Hepatocyte-specific knockout of HIF-2α cannot alleviate carbon tetrachloride-induced liver fibrosis in mice

Animals and treatments

We used the Cre-loxP system to generate hepatocyte-specific HIF-2α knockout mice. In principle, the albumin promoter drives the expression of cyclization recombination enzyme (Cre), leading to HIF-2α knockout (KO) located between two floxed (fl) sequences in hepatocytes. Floxed control wild-type (WT) mice were used as the control group. The mice used in this experiment were on the C57BL/6J background. Epas1tm1Mcs/J mice (HIF-2α^fl/fl) were purchased from the Jackson Laboratory (Bar Harbor, ME, USA; stock #008407), and B6.Cg-Speer6-ps1Tg 21Mgn/J mice (Alb-Cre^+/−) were purchased from Shanghai Model Organisms Center, Inc. (Shanghai, China; stock #JAX-003574). All mice were kept in an experimental condition with a 12-h light-to-dark cycle at 25 °C ± 2 °C and were fed a normal diet (ND) and water ad libitum. Animal studies were performed according to protocols approved by the Institutional Animal Care and Use Committee of the Fifth Affiliated Hospital of Sun Yat-sen University (approval number: 00158). All mice were treated humanely and with efforts to minimize suffering. Mice were deeply anesthetized with isoflurane and sacrificed by cervical dislocation. There were no surviving animals at the end of study.

Animal experiments were divided into two stages. In the first stage, eleven 7-week-old male mice (six KO and five WT mice) were fed a ND for 16 weeks to examine the possible effect of hepatocyte-specific HIF-2α deletion on the growth and metabolic indexes of mice, the body weight and food intake of mice were measured every twice week. In the second stage, thirty 7-week-old male mice (fifteen KO and fifteen WT mice) were divided into four groups [WT (n = 8), KO (n = 8), WT + CCl₄ (n = 7), and KO + CCl₄ (n = 7)] to evaluate whether hepatocyte-specific HIF-2α deletion could improve CCl₄-induced liver fibrosis in mice, the body weight and food intake of mice were measured every week. Groups treated with CCl₄ were intraperitoneally injected with 10% CCl₄ (CCl₄: olive oil = 1:9) at 5 ml/kg for 8 weeks, twice a week, to develop a liver fibrosis model, and the groups treated without CCl₄ were intraperitoneally injected with the corresponding amount of olive oil.

General condition and body composition analysis

An EchoMRI quantitative magnetic resonance (QMR) system (Nixon et al., 2010) (EchoMRI-500H; EchoMRI Houston, TX, USA) was used to analyze the body composition (fat mass, lean mass, total body water and free water) of mice in the awake state.

Glucose and insulin tolerance

We performed intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT) to evaluate glucose and insulin tolerance 2 weeks before the mice were sacrificed. Mice were injected intraperitoneally with 0.75 U/kg insulin after 4 h of fasting or 2.5 g/kg glucose after 12 h of fasting. Blood glucose from the tail tip was measured at 0, 30, 60, 90, and 120 min after injection using a Roche blood glucose monitoring system. Serum insulin levels were quantitatively measured by a commercial ELISA kit (Mercodia Mouse Insulin ELISA, 10-1247-10; Mercodia, Uppsala, Sweden). Insulin resistance index (HOMA-IR) was calculated using fasting serum glucose and fasting serum insulin according to the previously mentioned article (Yu et al., 2023).

Blood parameter analyses

After 8 h of fasting, all mice were sacrificed, and blood samples were collected. After centrifugation at 1,000×g for 15 min at 4 °C, serum samples were collected and stored at −80 °C until measurements. Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (TC), triglyceride (TG), high-density lipoprotein (HDL), and low-density lipoprotein (LDL) were measured according to the manufacturer’s protocols using commercial quantification assay kits (Nanjing Jiancheng Bioengineering Institution, Nanjing, China).

Liver hydroxyproline measurements

Hydroxyproline levels in the liver tissues were measured using commercial quantification assay kits from Nanjing Jiancheng Bioengineering Institution (Nanjing, China) according to the manufacturer’s protocols.

