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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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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.
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pone-0054962-g007: 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.

Mentions: Liver fibrogenesis is characterized by trans-differentiation of different cells into myofibroblasts, including HSCs. HSC-derived myofibroblasts are known to upregulate their α-SMA expression levels during the activation process. In order to address whether either the number of myofibroblasts in fibrous septae and/or the activation state of myofibroblasts might be affected by SPARC deficiency, liver tissue obtained from TAA-treated and BDL mice was immunostained with α-SMA. A significant reduction in the α-SMA+ immunostained area was found in TAA-treated SPARC−/− when compared to TAA-treated SPARC+/+ mice (0.11±0.01 vs. 0.45±0.02, respectively) (Figure 7A–F and 7I) and in SPARC−/− subjected to BDL compared to SPARC+/+ mice (0.1±0.03 vs. 0.7±0.19, respectively) (Figure 7G–H and 7I). These results suggest that a reduction in the number of activated myofibroblasts is likely involved in the inhibition of liver fibrogenesis found in SPARC deficient mice.


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)

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.
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pone-0054962-g007: 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.
Mentions: Liver fibrogenesis is characterized by trans-differentiation of different cells into myofibroblasts, including HSCs. HSC-derived myofibroblasts are known to upregulate their α-SMA expression levels during the activation process. In order to address whether either the number of myofibroblasts in fibrous septae and/or the activation state of myofibroblasts might be affected by SPARC deficiency, liver tissue obtained from TAA-treated and BDL mice was immunostained with α-SMA. A significant reduction in the α-SMA+ immunostained area was found in TAA-treated SPARC−/− when compared to TAA-treated SPARC+/+ mice (0.11±0.01 vs. 0.45±0.02, respectively) (Figure 7A–F and 7I) and in SPARC−/− subjected to BDL compared to SPARC+/+ mice (0.1±0.03 vs. 0.7±0.19, respectively) (Figure 7G–H and 7I). These results suggest that a reduction in the number of activated myofibroblasts is likely involved in the inhibition of liver fibrogenesis found in SPARC deficient mice.

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