**3. Results**

#### *3.1. Mitochondrial Dysfunction Is Present in the CCl4-Induced Mouse Model of Liver Fibrosis*

As has been reported for liver fibrosis, most forms of chronic liver diseases are associated with the accumulation of damaged mitochondria, which are responsible for abnormal ROS formation and respiratory complex alterations [23]. To investigate whether alterations in mitochondrial structure or functions in hepatocyte were associated with the progression of liver fibrosis, we established a mouse model of CCl4-induced liver fibrosis. As shown in Figure 1A, liver sections from the CCl4 group displayed a distorted architecture with extensive collagen deposition upon staining with H&E and Sirius Red. Further examination via TEM revealed obvious swelling in mitochondria, and the cristae disappeared in mouse livers during the progression of hepatic fibrosis, suggesting that the mitochondrial structure was damaged along with these fibrotic changes (Figure 1A). Based on these findings, we hypothesized that the structural alterations of mitochondria were due to mitochondrial damage and that mtDNA damage, such as damage-associated molecular patterns (DAMPs), accumulated in necrotic hepatocytes. Therefore, we performed a quantitative evaluation of damaged mtDNA and mtDNA content in CCl4-induced fibrotic livers. As in previous reports [21], we isolated genomic DNA, and then performed qPCR assay for two di fferent mtDNA, namely 79 bp fragment (damaged), and 230 bp fragment (undamaged). The structurally damaged mitochondria in CCl4-induced fibrotic model showed an increase in the damaged mtDNA at the ratios of the 79 bp fragment and 230 bp fragment (Figure 1B). In addition, we performed qPCR assay for two di fferent mtDNA markers, namely mt-ND1 and mt-COX1. The levels of these mtDNA markers were normalized against NADH dehydrogenase flavoprotein 1 (Ndufv1) levels to examine the relative mtDNA to nuclear DNA ratios as described previously [24]. As shown in Figure 1C, the copy numbers of *mt-ND1* and *mt-COX1* per *Ndufv1* were significantly increased by 2.2- and 2.3-fold, respectively in CCl4-induced fibrotic model. Based on these results, the increased damaged mtDNA and mtDNA content in CCl4-induced fibrotic livers can result in malfunctioning proteins and altered mtDNA replication and/or transcription e fficiency. Next, we examined the expression levels of mitochondrial respiratory and complex activity-encoded genes in mouse livers during the progression of hepatic fibrosis using qRT-PCR and western blot analysis. As shown in Figure 1D, the expression levels of nuclear-encoded subunit of complex IV *ATP5A* (Complex V) and *COX5B* (Complex IV) mRNA were decreased in CCl4-induced fibrotic livers. Furthermore, immunoblots illustrated that the levels of subunits of ATP5A and MTCO1 (Complex IV) were significantly decreased in CCl4-induced fibrotic livers, whereas those of the SDHB (Complex II) subunit were unchanged (Figure 1E). Therefore, these findings indicated that the pathogenesis of liver fibrosis is associated with mitochondrial damage or mitochondrial dysfunction.

**Figure 1.** Morphological and functional abnormalities in mitochondrial are present in the mouse model of CCl4-induced liver fibrosis. Male BALB/c mice were injected intraperitoneally with CCl4 biweekly for 8 weeks to establish a hepatic fibrosis mouse model. (**A**) Representative images of H&E and Sirius red staining (original magnification, X200) of liver sections. TEM analysis of hepatocyte showing nucleus (N) and mitochondria (M) (scale bar = 2 & 0.5 μm). (**B**) Damaged mtDNA levels of the 79 bp fragment (damaged) and 230 bp fragment (undamaged) in gDNA isolated from livers of mice were assessed by qPCR. Bars represent mean copy number ratios of mtDNA-79:230, normalized to 18S levels. (**C**) Analysis of mtDNA content to obtain the mtDNA/nDNA ratio. gDNA isolated from livers of mice was analyzed by qPCR for the indicated genes. (**D**) Total RNA isolated from livers of mice was analyzed by qRT-PCR for the indicated genes. (**E**) Western blot analysis of representative subunits of OXPHOS complexes expression.

