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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 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.
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pone-0054962-g004: 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.

Mentions: In order to characterize the profile of immune cells in the hepatic inflammatory infiltrate we performed immunohistochemistry for CD4+ T cells and observed that the amount of CD4+ cells was greatly decreased in SPARC−/− mice (Figure 4 A–D). Migration ability towards rCCL19 chemokine was explored in splenocytes derived from SPARC−/− and SPARC+/+ mice in vitro. We observed a reduced migration in SPARC−/− mice in response to CCL19 after 2 h of incubation (Figure 4E). Flow cytometry analysis of splenocytes showed similar expression of CCR7 receptor (not shown). In agreement with the microarrays results showing a decreased expression of CCL19 in SPARC−/− mice (Table 2), qPCR assay confirmed down-regulation of the transcript (Figure S2).


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 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.
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pone-0054962-g004: 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.
Mentions: In order to characterize the profile of immune cells in the hepatic inflammatory infiltrate we performed immunohistochemistry for CD4+ T cells and observed that the amount of CD4+ cells was greatly decreased in SPARC−/− mice (Figure 4 A–D). Migration ability towards rCCL19 chemokine was explored in splenocytes derived from SPARC−/− and SPARC+/+ mice in vitro. We observed a reduced migration in SPARC−/− mice in response to CCL19 after 2 h of incubation (Figure 4E). Flow cytometry analysis of splenocytes showed similar expression of CCR7 receptor (not shown). In agreement with the microarrays results showing a decreased expression of CCL19 in SPARC−/− mice (Table 2), qPCR assay confirmed down-regulation of the transcript (Figure S2).

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