Figure 1.
Immunohistochemistry of MMP-1, MMP-2 and MMP-9 expression in non-diseased and cirrhotic human liver injury.
Immunohistochemistry with NovaRED detection is shown for both non-diseased donor (Panels A, B, E, F, I, J, M and N) and cirrhotic HCV explant tissue (Panels C, D, G, H, K, L, O and P). The same result was seen for PBC, PSC, AIH and ALD tissue (not shown). MMP-1 was found both in non-diseased and cirrhosis in both hepatocytes and biliary structures located in the fibrous septa (Panels A–D). MMP-2 expression was not significant in non-diseased tissue (Panels E and F) but was seen in hepatocytes, bile ducts and HSC (Panels G and H) of cirrhotic tissue. A similar pattern was also observed for MMP-9 expression (Panels I-L). The isotype controls showed no significant staining (Panels M-P). Magnification is 10× and 40×.
Figure 2.
In-Situ Zymography of Matrix Metalloproteinase Activity and its Cellular Localisation in Liver Tissue.
Co-localisation in human liver of MMP activity (green) with hepatocyte marker CK18 (red - Panels A, B, D and M) and CD147 (red - Panels E, F, H and N), nuclei are stained with Dapi (blue). MMP activity (green) can be seen within the cells (Panels B, F, M and N). Confocal microscopy shows an absence of MMP activity (green) in non-diseased tissue (Panels A, E and I) but abundant hepatocyte MMP activity in CK18 positive hepatocytes (Panels B and M), these binuclear cells also express CD147 (Panels F and N). Importantly, α-SMA (red) an activated HSC-marker is not expressed in non-diseased liver HSCs (Panel I) but is expressed in cirrhosis (Panels J and L). α-SMA (red) does not co-localise with MMP activity in green (Panel J). The effect of addition of aminophenylmercuric acetate (APMA) is shown in the third group of confocal panels (Panels O–Q). There is no TIMP regulated MMP activity in non-diseased liver (Panel O). In cirrhosis, basal MMP activity (Panel P) is increased markedly with APMA addition (Panel Q compared to P). In panels B, F, J, P and Q the hepatic nodule and fibrotic septa have been labelled. In addition the arrowheads in panels F and J highlight the boundary between the nodule and septa. Magnification for panels A-L is 63× and for panels is O-Q 10×.
Figure 3.
Expression of MMP mRNA in human liver.
Quantitative PCR on whole liver tissue, non-diseased and HCV cirrhosis, of MMP-1, MMP-2, MMP-9 and MMP-14 mRNA (n = 4 per group). The expression of MMPs measured was significantly increased in cirrhosis compared to non-diseased controls, *p<0.05.
Figure 4.
Expression of CD147 mRNA in human liver.
Quantitative PCR on whole liver tissue, non-diseased as well as PBC, AIH and HCV cirrhosis of CD147 (splice variant 2) mRNA (n = 4 per group). CD147 was significantly increased in all cirrhotic specimens compared to non-diseased controls, *p<0.05.
Figure 5.
Immunohistochemistry of CD147 in Human Liver Tissue.
The α-CD147 antibody (MEM-6/1) was used to stain non-diseased (Panels A and D) and cirrhotic ALD tissue (Panels B and E). CD147 is expressed by hepatocytes and bile ducts. No significant staining of HSC is seen in the fibrous septa, the only structures within the septa that are CD147 positive are bile ducts (see arrows Panel B and E). Isotype controls are shown in Panels C and F.
Figure 6.
Co-localisation of CD147 and Liver Cell Markers in Human Liver Tissue.
Liver sections were stained with liver cell makers (red) including CK18 (Panels A, B and D), CK19 (Panels E, F and H), CD45 (Panels I, J and L), CD31 (Panels M, N and P) and α-SMA (Panels Q, R and T) as well as CD147 (green, all except D, H, L, P and T). CK-19 positive bile ducts (Panels E, F and H) co-localise with CD147 (Arrowheads Panel E and F) in both non-diseased and cirrhotic tissue. Similarly, CD45 positive leukocytes (Panels I, J and L) co-localise with CD147 in both non-diseased and cirrhotic tissue (Arrowheads in Panel I and J). Further, CD31 endothelial cells (Panels M, N and P) co-localise with CD147 (Panel M and N) in both non-diseased and cirrhotic tissue. Finally, in the α-SMA positive series of images (Panels Q, R and T) vascular structures are seen stained as indicated by the arrowhead in Panel Q and HSC in fibrotic septa in cirrhosis (Arrowhead in Panel R). Importantly, no co-localisation of α-SMA and CD147 was seen in cirrhosis (Panel R). Merged images show co-localisation of CD147 with the liver cell markers (yellow). Magnification 63×.