Liver histology

Liver tissues were fixed in 4% paraformaldehyde for 24 h, dehydrated and embedded in paraffin. Paraffin sections (4-μm thick) were stained with Masson’s trichrome to evaluate the degree of collagen distribution. Liver fibrosis was scored with blinding of genotype using the fibrosis ISHAK score. The ISHAK staging was determined according to the Ishak et al. (1995) protocol. Hematoxylin and eosin (HE) staining was used to evaluate the degree of necrotizing inflammatory liver injury. Immunohistochemistry (IHC) staining of the liver was used to determine the localization and expression of HIF-2α and α-SMA. The HIF-2α antibody (NB100-122; Novus, St. Louis, MO, USA) was used at a dilution of 1:100, and α-SMA antibody (ab124964; Abcam, Cambridge, UK) was used at a dilution of 1:1,000.

Polymerase chain reaction (PCR)

An alkaline lysis protocol was used to extract genomic DNA from the mouse toe/tail, and the genotype of the mice was confirmed by PCR analysis. The forward primer for HIF-2α was 5′-GAGAGCAGCTTCTCCTGGAA-3′, and the reverse primer for HIF-2α was 5′-TGTAGGCAAGGAAACCAAGG-3′. Two pairs of primers were used for Cre amplification. The first forward primer for Cre was 5′-ATTTGCCTGCATTACCGGTCG-3′, and the corresponding reverse primer was 5′-CAGCATTGCTGTCACTTGGTC-3′, which amplified a 310-bp band. The second forward primer for Cre was 5′-CAAATGTTGCTTGTCTGGTG-3′, and the corresponding reverse primer was 5′-GTCAGTCGAGTGCACAGTTT-3′, which produced 200-bp products. All PCR products were identified by agarose gel electrophoresis.

Quantitative real-time PCR (qRT–PCR)

Total RNA was extracted from liver tissues using a Tissue Total RNA Isolation Kit following the manufacturer’s instructions (Vazyme Biotech Co., Ltd., Nanjing, China) and then reverse-transcribed to complementary DNA. Finally, qRT‒PCR was performed by a QuantStudio 7 Flex Real-time PCR System. The forward primer for mouse HIF-2α was 5′-AAGGTGAAGAGCATCATAACCCT-3′, and the corresponding reverse primer was 5′-TCACGCCTTTCATAACACATTCC-3′. The forward primer for mouse β-actin was 5′-CTGGCACCACACCTTCTAC-3′, and the corresponding reverse primer was 5′-TCGTAGATGGGCACAGTGTGG-3′.

Bioinformatics analyses

RNA sequencing (RNA-seq) data of 31 normal human tissues in the Genotype-Tissue Expression (GTEx) database were downloaded from UCSC (https://xenabrowser.net/datapages/). The RNA-seq matrix and gene-expression array matrix of patients with alcoholic-related liver disease (GSE142530), chronic HBV-related liver disease (GSE84044), chronic HCV-related liver disease (GSE33650), and nonalcoholic fatty liver disease (GSE49541) were downloaded from the Gene Expression Omnibus database (https://www.ncbi.nlm.nih.gov/gds/). The RNA-seq matrix of CCl₄-induced liver fibrosis in mice was from GSE207855. Gene symbols were transformed according to their platform files, and the expression level of duplicate gene symbols in the same sample was taken from their average value. Gene expression levels are shown as transcripts per kilobase per million mapped reads (TPM) and relative mRNA levels for the RNA-seq data and gene-expression array data, respectively. Detailed information about these datasets is shown in Table S1.

Single-cell RNA-seq of hepatic nonparenchymal cells in normal and CCl₄-induced liver fibrotic mice was performed using GSE134037. The “Seurat” package was used to preprocess the single-cell RNA sequencing data (Butler et al., 2018). Genes expressed in fewer than five cells in a sample and cells that expressed fewer than 600 genes were excluded. Cells whose total feature RNA was more than 4,500, total unique molecular identifiers (UMIs) were more than 10,000, and mitochondrial gene content was >12% of the total UMIs were considered quality control standards. Data processing followed the standard process and used the default parameters. We retained 20 components for the merged object according to the elbow plot. To cluster these cells, we first applied the FindNeighbors function to construct a K-nearest neighbor graph according to the Euclidean distance in PCA space and then performed the FindClusters function to iterate group cells together with the resolution parameter set at 0.5. Furthermore, we contrasted cells from the subcluster to all other cells of that subcluster using the FindMarkers function. Biologically meaningful cell types were annotated according to the DEGs of each subcluster and canonical cell markers based on the “SingleR” package combined with manual work (Aran et al., 2019). All bioinformatics analyses were performed using R software (Version 4.2.0; R Core Team, 2022).