#### *3.2. HtrA2*/*Omi Expression Is Decreased in CCl4-Induced Fibrotic Mice and Patients with Liver Fibrosis*

We attempted to identify and characterize the role of the mitochondrial serine protease HtrA2/Omi in improving mitochondrial damage during the progression of hepatic fibrosis. We first used fibrotic mouse models to validate the association of HtrA2/Omi expression with liver fibrosis. We found that HtrA2/Omi expression was downregulated in CCl4-treated mouse livers via immunohistochemistry and western blotting. Staining assays revealed that HtrA2/Omi predominantly localized in the cytoplasm of hepatocytes and its levels were lower in fibrotic livers than in normal livers (Figure 2A). Likewise, immunoblotting demonstrated that HtrA2/Omi expression was markedly decreased in mouse livers during the progression of hepatic fibrosis (Figure 2B). We detected α-smooth muscle actin (α-SMA) expression as a marker of HSC activation in fibrotic liver tissues. These findings were consistent with results obtained via western blotting using liver tissue samples from patients with fibrosis and healthy controls (Figure 2C). Immunohistochemistry of HtrA2/Omi in human fibrotic liver revealed that liver tissue from patients with late-stage fibrosis (grade 4) had lower HtrA2/Omi expression than that from patients with early-stage fibrosis (grades 1–2) (Figure 2D). These results sugges<sup>t</sup> that HtrA2/Omi expression is downregulated in liver fibrosis with mtDNA alterations or mitochondrial dysfunction.

**Figure 2.** HtrA2/Omi expression is downregulated in CCl4-induced liver fibrosis and human fibrotic livers. (**A**) Representative images of H&E, Sirius red (original magnification, X200) and immunohistochemistry staining (X400) of liver sections. (**B**) Western blot analysis of HtrA2/Omi, α-SMA and β-actin expression. (**C**) The protein levels of HtrA2/Omi in liver tissue from patients with fibrosis and healthy controls, as evaluated by western blotting. (**D**) Immunohistochemical staining of HtrA2/Omi in human fibrotic liver (X200).

#### *3.3. HtrA2*/*Omi-Deficient Hepatocytes Cause Mitochondrial Accumulation and Structural Anomalies*

To investigate the direct relationship between HtrA2/Omi downregulation and mitochondrial function in hepatocyte during liver fibrogenesis, we transfected a plasmid encoding shRNA targeting HtrA2/Omi into FL83B mouse hepatocytes, which were named lenti-shHtrA2 cells. As shown in Supplemental Figure S1A, HtrA2/Omi protein levels were significantly lower in lenti-shHtrA2 cells than in negative control lenti-shNC cells. We previously reported that HtrA2/Omi deficiency causes mtDNA damage through mutation and ROS generation, which can lead to mitochondrial dysfunction and consequent cell death [12]. Therefore, we examined the effect of HtrA2/Omi depletion on intracellular total ROS and mitochondria ROS (mtROS) levels in lenti-shHtrA2 hepatocytes using CM-H2DCFDA and MitoSox. As shown in Figure 3A,B, lenti-shHtrA2 cells produced greater amounts of mtROS despite a decrease of total ROS than lenti-shNC cells. Recent reports suggested that mitochondria play a key role in regulating cell size by a ffecting the balance of cell growth and proliferation through metabolic activity [25]. Therefore, we measured the distributions of cell volume by analyzing forward (FSC) and side (SSC) light scatter as a cell-size and granularity index and then measured the mitochondrial mass via staining with a mitochondria-specific dye (MitoTracker FM) and intracellular voltage-dependent anion channel (VDAC) expression in HtrA2/Omi-depleted FL83B cells. In addition to the absence of changes in the cell-size and granularity index (Supplemental Figure S1C), the MitoTracker signal and VDAC expression revealed that the average mitochondrial mass was not changed in HtrA2/Omi-depleted hepatocytes (Supplemental Figure S1B). Next, the degree of mtDNA damage induced by mtROS was measured in HtrA2/Omi-depleted hepatocyte via qPCR assay. As shown in Figure 3C, the ratios of the 79 bp fragment and 230 bp fragment were significantly increased by 1.7-fold in HtrA2/Omi-depleted hepatocyte. Furthermore, an interesting observation was that *mt-ND1* mtDNA content per *Ndufv1* in HtrA2/Omi-depleted FL83B cells was significantly increased by approximately 6-fold compared with control levels, whereas *ND1* mRNA levels were significantly decreased (Figure 3D,E). These results are consistent with previous findings that MEF cells lacking HtrA2/Omi displayed increased mtDNA levels relative to paired control cell lines [12]. Mitochondrial oxidative stress-induced mtDNA damage was associated with a decrease in mitochondrial respiration [26]. Next, we examined the e ffect of HtrA2/Omi depletion on mitochondrial respiration by measuring the oxygen consumption rate (OCR) using an extracellular flux analyzer. Three basal OCRs were recorded, followed by the sequential injection of oligomycin, FCCP, and antimycin A. As shown in Figure 3F, HtrA2/Omi-depleted FL83B cells displayed impaired mitochondrial respiration either under basal or maximal oxygen consumption induced by FCCP treatment. Taken together, these results indicate that HtrA2/Omi-deficient hepatocytes exhibit mitochondrial dysfunction and a concomitant elevation of mtROS levels.

