Most organs (80%) used for liver transplantation (LT) are from brain-dead (BD) donors.1,2 Deceased donors may show steatosis, estimated in up to 30% of grafts.3,4 Both BD and steatosis are LT risk factors. BD produces an inflammatory response in liver grafts that affect the quality and function of the organ in the posttransplant period.5 Steatotic livers are more prone to damage caused by ischemia/reperfusion, resulting in an acute inflammatory response that can lead to significant tissue damage and organ dysfunction.5 Thus, many steatotic livers are discarded, exacerbating the critical shortage of liver donors.6
Clinical studies suggest that endocrine abnormalities in BD donors include a rapid decrease in hormones, such as growth hormone (GH), in the circulation.7,8 GH is released from the anterior pituitary gland, circulates in plasma, and binds to its receptors in the liver.9 Its availability is modulated by 2 hypothalamic hormones: GH-releasing hormone (GHRH), which stimulates both the synthesis and secretion of GH, and somatostatin, which inhibits GH release.9,10 In contrast to somatostatin, ghrelin, which is synthesized mainly in the stomach and brain, exerts a powerful effect on GH secretion.10,11
GH replacement treatment protects against inflammatory response in different pathologies including cardiovascular diseases.12 In addition to GH, considerable evidence suggests that epidermal growth factor (EGF) may be one of the key factors that reduce inflammation and initiate hepatocyte proliferation.13
In the present study, we aimed to elucidate the relevance of potential deficiencies in GH levels induced by BD in steatotic and nonsteatotic grafts used for LT, as well as evaluate any changes in the hormones regulating GH secretion and its effects on liver homeostasis. Given that (a) EGF modulates GH expression in different cell lines14 and (b) EGF and GH exert their effects on the same cell signaling pathways that are involved in cell proliferation and cell survival, that is, the phosphoinositide-3-kinase (PI3K)/protein kinase B (Akt) pathway,15,16 we aim to investigate the existence of a potential relation between EGF and GH in LT from BD donors as well as the underlying mechanisms of action.
MATERIALS AND METHODS
This study was performed using homozygous (obese [Ob]) and heterozygous (lean [Ln]) male Zucker rats (Iffa Credo, France) aged 10 to 11 weeks. Ob rats show moderate macrovesicular and microvesicular fatty infiltration in hepatocytes (40%–60% steatosis), whereas Ln rats show no evidence of steatosis.5 All procedures were approved by the Laboratory Animal Care and Use Committee of Barcelona University and by the Generalitat de Catalunya (DAAM 9353). European Union regulations (Directive 86/609 EEC) for animal experiments were respected.
Isoflurane anesthesia was used and rats were ventilated on a Harvard Rodent Ventilator 683 (Harvard Apparatus, MA) with a mixture of nitrous oxide and O2 for up to 30 minutes after BD induction (stroke rate of 60/min).5,17,18 Subsequently, all rats were ventilated with 30% O2 in air until the moment of termination. BD was induced using a standard animal method as previously described.5,17,18 Briefly, a frontolateral trepanation was performed in steatotic and nonsteatotic rats. A balloon catheter (Fogarty 14G; Baxter Health Care Corporation, CA) was introduced in the extradural space. The intracranial pressure was increased by inflating the balloon for 1 minute. The increase in intracranial pressure induced rapid brain injury leading to immediate BD, simulating a condition comparable to acute isolated cerebral trauma in humans. The BD state was confirmed 30 minutes after induction by the absence of corneal reflexes and the results of an apnea test. Normotension in all BD rats was obtained by colloid infusion (0.9% saline containing hydroxyethyl starch 5% and norepinephrine 0.01 mg/mL) through the tail vein at a maximum rate of 0.5 mL/min.5,17,18 A mean arterial pressure above 80 mm Hg was considered normotensive. The colloid infusion was started 15 minutes after BD induction, which allowed for a phase of hemodynamic stabilization after BD. The colloid saline volume used was similar in all groups in the study. Liver grafts were extracted from donors after 6 hours of normotensive BD.5,17,18
Surgical Procedure of LT
Liver grafts from BD or non-BD donors were flushed and stored in University of Wisconsin solution for 6 hours. Standard orthotopic LT in Ln Zucker rats was performed according to the cuff technique described by Kamada.19 The anhepatic phase was 17 to 20 minutes.5 The experimental groups, sample collection, and the biochemical and molecular analyses are described in Figure 1 and the Supplemental Material and Methods (SDC, http://links.lww.com/TP/B687), respectively.
