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Lack of the matricellular protein SPARC (secreted protein, acidic and rich in cysteine) attenuates liver fibrogenesis in mice.

Atorrasagasti C, Peixoto E, Aquino JB, Kippes N, Malvicini M, Alaniz L, Garcia M, Piccioni F, Fiore EJ, Bayo J, Bataller R, Guruceaga E, Corrales F, Podhajcer O, Mazzolini G - PLoS ONE (2013)

Bottom Line: Consistently, collagen deposits and mRNA expression levels were decreased in SPARC(-/-) mice when compared to SPARC(+/+) mice; in addition, MMP-2 expression was increased in SPARC(-/-) mice.A reduction in the number of activated myofibroblasts was observed.Overall our data suggest that SPARC plays a significant role in liver fibrogenesis.

View Article: PubMed Central - PubMed

Affiliation: Gene Therapy Laboratory, School of Medicine, Austral University, Derqui-Pilar, Buenos Aires, Argentina.

ABSTRACT

Introduction: Secreted Protein, Acidic and Rich in Cysteine (SPARC) is a matricellular protein involved in many biological processes and found over-expressed in cirrhotic livers. By mean of a genetic approach we herein provide evidence from different in vivo liver disease models suggesting a profibrogenic role for SPARC.

Methods: Two in vivo models of liver fibrosis, based on TAA administration and bile duct ligation, were developed on SPARC wild-type (SPARC(+/+)) and knock-out (SPARC(-/-)) mice. Hepatic SPARC expression was analyzed by qPCR. Fibrosis was assessed by Sirius Red staining, and the maturation state of collagen fibers was analyzed using polarized light. Necroinflammatory activity was evaluated by applying the Knodell score and liver inflammatory infiltration was characterized by immunohistochemistry. Hepatic stellate cell activation was assessed by α-SMA immunohistochemistry. In addition, pro-fibrogenic genes and inflammatory cytokines were measured by qPCR and/or ELISA. Liver gene expression profile was analyzed in SPARC(-/-) and SPARC(+/+) mice using Affymetrix Mouse Gene ST 1.0 array.

Results: SPARC expression was found induced in fibrotic livers of mouse and human. SPARC(-/-) mice showed a reduction in the degree of inflammation, mainly CD4+ cells, and fibrosis. Consistently, collagen deposits and mRNA expression levels were decreased in SPARC(-/-) mice when compared to SPARC(+/+) mice; in addition, MMP-2 expression was increased in SPARC(-/-) mice. A reduction in the number of activated myofibroblasts was observed. Moreover, TGF-β1 expression levels were down-regulated in the liver as well as in the serum of TAA-treated knock-out animals. Ingenuity Pathway Analysis (IPA) analysis suggested several gene networks which might involve protective mechanisms of SPARC deficiency against liver fibrogenesis and a better established machinery to repair DNA and detoxify from external chemical stimuli.

Conclusions: Overall our data suggest that SPARC plays a significant role in liver fibrogenesis. Interventions to inhibit SPARC expression are suggested as promising approaches for liver fibrosis treatment.

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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.
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pone-0054962-g005: 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.

Mentions: In order to quantify liver content of collagen, the ECM protein most abundantly accumulated in fibrous septae, tissue sections from 10 weeks TAA-treated animals were Sirius red stained and morphometric analysis was thereafter performed. A significant reduction in Sirius red+ area was found in SPARC−/− when compared to SPARC+/+ mice (Figure 5A–B). Consistently, α2(I) collagen mRNA expression levels were significantly reduced in SPARC deficient when compared to wild-type animals (3.31±0.64 vs. 6.48±0.95; SPARC−/− vs. SPARC+/+) (Figure 5C). Matrix metalloproteinases (MMPs) are known to be involved in ECM regulation. We observed that hepatic MMP-2 expression was significantly increased in SPARC−/− TAA-treated mice in comparison with SPARC+/+ (8.15±0.42 vs. 3.25±0.64, respectively) (Figure 5D).


