Unraveling the transcriptional determinants of liver sinusoidal endothelial cell specialization

Abstract

Liver sinusoidal endothelial cells (LSECs) are the first liver cells to encounter waste macromolecules, pathogens, and toxins in blood. LSECs are highly specialized to mediate the clearance of these substances via endocytic scavenger receptors and are equipped with fenestrae that mediate the passage of macromolecules toward hepatocytes. Although some transcription factors (TFs) are known to play a role in LSEC specialization, information about the specialized LSEC signature and its transcriptional determinants remains incomplete.Based on a comparison of liver, heart, and brain endothelial cells (ECs), we established a 30-gene LSEC signature comprising both established and newly identified markers, including 7 genes encoding TFs. To evaluate the LSEC TF regulatory network, we artificially increased the expression of the 7 LSEC-specific TFs in human umbilical vein ECs. Although Zinc finger E-box-binding protein 2, homeobox B5, Cut-like homolog 2, and transcription factor EC (TCFEC) had limited contributions, musculoaponeurotic fibrosarcoma (C-MAF), GATA binding protein 4 (GATA4), and MEIS homeobox 2 (MEIS2) emerged as stronger inducers of LSEC marker expression. Furthermore, a combination of C-MAF, GATA4, and MEIS2 showed a synergistic effect on the increase of LSEC signature genes, including liver/lymph node-specific ICAM-3 grabbing non-integrin (L-SIGN) (or C-type lectin domain family member M (CLEC4M)), mannose receptor C-Type 1 (MRC1), legumain (LGMN), G protein-coupled receptor 182 (GPR182), Plexin C1 (PLXNC1), and solute carrier organic anion transporter family member 2A1 (SLCO2A1). Accordingly, L-SIGN, MRC1, pro-LGMN, GPR182, PLXNC1, and SLCO2A1 protein levels were elevated by this combined overexpression. Although receptor-mediated endocytosis was not significantly induced by the triple TF combination, it enhanced binding to E2, the hepatitis C virus host-binding protein. We conclude that C-MAF, GATA4, and MEIS2 are important transcriptional regulators of the unique LSEC fingerprint and LSEC interaction with viruses. Additional factors are however required to fully recapitulate the molecular, morphological, and functional LSEC fingerprint.NEW & NOTEWORTHY Liver sinusoidal endothelial cells (LSECs) are the first liver cells to encounter waste macromolecules, pathogens, and toxins in the blood and are highly specialized. Although some transcription factors are known to play a role in LSEC specialization, information about the specialized LSEC signature and its transcriptional determinants remains incomplete. Here, we show that Musculoaponeurotic Fibrosarcoma (C-MAF), GATA binding protein 4 (GATA4), and Meis homeobox 2 (MEIS2) are important transcriptional regulators of the unique LSEC signature and that they affect the interaction of LSECs with viruses.

Keywords: endothelial heterogeneity; liver sinusoidal endothelial cell specification; transcription factors.

Graphical abstract

Fig. 1.

Establishment of a 30-gene (human) liver sinusoidal endothelial cell (LSEC) fingerprint. Brains, hearts, and livers were isolated from Tie2-green fluorescent protein (GFP) mice (A), which express GFP specifically in endothelial cells (ECs). Frozen cross-sections of these organs stained for α-smooth muscle actin (αSMA; red) and DAPI (as nuclear counterstain) confirmed GFP expression (green) in all ECs, and costaining for CD34 (red) and GFP (green) on liver cross-sections confirmed CD34 as a macrovascular EC marker. FACS analysis after colabeling of GFP⁺ ECs from Tie2-GFP livers with CD34 (left) or CD36 (right) revealed that <1% and >99% of the ECs represented macrovascular and microvascular ECs, respectively, such that the profiles of GFP⁺ cells reflected that of (sinusoidal) capillaries. A (human) LSEC-specific 30-gene signature consisting of genes encoding transcription factors (TFs) (left) and marker genes (right) arranged according liver EC probe set intensity (C) was established from comparative gene expression profiling using a four-step filtering process and by adding three established markers not retained by the filtering process (B). In C, the expression of the 30 genes in mouse livers (closed bars), hearts (dark shaded bars), and brains (light shaded bars) are shown as mean log2 probe intensities ± SE (n = 5 for each organ; *³P < 0.001 vs. heart or brain by ANOVA). Dotted horizontal lines in C indicate the expression threshold levels used in filter step three. Human liver cross-sections (female donor) immunostained (red) for Legumain (LGMN) (D), liver/lymph node-specific ICAM-3 grabbing non-integrin (L-SIGN) (E), G protein-coupled receptor 182 (GPR182) (F), mannose receptor C-type 1 (MRC1) (G), PlexinC1 (PLXNC1) (H), and solute carrier organic anion transporter family member 2A1 (SLCO2A1) (I). Diagram (J) displays annotations [gene ontology (GO) terms] identified by database for annotation, visualization, and integrated discovery analysis significantly (adjusted P-values: *P < 0.05, *²P < 0.01, or *³P < 0.001) associated with the identified LSEC markers, expressed as the % of genes from the 30-gene signature allocated to the corresponding terms. Scale bars = 100 μm in A and D–I. $Established markers.

Fig. 2.

