GIMAP5 maintains liver endothelial cell homeostasis and prevents portal hypertension

Abstract

Portal hypertension is a major contributor to decompensation and death from liver disease, a global health problem. Here, we demonstrate homozygous damaging mutations in GIMAP5, a small organellar GTPase, in four families with unexplained portal hypertension. We show that GIMAP5 is expressed in hepatic endothelial cells and that its loss in both humans and mice results in capillarization of liver sinusoidal endothelial cells (LSECs); this effect is also seen when GIMAP5 is selectively deleted in endothelial cells. Single-cell RNA-sequencing analysis in a GIMAP5-deficient mouse model reveals replacement of LSECs with capillarized endothelial cells, a reduction of macrovascular hepatic endothelial cells, and places GIMAP5 upstream of GATA4, a transcription factor required for LSEC specification. Thus, GIMAP5 is a critical regulator of liver endothelial cell homeostasis and, when absent, produces portal hypertension. These findings provide new insight into the pathogenesis of portal hypertension, a major contributor to morbidity and mortality from liver disease.

Figure 1.

Rare recessive LOF mutations in GIMAP5 identified in four kindreds. (A) Pedigrees depict four unrelated families. Affected and unaffected subjects are shown as black-filled and white-filled symbols, respectively. Mutations (p.I47T in kindred 1, p.L223P in kindred 2, p.P109L in kindred 3, and p.L204P in kindred 4) are homozygous in the available affected subjects and heterozygous in the parents. Consanguineous unions are denoted by a double line. (B) Schematic representation of human GIMAP5 protein with AIG1 domain depicted in dark gray, where all four missense mutations are located. Conservation of Ile47, Pro109, Leu204, and Leu223 positions across orthologues and GIMAP family members are shown. Amino acid positions identical to the human reference are highlighted in yellow. AIG1, GTP-binding AIG1 homology domain; TD, transmembrane domain; mut, mutant; wt, wild-type.

Figure S1.

Representative images of histological findings seen in liver biopsies from patients (P1-1, P2-1, P3-1, and P4-1) with biallelic mutations in GIMAP5. (A) P1-1’s liver biopsy shows lobular parenchyma devoid of any steatosis, hepatocytic ballooning, or acidophil necrosis. Minimal and focal lymphocytic infiltrates (arrow) are seen along with dilated channels at the periphery of the portal tracts, with some of them extending into the sinusoids (left panel; H&E stain). Middle panel reveals nodular architecture due to areas of regeneration consisting of thickened hepatic cell plates next to areas of atrophy without any intervening fibrosis (reticulin stain). Right panel shows few thin septa (in blue) in the left aspect of the picture (arrow) but lack of cirrhosis (trichrome stain). (B) P2-1’s liver biopsy shows largely unremarkable hepatocytes with occasional foci of lobular inflammation (arrow; left panel; H&E stain). Middle panel shows liver nodularity with zones of widened two-cell-thick plates consistent with hepatic regeneration bounded by narrow compressed liver cell plates (reticulin stain). Right panel shows periportal fibrosis (arrow; trichrome stain). (C) P3-1’s liver biopsy shows no significant abnormalities in the hepatocytes (left panel; H&E stain). Middle panel shows areas of regeneration consisting of thickened hepatic cell plates (reticulin stain). Right panel shows an area of hepatocyte loss with parenchymal collapse and fibrosis (arrow) but no evidence of cirrhosis (trichrome stain). (D) P4-1’s liver biopsy shows a portal tract with minimal and focal lymphocytic infiltrates (arrow). The lobular parenchyma is devoid of any steatosis, hepatocytic ballooning, or acidophil necrosis (left panel; H&E stain). Middle panel highlights an area of pericentral hepatocyte loss with parenchymal collapse and thin fibrous septa. The liver shows vague nodularity with areas of hepatic regeneration but no cirrhosis (reticulin stain). Right panel reveals few thin septa (blue) but lacks features of cirrhosis (trichrome stain). (E) Normal-appearing portal tract (oval) and a central venule (asterisk). The portal tract depicts a hepatic arteriole, bile duct, and portal venule. The portal venule is the largest structure with the widest lumen and a thin wall (H&E stain). Middle panel shows hepatic cords in a single thick cord plate. The central vein is seen near the center, and a portal tract is at the periphery (reticulin stain). Right panel shows normal architecture of the liver and no fibrosis (trichrome stain). Scale bars = 50 µm.

Figure 2.

Liver histology and CD34 expression in humans and mice with GIMAP5 deficiency. (A) Photomicrographs of abnormal CD34 immunostaining in liver sinusoids of four unrelated patients (P1-1, P2-1, P3-1, and P4-1) as compared with an unaffected control. (B) Photomicrographs of abnormal CD34 sinusoidal expression is shown in the liver sections from C57BL/6 Gimap5^sph/sph mice at 2, 3, and >7 wk old and in adult C57BL/6 Gimap5^sph/sphRag1^−/− mice as compared with Gimap5^sph/+ control mice. Histological data from 2-wk-old, 3-wk-old, adult Gimap5^sph/sph, and adult Gimap5^sph/sphRag1^−/− mice was verified at least four, two, seven, and three independent times, respectively. Littermates were used as controls. Scale bar = 50 µm.

