- Top of page
- Experimental Procedures
- Supporting Information
Partial liver transplantation represents an important potential means to expand the donor pool and increase the applicability of liver transplantation as the sole treatment for end-stage liver disease. Nevertheless, a major obstacle preventing broader use of partial grafts, including splitting a liver for two adults, is the fact that it may result in insufficient hepatic mass for the recipient, leading to the development of small-for-size syndrome (SFSS). SFSS is diagnosed clinically when cholestasis, coagulopathy, encephalopathy, ascites, gastrointestinal bleeding and/or renal failure arise in a liver representing <0.8% of the recipient's body weight or <40% of the recipient's standard liver volume (SLV), at the exclusion of another cause [1, 2]. The clinical presentation of SFSS is typically subacute; when it does occur, however, SFSS leads to graft failure within 2–3 months in up to 50% of recipients .
The pathogenesis of SFSS has been attributed to not only small functional liver mass but also excessive portal inflow and insufficient venous outflow and intragraft responses . We studied the acute phase of small-for-size (SFS) injury in a porcine model of 20% liver transplantation and found that portal vein flow (PVF) approximately fourfold higher than baseline and a significantly elevated portal venous pressure gradient (PVPG) were present upon reperfusion. These hemodynamic alterations caused direct mechanical injury to the sinusoidal endothelial cells (SEC) and were the chief stimulus for a subsequent series of intragraft events leading to acute failure . Using the same model, we demonstrated that a portocaval shunt (PCS) placed prior to reperfusion and calibrated to maintain twice-baseline PVF decompressed the splanchnic bed, prevented SEC injury and resulted in excellent recipient survival .
Though a calibrated PCS is effective at preventing acute SFS injury, it is a relatively permanent solution for a temporary problem, as the critical period of portal hyperperfusion-induced injury is limited . Hence, it is of interest to develop reversible means for reducing PVF in the immediate postreperfusion period while the partial graft is still adapting to the altered physiology of the recipient's body. Pharmacological portal inflow modification with short-acting drugs, such as somatostatin, is one such option.
Somatostatin is a naturally occurring tetradecapeptide that decreases portal venous pressure (PVP) and splanchnic blood flow in a dose-dependent manner [7, 8]. It induces vasoconstriction in the mesenteric arteries and portocollateral veins [9, 10] and inhibits the secretion of gut-derived vasodilatory peptides, such as glucagon, vasoactive intestinal peptide and substance P [11-13]. While the effects of somatostatin on the portomesenteric vasculature are well established, less is known about the role that somatostatin plays locally in the hepatic microvasculature, in particular among transplanted livers in the acute phase of injury. In the normal innervated liver, somatostatin-containing axons are present in the space of Disse and exert paracrine effects on nearby cells, depending on the expression of the five somatostatin receptor (SSTR) subtypes . Receptor expression is limited to cholangiocytes and a small number of endothelial cells lining the hepatic arterioles in the normal liver; in the cirrhotic liver, on the other hand, both hepatocytes and hepatic stellate cells (HSC) may express all five subtypes [15-17], indicating potential sites of action in chronic liver disease.
The hypothesis of the present work is that somatostatin is capable of reducing PVF in SFS liver transplantation, indirectly leading to less SEC injury and endothelin-1 (ET-1)-mediated activation of HSC and improved graft survival. Additionally, we hypothesize that somatostatin may have direct protective effects in certain key cells in the acute phase of injury in the partial liver, as well. To test these hypotheses, we perform a porcine model of 20% liver transplantation in which somatostatin-treated recipients are compared with untreated controls.
- Top of page
- Experimental Procedures
- Supporting Information
The porcine model is the most relevant means available to study SFSS outside the clinical setting. While rat and mouse models require relatively little material, personnel and space to perform, it is hard to perform hemodynamic studies in this context. Arterialization of the liver is difficult and often omitted in the case of rats, and the cuffed portal vein anastomosis may artificially decrease PVF [23-25]. Such a finding is not in any way representative of the clinical situation with partial liver transplantation and raises concern as to whether such a small-cuffed anastomosis truly allows unimpeded laminar flow through the portal vein. Large animal models, on the other hand, offer size and anatomy similar to humans. Vascular anastomoses may be performed end-to-end, as they are clinically, and are significantly less prone to artificial narrowing.
Using our porcine SFS liver transplant model, our group previously demonstrated that postreperfusion PVF increases considerably (fourfold with respect to baseline) in extremely small grafts . Herein, we demonstrate that somatostatin administered prior to and after reperfusion can significantly reduce this flow, improving posttransplantation outcomes. By decreasing portal flow and pressure, somatostatin has an indirect protective effect on the partial liver. However, the reduction in PVF and the PVPG produced by somatostatin was not as immediate or as important as that we achieved with calibrated PCS in our previous study, so we looked for other means by which somatostatin was able to offer hepatoprotection in this setting. For this reason, we performed in vitro studies, which demonstrated that somatostatin acts directly on and reduces the activation of HSC, as well. We also determined for the first time the effect of somatostatin on isolated, phenotypically injured SEC.