Statistical analyses

Data are presented as the mean ± standard deviation (SD). Group comparisons were performed using Student’s t test. Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software Inc., San Diego, CA, USA). A two-sided p value less than 0.05 was considered statistically significant.

Establishment of hepatocyte-specific HIF-2α knockout mice

Mice with the HIF-2α^fl/fl genotype were mated to Alb-Cre⁺ mice, and we obtained Alb-Cre⁺/HIF-2α^fl/wt mice from their offspring. Then, the Alb-Cre⁺/HIF-2α^fl/wt mice were mated to Alb-Cre⁻/HIF-2α^fl/fl mice to produce Alb-Cre⁺/HIF-2α^fl/fl (KO) mice and Alb-Cre⁻/HIF-2α^fl/fl (WT) mice (Fig. 1A). The mouse genotypes were identified by PCR. The HIF-2α^fl/fl mice produced a 220-bp band for HIF-2α amplification (Fig. 1B), and the Alb-Cre⁺ mice produced hybrid bands of 310 and 200 bp for Cre amplification (Fig. 1C). The relative HIF-2α mRNA level was obviously downregulated in the KO mice (Fig. 1D), which indicated that Alb-Cre^+/− mediated recombination of the HIF-2α^fl/fl allele disrupted the HIF-2α gene in the hepatocytes. Eleven 7-week-old male mice (six KO and five WT mice) were fed a ND for 16 weeks to examine the possible effect of hepatocyte-specific HIF-2α knockout on the growth of mice. We found that hepatocyte-specific HIF-2α knockout had no impact on food intake (Fig. 1E), body weight (Fig. 1F), liver/body weight (Fig. 1G), fat/body weight (Fig. 1H) or lean/body weight (Fig. 1I).

Hepatocyte-specific HIF-2α knockout does not impair metabolic indexes

To exclude the potential effect of hepatocyte-specific HIF-2α disruption on mouse metabolism, we compared the serum metabolic indexes between the KO and WT mice. As a result, we found that hepatocyte-specific HIF-2α deficiency resulted in no damage to serum ALT, AST, TC, TG, HDL, and LDL levels (Figs. 2A–2F). In addition, the IPGTT and IPITT indicated that hepatocyte-specific HIF-2α deficiency did not affect glucose and insulin tolerance in mice (Figs. 2G and 2H). These initial data showed that hepatocyte-specific HIF-2α knockout had no significant effect on metabolic indexes in mice fed a normal diet.

Hepatocyte-specific HIF-2α deletion does not alleviate liver injury in liver fibrosis mice

Thirty male mice (fifteen KO and fifteen WT mice) were divided into four groups: WT (n = 8), KO (n = 8), WT + CCl₄ (n = 7), and KO + CCl₄ (n = 7). One mouse in the KO + CCl₄ group died at the second week due to improper intraperitoneal administration. Therefore, twenty-nine mice that completed the entire experiment were finally used for analyses (Fig. 3A). The food intake of mice in the WT + CCl₄ group was obviously greater than that of mice in the other three groups (Fig. 3B), but their body weight and serum lipid levels were the lowest (Figs. 3C–3E), which suggested that the mice in this group expended more energy fighting against disease. Notably, the serum lipid levels in the KO group were significantly lower than those in the WT group, and the dyslipidemia in the KO + CCl₄ group was also better than that in the WT + CCl₄ group (Figs. 3D–3G). Hepatocyte-specific HIF-2α deficiency did not affect serum lipid levels in mice without any treatments (Figs. 1C and 1F), but it improved the serum TC and TG levels in mice injected intraperitoneally with olive oil (Figs. 3D–3E). Conversely, hepatocyte-specific HIF-2α deficiency protected the hepatic lipid synthesis function and caused a higher TC and TG levels in liver fibrosis mice (Figs. 3D–3E). This indicates that hepatocyte-specific HIF-2α knockout may regulate liver lipid synthesis and stabilize serum lipid levels. The liver/body weight in the KO + CCl₄ group was lower than that in the WT + CCl₄ group (Fig. 3H), but hepatocyte-specific HIF-2α deletion did not improve liver function (Figs. 3I and 3J) or glucose metabolism disorders (Figs. 3K–3M) in mice with CCl₄-induced liver fibrosis.

