Insulin-Like Growth Factor I (IGF-I) Expressed from an AAV1 Vector Leads to a Complete Reversion of Liver Cirrhosis in Rats

<div><p>IGF-I modulates liver tissue homeostasis. It is produced by hepatocytes and signals within the liver through IGF-I receptor expressed on hepatic stellate cells (HSCs). Liver cirrhosis is characterized by marked IGF-I deficiency. Here we compared the effect of two different gene therapy vectors encoding IGF-I as a potential treatment for cirrhotic patients. Rats with carbon tetrachloride-induced liver cirrhosis were treated with controls or with adeno-associated virus 1 (AAV) or simian virus 40 (SV40) vectors expressing IGF-I (AAVIGF-I or SVIGF-I) and molecular and histological studies were performed at 4 days, 8 weeks and 16 weeks. Increased levels of IGF-I were observed in the liver as soon as 4 days after vector administration. Control cirrhotic rats showed increased hepatic expression of pro-inflammatory and pro-fibrogenic factors including transforming growth factor beta (TGFβ), tumor necrosis factor-alpha (TNFα), connective tissue growth factor (CTGF), and vascular endothelial growth factor (VEGF) together with upregulation of α-smooth muscle actin (αSMA), a marker of HSC activation. In IGF-I-treated rats the levels of all these molecules were similar to those of healthy controls by week 8 post-therapy. Of note, the decline of TGFβ, CTGF, VEGF and αSMA expression was more rapid in AAVIGF-I treated animals reaching statistical significance by day 4 post-therapy. IGF-I-treated rats showed similar improvement of liver function tests in parallel with upregulation of hepatocyte nuclear factor 4α (HNF4α), a factor that promotes hepatocellular differentiation. A significant decrease of liver fibrosis, accompanied by upregulation of the hepatoprotective and anti-fibrogenic hepatocyte growth factor (HGF), occurred in all IGF-I-treated rats but complete reversal of liver cirrhosis took place only in AAVIGF-I group. Therefore, AAVIGF-I reverts liver cirrhosis in rats, a capability which deserves clinical testing.</p></div

Functional IGF-I is expressed in the liver after administration of AAVIGF-I or SVIGF-I.

<p>(a) Schematic of the experimental protocol used. Healthy animals were untreated, or used to induce liver cirrhosis by intragastric administration of CCl₄ once a week for eight weeks (w). One week after the end of the cirrhosis induction protocol, cirrhotic animals were treated with saline, AAVLuc, AAVIGF-I or SVIGF-I by intra-arterial administration. Animals were sacrificed 4 days, 8 weeks and 16 weeks after vector inoculation. Healthy animals were sacrificed in parallel as controls. Blood was collected and liver samples were processed for histology, and purification of RNA and proteins for further analysis. (b) Analysis of liver IGF-I expression and activity. Liver samples were obtained from healthy, cirrhotic animals treated with saline (Ci), AAVLuc (Ci+AAVLuc), AAVIGF-I (Ci+AAVIGF-I) or SVIGF-I (Ci+SVIGF-I). Total IGF-I protein (a) and IGF-I, IGFBP3 and IGF-IR mRNAs were quantified by ELISA and qRT-PCR in liver extracts. Transcript levels shown are relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels. Samples were obtained 4 days or 8 weeks after vector inoculation. Error bars denote standard deviations. Significant and non-significant (ns) differences are highlighted.</p

Analysis of HGF.

<p>Animals were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162955#pone.0162955.g001" target="_blank">Fig 1</a> and HGF mRNA (a) and protein (b) levels were evaluated by qRT-PCR or ELISA in liver extracts. Transcript levels shown are relative to GAPDH mRNA levels. Error bars denote standard deviations. Significant and non-significant (ns) differences are highlighted.</p

Analysis of liver damage, pro-inflammatory and pro-fibrogenic factors.

<p>Animals were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162955#pone.0162955.g001" target="_blank">Fig 1</a> and TGFβ (a), TNFα (b) and IL-6 (c) mRNA and protein levels were evaluated in liver extracts by qRT-PCR or ELISA. CTGF, VEGF and PDGF mRNA levels were also measured. Transcript levels shown are relative to GAPDH mRNA levels. (e) Transaminases AST, ALT and ALP were quantified in the serum of the animals 8 weeks after vector administration. Error bars denote standard deviations. Significant and non-significant (ns) differences are highlighted.</p

Analysis of liver fibrosis.

<p>Animals were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162955#pone.0162955.g001" target="_blank">Fig 1</a> and liver fibrosis was evaluated by quantification (b) of Sirius red staining 16 weeks after vector administration (a) or by qRT-PCR of collagen I and collagen IV mRNA performed in samples collected at the indicated times (c). Collagen mRNA levels shown are relative to GAPDH mRNA levels. Error bars denote standard deviations. Significant and non-significant (ns) differences are highlighted.</p

Analysis of MMPs and MMP inhibitors.

<p>Animals were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162955#pone.0162955.g001" target="_blank">Fig 1</a> and MMP-1, 2, 9 and 14 mRNAs (a) and MMP-2 and 9 proteins (b) were evaluated in liver extracts by qRT-PCR or ELISA respectively. Total MMP activity in liver extracts (c) and the levels of MMP inhibitor TIMP-2 mRNA (d) were also evaluated. Transcript levels shown are relative to GAPDH mRNA levels. Error bars denote standard deviations. Significant and non-significant (ns) differences are highlighted.</p

Patient characteristics and complementary data.

<p>* All the remaining variables including diabetes, dyslipidemia, heart failure, previous coronary heart disease, heart arrhythmia, neurologic illness, body mass index, temperature, pH, sodium, potassium, magnesium and GPT were not statistically significant. Some variables described as continuous were also studied as polychotomous and dichotomous ones.</p><p>¹ ICU: Intensive Care Unit.</p><p>² COPD: Chronic Obstructive Pulmonary Disease.</p><p>³ BP: Blood Pressure.</p><p>⁴ NIV: Non-invasive Ventilation.</p><p>⁵ ESRD: End-Stage Renal Disease.</p><p>⁶ SBP: Systolic Blood Pressure.</p><p>⁷ ImCU: Intermediate Care Unit.</p><p>Patient characteristics and complementary data.</p

Calibration curve based on the Hosmer and Lemeshow goodness-on-fit C test for SAPS II.

<p>Calibration curve based on the Hosmer and Lemeshow goodness-on-fit C test for SAPS II.</p

Performance of ImCUSS, SAPS II and SAPS 3.

<p>* SMR: Standardized Mortality Ratio.</p><p>^₸ GOF: Goodness-Of-Fit.</p><p>³ AUROC: Area Under Receiver Operating Characteristic Curve</p><p>¹ Performance in the validation cohort (n = 189).</p><p>² Performance in the whole cohort (n = 743).</p><p>Performance of ImCUSS, SAPS II and SAPS 3.</p

ImCUSS receiver operating characteristic curve (ROC) for mortality in the validation cohort (n = 189).

<p>ImCUSS receiver operating characteristic curve (ROC) for mortality in the validation cohort (n = 189).</p