Fig 2: c-Kit+ LSECs are rich in FGF1 production.(A) Knockout efficiency of c-Kit after tamoxifen induction by qPCR. n = 5 to 7. (B) Knockout efficiency of c-Kit after tamoxifen induction by Western blot. n = 3. (C) Knockout efficiency of c-Kit after tamoxifen induction and HFD feeding for 12 weeks by Western blot. n = 4. (D) c-Kit+ LSECs highly expressed genes were crossed with mouse secretome genes. (E) Heatmap of screened genes in chow c-Kit+/− LSEC RNA-seq. (F) FGF1 expression in LSECs from CV to PV (18). (G) FGF1 expression in LSECs from CV to PV (19). (H) ScRNA-seq cell clustering analysis of chow-fed mouse liver. (I) FGF1 expression in different cell types identified in (H). (J and K) Western blot and densitometric analysis of FGF1 in normal primary hepatic cell. n = 3. (L) qPCR analysis of FGF1 expressions in normal c-Kit+/− LSECs. n = 3. (M and N) Western blot and densitometric analysis of FGF1 in normal c-Kit+/− LSECs. n = 4. (O) FGF1 protein concentration in cultured medium of normal c-Kit+/− LSECs. n = 4. (P) FPKM comparison of FGF1 in chow c-Kit+/− LSEC RNA-seq. (Q) scRNA-seq analysis of LSEC FGF1 expression in chow- or HFD-fed mice. (R) qPCR analysis of FGF1 expressions in LSECs isolated from chow- or HFD-fed mice. n = 4. (S and T) Western blot and densitometric analysis of FGF1. n = 3. (U) FPKM comparison of FGF1 in HFD c-Kit+/− LSEC RNA-seq. (V and W) Western blot and densitometric analysis of FGF1 in c-Kit+/− LSECs isolated from HFD-fed mice. n = 3. Data are means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; ns, by one-way ANOVA (K), two-sample t test (A, C, L, N to P, R, T, U, and W), or Mann-Whitney U test (Q).

Fig 3: Endothelial c-Kit counteracts hepatic steatosis in an FGF-1-dependent manner.(A) qPCR analysis of FGF1 expressions in LSECs isolated from 8-week-old chow-fed c-Kit KO or c-Kit CT mice. n = 5 to 7. (B and C) Western blot and densitometric analysis of FGF1 in LSECs isolated from 8-week-old chow-fed c-Kit KO or c-Kit CT mice. n = 3 to 4. (D) FGF1 protein concentration in cultured medium of c-Kit+/− LSECs isolated from 8-week-old chow-fed mice. n = 3. (E and F) Western blot and densitometric analysis of FGF1 in livers of HFD-fed mice infused with c-Kit+/− LSECs isolated from 8-week-old chow-fed mice. n = 5. (G to R) 8-week-old c-Kit KO or c-Kit CT mice were fed HFD for 12 weeks. At the beginning of 10-week HFD, rFGF1 (0.5 mg/kg) was administrated intraperitoneally (i.p.) every other day for another 3 weeks. (G) Dosing regimen diagram of rFGF1. (H and I) Western blot and densitometric analysis of FGF1 in liver. n = 4. (J) Fasting blood glucose levels. n = 4 to 8. (K and L) MASLD severity and ORO staining was quantified by NAS and ORO staining areas. n = 4 to 8. (M) H&E and ORO staining in liver sections. Scale bar, 200 μm. n = 4 to 8. (N) F4/80 and αSMA immunostaining in liver sections. Scale bar, 200 μm. n = 4 to 8. (O and P) F4/80 and αSMA immunostaining was quantified by F4/80 and αSMA staining areas. n = 4 to 8. (Q) Liver TC and TG concentration. n = 4 to 8. (R) Serum ALT and AST levels. n = 4 to 8. Data are means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; ns, by two-sample t test (A, C to E, H, and J) or one-way ANOVA (K, L, and O to R).