**Figure 3.** Lentivirus-mediated HtrA2/Omi depletion in hepatocyte lead to impaired mitochondrial function and metabolism.

(**A**) Intracellular ROS levels were measured by FACS-analysis using CM-H2DCFDA staining. (**B**) Mitochondrial superoxide level was measured by FACS-analysis using MitoSOX red staining. The fluorescence mean intensity of MitoSOX red per cell was quantified. ( **C**) Damaged mtDNA levels of the 79 bp fragment (damaged) and 230 bp fragment (undamaged) in gDNA isolated from cells were assessed by qPCR. Bars represent mean copy number ratios of mtDNA-79:230, normalized to 18S levels. (**D**) Real time qPCR analysis of *mt-ND1* DNA normalized to nuclear *Ndufv1* DNA. (**E**) *ND1* mRNA expression, as determined by qRT-PCR. (**F**) OCR measurements were obtained using an extracellular flux analyzer. Results for mitochondrial respiration were normalized to the total protein content.

#### *3.4. Loss of HtrA2*/*Omi Protease Activity in Hepatocytes Results in the Accumulation of Dysfunctional Mitochondria and Oxidative Stress*

We attempted to confirm the association of mitochondrial dysfunction with mtROS levels following the loss of HtrA2/Omi mitochondrial protease activity as well as HtrA2/Omi depletion in hepatocytes. Motor neuron degeneration 2 (mnd2)-mutant mice carry a single missense mutation (Ser276Cys) in the HtrA2/Omi gene that inactivates the protease activity of HtrA2/Omi [27]. Consistent with previous studies [28], mnd2-mutant mice exhibited striatal neuron loss; severe muscle wasting; weight loss; general decreases in the sizes of organs such as the liver, thymus, heart, and spleen; and death before 40 days of age (Supplemental Figure S2A). We next examined whether HtrA2/Omi mutation influences the quantity and function of mitochondria in hepatocytes as well as liver fibrogenesis. We observed mitochondrial morphology using TEM in liver tissue from wild-type (WT) or mnd2-mutant mice at postnatal day 32. Compared with the findings in WT mice, the liver tissue sections from mnd2-mutant mice appeared to have greater numbers of mitochondria and a slightly larger volume (Supplemental Figure S2B). In addition, the mitochondria in the liver tissue sections from mnd2-mutant mice were swollen compared with those in WT mice. These findings indicate that HtrA2/Omi mutation induces mitochondrial accumulation, mitochondrial swelling, and disruption of the cristae, and the results are similar for primary hepatocytes isolated from mnd2-mutant mouse livers (Supplemental Figure S2C).

