Aging Influences Hepatic Microvascular Biology and Liver Fibrosis in Advanced Chronic Liver Disease
Raquel Maeso-Díaz1, Martí Ortega-Ribera1, Erica Lafoz1, Juan José Lozano2, Anna Baiges1,2, Rubén Francés2,3, Agustín Albillos2,4, Carmen Peralta2,5, Juan Carlos García-Pagán1,2, Jaime Bosch1,2,6, Victoria C Cogger7, Jordi Gracia-Sancho1,2,6,*
1Liver Vascular Biology Research Group, Barcelona Hepatic Hemodynamic Laboratory, IDIBAPS Biomedical Research Institute, University of Barcelona Medical School, Barcelona, Spain 2Biomedical Research Network Center in Hepatic and Digestive Diseases (CIBEREHD), Madrid, Spain 3Instituto de Investigación Sanitaria y Biomédica de Alicante (ISABIAL -Fundación FISABIO), Alicante, Spain 4 Department of Gastroenterology and Hepatology, Hospital Universitario Ramón y Cajal, IRYCIS, Universidad de Alcalá, Madrid, Spain 5Protective Strategies Against Hepatic Ischemia-Reperfusion Group, IDIBAPS, Barcelona, Spain 6Hepatology, Department of Biomedical Research, Inselspital, Bern University, Switzerland 7Centre for Education and Research on Ageing & ANZAC Research Institute, University of Sydney and Concord Hospital, Sydney, Australia
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.
Table 1 Biometric, biochemical and hemodynamic characteristics in aCLD-young and aCLD-old rats.
Figure 1. Hepatocyte phenotype markers in 4 months-young and 20 months-old rats with aCLD
(A) Representative transmission electron microscopy images and corresponding quantification of numbers of sinusoids (S), lack of microvilli (M), big space of Disse (D), peliosis(P), basal lamina deposition and number of necrotic hepatocytes (H) in liver tissue from 4 months-young and 20 months-old rats with aCLD. (B) HNF4α, Oct1, Mrp2 and Mrp3 mRNA expression in livers described in A. n=3 (A) and (B) n=7 per group. Results represent mean ± S.E.M. All images: 3000X, scale bar=20μm.
Figure 2. LSEC phenotype markers in aged rats with aCLD
The following markers of sinusoidal endothelial phenotype were analysed in liver tissue from 4 months-young and 20 months-aged rats with aCLD. (A) mRNA expression of KLF2 and CD32b. (B) Representative images of CD32b immunehistochemistry and corresponding quantification. (C) Representative images of eNOS immunohistochemistry and corresponding quantification. (D) Representative images of vWF immunohistochemistry and corresponding quantification. (E) mRNA expression of HGF, Wnt2, Hamp and Axin2. (F) Representative scanning electron microscopy images & quantification of porosity, fenestration frequency and fenestration diameter. n=7 (A-E) and n=3 (F) per group. Results represent mean ± S.E.M. Images from B-D: 400X, scale bar=50μm. Images from F: 15000X, scale bar=1μm.
Fibrotic content, HSC phenotype and macrophage infiltration and phenotype were evaluated in young and aged rats with aCLD. (A) Representative images of fibrotic content measured as positive area for Sirius Red with their corresponding quantifications. (B) Representative western blot of Collagen I normalized to GAPDH. (C) Representative western blot of α-SMA normalized to GAPDH. (D) Representative western blots of moesin and p-moesin and corresponding quantification. (E) Left, representative images of CD68 immunofluorescence in liver tissue and corresponding quantification. Right, representative images of CD163 immunohistochemistry in liver tissue and its quantification. (F) Expression of TNF-α, iNOS, and IL-6 as pro-inflammatory markers (left) and Mrc1, Arg1 and IL-10 as anti-inflammatory markers (right) in liver tissue from young and old rats with aCLD. n=7 per group. Results represent mean ± S.E.M. All images: 400X, scale bar=50μm.
Figure 4. Aged-related changes in the gene signature of cirrhotic human liver
Gene expression analysis in cirrhotic young and old human livers. (A) Left, fold enrichments (log2) are plotted in a heatmap using red colour for transcripts that are increased or using green colour for transcripts that are decreased in old cirrhotic humans. Right, pathway enrichment analysis results for genes upregulated (red) and downregulated (green) are summarized. (B) Representative gene sets upregulated (red) or downregulated (green) related to microcirculatory function in old cirrhotic humans, full description of top ten gene sets can be found in supplementary materials. FDR < 10%, n=7 per group. Clinical characteristics of donors are described in Supplementary table 1.