Identification of transcription factors (TFs) regulating liver sinusoidal endothelial cell (LSEC) marker gene expression (A). Expression of LSEC-specific TFs in freshly isolated (t = 0 h) primary mouse LSECs (closed bars), primary mouse LSECs cultured for 12 h (open bars), or primary mouse LSECs cultured for 24 h (shaded bars). Data were analyzed by ANOVA and are expressed as mean ± SE and represent expression levels relative to t = 0 h (n = 3; *P < 0.05; *²P < 0.01; *³P < 0.001). LSEC signature genes induced after 6 days of lentiviral overexpression in human umbilical vein endothelial cells of Zinc finger E-box-binding protein 2 (ZEB2) (B), homeobox B5 (HOXB5) (C), Cut-like homolog 2 (Cux2) (D), transcription factor EC (TCFEC) (E), musculoaponeurotic fibrosarcoma (C-MAF) (F), MEIS homeobox 2 (MEIS2) (G), or GATA binding protein 4 (GATA4) (H). Genes with significant (*P < 0.05) induction, borderline significant (#0.05 < P < 0.1) induction or at least twofold not significant (P > 0.1) induction are indicated in closed squares, shaded triangles or lightly shaded diamonds, respectively. Insets: pie charts of corresponding distributions of these four categories of B–H. Data are expressed as means ± SE and represent fold increase relative to control virus (n = 6–8 from 2 independent experiments; Wilcoxon signed ranked test).

Fig. 3.

Identification of a transcription factor (TF) combination regulating liver sinusoidal endothelial cell (LSEC) marker gene expression. A: expression of LSEC signature genes (arranged according to the fold increase) after 6 days of combined lentiviral overexpression of musculoaponeurotic fibrosarcoma (C-MAF), GATA binding protein 4 (GATA4) and MEIS homeobox 2 (MEIS2) in human umbilical vein endothelial cells (HUVECs). Genes with at least twofold significant (*P < 0.05) induction, borderline significant (#0.05 < P < 0.1) induction, at least twofold not significant (P > 0.1) induction, or no induction are indicated in closed squares, shaded triangles, lightly shaded diamonds, or open circles, respectively. Corresponding distributions of these four categories are shown as a pie chart (upper right inset). Data are expressed as means ± SE and represent fold increase relative to control virus-transduced HUVECs from the same donor (n = 6–8 from 2 independent experiments; Wilcoxon signed ranked test). B: table schematically representing the effect of each TF or the triple combination on expression of the signature genes and corresponding pie charts (color codes same as those in A). C: annotations [gene ontology (GO) terms] identified by database for annotation, visualization, and integrated discovery analysis significantly (adjusted P values: *P < 0.05; *²P < 0.01) associated with the identified LSEC markers, expressed as the % of genes from the 30-gene signature allocated to the corresponding terms. Genes only statistically significantly induced by the three TF combination or still not induced by the three TF combination are underlined or shown in smaller font size, respectively. ND, not detectable.

Fig. 4.

Liver sinusoidal endothelial cell (LSEC) marker protein expression induced by combined musculoaponeurotic fibrosarcoma (C-MAF), GATA binding protein 4 (GATA4), and MEIS homeobox 2 (MEIS2) overexpression. Western blots showing protein expression in human umbilical vein endothelial cells (HUVECs) transduced with a combination of C-MAF, GATA4, and MEIS2 coding lentiviruses [transcription factors (TFs)] (closed bars) or with control virus (C) (shaded bars) for (pro)-legumain (LGMN) (A), mannose receptor C-Type 1 (MRC1) (B), liver/lymph node-specific ICAM-3 grabbing non-integrin (L-SIGN) (C), G-Protein-Coupled Receptor 182 (GPR182) (D), Solute Carrier Organic Anion Transporter Family Member 2A1 (SLCO2A1) (E), and PlexinC1 (PLXNC1) (F). GAPDH is used as loading control (the same GAPDH loading control was used for GPR182 and SLCO2A1 because both proteins were detected on the same blot). Data represent mean band intensity ± SE, expressed as arbitrary units (AUs) (n = 4, representative of 2–3 independent experiments) (*P < 0.05; *²P < 0.01 vs. control virus by Student’s t test). Representative pictures of HUVECs treated with the triple TF combination (MGM) or with control virus and stained (red) for pro-LGMN (G and H), MRC1 (I and J), L-SIGN (K and L), GPR182 (M and N), SLCO2A1 (O and P), and PLXNC1 (Q and R). Scale bars = 250 μm.

Fig. 5.

Molecular and functional specialization on combined musculoaponeurotic fibrosarcoma (C-MAF), GATA binding protein 4 (GATA4), and MEIS homeobox 2 (MEIS2) overexpression. Scanning electron micrographs of freshly isolated rat liver sinusoidal endothelial cells (LSECs) (A), human umbilical vein endothelial cells (HUVECs) transduced with a combination of C-MAF, GATA4, and MEIS2 coding lentiviruses (MGM) (B), or HUVECs transduced with control virus (C). Diagrams representing cell-associated and degraded (endocytosed) formaldehyde-treated serum albumin (FSA) (D), RNAseB (E), or aggregated gamma globulin (AGG) (F) after 1 h incubation with freshly isolated rat LSECs (open bars), HUVECs transduced with a combination of C-MAF, GATA4, and MEIS2 coding lentiviruses (closed bars), or HUVECs transduced with control virus (shaded bars). Data represent means ± SE (n = 1 for rat LSECs; n = 4 for HUVECs). Diagram (G) representing quantification of the fraction of transduced [green fluorescent protein positive (GFP⁺)] cells (quadrant 1 + 2) that was bound to the E2 protein of the hepatitis C virus (HCV-E2) in HUVECs transduced with a combination of C-MAF, GATA4, and MEIS2 coding lentiviruses (closed bar) or control virus (shaded bar). Data represent mean fraction of E2⁺ cells ± SE, expressed relative to the total amount of GFP⁺ cells (n = 3 from 3 independent experiments; *P < 0.01 vs. control virus by Student’s t test). Scale bars in A–C = 200 nm.