Effects of aging on liver microcirculatory function and sinusoidal phenotype

Abstract The socioeconomic and medical improvements of the last decades have led to a relevant increase in the median age of worldwide population. Although numerous studies described the impact of aging in different organs and the systemic vasculature, relatively little is known about liver function and hepatic microcirculatory status in the elderly. In this study, we aimed at characterizing the phenotype of the aged liver in a rat model of healthy aging, particularly focusing on the microcirculatory function and the molecular status of each hepatic cell type in the sinusoid. Moreover, major findings of the study were validated in young and aged human livers. Our results demonstrate that healthy aging is associated with hepatic and sinusoidal dysfunction, with elevated hepatic vascular resistance and increased portal pressure. Underlying mechanisms of such hemodynamic disturbances included typical molecular changes in the cells of the hepatic sinusoid and deterioration in hepatocyte function. In a specific manner, liver sinusoidal endothelial cells presented a dysfunctional phenotype with diminished vasodilators synthesis, hepatic macrophages exhibited a proinflammatory state, while hepatic stellate cells spontaneously displayed an activated profile. In an important way, major changes in sinusoidal markers were confirmed in livers from aged humans. In conclusion, our study demonstrates for the first time that aging is accompanied by significant liver sinusoidal deregulation suggesting enhanced sinusoidal vulnerability to chronic or acute injuries.

demonstrates for the first time that aging is accompanied by significant liver sinusoidal deregulation suggesting enhanced sinusoidal vulnerability to chronic or acute injuries.

| INTRODUCTION
Societies in developed countries are getting older due to the increase in life expectancy. Nowadays, 14% of European citizens are aged over 65, and by 2030, they are expected to constitute 23% (He, Goodkind, & Kowal, 2016). Therefore, the pace of population aging represents an important healthcare and social issue and it is therefore essential to understand the molecular basis of aging to identify possible approaches for therapeutic intervention.
The liver plays essential roles in metabolism, toxicants clearance, regulation of inflammation, and molecule biosynthesis. To fulfill these complex tasks, it requires an adequate microcirculation and a correct coordinated function of all hepatic cell types (Arias et al., 2009).
Although it is well known that hepatocytes constitute the main cell type contributing to the metabolic and synthetic capacities of the liver, their function deeply depends on an efficient exchange of substances with the blood stream and a proper communication with other hepatic cells.
Liver sinusoidal endothelial cells (LSEC), hepatic stellate cells (HSC), and Kupffer cells (KC) are the major components of the hepatic sinusoid, which collaborate to maintain the integrity and functionality of the unique liver microcirculatory system (Marrone, Shah, & Gracia-Sancho, 2016). LSEC are a very specialized fenestrated endothelial cell forming the capillary bed of the sinusoids, being separated from hepatocytes through the space of Disse. HSC are vitamin A storing cells located in the space of Disse, surrounding LSEC, and represent the liver pericytes. At last, KC, the resident macrophages of the liver, are located at the luminal side of the endothelial lining Friedman, 2008;Ju & Tacke, 2016).
Previous studies characterized single variables in the liver during aging and showed reduction in liver mass and hepatic blood flow, partial loss of endothelial fenestration, and possibly activation of HSC (Le Couteur et al., 2001;Vollmar, Pradarutti, Richter, & Menger, 2002). Nevertheless, studies analyzing in depth the hepatic sinusoid, its cellular components, and the hepatic microcirculatory function in aging are limited.
This study aimed at comprehensively characterizing the phenotype of the sinusoid in aged liver using a preclinical rat model of healthy aging, particularly focusing on the microcirculatory function and the molecular profile of each major liver cell type. In addition, our study further compared these findings to the key cellular modifications related to aging in livers from young and aged humans.
2 | RESULTS 2.1 | Aged rat model: Baseline characteristics and biochemical parameters As shown in Table 1, aged rats (20 months old) presented significantly increased body and liver weight compared to 3-month young rats, however, liver-body weight ratio was moderately diminished in old rats. In addition, elderly animals exhibited certain decline in liver function as suggested by reductions in albumin levels and bile production, although no significant differences in transaminases (as a marker of liver cell injury) were observed. Evaluation of plasma lipids revealed an increase in cholesterol and LDL cholesterol, without significant changes in HDL cholesterol, triglycerides, and free fatty acids. Moreover, aged rats had higher hepatic lipid accumulation as compared to young rats by oil red O staining. Hepatic malondialdehyde (MDA) in old rats was elevated, suggesting an increase in oxidative stress secondary to lipid peroxidation process.
As expected, analysis of senescence markers exhibited differences between young and old animals (Supporting Information Figure S1). P16, a protein involved in cell cycle regulation, was up-regulated in liver tissue and in LSEC freshly isolated from aged rats. SIRT1, an enzyme related with longevity, was decreased in the aged liver. At last, we also observed hepatic telomere length attrition in old animals.