Fig 4: Endothelial cell-specific FGF1 KO abrogated the therapeutic effects mediated by c-Kit+ LSECs.(A) Total RNA was extracted from primary LSECs isolated from EC-specific FGF1 CT and FGF1 KO mice after tamoxifen induction and qRT-PCR was performed to examine FGF1 mRNA expression. n = 5. (B and C) Western blot and densitometric analysis of FGF1 in livers of LSEC-specific FGF1 KO or FGF1 CT mice after tamoxifen induction. n = 3. (D and E) Western blot and densitometric analysis of FGF1 in livers of LSEC-specific FGF1 KO or FGF1 CT mice. n = 4. (F to I) Eight-week-old LSEC-specific FGF1 KO or FGF1 CT mice were fed HFD for 12 weeks. (F) H&E, ORO, F4/80, and αSMA immunostaining in liver sections. Scale bar, 200 μm. n = 4. (G) MASLD severity, ORO, F4/80, and αSMA immunostaining were quantified by NAS and ORO, F4/80, and αSMA staining areas. n = 4. (H) Hepatic TC and TG. n = 4. (I) Serum ALT and AST levels. n = 4. (J to M) Eight-week-old wild-type mice were fed HFD for 10 weeks and infused c-Kit+/− LSECs isolated from 8-week-old chow-fed FGF1flox/flox mice or c-Kit+ LSECs isolated from 8-week-old chow-fed Cdh5creERT FGF1flox/flox mice three times per week for another 2 weeks. n = 6. (J) H&E and ORO staining, F4/80, and αSMA immunostaining in liver sections. Scale bar, 200 μm. (K) MASLD severity and ORO staining was quantified by NAS and ORO staining areas. F4/80 and αSMA immunostaining was quantified by F4/80 and αSMA staining areas. (L) Liver TC and TG concentration. (M) Serum ALT and AST levels. Data are means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; ns, by two-sample t test (A, C, E, and G to I) or one-way ANOVA (K to M).

Fig 5: c-Kit and RA improved hepatic steatosis via RXRG-FGF1 axis.(A) Transcription factors prediction using JASPAR, Cistrome DB, and AnimalTFBS database. (B) qPCR analysis of RXRs in c-Kit KO or c-Kit CT LSECs. n = 3 to 4. (C) qPCR analysis of RXRs in primary c-Kit+/− LSECs. n = 3. (D) qPCR analysis of RXRs in LSECs isolated from chow- or HFD-fed mice. n = 4. (E and F) Western blot and densitometric analysis of RXRG. n = 3. (G and H) qPCR analysis of FGF1 expression in LSECs treated with the RXR agonist bexarotene or DMSO at different time points and doses. n = 3. (I) ChIP-seq analysis of RXR binding sites on FGF1 gene. (J) Dual-luciferase reporter gene assay analyzing the transcriptional regulatory effect of RXRG on FGF1. (K) qPCR analysis of FGF1 in LSECs treated with DMSO or RA at different time points and doses. n = 3. (L to W) HFD-fed mice were administered RA or vehicle and chow-fed mice with vehicle served as controls. (L and M) Serum ALT, AST, TC, and TG levels. n = 5 to 6. (N) Fasting blood glucose levels. n = 5 to 6. (O) Liver TC and TG concentration. n = 5 to 6. (P to T) H&E, ORO, F4/80, and αSMA immunostaining in liver sections and quantitative analysis. Scale bar, 200 μm. n = 5 to 6. (U to W) qPCR analysis, Western blot, and densitometric analysis of FGF1 in LSECs isolated from RA or vehicle-treated mice. n = 3. (X) RXRG expression in LSECs from CV to PV (18). (Y) RXRG expression in LSECs from CV to PV (19). Data are means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; ns, by two-sample t test (B to D, F, U, and V) or one-way ANOVA (G, H, J to O, and Q to T).