20, 2020. ; https://doi.org/10. 1101/2020