In this study, we demonstrate that atmospheric oxygen levels represent a deleterious environment for LSECs, and that different oxygen environments induce significant functional and structural changes in these cells. LSECs are known to be particularly vulnerable to variations of oxygen levels, as demonstrated by ischemia-reperfusion challenges of livers, or in anoxia-reoxygenation experiments in vitro . During the re-oxygenation periods LSECs, KCs and hepatocytes produce large amounts of oxygen radicals, and LSECs gradually go into apoptosis induced by oxidative stress . These findings suggest that LSECs are poorly equipped to adapt to hyperoxic conditions. It is therefore a curious fact that all published studies using cultured LSECs have been conducted with cells maintained under atmospheric or hyperoxic oxygen tension. We here demonstrate that LSECs cultured under moderately low oxygen tension exhibit improved survival and maintain their in vivo characteristics better as compared to LSECs cultured under atmospheric oxygen tension.
At physiologic conditions the ratio of portal vs. arterial blood flow entering the liver is around 4:1. In the rat system, most arterial blood reaches the sinusoids indirectly, via initial anastomosis between the terminal hepatic arteriole and the portal venule . In average, the oxygen content of hepatic blood is rather low (~55 mmHg). However, oxygen gradients normally exist between the periportal and the perivenous areas of the liver lobule, ranging from 60–70 mmHg O2 in the periportal area to 25–35 mmHg O2 in the perivenous areas . Some laboratories have developed complex bioreactor systems that allow the formation of steady state oxygen gradients in culture . Zonal heterogeneity with regard to the oxygen tension has been studied in hepatocyte cultures but not in cultured LSECs. Practically all published in vitro studies on LSECs are conducted in static culture systems where the levels of oxygen have not been an issue.
Many research groups use long-term cultures of LSECs to explore these cells [13, 14]. In spite of this fact, surprisingly few studies have focused on identifying changes induced on LSEC functions, morphology or viability during normal in vitro culture. Nevertheless, some laboratories have reported that signature LSEC functions such as endocytic capacity or fenestration are drastically reduced after 1 or 2 days in culture [4, 1]. Some research groups have attempted to develop protocols to extend the in vitro lifetime of functionally intact LSECs. Most strategies have used specially designed culture media, including tumour-conditioned medium , hepatocyte-conditioned medium , autologous rat serum , phorbol ester supplemented medium, vascular endothelial growth factor-containing medium  or orthovanadate . In the present study we used a serum-free medium developed previously in our laboratory . With this medium, we were able to maintain fuctionally intact rat LSEC cultures during 48 h. However, from the third day of incubation, the cultures gradually lost their integrity. This loss was nevertheless prevented by incubation of cultures under low oxygen tension, enabling maintenance of intact morphology after six days of culture. This demonstrates that atmospheric oxygen levels itself is a disfavourable environment for LSECs.
Several reports have shown that a major biological function of LSECs is to rid the blood of an array of naturally occurring soluble macromolecular and colloidal waste substances, via clathrin-mediated endocytosis (for review see ). Based on the knowledge that receptor-mediated endocytosis represents a characteristic function of LSECs, we compared the ability of the cells to internalize and degrade formaldehyde treated serum albumin (FSA), that is specifically taken up by the scavenger receptor of LSEC, in high and low oxygen environments. Although the endocytic capacity decreased gradually over time in both conditions, the total uptake measured at 24, 48 and 72 h was significantly higher under physiological oxygen conditions than under hyperoxic conditions. Yet, culturing of LSECs under low oxygen levels per se was not enough to maintain the endocytic capacity at the same level as measured in freshly prepared cultures. The reason for this is at least three-fold, based on the fact that LSEC monocultures lack: i) essential factors produced locally or brought to the cells via the portal circulation, ii) interaction with other liver cells, and/or iii) interaction with native extracellular matrix. Focusing in the present work on the impact of oxygen tension, we show here that a low oxygen level in vitro, approaching that of the local sinusoidal environment in vivo, significantly prolong the naturally high scavenger activity of LSECs when compare to traditional cultures established at atmospheric oxygen levels.
Another important physiological function carried out by LSECs in vivo is the filtration of numerous plasma components towards the liver parenchyma through a well organized net of transcytoplasmic holes called fenestrae. This fenestration represents a hallmark of intact mammalian LSEC. It is noteworthy that several reports conclude that the cells undergo a rapid defenestration after isolation and culture . Assessing fenestration using scanning electron microscopy, we found that this feature is gradually lost over time independent of the culture conditions used. Of note, LSECs lost fenestration more rapidly when seeded on fibronectin-coated dishes than on collagen-coated ones. This shows that the key factors which enable the maintenance of the LSEC fenestrae in the intact liver are lost upon cultivation, regardless the oxygen levels.
Adaptation to hypoxia is known to induce changes in the energy metabolic routes of cells, most commonly shifting from the oxidative phosphorylation pathways to the glycolytic routes. Morphometric studies of LSECs in the intact liver have shown that the cells contain unusually few mitochondria [19, 20]. This observation, along with the fact that rat LSECs produce large amounts of lactate and acetate, even when cultured at high oxygen levels, strongly suggest that these cells are geared to a largely anaerobic type of metabolism. Conceivingly, LSECs perform less oxidative phosphorylation compared to most other cells types, and it has been suggested that in LSECs, glutamine and fatty acid oxidation are the main sources of energy . An alternative path is the anaerobic conversion of pyruvate, originating from the catabolism of glucogenic aminoacids from the growth medium, into lactate . Examining glucose consumption and lactate production under different oxygen tensions to explore possible variations in the energy sources of the cells, we found that glucose was not consumed by LSECs under hyperoxic or normoxic conditions. This suggests that the energy metabolic routes are independent of the oxygen levels. In contrast, the amount of lactate generated by LSECs under normoxic conditions was enhanced almost three times, suggesting that metabolic energy reactions are driven more efficiently under low oxygen.
As a rule, procedures used to isolate and cultivate cells induce cell activation to some extent. Desirable in vitro models should be based on non-activated or low-activated cells. In the present study we measured the production of inflammatory cytokines, reactive oxygen species and the expression of adhesion molecules after cell cultivation as indicators of cell activation. Our results confirm that LSECs produce high levels of IL-6 when cultured under "standard" high (20%) oxygen pressure. Notably, the expression of this cytokine was reduced by 50% when the cells were maintained at low oxygen levels. In contrast, the production of IL-10, an anti-inflammatory mediator, was enhanced when the cells were incubated at 5% oxygen. Flow cytometric analysis of ICAM-1 expression at the cell surface show strong signal on LSECs cultured for 24 h at 20% oxygen. These values are however slightly reduced upon incubation of cells at low oxygen tensions. The overall results indicate that LSECs have a less activated phenotype when they are incubated at low oxygen levels.
The transfer of LSECs from an in vivo low oxygen tension to an in vitro high oxygen tension may exert effects on the cells similar to those observed in LSECs during hypoxia-reoxygenation of the intact liver. Indeed, we observed large production of hydrogen peroxide by LSECs when the cells were kept at atmospheric oxygen conditions. Of note, this production was much lower at low oxygen tension. Hydrogen peroxide induces toxic effects on LSECs, mostly because these cells are not well equipped to metabolize this reactive substance [8, 9]. Addition of catalase to the medium, an enzyme that mediates directly the catabolism of H2O2, was able to abrogate to a large extent the formation of H2O2, and had beneficial effects on the morphology and survival of LSEC cultures. We thus entertain the idea that endogenous H2O2 may be largely responsible for the rapid culture decline observed during high oxygen incubation.