(A) LSEC-specific Col4a1-mutant mice were generated by crossing Col4a1^fl/wt mice with Cdh5^CreERT2 mice. Col4a1 mutation was induced by deleting exon 41 to produce a truncated dysfunctional COL4 protein. LSECs were isolated from Col4a1^fl/wt and Col4a1^fl/wt Cdh5^CreERT2 mice after in vivo tamoxifen administration to extract DNA for PCR. Post-PCR gel showed a lower–molecular weight band after Cre recombinase treatment to excise the floxed exon 41 sequence in Col4a1^fl/wt Cdh5^CreERT2 mouse LSECs (red arrow). (B) COL4 immunogold staining of mouse liver tissues showed that COL4 protein was secreted and deposited in liver sinusoids from Col4a1^fl/wt mice (red arrow), whereas LSEC-specific Col4a1 mutation inhibited COL4 deposition. Bottom images are 100 nm magnifications of red boxes on top 500 nm images. (C) To induce PHTN, Col4a1^fl/wt and Col4a1^fl/wt Cdh5^CreERT2 mice were subjected to 6 weeks of CCl₄ administration (or olive oil for control). CCl₄ injection increased portal pressure by 2-fold in Col4a1^fl/wt mice, whereas this increase was abrogated by Col4a1 mutation in LSECs (n = 8–13/group). (D) The ratio of spleen weight to body weight was increased after CCl₄ administration, which was blocked by LSEC Col4a1 mutation (n = 7–10/group). (E) To explore liver sinusoidal permeability, 4 kDa FITC-dextran was administered via the tail vein. It passed through LSECs and was endocytosed by hepatocytes (red stars) in healthy livers. In contrast, FITC-dextran failed to cross the membrane of CCl₄-induced LSECs, gathering at the sinusoidal line (red arrows). FITC-dextran was noticeable in hepatocytes from Col4a1-mutated livers even with CCl₄ treatment, which indicates that COL4 blocks sinusoidal permeability (n = 4/group). Scale bars: 10 μm. Graphs represent mean ± SEM. *P < 0.05, **P < 0.01, 1-way ANOVA followed by Bonferroni’s posttest.