Fig 1: Loss of par1 function decreases p-Erk1/2 activity and flt4 expression in the PCV of zebrafish embryos(A) Immunostaining of phosphoErk1/2 (p-Erk1/2) activity in Tg(fli1a:EGFP) siblings and par1 homozygous mutants at 28 hpf. Blue arrowheads with white dashed lines indicate positive p-Erk1/2 staining; DA and PCV (in brackets) area are noted. Scale bars: 100 μm.(B) Quantification of p-Erk1/2 expression staining in the PCV of siblings (n = 15 embryos) and par1 mutants (n = 15 embryos); 8 somites/embryos were used for quantification.(C) Relative mRNA expression of VEGFR3 in HDLECs after ctr-siRNA (control) and PAR1-siRNA transfection.(D) Western blot analysis of p-ERK1/2 activity in HDLECs transfected with ctr-siRNA or PAR1-siRNA, followed by VEGFC treatment.(E) Relative mRNA expression of the flt4 gene in siblings and par1 homozygous zebrafish embryo mutants.(F) WISH of flt4 gene expression at 28 hpf in wild-type and par1 homozygous mutant embryos. The white arrowhead indicates the PCV area.(G) WISH of hhex gene expression at 28 hpf in wild-type and par1 mutant embryos. The white arrowhead indicates the PCV area. In (B, C, and, E), values represent means ± SEMs. ∗p ≤ 0.05, ∗∗p ≤ 0.001, ∗∗∗p ≤ 0.0001 in the Student's t test.
Fig 2: mLV density increased in liver cirrhosis.(A) IHC with Pdpn antibody was performed in mesentery tissue of control and CCl4 model of liver cirrhosis. Scale bar: 500 μm. (B) Number of the pdpn + mLVs was quantified using ImageJ. p = 0.0240. (C) Diameter of mLVs in control and CCl4 rats was measured using ImageJ’s diameter plugin. n = 5 each. p = 0.0119. (D) mRNA levels for Prox1, LyVE1, VEGF-C, CCL21, eNOS, and COX2 were quantified in mesentery tissue. n = 5 each. p = 0.05–0.01. The dotted line represents control. (E) Immunofluorescence staining for VEGFR-3, and DAPI was performed in mesentery tissue of control and CCl4 rats. Scale bar: 186.2 μm. (F) VEGFR-3 expression was quantified using ImageJ. n = 5 each. p = 0.0147. Data expressed as mean ± SD. Two-tailed, unpaired t test.∗p < =0.05. IHC, immunohistochemistry; LV, lymphatic vessel; MFI, mean fluorescence intensity; mLV, mesenteric LV; Pdpn, podoplanin; VEGF-C, vascular endothelial growth factor-C; VEGFR-3, vascular endothelial growth factor receptor-3.
Fig 3: Effect of E-VEGF-C on gene expression profiling and permeability of LyECs.(A) Schema of workflow. LyECs were isolated from the mesentery and MLNs using FACS. RNA isolation and qRT-PCR analysis were performed for different marker genes of LyECs. (B) Relative gene expression of LyVE1, Prox1, VCAM1, VE-Cad, MHC-II, CD86, CCL21, and COX2 genes in CCl4-V and E-VEGF-C-treated rats were plotted. Dotted lines represent control (C) Transwell permeability assay using BODIPY in cultured mesenteric LyECs from control, CCL4-V, and E-VEGF-C-treated rats. The concentration of BODIPY in collected media after 4, 6, 10, and 24 h n = 3 each. Data expressed as mean ± SD. One-way ANOVA with Tukey’s post hoc test was performed. ∗p <0.05; ∗∗p <0.01 (comparison with CCl4-V). CCl4, carbon tetrachloride; E-VEGF-C, engineered VEGF-C; LyEC, lymphatic endothelial cell; MLN, mesenteric lymph node; ns, not significant; qRT-PCR, quantitative reverse-transcription PCR; VE-Cad, vascular endothelial-cadherin; VEGF-C, vascular endothelial growth factor-C.