Figure 5. Effects of simvastatin on hepatocytes and microcirculatory function
(A) HNF4α, Oct1, Mrp2 and Mrp3 mRNA expression in livers from aged rats with aCLD treated with simvastatin or vehicle. (B) Cytochrome P4503A4 activity in hepatocytes isolated from livers described in A. (C) Representative transmission electron microscopy images and corresponding quantification of numbers of sinusoids (S), lack of microvilli (M), big space of Disse (D), peliosis and number of necrotic hepatocytes (H). (D) Microvascular function evaluation in livers from aged rats with aCLD treated with simvastatin or vehicle. (E) Representative images of vWF immunohistochemistry and corresponding quantification from livers described in A. (F) Representative scanning electron microscopy images & quantification of porosity, fenestration frequency and fenestration diameter. n=10 (A-B, D-E), n=5 (B) and n=3 (C and F) per group. Results represent mean ± S.E.M. Images from C: 3000X, scale bar=20μm. Images from E: 400X, scale bar=50μm. Images from F: 15000X, scale bar=1μm.
Figure 6. Simvastatin promotes decreased fibrosis deposition and HSC de-activation
(A) Representative images of fibrotic content, α-SMA and desmin with their corresponding quantifications. (B) α-SMA and Collagen I protein expression in total liver tissue, normalized to GAPDH. (C) Representative western blots of moesin and p-moesin and corresponding quantification. n=10 (A-C). Results represent mean ± S.E.M. All images 400X, scale bar=50μm.
Body weight (g)
655 ± 31
681 ± 18
16.6 ± 1.0
19.1 ± 1.1
Liver-body weight ratio (%)
2.58 ± 0.19
2.81 ± 0.15
188 ± 34
155 ± 28
61 ± 5
57 ± 6
64 ± 13
47 ± 10
0.23 ± 0.06
0.10 ± 0.00
Bile production (µL/min*100g bw)
23.5 ± 7.1
42.0 ± 16.5
22.0 ± 1.1
24.2 ± 0.8
Plasma cholesterol (mg/dL)
86 ± 5
79 ± 10
Plasma LDL cholesterol (mg/dL)
64 ± 5
55 ± 7
Plasma HDL cholesterol (mg/dL)
14.0 ± 1.5
15.6 ± 2.6
Plasma triglycerides (mg/dL)
38.0 ± 5.1
42.1 ± 7.1
Oil red O-staining (%)
1.33 ± 0.22
1.64 ± 0.39
MDA (nmol/mg protein)
2.68 ± 0.74
2.35 ± 0.30
1.49 ± 0.18
1.62 ± 0.13
15.9 ± 1.4
11.9 ± 0.8
1.28 ± 0.17
1.13 ± 0.16
13.9 ± 2.4
12.4 ± 1.5
Ex vivo HVR (mmHg*min/mL*g)
0.39 ± 0.04
0.32 ± 0.03
97 ± 6
110 ± 5
332 ± 22
381 ± 25
Table 2 Biometric, biochemical and hemodynamic characteristics in aCLD-old rats treated with simvastatin or vehicle.
He W, Goodkind D, Kowal P (2016). An Aging World: 2015 International Population Reports. US Gov Publ Off Washington, DC P95/16-1.
Le Couteur DG, McLean AJ (1998). The aging liver: Drug clearance and an oxygen diffusion barrier hypothesis. Clin Pharmacokinet, 34: 359-373.
Maeso-Díaz R, Ortega-Ribera M, Fernández-Iglesias A, Hide D, Muñoz L, Hessheimer AJ, et al. (2018). Effects of aging on liver microcirculatory function and sinusoidal phenotype. Aging Cell, 17: e12829.
Blachier M, Leleu H, Peck-Radosavljevic M, Valla DC, Roudot-Thoraval F (2013). The burden of liver disease in Europe: A review of available epidemiological data. J Hepatol, 58: 593-608.
Marcellin P, Kutala BK (2018). Liver diseases: A major, neglected global public health problem requiring urgent actions and large-scale screening. Liver Int, 38: 2-6.
Angulo P, Keach JC, Batts KP, Lindor KD (1999). Independent predictors of liver fibrosis in patients with nonalcoholic steatohepatitis. Hepatology, 30: 1356-1362.
Sheedfar F, Biase S Di, Koonen D, Vinciguerra M (2013). Liver diseases and aging: Friends or foes? Aging Cell, 12: 950-954.
Ramirez T, Li YM, Yin S, Xu MJ, Feng D, Zhou Z, et al. (2017). Aging aggravates alcoholic liver injury and fibrosis in mice by downregulating sirtuin 1 expression. J Hepatol, 66: 601-609.
