Volume 126, Issue 7, Pages 1795-1808 (June 2004) Spontaneous recovery from micronodular cirrhosis: Evidence for incomplete resolution associated with matrix cross-linking  Razao Issa, Xiaoying Zhou, Christothea M. Constandinou, Jonathan Fallowfield, Harry Millward-Sadler, Marianna D.A. Gaca, Emma Sands, Ibnauf Suliman, Nathan Trim, Andreas Knorr, Michael J.P. Arthur, R.Christopher Benyon, John P. Iredale  Gastroenterology  Volume 126, Issue 7, Pages 1795-1808 (June 2004) DOI: 10.1053/j.gastro.2004.03.009

Figure 1 Histologic appearance of representative livers harvested at peak fibrosis after induction of micronodular cirrhosis by CCl4 injection for (A) 12 weeks, (B) after 84 days, and (C) after 366 days of spontaneous recovery after staining with Sirius red. At peak fibrosis, there is an established micronodular cirrhosis with dense mature septa bridging hepatic veins and linking these areas with the portal tracts. The septa are highly cellular. (A) Fine collagen fibrils are observed extending into the parenchyma from the fibrotic areas. (B) Little matrix remodeling was observed during the first 28 days of recovery, but from 28 to 84 days of recovery, progressive remodeling of matrix was observed, most obvious with the loss of the most recently formed bridging fibrils and areas of perisinusoidal fibrosis; regenerative nodules were also clearly present and in some areas were no longer bounded by fibrotic tissue. (C) Further recovery to 168 days and 366 days showed an additional loss of matrix to yield an attenuated macronodular cirrhotic pattern with prominent regenerating nodules. (C) Ultimately, residual thin attenuated septa were left (366 days), representing an established macronodular pattern of cirrhosis. Gastroenterology 2004 126, 1795-1808DOI: (10.1053/j.gastro.2004.03.009)

Figure 2 (A) Total liver collagen was determined by image analysis after Sirius red staining of representative liver sections as described in the Materials and Methods section. Progressive treatment with CCl4 resulted in increased areas of positive staining with Sirius red from 6 through 12 weeks of treatment. During recovery, there was a progressive decrease in Sirius red staining to 366 days of recovery. (B) Hydroxyproline analysis also was undertaken to provide an indirect biochemical measurement of liver collagen and was expressed relative to normal (untreated) liver (control). By this method, a similar pattern of increasing liver collagen content was observed in the progressive phase of injury. Recovery was associated with a decrease in liver hydroxyproline content to levels approaching that observed in untreated liver. Gastroenterology 2004 126, 1795-1808DOI: (10.1053/j.gastro.2004.03.009)

Figure 3 Analysis of total liver RNA was undertaken as described in the Materials and Methods section by Taqman PCR in normal, untreated liver and at peak fibrosis 3 days after the last injection of CCl4 (peak fibrosis [PFO]) during 366 days of recovery (28, 84, 168, and 366 days) after 12 weeks of CCL4 intoxication as described in the Materials and Methods section. (A) The expression of procollagen-1 initially was constant and showed a progressive decrease during the recovery period only after 28 days of recovery. Expression of (B) TIMP-1 and (C) TIMP-2 showed a similar pattern with significant reductions in mRNA expression only occurring after 28 days of recovery. (D) Expression of MMP-13 showed a sudden decrease in expression over the first 28 days of recovery, after which expression remained relatively constant through 366 further days of recovery. (Data are presented as mean ± SEM for n = 4 representative samples at each time point analyzed in triplicate. ∗P < 0.05, ∗∗P < 0.01 by Mann Whitney.) Gastroenterology 2004 126, 1795-1808DOI: (10.1053/j.gastro.2004.03.009)

Figure 4 (A) Representative examples of gelatinase A expression determined by zymography as described in the Materials and Methods section. Expression of gelatinase A was increased relative to normal (con, control) after 12 weeks of CCl4 treatment (0). In particular, the 66-kilodalton form of gelatinase A also was found to be increased. During recovery, expression of both active and proform of gelatinase A remained increased at 84 days (84d) but by 168 days (168d) expression had decreased to levels comparable with untreated normal controls in all livers studied. (B) Representative examples of MT1-MMP expression was determined by Western blot analysis as described in the Materials and Methods section. MT1-MMP showed an essentially identical expression pattern to gelatinase A, being expressed at high levels through peak fibrosis to 28 days of recovery, whereafter MT1-MMP levels decreased to levels comparable with untreated control (data not shown) by 168 days of recovery. Gastroenterology 2004 126, 1795-1808DOI: (10.1053/j.gastro.2004.03.009)