Fig 4: Effect of E-VEGF-C on priming of immune cells after bacterial challenge.(A) Schema of workflow, 109 GFP + bacteria were given to rats orally, and tissue was collected in sterile conditions after 48 h of gavage. Afterward, 100 mg of MLN tissue was used for bacterial load quantification. Cells were isolated from MLNs for quantification using flow cytometry. (B) GFP+ Salmonella typhimurium colonies in 100 mg of MLN tissue extract of each group were visualised using a UV transilluminator. (C) Quantitative analysis of CFU/gm of MLN tissue for GFP + Salmonella typhimurium. n = 3 each. p <0.001. (D) Dot plots of T-cell subsets in cells isolated from MLNs of control, CCl4-V and E-VEGF-C treated rats after bacterial challenge. (E) Percentage population of CD4 Th cells, CD8 Tc cells, and CD134+ recently activated Th and Tc cells. (F) Dot plots of CD25+ regulatory Th cells. (G) Percentage population of Treg cells positive for CD3, CD4/CD8, and CD25. n = 4 each. Data expressed as mean ± SD. One-way ANOVA with Tukey’s post hoc test was performed. ∗p <0.05; ∗∗p <0.01; ∗∗∗p <0.001 (comparison with CCl4-V). #p <0.05 (comparison with control). CCl4, carbon tetrachloride; CFU/gm, colony forming unit–granulocyte/macrophage; E-VEGF-C, engineered VEGF-C; GFP, green fluorescent protein; MLN, mesenteric lymph node; ns, not significant; Tc, cytotoxic T; Th, helper T; Treg, regulatory T; VEGF-C, vascular endothelial growth factor-C.
Fig 5: Effect of E-VEGF-C on proliferation and drainage of mLVs.(A) Whole-mount immunostaining of mesentery for visualisation of mLVs in the control, CCl4-V, and E-VEGF-C study groups. The upper two panels represent collecting mLVs, and the lower two represent lymphatic capillaries. Scale bar: 309.4 μm. (B) Diameter of collecting mLVs was measured using the ImageJ diameter plugin. n = 3 or 4 rats in each group. p <0.0001 for control vs. CCl4-V and p = 0.0183 for CCl4-V vs. E-VEGF-C. (C) Mesentery tissue of CCl4-V and E-VEGF-C rats. Arrowheads indicated proliferation and branching of mLVs in the mesentery of E-VEGF-C-treated rats. (D) Quantitative analysis of branching points in the mesentery in control, CCl4-V, and E-VEGF-C rats. n = 3–4 rats in each group. p >0.05 for each comparison. (E) Whole-mount images of mLVs 2 h after BODIPY FL-C16 administration in control, CCl4-V, and E-VEGF-C rats. Scale bar: 309.1 μm. Representative graphs for characterisation of functional mLVs. Mean of three points from each field was taken. (F) Diameter of LVs was measured using ImageJ diameter plugin. (G) Drainage of LVs was measured by quantifying fluorescence intensity inside the vessels using ImageJ. (H) Leakage from mLVs was quantified by measuring fluorescence intensity in the extraluminal space of mLVs using ImageJ. n = 4 or 5 rats in each group. (I) Whole-mount images of mLVs 2 h after BODIPY FL-C16 administration in TAA-V, TAA + SAR, and TAA + E-VEGF-C rats. Scale bar: 200 μm Representative graphs for characterisation of functional mLVs. A mean of three points from each field was taken. (J) Diameter of LVs was measured in μm using ImageJ diameter plugin. (K) Drainage of LVs was measured by quantifying fluorescence intensity inside the vessels using ImageJ. (L) Leakage from mLVs was quantified by measuring fluorescence intensity in the extraluminal space of mLVs using ImageJ. n = 4 or 5 rats in each group. Data expressed as mean ± SD. One-way ANOVA with Tukey’s post hoc test was performed. ∗p <0.05; ∗∗p <0.01; ∗∗∗p <0.001. CCl4, carbon tetrachloride; E-VEGF-C, engineered VEGF-C; LV, lymphatic vessel; mLV, mesenteric LV; ns, not significant; TAA, thioacetamide; VEGF-C, vascular endothelial growth factor-C.
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