Collins BH, Holzknecht ZE, Lynn KA, Sempowski GD, Smith CC, Liu S, et al. (2013). Association of age-dependent liver injury and fibrosis with immune cell populations. Liver Int, 33: 1175-1186.
Kim IH, Xu J, Liu X, Koyama Y, Ma HY, Diggle K, et al. (2016). Aging increases the susceptibility of hepatic inflammation, liver fibrosis and aging in response to high-fat diet in mice. Age (Omaha), 38: 291-302.
Trebicka J, Hennenberg M, Laleman W, Shelest N, Biecker E, Schepke M, et al. (2007). Atorvastatin lowers portal pressure in cirrhotic rats by inhibition of RhoA/Rho-kinase and activation of endothelial nitric oxide synthase. Hepatology, 46: 242-253.
Abraldes JG, Rodríguez-Vilarrupla A, Graupera M, Zafra C, García-Calderó H, García-Pagán JC, et al. (2007). Simvastatin treatment improves liver sinusoidal endothelial dysfunction in CCl4cirrhotic rats. J Hepatol, 46: 1040-1046.
Marrone G, Maeso-Díaz R, García-Cardena G, Abraldes JG, García-Pagán JC, Bosch J, et al. (2015). KLF2 exerts antifibrotic and vasoprotective effects in cirrhotic rat livers: Behind the molecular mechanisms of statins. Gut, 64: 1434-1443.
Meireles CZ, Pasarin M, Lozano JJ, García-Calderó H, Gracia-Sancho J, García-Pagán JC, et al. (2017). Simvastatin attenuates liver injury in rodents with biliary cirrhosis submitted to hemorrhage/resuscitation. Shock, 47: 370-377.
Tripathi DM, Vilaseca M, Lafoz E, Garcia-Caldero H, Haute GV, Fernández-Iglesias A, et al. (2018). Simvastatin Prevents Progression of Acute on Chronic Liver Failure in Rats With Cirrhosis and Portal Hypertension. Gastroenterology, 155: 1564-1577.
Abraldes JG, Villanueva C, Aracil C, Turnes J, Hernandez-Guerra M, Genesca J, et al. (2016). Addition of Simvastatin to Standard Therapy for the Prevention of Variceal Rebleeding Does Not Reduce Rebleeding but Increases Survival in Patients with Cirrhosis. Gastroenterology, 150: 1160-1170.
Bosch J, Abraldes JG, Fernández M, García-Pagán JC (2010). Hepatic endothelial dysfunction and abnormal angiogenesis: New targets in the treatment of portal hypertension. J Hepatol, 53: 558-567.
Mohanty A, Tate JP, Garcia-Tsao G (2016). Statins Are Associated with a Decreased Risk of Decompensation and Death in Veterans with Hepatitis C-Related Compensated Cirrhosis. Gastroenterology, 150: 430-440.
Gracia-Sancho J, Laviña B, Rodríguez-Vilarrupla A, Brandes RP, Fernández M, Bosch J, et al. (2007). Evidence Against a Role for NADPH Oxidase Modulating Hepatic Vascular Tone in Cirrhosis. Gastroenterology, 133: 959-966.
Russo L, Gracia-Sancho J, García-Calderó H, Marrone G, García-Pagán JC, et al. (2012). Addition of simvastatin to cold storage solution prevents endothelial dysfunction in explanted rat livers. Hepatology, 55: 921-930.
De Mesquita FC, Guixé-Muntet S, Fernández-Iglesias A, Maeso-Díaz R, Vila S, Hide D, et al. (2017). Liraglutide improves liver microvascular dysfunction in cirrhosis: Evidence from translational studies. Sci Rep, 7: 1-10.
Le Couteur DG, Cogger VC, Markus AMA, Harvey PJ, Yin ZL, Ansselin AD, et al. (2001). Pseudocapillarization and associated energy limitation in the aged rat liver. Hepatology, 33: 537-543.
Gracia-Sancho J, Laviña B, Rodríguez-Vilarrupla A, García-Calderó H, Fernández M, et al. (2008). Increased oxidative stress in cirrhotic rat livers: A potential mechanism contributing to reduced nitric oxide bioavailability. Hepatology, 47: 1248-1256.
Hide D, Ortega-Ribera M, Fernández-Iglesias A, Fondevila C, Salvadó MJ, Arola L, et al. (2014). A novel form of the human manganese superoxide dismutase protects rat and human livers undergoing ischaemia and reperfusion injury. Clin Sci, 127: 527-537.