| Aged animals present mild hepatic microcirculatory dysfunction
When compared to young animals, old rats exhibited significantly higher hepatic vascular resistance (HVR) in vivo, associated with a reduction in liver perfusion, altogether leading to a moderately increased portal pressure (PP), without meeting criteria for portal hypertension (Figure 1).
Mean arterial pressure was also significantly elevated, with no significant changes in heart rate (308 ± 9 vs. 319 ± 13 bpm).
Ex vivo perfusion experiments analyzing the liver endothelial-dependent vasodilatory capacity showed no significant differences between groups (data not shown).
Mechanisms responsible of the observed age-associated increased HVR were investigated in succeeding experiments.

| Aging is associated with mild hepatocyte injury and dysfunction
Liver architecture was overall preserved in aged animals (Supporting Information Figure S2); however, evaluation of the severity of hepatic injury using a histological damage score revealed mild liver dysfunction in aged rats in comparison with young animals (Supporting Information Table S1). Indeed, injury analysis showed differences between groups in all of the evaluated parameters including cytoplasmic vacuolation, nuclear pyknosis, cytoplasmic hypereosinophilia, loss of intercellular borders, necrosis, neutrophil infiltration, and fat accumulation. Ultrastructural analysis of liver damage using transmission electron microscopy (TEM) evidenced a decrease in the number of sinusoids with no changes in other parameters. In addition, aged rats showed higher hepatic cell death as demonstrated by the TUNEL staining (Supporting Information Figure S3a), with no significant changes in apoptotic proteins c-caspase-3 and BAD (data not shown).
Liver tissue analyses were complemented examining the phenotype of hepatocytes freshly isolated from both groups of animals (Supporting Information Figure S3b-d). In agreement with the in vivo biochemical parameters, aged hepatocytes exhibited slight but not significant lower urea and albumin synthetic capacity. This was associated with deregulation in different specific markers including Oct1 and Mrp3, with no differences in Mrp2 or HNF4α. At last, hepatocyte cytochrome P4503A4 activity tended to be higher in aged animals.
2.4 | The aged hepatic endothelium is pseudocapillarized and procontractile Analysis of the liver sinusoid using scanning electron microscopy confirmed pseudocapillarization of aged LSEC in this experimental model of aging. As seen in Figure 2a, fenestrae porosity was markedly diminished in the aged hepatic sinusoid. In accordance, aged livers exhibited reduced expression of VEGFR2, a protein involved in fenestrae formation and maintenance, and CD32b, a well-established marker of LSEC differentiation (Figure 2b,c).
Intrahepatic neovascularization analysis did not show differences between groups (Figure 2d). Nevertheless, the number of sinusoids seemed to be decreased in aged livers (Figure 2e), which correlates with the reduced CD32b expression described above.
Detrimental effects of aging on LSEC phenotype were not lim- A trend for increased HSC in aged livers was supported by incremented desmin protein expression and significant increase in the proliferative HSC-related growth factor PDGFRβ. In addition, aging was associated with slight, but significant, activation of HSC as demonstrated by increments in the mRNA and protein expression of different activation markers including α-SMA, collagen1α1, collagen1α2, PDGFRβ, and p-moesin, together with changes in some matrix remodeling genes including TIMP-2 and MMP9. We observed a moderate but not significant increase in cellular and mitochondrial superoxide levels in aged HSC in comparison with young HSC. In an interesting manner, analysis of vitamin A metabolism pathways showed alterations in aged livers; indeed, PNPLA3 expression seemed to be increased while CRBP-1 protein levels were diminished.