Based on the abnormal mitochondrial morphology and biogenesis in the livers of mnd2-mutant mice, HtrA2/Omi mutation in hepatocytes is expected to lead to mitochondrial dysfunction. We hypothesized that pathologic changes in mitochondria caused by HtrA2/Omi mutation result from abnormalities of respiratory complex subunits. As mentioned previously, mnd2-mutant mice displayed decreases in liver size, and thus, we compared the cell-size and granularity index of primary hepatocytes isolated from WT and mnd2-mutant mouse livers at postnatal day 32 via FSC and SSC analysis (Figure 4A). FACS analysis illustrated that the cell-size and granularity index was lower for hepatocytes from mnd2 mutant mice. A previous study found that mitochondrial activity changes with cell size, resulting in allometric scaling of metabolism at the cellular level [25,29]. To determine whether HtrA2/Omi mutation in hepatocytes a ffects mitochondrial mass and mtROS, we assessed VDAC expression, MitoTracker Green, and MitoSOX staining in primary hepatocytes isolated from mnd2-mutant mouse livers via western blot, flow cytometry, and confocal microscopy. As shown in Figure 4B, mtROS levels to be higher in mnd2-mutant hepatocytes than in WT hepatocytes. However, no di fferences in mitochondrial mass by VDAC expression and MitoTracker staining were observed between hepatocytes isolated from mnd2-mutant and WT mouse livers. Furthermore, confocal microscopy revealed that mnd2-mutant hepatocytes had smaller mitochondrial areas, but higher mitochondrial fluorescence intensity for MitoSOX Red than WT hepatocytes (Figure 4C). We also measured intracellular ROS levels using the intensity of CM-H2DCFDA fluorescence in H2O2-treated hepatocytes. As shown in Figure 4D, intracellular ROS levels were increased in mnd2-mutant mouse hepatocytes treated with 1.5 mM H2O2. ROS levels were higher in mnd2-mutant mouse hepatocytes, and mnd2-mutant mouse hepatocytes are more sensitive to H2O2-induced oxidative stress than in WT hepatocytes. These findings sugges<sup>t</sup> that the number of mitochondria per cell was not changed by mnd2 mutation in primary hepatocytes, whereas mtROS and intracellular ROS levels were elevated.

**Figure 4.** Loss of HtrA2/Omi in hepatocyte results in the accumulation of dysfunctional mitochondria and oxidative stress. (**A**) Exemplary flow cytometry plot and histogram of showing populations of primary hepatocyte in light scatters. (**B**) Mitochondrial morphology and superoxide levels in isolated hepatocyte were measured by FACS-analysis using MitoTracker green and MitoSOX red staining. The fluorescence mean intensity per cell was quantified. Western blotting for VDAC, mitochondrial mass proteins in WT and mnd2-mutant hepatocytes, with β-actin as a loading control. (**C**) Confocal microscopy of Mitotracker and MitoSOX red staining in primary hepatocyte. Nuclei were stained blue by DAPI. (**D**) Intracellular ROS levels in primary hepatocyte were measured by FACS-analysis using CM-H2DCFDA staining.

To clarify whether increased mtROS levels are involved in mitochondrial dysfunction in primary hepatocytes isolated from mnd2-mutant mouse livers, we assessed damaged mtDNA and mtDNA content using real-time qPCR. As shown in Supplemental Figure S2D,E, *mt-ND1* and *mt-COX1* were detectable at higher levels in mnd2 hepatocyte, whereas their mRNA levels were significantly lower. In addition, the ratios of the 79 bp fragment and 230 bp fragment were significantly increased by 5.5-fold in mnd2 hepatocyte. These results are consistent with those in HtrA2/Omi-deficient hepatocytes and CCl4-induced fibrotic livers. In addition, mitochondrial respiration (*ATP5A, COX5B*) and biogenesis (*ERR*α) genes were downregulated in mnd2-mutant hepatocytes compared with their levels in WT hepatocytes (Supplemental Figure S2F). These results reveal statistically significant associations between mtDNA damage and the concomitant elevation of mtROS levels in mnd2-mutant hepatocytes.

#### *3.5. Loss of HtrA2*/*Omi Mitochondrial Protease Activity in mnd2-Mutant Mice Promotes Liver Fibrosis*

The abnormal mitochondrial shape and mitochondrial dysfunction observed in HtrA2/Omi-deficient liver tissue might be closely linked to liver fibrogenesis. Next, we induced

chronic liver injury in mnd2 heterozygous (mnd2/+) mice via repetitive CCl4 injections to observe the development of extensive bridging fibrosis and substantial collagen deposits. As shown in Supplemental Figure S3A, we demonstrated that the heterozygous (mnd2/+) mice for this deletion showed a 50–70% reduction in mitochondrial protease activity. The mnd2/+ mice were used because mnd2 homozygous (mnd2/mnd2) mice die before 40 days of age. After 8 weeks of CCl4 treatment, mnd2/+ mice displayed significant hepatic fibrosis, as demonstrated by quantification of Sirius-red positive area (Supplemental Figure S3B,C), compared with the findings in WT mice. Moreover, the concentration of hydroxyproline in mnd2/+ mouse livers was also increased after CCl4 injection compared with the levels in WT mice (Supplemental Figure S3D). These results sugges<sup>t</sup> that loss of HtrA2/Omi mitochondrial protease activity in mnd2-mutant mice promotes liver fibrosis by increasing mtDNA damage and mtROS levels.