Vilaseca M, García-Calderó H, Lafoz E, Ruart M, López-Sanjurjo C, Murphy MP, et al. (2017). Mitochondria-targeted antioxidant mitoquinone deactivates human and rat hepatic stellate cells and reduces portal hypertension in cirrhotic rats. Liver Int, 37: 1002-1012.
Du P, Kibbe WA, Lin SM (2008). lumi: A pipeline for processing Illumina microarray. Bioinformatics, 24: 1547-1548.
Ritchie M, Phipson B, Wu D, Hu Y, Law C, Shi W, et al. (2015). limma powers di erential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res, 43: e47.
Yamazaki H, Oda M, Takahashi Y, Iguchi H, Yoshimura K, Okada N, et al. (2013). Relation between Ultrastructural Localization, Changes in Caveolin-1, and Capillarization of Liver Sinusoidal Endothelial Cells in Human Hepatitis C-Related Cirrhotic Liver. J Histochem Cytochem, 61: 169-176.
Frith J, Jones D, Newton JL (2009). Chronic liver disease in an ageing population. Age Ageing, 38: 11-18.
Poynard T, Ratziu V, Charlotte F, Goodman Z, McHutchison J, Albrecht J (2001). Rates and risk factors of liver fibrosis progression in patients with chronic hepatitis C. J Hepatol, 34: 730-739.
Thabut D, Le Calvez S, Thibault V, Massard J, Munteanu M, Di Martino V, et al. (2006). Hepatitis C in 6,865 patients 65 yr or older: A severe and neglected curable disease? Am J Gastroenterol, 101: 1260-1267.
Davis GL, Alter MJ, El-Serag H, Poynard T, Jennings LW (2010). Aging of hepatitis C virus (HCV)-infected persons in the United States: a multiple cohort model of HCV prevalence and disease progression. Gastroenterology, 138: 513-521.
Gracia-Sancho J, Marrone G, Fernández-Iglesias A (2018). Hepatic microcirculation and mechanisms of portal hypertension. Nat Rev | Gastroenterol Hepatol, doi: .
Canbay A, Taimr P, Torok N, Higuchi H, Friedman S, Gores GJ (2003). Apoptotic body engulfment by a human stellate cell line is profibrogenic. Lab Investig, 83: 655-663.
Natori S, Higuchi H, Contreras P, Gores GJ (2003). The caspase inhibitor IDN-6556 prevents caspase activation and apoptosis in sinusoidal endothelial cells during liver preservation injury. Liver Transplant, 9: 278-284.
Mitchell SJ, Huizer-Pajkos A, Cogger VC, McLachlan AJ, Le Couteur DG, Jones B, et al. (2011). Age-related pseudocapillarization of the liver sinusoidal endothelium impairs the hepatic clearance of acetaminophen in rats. Journals Gerontol - Ser A Biol Sci, 66 A: 400-408.
Fernández-Iglesias A, Gracia-Sancho J (2017). How to Face Chronic Liver Disease: The Sinusoidal Perspective. Front Med, 4: 1-10.
Mahrouf-Yorgov M, de l’Hortet AC, Cosson C, Slama A, Abdoun E, Guidotti J-E, et al. (2011). Increased Susceptibility to Liver Fibrosis with Age Is Correlated with an Altered Inflammatory Response. Rejuvenation Res, 14: 353-363.
Wali M, Harrison RF, Gow PJ, Mutimer D (2002). Advancing donor liver age and rapid fibrosis progression following transplantation for hepatitis C. Gut, 51: 248-252.
Tacke F (2017). Targeting hepatic macrophages to treat liver diseases. J Hepatol, 66: 1300-1312.
Nieto N (2006). Oxidative-stress and IL-6 mediate the fibrogenic effects of rodent Kupffer cells on stellate cells. Hepatology, 44: 1487-1501.
Moctezuma-Velázquez C, Abraldes JG, Montano-Loza AJ (2018). The Use of Statins in Patients With Chronic Liver Disease and Cirrhosis. Curr Treat Options Gastroenterol, 16: 226-240.
Rodríguez S, Raurell I, Torres-Arauz M, García-Lezana T, Genescà J, Martell M (2017). A Nitric Oxide-Donating Statin Decreases Portal Pressure with a Better Toxicity Profile than Conventional Statins in Cirrhotic Rats. Sci Rep, 7: 1-12.
Reagan-Shaw S, Nihal M, Ahmad N (2007). Dose translation from animal to human studies revisited. FASEB J, 22: 659-661.
Briones AM, Rodríguez-Criado N, Hernanz R, García-Redondo AB, Rodrigues-Díez RR, Alonso MJ, et al. (2009). Atorvastatin prevents angiotensin II-induced vascular remodeling and oxidative stress. Hypertension, 54: 142-149.