| Bacterial translocation and intestinal inflammation are not affected in elderly rats
In comparison with young rats, aged animals did not present discrepancies in gut bacterial translocation to mesenteric lymph nodes, feces bacterial load, nor in the expression of the cytokines IFN-γ, TNFα, and IL17α measured in the ileum (data not shown). In contrast, a minor increase in LPS plasmatic levels was observed in aged rats (Table 1).

| The aged human liver displays similar features of a dysfunctional sinusoid
Characterization of the liver sinusoid in young and old human tissues corroborated most of the molecular changes observed in the animal model ( Figure 6). Indeed, aged livers exhibited features of LSEC dedifferentiation as suggested by significant reductions in angiocrine and vasodilatory gene expression levels, together with partial activation of HSC (Supporting Information Figure S4). As expected, lipid metabolism and senescence pathways were increased in aged patients.

| DISCUSSION
The socioeconomic and medical care improvements occurring during the last decades have led to a relevant increase in the elderly, especially in developed countries (He et al., 2016). Therefore, it is critical to understand the molecular basis of aging and to identify possible approaches for therapeutic intervention in case major abnormalities In the present study, we demonstrate for the first time that healthy aged rats exhibit a significant increase in the hepatic vascular resistance, which leads to reduced liver perfusion and to a mo-derate increment in portal pressure. Although a reduction in portal blood inflow was previously reported in humans and rodents (Vollmar et al., 2002), we herein describe a deregulation in the intra-hepatic vascular system due to diverse molecular changes in the cells of the hepatic sinusoid, together with deterioration in hepatocyte function.
Analysis of hepatocytes revealed mild liver dysfunction in aged animals, which could likely be a consequence of altered oxygen and nutrient supply following the alterations in the vascular system. We  (Iwakiri & Kim, 2015), and maintaining neighboring cells phenotype (Marrone et al., 2016;Xie et al., 2013). Reduced nitric oxide bioavailability might, at least in part, derive from diminished eNOS activity, which may be due to reduced VEGF-p-eNOS pathway (Kroll & Waltenberger, 1998), and from increased scavenging due to elevated oxidative stress (Gracia-Sancho et al., 2008). An additional mechanism contributing to VEGF-p-eNOS pathway depletion may be the increase in the senescence marker p16, which exerts an inhibitory effect on VEGF (Kawagishi et al., 2010). Nevertheless, a previous study reported no changes in hepatic VEGF expression in aging (Le . Therefore, this issue would require further investigation to confirm the current findings. Similar to nitric oxide, the observed diminution in HO-1 expression in the aged endothelium may contribute to the increased HVR (Van Landeghem et al., 2009). Altogether our results strongly suggest that vasodilatory pathways in LSEC are profoundly affected by age and this might be one of the mechanisms responsible of aged-associated increase in HVR.
A previous report described increased number of fatty HSC in the aged liver (Warren et al., 2011); however, no comprehensive analysis of stellate cells phenotype in aging has been conducted.
Herein, we confirm lipid accumulation within aged HSC and additionally describe increments in other mediators of HSC activation including intracellular oxidative stress and p16 (Bataller et al., 2003).
In fact, analysis of cellular phenotype revealed a slight, but significant, activation of HSC, supported by increments in different activation markers including α-SMA or collagen I. Underlying mechanisms explaining HSC activation in aging revealed no modification in different proinflammatory mediators including TLR4, NFκB, and TGFβ (data not shown), but interestingly suggested alterations in retinoid F I G U R E 3 LSEC phenotype in aged livers. The phenotype of LSEC freshly isolated from aged and young rats was characterized as follows.  (Bruschi et al., 2017;Uchio et al., 2002).
Very few data regarding inflammation and KC phenotype in aging are available, and the data are inconclusive. For instance, while one study reported that KC lose their phagocytic capacity with aging (Brouwer & Knook, 1983), a subsequent study reported increased phagocytic activity in KC from aged animals (Hilmer, Cogger, & Le Couteur, D. G., 2007). In our evaluation, we noticed an increase in the recruitment of proinflammatory cells with a concomitant increment in inflammatory markers, altogether suggesting that aging may analyses revealed no significant differences in most of these parameters when comparing both groups of rats, except for LPS plasmatic levels that were increased in aged rats. We therefore cannot discard that the observed minimal aged-related endotoxemia may contribute to the abnormalities of sinusoidal cells.
At last, and although an undeniable degree of heterogeneity was found in each group analyzed, characterization of liver biopsies from healthy young and old humans confirmed the overall trend of pathway deregulation, therefore suggesting that sinusoidal vulnerability hereby described in old rats is also of relevance in human aging.
Many efforts are currently being directed toward the development of novel therapeutic approaches for portal hypertension . Most preclinical studies on the effects of different substances and drugs on the liver circulation have been conducted in young animals. Our current work revealed striking differences between young and aged liver, including alterations in vasoactive pathways, indicating that studies performed in young animals may not entirely reflect what should be expected from the cli-nical use of most interventions, as vasoactive drugs are and will be mainly prescribed in the elderly.
We are aware that our study is descriptive in nature. However, this work represents an important cornerstone characterization of liver aging. While single variables have been examined previously, no preceding report fully described the liver microcirculatory phenotype in a preclinical model of healthy aging, additionally including validation in human samples. These data provide important evidence for the changing biology of the liver in aging and create new research avenues to comprehend the effects of acute or chronic liver injury in the elderly. It is important to acknowledge that aged animals exhibited some degree of steatosis; therefore, part of the results could be attributable to intracellular fat instead of age. Nevertheless, a F I G U R E 6 Aged-related changes in the human liver. Gene expression analysis in healthy young and old human livers. Data expressed as fold enrichment (log 2 ) old relative to young (0.0). The fold enrichments are plotted using positive values for transcripts that are increased or using negative values for transcripts that are decreased in old humans. *p-value <0.05, n = 14 young and 13 old livers. Clinical characteristics of donors are described in Supporting Information Table S2 subanalysis of animals without evidence of significant steatosis confirmed the same hemodynamic and molecular results than in the group of animals exhibiting steatosis (data not shown), therefore ensuring that the results of this study describe the effects of aging per se on liver microcirculatory phenotype.
In conclusion, the present study demonstrates for the first time that aging is accompanied by significant liver sinusoidal deregulation, both in rodents and humans, suggesting sinusoidal vulnerability in front of subsequent chronic or acute injuries.