Figure S2.

Liver pathology of Gimap5^sph/+Rag1^−/− and Gimap5^sph/sphRag1^−/− mice. (A) Gross liver morphology of adult Gimap5^sph/+Rag1^−/− (smooth) and Gimap5^sph/sphRag1^−/− mice (nodular). (B) Reticulin and trichrome stains of liver sections from Gimap5^sph/+Rag1^−/− (left panels) and Gimap5^sph/sphRag1^−/− (right panels) show two-cell-thick plate and increased collagen (blue) deposition solely in mutant mice. (C) Flow cytometric analysis shows increased CD34 expression in liver endothelial cells (DAPI⁻CD45⁻CD31⁺) isolated from Gimap5^sph/sphRag1^−/− mice as compared with Gimap5^sph/+Rag1^−/− mice. Experimental data were verified in at least two independent experiments, and littermates were used as controls. Scale bars = 50 µm.

Figure 3.

Genetic deficiency of GIMAP5 causes liver endothelial cell abnormalities. (A) Gimap5 mRNA expression in sorted liver endothelial cells (LECs; DAPI⁻CD45⁻CD31⁺) from C57BL/6 WT and Rag1^−/− mice, and splenocytes, sorted Kupffer cells (DAPI⁻CD45⁺CD115⁺F4/80⁺), and hepatocytes from C57BL/6 mice. (B) Immunoblot for GIMAP5 in Gimap5^sph/+ and Gimap5^sph/sph splenocytes and hepatocytes and in sorted LECs and liver CD45⁺ cells from Gimap5^sph/+ mice. GAPDH is shown as a loading control. (C) Confocal microscopy of LECs isolated from Gimap5^sph/+ and Gimap5^sph/sph and stained for GIMAP5 (green), Lamp-1 (red), and cytochrome-c (magenta) and counterstained with DAPI (blue). Scale bars = 5 µm. (D) LECs isolated from Gimap5^sph/+, Gimap5^sph/sph, and Gimap5^sph/sphRag1^−/− livers (left panels) and respective Ly6a and CD34 surface expression (right panels). (E) Absolute number of LECs that express CD34 or not in Gimap5^sph/+ (n = 6) and Gimap5^sph/sph (n = 5) livers. (F) Histological and flow cytometric analysis of sinusoidal CD34 expression in tamoxifen-treated adult Gimap5^flx/flxxCdh5(PAC)-CreERT2 and Gimap5^flx/flxxVav1-Cre mice (upper and bottom panels, respectively). Experimental data were verified in at least two independent experiments, and littermates were used as controls. Scale bars = 50 µm. Numbers depict the percentage of total cells. Student’s two-tailed t test was used. *, P < 0.05; **, P < 0.005; ***, P < 0.0005. Adult mice, >7 wk old. APC, allophycocyanin; Cy, cyanine.

Figure S3.

Subcellular localization of Gimap5 in sorted liver endothelial cells (LECs; DAPI⁻CD45⁻CD31⁺). (A and B) Confocal microscopy of isolated LECs from Gimap5^sph/+ (A) and Gimap5^sph/sph (B) and stained for GIMAP5 (green), lysosomal marker Lamp-1 (red), and mitochondrial marker cytochrome-c (magenta) and counterstained with DAPI (blue). Experimental data were verified in at least two independent experiments. Scale bars = 5 µm. Images shown in Fig. 3 C are also included in the panels above.

Figure 4.

scRNA-seq analysis of liver endothelial cells from Gimap5^sph/+ and Gimap5^sph/sph mice. (A) Clustering of endothelial cells from Gimap5^sph/+ and Gimap5^sph/sph livers using the Louvain method with a resolution parameter 0.4. Uniform Manifold Approximation and Projection (UMAP). Cell identities were inferred from marker genes. (B) Absolute number of cells in each endothelial subpopulation. (C) Overlay of endothelial cell cluster maps annotated by genotype: Gimap5^sph/+ and Gimap5^sph/sph. (D) Pecam1, Clec4g, Dnase3l1, Ly6a, Cd34, and Gata4 expression in combined subpopulations of LSECs and CECs isolated from Gimap5^sph/+ (blue) and Gimap5^sph/sph (red) mice. ECs, endothelial cells. (E) Enrichment plot from GSEA of preranked list of genes differentially expressed in GIMAP5-deficient and sufficient liver endothelial cells as compared with a background list of mouse Gata4-dependent liver endothelial cell regulated genes. NES, normalized enrichment score. (F) Cluster of LSECs, CECs, and macrovascular-like cells with trajectory analysis performed in Monocle.