Effects of Exogenous EGF-GH When These Drugs Are Administered Only in the BD Donor
Effect of EGF on GH Levels in Liver Grafts From BD Donors After LT
Reduced circulating GH levels and increased hepatic GH levels were observed in recipients of steatotic grafts from BD donors (BD + LT group) when compared with the LT group (Figure 2A). This was associated with reduction in hepatic EGF levels (Figure 2B). In recipients of nonsteatotic grafts from BD donors (BD + LT group), reduced circulating GH levels and no changes in either hepatic GH or EGF levels were observed. Interestingly, EGF treatment (BD + EGFD + LT group) reduced circulating and hepatic GH levels in recipients of steatotic grafts from BD donors compared with the BD + LT group (Figure 2A). In contrast, exogenous EGF increased both the circulating and hepatic GH levels in recipients of nonsteatotic grafts from BD donors when compared with the BD + LT group. EGF treatment (BD + EGFD + LT group) increased circulating levels of somatostatin only in recipients of steatotic grafts, whereas increased ghrelin levels were only observed in recipients of nonsteatotic grafts. Plasma GHRH levels were unaffected in recipients of steatotic or nonsteatotic liver grafts from BD donors (BD + LT group) (Figure 2C).
Relevance of the Changes in GH Levels Induced by EGF in LT
EGF treatment (BD + EGFD + LT group) resulted in attenuation of the hepatic damage in recipients of steatotic livers, but induced injurious effects in recipients of nonsteatotic livers (Figure 3A). In recipients of steatotic grafts from BD donors, histological analysis showed severe, extensive, and confluent areas of coagulative necrosis in the steatotic liver (BD + LT group) (Figure 4). Importantly, treatment with EGF (BD + EGFD + LT group) decreased the extent and number of necrotic areas in the steatotic livers, since there were only moderate areas of coagulative necrosis with neutrophil infiltration. In recipients of nonsteatotic grafts, moderate and multifocal areas of coagulative necrosis and neutrophil infiltration were observed in nonsteatotic livers (BD + LT group). The extent and number of necrotic areas in the nonsteatotic livers of the BD + EGFD + LT group were increased when compared with the histological results of the BD + LT group.
EGF (BD + EGFD + LT group) induced benefits regarding hepatic regeneration in recipients of steatotic grafts, increasing the number of proliferate cell nuclear antigen (PCNA)-positive hepatocytes and cyclin D1 when compared with the BD+LT group (Figures 3B and 5). However, EGF (BD + EGFD + LT group) elicited deleterious effects regarding regeneration in recipients of nonsteatotic livers, with fewer PCNA-positive hepatocytes and lower levels of cyclin D1 in nonsteatotic liver compared with the BD + LT group.
In the absence of BD in the donors, recipients of steatotic and nonsteatotic grafts (LT group) had survival rates of 30% and 80%, respectively (Figure 3C). BD in the donors increased mortality in the recipients of steatotic and nonsteatotic liver grafts (BD + LT group), with survival rates of 20% and 50%, respectively. The administration of EGF (BD + EGFD + LT group) increased the survival rate of the recipients of steatotic liver grafts (70%) but decreased the survival rate of the recipients of nonsteatotic livers (20%) (Figure 3C).
We lastly evaluated whether GH exogenous may abrogate the beneficial effects induced by EGF. In terms of hepatic damage, GH administration (BD + GHD + LT group) increased hepatic GH levels in recipients of steatotic grafts (Figure 2A) and exacerbated hepatic damage when compared with the BD + LT group (Figures 3A and 4). This was associated with a reduction in the number of PCNA-positive hepatocytes (Figures 3B and 5) and deleterious effects on the survival rates of recipients (10% [1 in 10] survival rate) (Figure 3C). Moreover, the beneficial effects of EGF were abolished when GH was administered. Indeed, the administration of EGF together with GH (BD + EGFD + GHD + LT group) produced transaminase levels, damage score values, a percentage of PCNA-positive hepatocytes, and a survival rate that were similar to those of the BD + LT group in recipients of steatotic livers (Figures 3–5). GH administration (BD + GHD + LT group) exacerbated hepatic damage and regenerative failure in recipients of nonsteatotic livers and all the recipients died within the first day after LT.
