Aging Influences Hepatic Microvascular Biology and Liver Fibrosis in Advanced Chronic Liver Disease

Advanced chronic liver disease (aCLD) represents a major public health concern. aCLD is more prevalent and severe in the elderly, carrying a higher risk of decompensation. We aimed at understanding how aging may impact on the pathophysiology of aCLD in aged rats and humans and secondly, at evaluating simvastatin as a therapeutic option in aged animals. aCLD was induced in young (1 month) and old (16 months) rats. A subgroup of aCLD-old animals received simvastatin (5 mg/kg) or vehicle (PBS) for 15 days. Hepatic and systemic hemodynamic, liver cells phenotype and hepatic fibrosis were evaluated. Additionally, the gene expression signature of cirrhosis was evaluated in a cohort of young and aged cirrhotic patients. Aged animals developed a more severe form of aCLD. Portal hypertension and liver fibrosis were exacerbated as a consequence of profound deregulations in the phenotype of the main hepatic cells: hepatocytes presented more extensive cell-death and poorer function, LSEC were further capillarized, HSC over-activated and macrophage infiltration was significantly increased. The gene expression signature of cirrhosis significantly differed comparing young and aged patients, indicating alterations in sinusoidal-protective pathways and confirming the pre-clinical observations. Simvastatin administration for 15-day to aged cirrhotic rats improved the hepatic sinusoidal milieu, leading to significant amelioration in portal hypertension. This study provides evidence that aCLD pathobiology is different in aged individuals. As the median age of patients with aCLD is increasing, we propose a real-life pre-clinical model to develop more reliable therapeutic strategies. Simvastatin effects in this model further demonstrate its translational potential.


Liver endothelial function
Rat livers were isolated and perfused at 35 mL/min with Krebs' buffer. The perfused rat liver preparation was allowed to stabilize for 20 min before vasoactive substances were added. The intrahepatic microcirculation was pre-constricted by adding the α1-adrenergic agonist methoxamine (Mtx; 10 -4 M; Sigma) to the reservoir. After 5 min, concentrationresponse curves to cumulative doses of acetylcholine (Ach; 10 -7 -10 -5 M; Sigma) were evaluated. The concentration of Ach was increased by 1log unit every 1.5 min interval. Responses to Ach were calculated as the percentage change in portal perfusion pressure [2]. The gross appearance of the liver, stable perfusion pressure, bile production over 0.4µl/min/g of liver and stable buffer pH (7.4 ± 0.5) were monitored during this period. If any viability or stability criteria were not satisfied, the experiment was discarded.

Electron microscopy
Liver sinusoidal ultrastructure was characterized using electron microscopy as previously described [3]. Briefly, livers were perfused through the portal vein with a fixation solution containing 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1M cacodylate buffer 0.1% sucrose and fixed overnight at 4º C. Samples were washed 3 times with 0.1M sodium cacodylate buffer. Liver sections were post-fixed with 1% osmium in cacodylate buffer and dehydrated in an ethanol gradient to 100%.
For scanning electron microscopy 6 to 8 liver blocks per sample were mounted on stubs, sputter coated with gold and examined using a Jeol 6380 scanning electron microscope. Measurements of fenestrae size, number and density were carried out. Fenestrations were defined as open pores with diameters <300nm. Diameter was defined as the major length of each fenestration or gap. Porosity was defined as the sum area of fenestrations/total quantified area. Frequency was defined as number of fenestrae per μm 2 . At least 10 images per animal were taken.
For transmission electron microscopy, fixed liver tissue was embedded in Spurr resin, cut in 50nm ultrathin sections, counterstained with uranyl acetate and lead citrate and examined using microscope. 10 micrographs per sample were taken to estimate % of necrotic hepatocytes and % of sinusoids presenting each of the following parameters: lack of microvilli, big space of Disse, peliosis and basal lamina deposition.

