Figure 1.
Induction of SPARC mRNA expression during liver fibrogenesis.
(A) Quantitative data showing differences in SPARC mRNA expression levels in human cirrhotic (Pt#1 to Pt#5) with fibrosis degree F4 and non-cirrhotic liver samples as measured by qPCR. *p<0.05, **p<0.01 compared with healthy liver samples. Mann-Whitney test. (B) qPCR analyses of liver samples from TAA and BDL mice. Complementary DNA was synthesized and was subjected to qPCR for the expression of SPARC transcripts. The relative amount of the PCR product (AU, arbitrary units) amplified from control liver samples was set at 1. *p<0.05 versus control (−TAA), **p<0.01 versus control (−BDL), Mann-Whitney test.
Figure 2.
Patterns of SPARC expression during liver fibrogenesis.
(A–F) Representative images taken from SPARC+/+ mice liver sections stained for SPARC (B, E; red) and SMA (A; green) or vWF (D; green) and merge of both images (C, F). (G–L). Representative images taken from 10 weeks TAA-treated SPARC+/+ mice liver sections (n = 4–6) stained for SPARC (H, K; red) and SMA (G; green) or vWF (J; green). Co-localization of SPARC and SMA (I) or SPARC and vWF (L). (M–R) Representative images taken from 7 days BDL SPARC+/+ mice liver stained for SPARC (N, Q; red) and SMA (M; green) or vWF (P; green). Arrows: co-expression of the two markers; dotted arrows: autofluorescence due to hepatic ceroid-laden macrophages. Original magnification 400X (A–F) or 1000X (G–R).
Figure 3.
Reduced liver damage in SPARC deficient mice.
(A–F) Representative photomicrographs of liver sections from untreated SPARC+/+ (A) or 10 weeks TAA-treated SPARC+/+ or SPARC−/− mice (n = 6–8), stained with H&E (A–C) or Masson’s trichrome (D–F). (G–J) Representative photomicrographs of liver sections from SPARC+/+ and SPARC−/− mice subjected to BDL, stained with H&E (G–H) or Masson’s trichrome (I–J). Original magnification 200X. PT, portal tract; CV, central vein. (K) Serum ALT and AST levels were measured at the indicated time in TAA-treated and BDL mice. Dotted lines, upper normal limited. **p<0.05 versus treated SPARC+/+.
Table 1.
Severity of necroinflamatory activity and fibrosis in SPARC+/+ and SPARC−/− mice after 10 weeks of TAA treatment (n = 6–8) or in SPARC+/+ and SPARC−/− BDL mice (n = 5–6).
Figure 4.
Reduced hepatic inflammatory infiltration and migratory capacity in SPARC deficient mice.
Photomicrographs of representative liver sections from TAA-treated animals. SPARC+/+ mice showed an increased CD4+ cells infiltration in hepatic parenchyma, especially around portal tracts (A,B); while in TAA-treated SPARC−/− mice CD4+ cells are scarce and located near the sinusoids (C,D). Arrows indicate CD4+ cells. Original magnification 400X. (E) Migratory response of splenocytes towards CCL19 chemokine. Percentage of cells that migrated relative to respective controls for SPARC+/+ and SPARC−/− splenocytes (n = 3 replicates) in a Boyden Chamber system. Splenocytes were placed into the upper well, separated from the lower by a 5-µm porosity membrane. The bottom well contained either DMEM or DMEM with 10 ng/µl rCCL19 and cells were allowed to migrate during 2 h. ***p<0.001, Mann Whitney test.
Table 2.
IPA® top molecules which were significantly alters in SPARC−/− versus SPARC+/+ mice and those modified in SPARC−/− versus SPARC+/+10 weeks TAA treated mice.
Figure 5.
Reduced liver fibrosis in SPARC deficient mice.