Mechanisms of Action of EGF and GH in Steatotic Liver Grafts From BD Donors After LT
Hepatic protein levels of PI3K/Akt, suppressors of cytokine signaling 1 (SOCS1), and SOCS3 (Figure 6A and B) were lower in recipients of steatotic grafts from BD donors (BD + LT group) when compared with the LT group. Treatment with EGF (BD + EGFD + LT group) induced higher levels of PI3K and pAkt, SOCS1, and SOCS3 in steatotic livers than in those of the BD + LT group. This affected the inflammatory response since the malondialdehyde and myeloperoxidase levels as well as edema were reduced, whereas HMGB1 levels were increased when compared with the BD + LT group (Figure 6C). The administration of GH (BD + GHD + LT group), which resulted in low hepatic PI3K/Akt expression, reduced the hepatic expression of SOCS1 and SOCS3 proteins in recipients of steatotic grafts from BD donors when compared with the BD + LT group. This was also associated with reduced hepatic HMGB1 levels and increased edema as well as elevated malondialdehyde and myeloperoxidase levels.
Effect of EGF on GH Levels in Steatotic and Nonsteatotic Liver Grafts Before Retrieval From BD Donors
Our results showed that the changes in circulating and hepatic GH and EGF levels detected in recipients of steatotic or nonsteatotic grafts from BD donors (Figure 2) were also detected in steatotic and nonsteatotic BD donors after 6 hours of normotensive BD (BD group) (Figure S1, SDC, http://links.lww.com/TP/B687). In addition, the effects of exogenous EGF-GH detected in recipients of steatotic or nonsteatotic grafts (Figure 3) were also detected in steatotic and nonsteatotic BD donors after 6 hours of normotensive BD and immediately before retrieval of the liver grafts from the donors (Figure S1, SDC, http://links.lww.com/TP/B687). Thus, the administration of EGF alone (BD + EGFD) protected against hepatic damage in steatotic BD donors after 6 hours of normotensive BD, whereas the administration of GH alone or combined with EGF (BD + GHD and BD + EGFD + GHD groups) exacerbated hepatic damage. However, the administration of either EGF or GH in nonsteatotic BD donors increased damage in this type of liver (Figure S1, SDC, http://links.lww.com/TP/B687).
Effects of Exogenous EGF-GH When These Drugs Are Administered in the Recipient Alone and in Both the Donor and the Recipient
In our hands, the protection conferred by exogenous EGF in steatotic LT from BD donors, when EGF was administered in donors (BD + EGFD + LT group), was stronger than that obtained by EGF administration in the recipients of steatotic grafts (BD + EGFR + LT group). Indeed, our results indicated that, when EGF was administered only in the recipients of steatotic grafts (BD + EGFR + LT group), GH levels, hepatic damage, or regenerative failure associated with LT from BD donors were not affected (Figure S2, SDC, http://links.lww.com/TP/B687). In addition, in steatotic LT from BD donors, the administration of EGF in both donors and recipients (BD + EGFDR + LT group) resulted in similar levels of GH, hepatic damage, and regeneration to those observed when exogenous EGF was only administered in donors (BD + EGFD + LT group). The administration of exogenous GH in the donor (BD + GHD + LT group), recipient (BD + GHR + LT group), as well as in both the donor and the recipient (BD + GHDR + LT group) exacerbated hepatic damage and regenerative failure compared with the results of the BD + LT group (Figure S2, SDC, http://links.lww.com/TP/B687). In nonsteatotic LT from BD donors, the administration of EGF in both donors and recipients (BD + EGFDR + LT group) resulted in similar levels of GH, EGF, hepatic damage, and regeneration to those observed when exogenous EGF was only administered in donor (BD + EGFD + LT group). In addition, BD + GHDR + LT group resulted in GH, hepatic damage, and regeneration values similar to those of the BD + GHD + LT group. Furthermore, the administration of either EGF or GH only in the recipient (BD + EGFR + LT and BD + GHR + LT groups) induced injurious effects regarding damage and regeneration in recipients of nonsteatotic livers compared with the BD + LT group (Figure S2, SDC, http://links.lww.com/TP/B687).