Figure 5 (A–B) To detect elastin and matrix cross-linking, representative liver sections were analyzed after orcein staining. (C–H) Further staining for cross-linking with a specific antibody to matrix cross-links was undertaken and (I–K) homogenates from these livers were subjected to Western blot analysis for matrix cross-links. (A–B) shows a representative result after orcein staining. (A) Evidence of cross-linking and elastin was observed in the central portion of the most mature fibrotic bands after 12 weeks of CCl4 treatment (orcein-positive fibrils, arrow). (B) During the 366 days of recovery, there was a loss of matrix, leaving a residual attenuated macronodular cirrhosis in which the residual fibrotic bands showed some orcein positivity (arrow). (C–H) Representative liver sections were stained with an antibody directed against ε-(γ-glutamyl) lysine matrix (tTg-mediated) cross-links. The central portion of the most mature septa was found to be positive for cross-links in the 12-week peak fibrosis samples (C × 40, D with propidium iodide (PI) counterstain × 20). Negative control samples were entirely negative (representative example E × 10). Positive staining for tTg was observed in areas of fibrosis at 12 weeks peak fibrosis (F × 10). After 168 days of recovery, cross-links were immunolocalized to the persistent areas of fibrosis within liver (G × 10, H × 40). (I–K) Western blot analysis of liver homogenates was undertaken by using a cross-link-detecting antibody as described in the Materials and Methods section (I, J, and K represent individual representative Western blot analyses). No binding was detected with an antibody directed against matrix cross-links in normal untreated liver (Normal) and minimal binding was observed at 6 weeks peak fibrosis (I, day 0). This signal was no longer detectable after 15 days of spontaneous recovery in the 6-week fibrosis model (I, day 18). In contrast, a band of 160 kilodaltons was detected after Western blot analysis for matrix cross-links in representative liver homogenates from 12 weeks peak fibrosis (J and K, day 0). This signal remained detectable even at 366 days of recovery (K, day 366). Gastroenterology 2004 126, 1795-1808DOI: (10.1053/j.gastro.2004.03.009)

Figure 6 Protein extracts from activated HSCs were subjected to Western analysis for tTg. tTg with a molecular weight of 77 kilodaltons was detected in activated HSCs. Lanes 1–3 represent increasing concentrations of cell lysates from activated HSCs. Gastroenterology 2004 126, 1795-1808DOI: (10.1053/j.gastro.2004.03.009)

Figure 7 (A) The liver levels of α-SMA mRNA were determined by Taqman PCR of total liver RNA as described in the Materials and Methods section. No significant change in α-SMA expression was observed during the first 28 days of recovery after 12 weeks of CCl4 treatment. However, from 28 through 366 days of recovery, there was a significant decrease in α-SMA mRNA (n = 4 for each time point, ∗P < 0.05 and ∗∗P < 0.01 by Mann-Whitney). After immunostaining of representative sections, the number of α-SMA-positive cells were counted over 20 random high-powered fields after 6 weeks of CCl4 (6Wk PFO), 8 weeks of CCl4 (8Wk PFO), 12 weeks of CCl4 (12Wk PFO), and at time points during recovery (28–366 days) after 12 weeks of CCl4. Recovery from fibrosis was associated with a significant diminution in α-SMA-positive cells over the 366-day time period. (n = 4–6 for each time point. ∗P < 0.05 and ∗∗P < 0.01 by Mann-Whitney.) Gastroenterology 2004 126, 1795-1808DOI: (10.1053/j.gastro.2004.03.009)

Figure 8 Representative liver sections were dual stained with TUNEL and α-SMA as described in the Materials and Methods section. In addition, the total number of apoptotic cells (TUNEL positive) within each fibrotic band were determined at PFO and 168 (168d) and 366 (366d) days of recovery. (A) At each recovery time point, there is evidence of apoptosis of cells within the fibrotic septa (arrow), TUNEL-positive cells were detected with red chromogen, α-SMA cells were detected with blue chromogen at PFO after 12 weeks of CCl4. (A) However, the α-SMA-negative cells, which predominated in the center of the mature fibrotic bands, showed fewer apoptotic figures. (B) The total number of apoptotic figures within fibrotic septa at each time point was determined and is shown. (n = 4–6 for each time point data expressed as mean ± SEM. ∗∗P < 0.01 by Mann-Whitney.) Gastroenterology 2004 126, 1795-1808DOI: (10.1053/j.gastro.2004.03.009)

Figure 9 Decreasing cellularity of residual scar tissue occurs with recovery. Sections were stained with H&E and Sirius red and subjected to image analysis as described in the Materials and Methods section. By this method, there was a significant decrease in the cellularity of the scar per unit area of Sirius red-positive tissue comparing PFO after 12 weeks of CCL4 with 366 days (366d) of recovery. (n = 4 for each time point, data are expressed as mean ± SEM. ∗P < 0.05 by Mann-Whitney.) Gastroenterology 2004 126, 1795-1808DOI: (10.1053/j.gastro.2004.03.009)

Figure 10 The intermediate filament expression of the cells populating the septa was determined by staining for α-SMA and desmin as described in the Materials and Methods section. (A) Cells in the most recently formed septa and bridging septa, perisinoisodal areas, and at the margins of mature septa were found to be α-SMA positive, however, (B) the cells at the center of the most mature septa generally were found to be α-SMA negative. (B) Nevertheless, all these cells were shown to be desmin positive. By 366 days recovery, the residual cells within the fibrotic bands were found to be almost uniformly α-SMA negative. (C) α-SMA staining (left panel top and bottom) with parallel analysis for Sirius red (right panel top and bottom) in pericentral fibrotic areas (top) and attenuated residual septa (bottom) are shown. (D) Residual septa at 366 days of recovery were uniformly desmin positive. Gastroenterology 2004 126, 1795-1808DOI: (10.1053/j.gastro.2004.03.009)