Figure 7.
The Effect of Inhibition of CD147 on Matrix Metalloproteinase Expression and Activities in pH5CH8 Hepatocytes.
Downregulation of MMPs with CD147 knockdown in a hepatocyte cell line in-vitro. pH5CH8 cells were transfected with siCtrl (C) or siCD147 (Si) oligonucleotides. Transfected cells and mock controls (M) were cultured in serum free conditions for 48 hrs. Shown in panel A are representative immunoblots of CD147 with the higher (HG CD147) and lower molecular weight (LG CD147) glycoforms, MMP-14 and GAPDH as loading control. Gelatin zymography of MMP-2 and -9 on the conditioned media from the same cells are shown in panel B. Densitometry was performed on the CD147 immunoblots and the results are shown as HG CD147 and LG CD147 forms normalised for GAPDH (n = 3, Panels C and D). Densitometric analysis of gelatin zymography of MMP-9 and MMP-2 are shown (Panels E and F). *p<0.05 using Mann-Whitney U t-test, compared to Mock (n = 3 for all groups).
Figure 8.
MMP and TIMP expression in whole liver and primary hepatocytes from CCl4 induced liver injury.
Quantitative PCR of MMP and TIMP expression in whole liver and isolated primary hepatocytes. Cirrhosis was induced in a mouse model of liver injury (C57bl/6) with CCl4. RNA extracted from primary hepatocytes as well as whole liver were assessed at commencement of injury and weeks 1, 2 and 4. The expression of MMP-2 (Panel A), MMP-9 (Panel B), MMP-13 (Panel C), MMP-14 (Panel D), TIMP-1 (Panel E) and TIMP-2 (Panel F) was assessed by quantitative PCR (n = 4 per group, data expressed as mean and SEM. *p<0.05 and **p<0.01 relative to untreated).
Figure 9.
Injury in a mouse model of fibrosis with 4 weeks CCl4 and α-CD147 antibody intervention.
Cirrhosis was induced in a mouse model of liver injury (Balb/c) with CCl4. An antibody targeting CD147 (RL73.2) was given twice weekly for 4 weeks, mice given an equal amount of IgG were used as control. There was no appreciable phenotype with either α-CD147 antibody or IgG control treatment in the absence of CCl4 (not shown). The injury groups are CCl4 (+/− isotype control) and CCl4 with antibody targeting CD147. Quantitative data of PSR staining is shown in panel A. Injury as assessed by AST (Panel B), α-SMA (Panel C), Collagen I (Panel D), TGF-β (Panel E) and Collagen IV (Panel F) were all significantly reduced in CCl4 injury with α-CD147 antibody intervention compared to the IgG control treated with CCl4. All groups are n = 4−6 with data expressed as mean and SEM. *p<0.05.
Figure 10.
MMP expression in CCl4 induced liver injury with α-CD147 antibody intervention.
Cirrhosis was induced in a mouse model of liver injury (Balb/c) with CCl4. An antibody targeting CD147 (RL73.2) was given twice weekly for 4 weeks, mice given an equal amount of IgG were used as control. There were no appreciable changes in MMP expression with either α-CD147 antibody or IgG control treatment in the absence of CCl4 (not shown). Tissue hydroxyproline was significantly increased with α-CD147 antibody treatment (Panel A). Compared with control MMP expression is significantly increased in the CCl4 injury with IgG group and reversed by intervention with α-CD147 antibody (Panels B–E). TIMP-1 expression was upregulated in injury but unaffected by intervention with α-CD147 antibody (Panel F) (n = 5−7 per group data expressed as mean and SEM. *p<0.05, **p<0.01 and ***p<0.001).