#### *3.6. HtrA2*/*Omi Deficient Hepatocyte Derived-mtDNA Induces Liver Fibrogenesis*

Based on the aforementioned results, we demonstrated a direct link between increased mtDNA damage in HtrA2/Omi-deficient or HtrA2/Omi-mutated hepatocytes and liver fibrogenesis. HtrA2/Omi was predominantly expression in hepatocyte, but there is little information available regarding the effects of potential paracrine stimulation by hepatocyte-derived mtDNA or mtROS on HSC activation either in vivo or in culture. Therefore, we hypothesized that the accumulation of damaged mtDNA in hepatocyte may serve as DAMPs to link the HSC activation. We isolated damaged mtDNA from HtrA2/Omi-deficient or HtrA2/Omi-mutated hepatocytes and examined whether mtDNA can induce HSC activation as DAMP molecules. Forty-eight hours after adding the same concentration (500ng) of mtDNA extracted from HtrA2/Omi-deficient hepatocytes, upregulation of the mRNA transcripts for *collagen 1* and α*-SMA* was observed in inactivated LX-2 cells with serum-free medium (Figure 5A). Furthermore, LX-2 cells underwent morphological changes in response to damaged mtDNA treatment, as shown in stained images (Figure 5B). Consistent with the upregulation of *collagen 1* and α*-SMA* transcripts in LX-2 cell treated with mtDNA from HtrA2/Omi-mutated hepatocytes, *collagen 1* and α*-SMA* expression was upregulated in LX-2 cell treated with mtDNA from primary hepatocytes isolated from mnd2-mutant mice (Figure 5C,D). These results, together with the previously mentioned data, strongly suggested that the accumulation of damaged mtDNA due to a loss of HtrA2/Omi in hepatocyte is associated with liver fibrosis through crosstalk with the activation of HSC.

**Figure 5.** Loss of HtrA2/Omi mitochondrial protease activity promotes liver fibrosis. (**A**,**C**) Collagen 1 and α-SMA mRNA expression in LX-2 cell treated with the same concentration (500 ng) of hepatocyte derived-mtDNA, as determined by qRT-PCR. (**B**,**D**) Immunofluorescence staining for α-SMA (green) performed in LX-2 cell treated with hepatocyte derived-mtDNA. Nuclei were stained with DAPI (blue). The relative intensity measurement of immunofluorescence is shown as histogram for α-SMA. Original magnification, X400. CM: cultured LX-2 cells in completed media condition; S.F.: cultured LX-2 cells in serum free media condition.

#### *3.7. Restoration of HtrA2*/*Omi Expression Rescues CCl4-Induced Liver Fibrosis and Reverses Mitochondrial Dysfunction in Hepatocyte*