P values for all statistical analysis regarding results shown in Figures 2–6 and Figures S1 and S2 (SDC, http://links.lww.com/TP/B687) are shown in Table S1 (SDC, http://links.lww.com/TP/B687).
Our study demonstrates the endocrine abnormalities in LT from BD donors that are specific for steatotic and nonsteatotic livers, including alterations in the levels of GH, EGF, and the hormones regulating GH secretion.
In line with clinical studies,8 our results indicated dysfunctions caused by BD in the hypothalamic-pituitary axis since BD by itself reduced circulating GH levels in steatotic and nonsteatotic LT. Unexpectedly, lower circulating GH levels did not translate into low hepatic GH levels since GH levels were increased or unaffected after BD induction in steatotic and nonsteatotic livers, respectively. Thus, whereas nonsteatotic livers maintain local GH availability under BD conditions by limiting hepatic GH exposure to avoid its deleterious effects, GH is accumulated in steatotic liver grafts. This GH accumulation in steatotic grafts might be explained, at least partly, by an increase in GH uptake from the circulation. In addition, differences in GH clearance and/or regulation between steatotic and nonsteatotic LT should not be ruled out. According to previous data,20 the pathological factors that regulate, suppress, or stimulate GH release might differ between obese and healthy subjects. Indeed, we observed changes induced by EGF in the circulating levels of GH-regulating hormones, such as somatostatin and ghrelin, that were dependent on the type of donor liver.
The goal of GH replacement therapy is to increase height in children with GH deficiency and to treat adult GH deficiency syndrome, which is characterized by an increased risk of cardiovascular disease and perturbations in body composition and bone mineral density.21 Currently, guidelines approved by both Endocrine Society and the American Association of Clinical Endocrinologists22,23 on GH replacement therapies for these pathologies do not focus on the routine evaluation of the effect of exogenous GH on the liver. In the present experimental study, we report a clear deleterious effect of exogenous GH on steatotic and nonsteatotic LT from BD donors. Thus, although treatment with GH might appear to be generally safe in the above-mentioned pathologies, in our view its deleterious effect on the liver should be considered and certain requirements should be met, such as careful monitoring of hepatic function.
The adverse effects of GH, especially on nonsteatotic LT, were not related to the dose used herein, that is, 1.5 mg/kg. In fact, we used a notably low dose of GH, as indicated by the fact that circulating GH levels were similar to those of the obese Sham group. Our aim was to restore the BD-induced decrease in circulating GH levels to investigate its effects on LT. Indeed, our preliminary results indicate that lower doses fail to reach the circulating levels found in Sham animals. Furthermore, the dose used herein (1.5 mg/kg) has been reported to be beneficial in different pathologies.24 Moreover, the adverse effects of GH were observed only at the dose of 5 mg/kg.25 The administration of exogenous GH does not mimic the endogenous pulsatile pattern of hormone secretion.26 However, our findings (not presented) indicated that electrical stimulation, which physiologically simulates GH secretion, produced hepatic damage, proliferation, and lethality similar to that observed after exogenous GH administration.
To explain why nonsteatotic grafts were more vulnerable to the deleterious effects of GH, the following should be considered: (a) tolerance to the same GH dose is known to vary among patients21; (b) GH therapy can interfere with other pituitary deficits12 and that the dysfunctions in the hypothalamic-pituitary axis induced by BD might be specific for each type of liver, as reported in the current study; and (c) adipose tissue in obese subjects has a higher density of GH receptors27 that might sequester more GH from the circulation, consequently reducing the liver uptake of GH and counteracting its adverse effects.
EGF-mediated changes in GH levels have been described in isolated cells.14 GH is released from the anterior pituitary gland, circulates in plasma, and binds to its receptors in the liver.9 On the basis of these observations, we propose that EGF might exert its effects on the hypothalamic-pituitary axis and consequently induce changes in circulating and hepatic GH levels. Several studies have shown a direct correlation between EGF administration and increased somatostatin release.28 The inhibitory role of somatostatin in GH secretion has been widely reported.29 EGF treatment increased somatostatin levels in steatotic LT from BD donors, which would explain, at least partly, the reduction in GH levels. The inhibitory effect of somatostatin is enacted by 3 mechanisms: (a) antagonism of the effect of GHRH through membrane hyperpolarization, thereby inhibiting GHRH-mediated GH release30; (b) reductions in GH secretion30; and (c) reductions in GHRH mRNA expression.31 As we did not observe any changes in GHRH levels, lower GHRH mRNA expression is unlikely to have contributed to the effects of EGF on GH levels. However, we should not rule out the direct suppression of GH by somatostatin, the antagonistic effect of somatostatin on GHRH, or even the combination of both in steatotic LT from BD donors.