Histological Analysis
Liver samples were fixed in 10% formalin, embedded in paraffin, sectioned, and slides were stained with Sirius Red for liver fibrosis evaluation [5]. Frozen sections were cut to 10 µm and stained with Oil Red O (Sigma Aldrich) for lipid analysis. Lipid droplets were evaluated as the red-stained area per total area using ImageJ software.

Immunohistochemistry
Liver samples were fixed in 10% formalin, embedded in paraffin, sectioned and processed for immunohistochemistry (IHC) or immunofluorescence (IF) as previously described [6].
For IHC liver sections were incubated with antibodies against CD32b (sc -13271, Santa Cruz), eNOS (sc-654, Santa Cruz), vWF (A0082, Dako), -SMA (M0851, Dako), or CD163 (MCA342R, Biorad). After incubation with corresponding secondary antibodies, color development was induced by incubation with a DAB kit (Dako) and counterstained with hematoxylin. Sections were dehydrated and mounted. The specific staining was visualized and fifteen images per liver were acquired using a microscope equipped with a digital camera and the assistance of Axiovision software. The relative volume was calculated by dividing the number of points positive in sinusoidal areas by the total number of points over liver tissue [7].

Cytochrome 4503A4 activity
Phase I detoxification capacity of hepatocytes was analyzed using P450-Glo™ CYP4503A4 Assay following manufacturer's instructions (V8901, Promega) [8]. Briefly, hepatocytes cultured for 24 hours were rinsed twice with DPBS and incubated with culture media containing 50 µM Luciferin-PFBE at 37°C for 4h. Then supernatant was collected and neutralized with Luciferin Detection Reagent. After incubation for 30 min at RT, plate luminescence was read in a luminometer (Orion II Microplate Luminometer, Germany). Samples luminescence was corrected subtracting background luminescence.

Human HSC isolation and contraction assay
Human hepatic stellate cells were isolated from remnant liver tissue approximately weighting 20 g obtained from discarded tissue after liver transplantation (chronic ethanol etiology). Ethics Committee of the Hospital Clí nic de Barcelona approved the experimental protocol (HCB/2015/0624) and in all cases patients received and agreed informed consent. Median age of liver donors for cell isolation was 70 ± 3 years.
Contraction of aged human HSC was performed as previously described [2]. Culture plates were incubated with 1% BSA-PBS and afterwards filled with a mix of collagen (2 mg/mL) and human HSC (1-2 × 10 5 cells/mL). Once the gels were solidified, serum free IMDM with 10 μM simvastatin or vehicle was added. After 24 h, contraction was induced by adding 10% FBS for 24 h. Finally, the contraction area was digitalized and measured with ImageJ software. The results are expressed as % of contraction relative to the initial area of the gel.

Endotoxemia quantification
Endotoxemia was quantified using a quantitative chromogenic limulus amoebocyte lysate (LAL) test (BioWhittaker, Nottingham, UK), following manufacturer's instructions. Due to LPS ubiquity, samples and reagents were handled in an airflow chamber and processed with pyrogen-free material tested by manufacturers. E. coli lyophilized endotoxin (22 UE/mL) provided by the kit was used to set standard endotoxin concentrations ranging from 5.0 UE/mL to 0.1 UE/mL. To verify the lack of product inhibition by plasma protein, a dilution/heating inactivation protocol was followed prior to endotoxin measurement. A pooled E. coli endotoxin spike solution (0.4 UE/mL) was prepared with serum samples. Dilutions ranging from 1/2 to 1/20 were performed over spiked and unspiked serum samples. All test samples were then incubated at 60°C during 30 min [9]. The LAL test was performed after this period. The non-inhibitory dilution was established when the difference between spiked and unspiked endotoxin values was equal to the known concentration of the spike ± 25%, as detailed by the manufacturer. Final sample dilutions used were 1/10 (spike recovery after correction of dilution: 0.34 UE/mL). All samples were tested in triplicate and read at 405 nm in a Tecan Sunrise microplate reader (Mä nnedorf, Switzerland).

Supplementary references
Supplementary