(A) Representative photomicrographs of liver sections stained with picrosirius red. SPARC+/+ mice show staining limited to periportal areas (left panel), while liver sections from TAA-treated SPARC+/+ mice exhibits marked portal fibrosis and portal-portal bridges (central panel) and those from TAA-treated SPARC−/− mice present weak fibrotic response (right panel). (B) Morphometric quantification of Sirius red stained area showing a significant attenuation of the fibrotic process in TAA-treated SPARC−/− mice when compared to treated wild-type mice. **p<0.01, Mann-Whitney test. (C–D) Quantitative data from qPCR analysis of collagen (COL1A2) and MMP-2 mRNA expression. *p<0.05, **p<0.01 versus SPARC+/+ TAA 10 weeks, Mann-Whitney test. (E) Representative pictures taken from liver sections of from SPARC+/+ or SPARC−/− mice, at 7 days after BDL. Original magnification 200X. PT, portal tract; CV, central vein. (F) Morphometric quantification of Sirius red stained area showing a significant attenuation of the fibrotic process in SPARC−/− mice at day 7 after BDL when compared to wild-type mice. **p<0.01, Mann-Whitney test. (G) qPCR analysis of collagen mRNA expression in SPARC+/+ and SPARC−/− mice subjected to BDL. *p<0.05, versus BDL SPARC+/+ Mann-Whitney test.
Figure 6.
Reduced maturation of SPARC−/− collagen fiber deposits.
Representative pictures showing picrosirius red stained liver sections obtained from SPARC+/+ (A,C,E) or SPARC−/− (B,D,F) mice (n = 6–8) observed under polarized light. Animals were left untreated (A,B), or were TAA-treated during 10 weeks (C,D) or subjected to BDL (E, F). Note the predominant mature and compacted nature of collagen fibers in wild-type treated mice and their immature and thin appearance in SPARC−/− animals. Original magnification 400X.
Figure 7.
Reduction in the number of active myofibroblasts and in liver and serum TGF-β1 levels in SPARC deficient fibrotic mice.
(A–H) Representative pictures taken from liver sections of untreated SPARC+/+ and SPARC−/− (A,B), 10 weeks TAA treated SPARC+/+ (C,E) andSPARC−/− (D,F), or BDL SPARC+/+ (G) and SPARC−/− (H) mice immunostained for α-SMA. (E,F) are higher magnification images from box areas in C,D respectively. (I) Quantitative data of densitometric analyses of αSMA immunostained area from TAA-treated and BDL SPARC−/− or SPARC+/+ mice. **p<0.01, Mann-Whitney test. (J) Quantitative data of TGF-β1 mRNA levels obtained by qPCR analysis from 10 weeks TAA-treated and BDL SPARC+/+ or SPARC−/− mice (n = 6–8). Data are expressed as relative values to those of wild-type mice without treatment. *p<0.05, SPARC+/+ treated vs SPARC−/− treated. Mann-Whitney test. (K) Serum levels of TGF-β1 were measured after 10 weeks of TAA treatment. nd, non-detectable. **p<0.01, Mann-Whitney test.
Figure 8.
The top network of differentially expressed genes.
The networks are presented as graphical displays where genes appear as nodes and the molecular relationships are represented by lines. Up-regulated and down-regulated genes in SPARC−/− mice are shown as red spot or green spot, respectively. The top network of differentially expressed genes in SPARC−/− versus SPARC+/+ mice (A) or SPARC−/− after 10 weeks of TAA treatment versus SPARC+/+ TAA treated mice (B), as identified by IPA analysis. Intensity of the red or green color shows the level of gene expression. Gray represents a gene found which is related to the others but did not meet the cutoff criteria. A Activation, E Expression (includes metabolism/synthesis for chemicals), I Inhibition, L Proteolysis (includes degradation for Chemicals), LO Localization, M Biochemical Modification, MB Group/complex Membership, P Phosphorylation/Dephosphorylation, PD Protein-DNA binding, PP Protein-Protein binding, RB Regulation of Binding,T Transcription,TR Translocation.