The increased circulating levels of ghrelin after EGF administration might partly explain the increases in GH levels observed in nonsteatotic LT. This is consistent with several studies that have proposed a correlation between EGF and ghrelin levels.32,33 Ghrelin elicits GH secretion by directly targeting the pituitary and modulating GHRH and somatostatin levels in the hypothalamus.34,35 In nonsteatotic LT from BD donors, GHRH and somatostatin were not altered by EGF administration, suggesting that ghrelin directly acts on GH release.
From a therapeutic perspective, we herein demonstrate that EGF pretreatment in steatotic BD donors counteracts the detrimental effects of BD in this liver type, possibly by decreasing GH levels and consequently protecting against inflammation and damage, increasing the survival rates of the recipients. Both EGF and GH induce changes in the protein expression of SOCS in a PI3K/Akt-dependent manner in different cell types.36–38 Endogenous SOCS proteins are key regulators of the inflammatory response in different liver diseases and are essential for maintaining normal cellular homoeostasis.37–39 Herein, we show that an EGF-induced reduction in GH levels upregulated the expression of PI3K-Akt, which promotes cell survival in steatotic grafts. This activation was associated with increased hepatic SOCS1 and SOCS3 levels, a decrease in neutrophil accumulation, oxidative stress, and edema, and regulation of HMGB1 levels. It is well known that the loss of HMGB1 in liver cells elicits an exaggerated inflammatory response and damage.40 However, these benefits of EGF were not observed in nonsteatotic LT from BD donors, since EGF administration increased GH levels and was associated with inflammation, hepatic damage, and lower survival rates.
Interestingly, we report that the injurious effects of BD as well as the effects of EGF-GH in recipients of steatotic or nonsteatotic grafts from BD donors were initiated in the BD donors. Of clinical interest, methods to diminish the detrimental effects of BD before liver grafts are retrieved from BD donors may be needed to promote better preservation of the organ and to improve outcome after LT. In our experimental conditions (steatotic liver grafts maintained in the donor after BD induction for 6h before their retrieval from the donor), we show that EGF treatment in donors reduced the adverse effects of BD and improved the quality of steatotic liver grafts before their retrieval from the donors, as well as the postoperative outcomes and the survival rate of recipients after LT from BD donors. Indeed, our experimental results indicate that the most convenient strategy to reduce the damage due to BD in LT would be to administer exogenous EGF only in the donor.
Clinically, liver donors are often kept in the intensive care unit for longer periods than 6 hours after diagnosis of BD, a period during which inflammatory alterations occur, overall negatively influencing the organ quality for LT.17 In line with suggestions made by Van der Hoeven,17,41 in our view, the time frame between the declaration of BD and organ retrieval provides an important window for cytoprotective intervention, which may counteract the detrimental effects of BD. The results of the current study and further investigations might help in the development of novel and useful strategies in LT from deceased donors. We acknowledge potential limitations of the present study including small numbers of animals per group. Nevertheless, in the experimental design, sample size was calculated to determine the number of rats required to achieve statistical significance with a power of 90% and alpha level of 0.05, bearing in mind to maintain the lower number of animals to adhere to the 3Rs policy. In addition, the use of hydroxyethyl starches to maintain the normotension in BD rats is contraindicated in organ donors because they are associated with acute kidney injury and early graft dysfunction in the transplanted kidneys42,43; therefore, further investigations will be required to evaluate its effects on liver grafts.
In conclusion, the results presented herein indicate that in the setting of experimental LT from BD donors, the EGF-GH molecular axis plays a key role in LT-related pathophysiology (Figure 7). We show the effectiveness of EGF treatment in steatotic LT, whereas its use in nonsteatotic livers had adverse effects on posttransplant outcomes.
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