Liver sinusoidal endothelial cells contribute to portal hypertension through collagen type IV–driven sinusoidal remodeling
Can Gan, Usman Yaqoob, Jianwen Lu, Man Xie, Abid Anwar, Nidhi Jalan-Sakrikar, Sofia Jerez, Tejasav S. Sehrawat, Amaia Navarro-Corcuera, Enis Kostallari, Nawras W. Habash, Sheng Cao, Vijay H. Shah
Can Gan, Usman Yaqoob, Jianwen Lu, Man Xie, Abid Anwar, Nidhi Jalan-Sakrikar, Sofia Jerez, Tejasav S. Sehrawat, Amaia Navarro-Corcuera, Enis Kostallari, Nawras W. Habash, Sheng Cao, Vijay H. Shah
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Research Article Cell biology Hepatology

Liver sinusoidal endothelial cells contribute to portal hypertension through collagen type IV–driven sinusoidal remodeling

Abstract

Portal hypertension (PHTN) is a severe complication of liver cirrhosis and is associated with intrahepatic sinusoidal remodeling induced by sinusoidal resistance and angiogenesis. Collagen type IV (COL4), a major component of basement membrane, forms in liver sinusoids upon chronic liver injury. However, the role, cellular source, and expression regulation of COL4 in liver diseases are unknown. Here, we examined how COL4 is produced and how it regulates sinusoidal remodeling in fibrosis and PHTN. Human cirrhotic liver sample RNA sequencing showed increased COL4 expression, which was further verified via immunofluorescence staining. Single-cell RNA sequencing identified liver sinusoidal endothelial cells (LSECs) as the predominant source of COL4 upregulation in mouse fibrotic liver. In addition, COL4 was upregulated in a TNF-α/NF-κB–dependent manner through an epigenetic mechanism in LSECs in vitro. Indeed, by utilizing a CRISPRi-dCas9-KRAB epigenome-editing approach, epigenetic repression of the enhancer-promoter interaction showed silencing of COL4 gene expression. LSEC-specific COL4 gene mutation or repression in vivo abrogated sinusoidal resistance and angiogenesis, which thereby alleviated sinusoidal remodeling and PHTN. Our findings reveal that LSECs promote sinusoidal remodeling and PHTN during liver fibrosis through COL4 deposition.

Authors

Can Gan, Usman Yaqoob, Jianwen Lu, Man Xie, Abid Anwar, Nidhi Jalan-Sakrikar, Sofia Jerez, Tejasav S. Sehrawat, Amaia Navarro-Corcuera, Enis Kostallari, Nawras W. Habash, Sheng Cao, Vijay H. Shah

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Figure 8

COL4 contributes to sinusoidal remodeling via activating angiogenic sprouting of LSECs.

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COL4 contributes to sinusoidal remodeling via activating angiogenic spro...
(A) Representative IF images of isolated LSECs stained for Col4a1 (red) and Hsp47 (green) (DAPI, blue, indicates nucleus). Mice with PHTN showed Col4a1 production and secretion in LSECs (white arrows), whereas Col4a1 mutation in LSECs led to dysfunctional Col4a1 retained in endoplasmic reticulum (ER, red arrows). Hsp47 is a chaperone located in ER to help collagen synthesis. Images in the first column are 10 μm magnifications of white boxes in the second column (20 μm, n = 3/group). (B and C) Human LSECs were transfected with COL4A1 small interfering RNA (siRNA) to knock down the expression of COL4. Scramble siRNA was used as a negative control. (B) qPCR results showed decreased COL4 expression in human LSECs after COL4A1 siRNA transfection. (C) COL4A1 siRNA–transfected human LSECs were plated in 3D fibrin gel and treated with TNF-α. Representative IF images of fibrin gel stained for COL4 (green) showed it to be secreted extracellularly and organized into COL4 bundles and tubules. These formed tubes in the scramble siRNA group, shown by increased branch points, but COL4A1 knockdown inhibited tube formation due to the lack of COL4 production and secretion (n = 3, biologically independent samples). Green indicates COL4 stained with anti-COL4 antibody; red, phalloidin staining of actin filaments; blue, DAPI staining of DNA. Scale bars: 200 μm (top), 20 μm (bottom). Graphs represent mean ± SEM. *P < 0.05, ***P < 0.001, 2-tailed unpaired t test.