Because HtrA2/Omi expression is downregulated during hepatic fibrogenesis, it was expected that HtrA2/Omi plays a protective role in our CCl4-induced liver fibrosis model. To further confirm the protective role of HtrA2/Omi, we examined the effects of the hydrodynamic gene delivery of pFLAG-HtrA2/Omi in the CCl4-induced liver fibrosis model. In a standard CCl4-induced mouse model of liver fibrosis, serum ALT activity was significantly changed in mice treated with CCl4 twice weekly for 8 weeks. The hydrodynamic injection of HtrA2/Omi attenuated the elevation of ALT activity in CCl4-treated mice (Figure 6B). Subsequent experiments were performed to analyze liver histological alterations occurring in response to HtrA2/Omi injection in CCl4-treated mouse livers. Semiquantitative IHC detection of HtrA2/Omi expression cells confirmed a transient increase in the HtrA2/Omi-injected liver than in control CCl4- treated livers (Figure 6C). Intriguingly, pFLAG-HtrA2/Omi administration inhibited the development of hepatic fibrosis, as confirmed by H&E and Sirius red staining (Figure 6A). Quantification indicated that the Sirius red-positive area was smaller (by 5.8%) in fibrotic livers from mice injected with pFLAG-HtrA2/Omi plasmids than in livers from CCl4-treated mice (Figure 6D). The hydroxyproline content was significantly lower in livers treated with pFLAG-HtrA2/Omi (1.1 μg/mg, *p* < 0.01) than in control CCl4- treated livers (2.17 μg/mg) (Figure 6E). Hepatic HtrA2/Omi expression significantly decreased the hepatic hydroxyproline level. Immunohistochemical staining using α-SMA antibody illustrated that strong α-SMA expressions was limited to scarred areas in the CCl4 treatment group, but pFLAG-HtrA2/Omi administration decreased α-SMA expression in fibrotic areas (Figure 6A). As previous studies reported that HtrA2/Omi directly contributes to apoptosis [9], we evaluated apoptosis using the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay in HtrA2/Omi-injected tissues. In the CCl4-treated group, 76% of cells were TUNEL-positive, versus 67% of cells in the HtrA2/Omi-injected group (Supplemental Figure S4A,B). Consistent with the percent of TUNEL-positive apoptotic cells, we found that cleaved caspase 3 levels were reduced in the HtrA2/Omi-injected group compared with those in CCl4-treated group (Supplemental Figure S4A,C). These results sugges<sup>t</sup> that HtrA2/Omi expression appears to reverse, or at least prevent, further progression of liver fibrosis.

**Figure 6.** HtrA2/Omi expression in mouse model of CCl4-induced liver fibrosis alleviates liver fibrosis by protecting hepatocytes damage. HtrA2/Omi expression in CCl4-treated mice following the hydrodynamic tail vein injection of 30 μg HtrA2/Omi-encoding plasmid DNA (*n* = 7) compared with the mock control group (*n* = 5) over 8 weeks at 4-day intervals. Hepatic fibrosis was induced by injection of CCl4 two times per week for 8 weeks. (**A**) Representative images of H&E, Sirius red (original magnification, X200) and immunohistochemistry staining (X400) of liver sections. (**B**) Effects of HtrA2/Omi expression on serum ALT. (**C**) IHC quantification of HtrA2/Omi positivity and (**D**) semi-quantitative analysis of Sirius red staining. (**E**) The hydroxyproline content in mouse livers.

Furthermore, to determine whether HtrA2/Omi expression could reverse mitochondrial dysfunction induced by CCl4 in hepatocyte, we compared the mitochondrial ultrastructure and mtDNA content in the fibrotic livers of mice injected with pFLAG-HtrA2/Omi plasmids. Additional analyses revealed that mitochondria more frequently had a normal structure within hepatocyte from the HtrA2/Omi-injected group (Figure 7A). As shown in Figure 7B, the ratios of the 79 bp fragment and 230 bp fragment were decreased in the HtrA2/Omi-injected group than in the CCl4-treated control group. The *mt-ND1* and *mt-COX1* mtDNA contents were significantly lower in the HtrA2/Omi-injected group than in the CCl4-treated control group (Figure 7C). Conversely, there was no difference in *ATP5A* and *COX5B* mRNA levels between the HtrA2/Omi-injected and CCl4-treated groups

(Supplemental Figure S4D), but MnSOD and CuZnSOD expression in antioxidant enzymes was restored by HtrA2/Omi treatment (Figure 7D). Taken together, these data suggested that HtrA2/Omi has an important role in maintaining mitochondrial homeostasis that might decrease vulnerability to liver injury and the development of liver fibrosis.

**Figure 7.** HtrA2/Omi expression in CCl4-induced liver fibrosis protects mitochondrial damage of hepatocyte. (**A**) TEM analysis of hepatocyte showing nucleus (N) and mitochondria (M) (scale bar = 2 & 0.5 μm). (**B**) Damaged mtDNA levels of the 79 bp fragment (damaged) and 230 bp fragment (undamaged) in gDNA isolated from liver tissue were assessed by qPCR. Bars represent mean copy number ratios of mtDNA-79:230, normalized to 18S levels. (**C**) qPCR analysis of *mt-ND1* and *mt-COX1* DNA normalized to nuclear *Ndufv1* DNA. (**D**) Western blot analysis of MnSOD and CuZnSOD expression in liver tissue from pFLAG-HtrA2/Omi treated fibrotic mice compared to mock vector treated fibrotic mice.