Hepatocyte-specific HIF-2α deficiency does not improve fibrogenesis in liver fibrosis mice

Mice injected intraperitoneally with CCl₄ developed obvious hepatocyte necrosis, inflammatory cell infiltration, and fiber deposition in the liver according to HE and Masson staining (Figs. 4A and 4B). However, there were no significant differences in the positive fibrotic area (%) and ISHAK scores between the KO + CCl₄ group and the WT + CCl₄ group (Fig. 4C). The α-SMA content (Figs. 4D and 4E) and hydroxyproline content (Fig. 4F), which reflected the severity of liver fibrosis, also showed no significant difference between the two groups. These results suggest that hepatocyte-specific HIF-2α knockout could not improve CCl₄-induced liver fibrosis in mice. Immunohistochemistry staining showed that liver HIF-2α expression increased significantly in mice injected intraperitoneally with CCl₄, but no difference was observed between the KO + CCl₄ group and the WT + CCl₄ group (Figs. 4G and 4H). Notably, we found that increased HIF-2α expression mainly occurred in the portal area and hepatic sinusoids but not in hepatocytes in mice injected intraperitoneally with CCl₄ (Fig. 4G), which indicates that the hypoxia sites of CCl₄-induced liver fibrosis are mainly concentrated in the portal area and hepatic sinusoids but not in hepatocytes. This may be the main reason why hepatocyte-specific HIF-2α knockout could not alleviate liver fibrosis. In addition, the increased HIF-2α protein expression was distributed along the hepatic sinuses. Locations close to the portal area demonstrated robust HIF-2α protein expression in the cytoplasm and nucleus of sinus endothelial cells in the fibrotic liver.

Hepatocyte-specific HIF-2α may not be a key fibrogenic factor in liver fibrosis

HIF-2α has a broad human tissue distribution, with the highest expression in lung and the lowest expression in blood, as suggested in the GETx database (Fig. 5A). However, obviously, it is not a liver-specific protein. In addition, severe liver fibrosis did not result in a significant change in liver HIF-2α transcription levels in patients with alcoholic-related liver disease, chronic HBV-related liver disease, chronic HCV-related liver disease, and nonalcoholic fatty liver disease (Figs. 5B–5E). Similarly, HIF-2α transcription in the liver showed no significant fluctuations in CCl₄-induced liver fibrosis mice (Fig. 5F), which was different from its protein expression trend, as suggested by our results (Figs. 4G and 4H). This may be because hypoxia affects the protein expression level of HIF-2α rather than its transcription level since pVHL-mediated HIF degradation depends on ambient oxygen.

We further used the single-cell RNA-seq data of liver nonparenchymal cells from CCl₄-induced fibrosis mice to examine the expression of HIF-2α in different hepatic cells. In terms of total hepatic nonparenchymal cells, there was no difference in HIF-2α transcription between the intraperitoneal injection of CCl₄ or oil (Fig. 5G). The liver nonparenchymal cells were divided into 28 cell clusters and annotated to 15 cell types (Figs. 5H and 5I). Residual hepatocytes remained in the mice injected intraperitoneally with olive oil, but this did not affect our judgment that the volume of hepatic satellite cells and fibroblasts increased significantly in CCl₄-induced liver fibrosis mice (Fig. 5I). Notably, HIF-2α mainly existed in sinusoidal endothelial cells but rarely in other cell types (Figs. 5J and 5K). The above results revealed that HIF-2α expression is not liver specific or hepatocyte specific, and knockout of HIF-2α in sinusoidal endothelial cells instead of hepatocytes may improve liver fibrosis.