Lack of the matricellular protein SPARC (secreted protein, acidic and rich in cysteine) attenuates liver fibrogenesis in mice.

Atorrasagasti C, Peixoto E, Aquino JB, Kippes N, Malvicini M, Alaniz L, Garcia M, Piccioni F, Fiore EJ, Bayo J, Bataller R, Guruceaga E, Corrales F, Podhajcer O, Mazzolini G - PLoS ONE (2013)

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.
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pone-0054962-g005: 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.
Mentions: In order to quantify liver content of collagen, the ECM protein most abundantly accumulated in fibrous septae, tissue sections from 10 weeks TAA-treated animals were Sirius red stained and morphometric analysis was thereafter performed. A significant reduction in Sirius red+ area was found in SPARC−/− when compared to SPARC+/+ mice (Figure 5A–B). Consistently, α2(I) collagen mRNA expression levels were significantly reduced in SPARC deficient when compared to wild-type animals (3.31±0.64 vs. 6.48±0.95; SPARC−/− vs. SPARC+/+) (Figure 5C). Matrix metalloproteinases (MMPs) are known to be involved in ECM regulation. We observed that hepatic MMP-2 expression was significantly increased in SPARC−/− TAA-treated mice in comparison with SPARC+/+ (8.15±0.42 vs. 3.25±0.64, respectively) (Figure 5D).

Bottom Line: Consistently, collagen deposits and mRNA expression levels were decreased in SPARC(-/-) mice when compared to SPARC(+/+) mice; in addition, MMP-2 expression was increased in SPARC(-/-) mice.A reduction in the number of activated myofibroblasts was observed.Overall our data suggest that SPARC plays a significant role in liver fibrogenesis.

View Article: PubMed Central - PubMed

Affiliation: Gene Therapy Laboratory, School of Medicine, Austral University, Derqui-Pilar, Buenos Aires, Argentina.

ABSTRACT

Introduction: Secreted Protein, Acidic and Rich in Cysteine (SPARC) is a matricellular protein involved in many biological processes and found over-expressed in cirrhotic livers. By mean of a genetic approach we herein provide evidence from different in vivo liver disease models suggesting a profibrogenic role for SPARC.

Methods: Two in vivo models of liver fibrosis, based on TAA administration and bile duct ligation, were developed on SPARC wild-type (SPARC(+/+)) and knock-out (SPARC(-/-)) mice. Hepatic SPARC expression was analyzed by qPCR. Fibrosis was assessed by Sirius Red staining, and the maturation state of collagen fibers was analyzed using polarized light. Necroinflammatory activity was evaluated by applying the Knodell score and liver inflammatory infiltration was characterized by immunohistochemistry. Hepatic stellate cell activation was assessed by α-SMA immunohistochemistry. In addition, pro-fibrogenic genes and inflammatory cytokines were measured by qPCR and/or ELISA. Liver gene expression profile was analyzed in SPARC(-/-) and SPARC(+/+) mice using Affymetrix Mouse Gene ST 1.0 array.

Results: SPARC expression was found induced in fibrotic livers of mouse and human. SPARC(-/-) mice showed a reduction in the degree of inflammation, mainly CD4+ cells, and fibrosis. Consistently, collagen deposits and mRNA expression levels were decreased in SPARC(-/-) mice when compared to SPARC(+/+) mice; in addition, MMP-2 expression was increased in SPARC(-/-) mice. A reduction in the number of activated myofibroblasts was observed. Moreover, TGF-β1 expression levels were down-regulated in the liver as well as in the serum of TAA-treated knock-out animals. Ingenuity Pathway Analysis (IPA) analysis suggested several gene networks which might involve protective mechanisms of SPARC deficiency against liver fibrogenesis and a better established machinery to repair DNA and detoxify from external chemical stimuli.

Conclusions: Overall our data suggest that SPARC plays a significant role in liver fibrogenesis. Interventions to inhibit SPARC expression are suggested as promising approaches for liver fibrosis treatment.

Show MeSH
Related in: MedlinePlus