| EXPERIMENTAL PROCED URES
A complete description of Materials and Methods can be found in Appendix S1.

| Animal model
Male Wistar rats at the age of 20 months old were used to evaluate aging and compared to young animals of 3 months old (n = 12 animals per group) (Steppan et al., 2014;Wang, Wehling-Henricks, Samengo, & Tidball, 2015). Animals were kept in environmentally controlled animal facilities at IDIBAPS. All procedures were approved by the Laboratory

| In vivo hemodynamic measurements
Mean arterial pressure (MAP), portal pressure (PP), and portal blood flow (PBF) were measured in old and young rats using microcatheters and flow probes (Marrone et al., 2015). Hepatic vascular resistance (HVR) was calculated as PP/PBF.

| Electron microscopy
Liver sinusoidal ultrastructure was characterized using electron microscopy as previously described (Le Couteur et al., 2001).

| Statistical analysis
Statistical analysis was performed with the SPSS for Windows statistical package (IBM, Armonk, New York, USA). All results are expressed as mean ± standard error of the mean (SEM). Comparisons between groups were performed with Student's t test. Differences were considered significant at a p value <0.05.

ACKNOWLEDGMENTS
This study was carried out at the Esther Koplowitz Center-IDI-BAPS. Authors are indebted with Sergi Guixé, María Úbeda, Javier Muñoz, and Juanjo Lozano for their assistance and critical discussions during the development of this study, and with Héctor García, Montse Monclús, and the Electron Microscopy Unit (TEM/SEM) CCi-TUB for technical assistance.

CONF LICT OF I NTEREST
Authors have no conflict of interest.