Another group looked at the effects of somatostatin on activated HSC in vitro and found significant decreases in α-SMA gene expression and de novo protein synthesis but no differences in the cellular contractile apparatus or overall levels of α-SMA protein . When we re-dosed somatostatin every 6 h, as was done in the previous study, we, too, observed no differences in α-SMA protein expression versus vehicle. Only when somatostatin was given 1 h before HSC were lysed for protein extraction did we observe a significant decrease in α-SMA protein expression, regardless of the overall duration of incubation. This is an important finding that supports the results of our porcine studies by confirming the fact that the effect of somatostatin on the HSC contractile apparatus is immediate and offers further proof that somatostatin may play a key role in overcoming early SFS liver injury through a direct hepatoprotective mechanism, independent of the reduction in PVF.
In addition to its direct effect on HSC, we have been able to demonstrate a protective effect for SEC in grafts treated with somatostatin in vivo, as reflected by decreased immunostaining and circulating levels of ET-1. Other authors have similarly observed down-regulated expression of ET-1 in partial grafts treated with somatostatin in both rodents and humans, although they did not investigate the specific mechanism through which this down-regulation occurred [27, 28]. Given the results of the in vitro studies in which we were unable to demonstrate a reduction in ET-1 expression in phenotypically injured SEC incubated with somatostatin, both in isolation and co-cultured with similarly treated HSC, it appears that the reduction in ET-1 expression in vivo is an effect of the splanchnic vasoconstriction and decrease in PVF. That is to say, there does not appear to be any direct protective effect of somatostatin on SEC. No other studies have looked at the direct effects of somatostatin on hepatic SEC in culture, so there is no other data with which to compare our results.
Postoperatively, biochemical parameters were improved in somatostatin-treated animals and tended to be the best among those receiving 15 μg/kg/h, indicating that liver failure was not the cause of the death among these animals. An effect of the learning curve was not to blame, either, as our group had extensive experience in this model when this study was undertaken. The lower dose of somatostatin we used was chosen based on how the drug is typically administered clinically in the context of bleeding esophageal varices: an initial 250–500 μg bolus (the equivalent of about 4–8 μg/kg, based on a 60–70 kg adult) followed by 250–500 μg/h over the course of 5 days . With respect to our choice of 15 μg/kg, we performed a preliminary set of experiments in anesthetized pigs and observed a more immediate response in portal hemodynamic parameters with an initial bolus of 15 µg/kg versus 8 μg/kg, although some slowing of the heart rate with a 15 μg/kg/h infusion was also observed. At standard doses somatostatin is considered to be a very safe drug [30, 31], but high doses may provoke bradypnea, bradycardia, hypotension and even apnea [32-35]. Thus, while it was effective at decreasing splanchnic blood flow and PVP and even led to some improvements over the more standard dose in terms of the hepatic biochemical profile, we believe that the high-dose somatostatin infusion we used in the SST-15 group may have had the adverse effect in this model of provoking terminal cardiorespiratory events.
Given the adverse effects of high-dose somatostatin, one might wonder whether it could potentially be administered through the portal vein or its venous tributaries and act on the liver directly while avoiding some of its systemic effects due to first-pass metabolism. Administration of the drug in this manner, however, means that it would have much less of a vasoconstrictive effect on mesenteric arterial vasculature, and the reduction in portal inflow would be less. Similarly, the administration of longer-acting somatostatin analogs such as octreotide and lanreotide, which are more resistant to endogenous peptidases, might be appealing. Native somatostatin and its analogs, however, have different affinities for the five SSTR subtypes: the former binds all five, while octreotide binds only SSTR2 and SSTR5 . Previous studies have shown that relaxation of cultured HSC is mediated by SSTR1 [15, 17, 37], indicating that octreotide would perhaps not offer a direct hepatoprotective effect in the setting of acute SFS injury.
Based on the fact that it lowers PVF and suppresses the release and action of several hepatotrophic factors, somatostatin is known to suppress hepatic regeneration [38-40]. While some authors argue that SFSS arises primarily due to the failure of a partial liver to regenerate [41, 42], other groups have made observations to the contrary. In the clinical setting, good restoration of hepatic size on computer tomographic volumetry is observed in patients who simultaneously demonstrate clinical signs of liver insufficiency [43, 44], and small grafts have been shown to have greater regenerative capacity compared with larger ones . In our own preclinical experience, we have consistently observed a significantly greater increase in mass and TK activity among failing SFS grafts versus those with initial good function, in particular those treated with measures to reduce PVF and suppress regeneration (e.g. an appropriately calibrated portocaval flow derivation or, in the present case, somatostatin) [4, 5].
Events occurring at the cellular level help explain why livers that regenerate rapidly might still be SFS. After major hepatic resection, entry into the cell cycle and DNA synthesis occur much earlier in hepatocytes versus nonparechymal cells. In the rat, DNA synthesis in hepatocytes begins to increase after 12 h and peaks at 24 h, whereas it begins around 48 h for Kupffer cells and cholangiocytes and 96 h for SEC [46, 47]. This order of events results in the formation of avascular clusters of 10–14 hepatocytes devoid of extracellular matrix, the so-called “hepatocyte islands” [48-50], which persist until SEC are replicated and the normal extracellular matrix restored. Consequently, reformation of the normal liver microarchitecture occurs only after the original mass has been replaced. Hepatocyte islands are considered less functional than normal single-cell-wide hepatocyte plates due to the fact that there are fewer sinusoids and bile canaliculi for each hepatocyte. Therefore, it follows that the smaller the graft, the greater is the proportion of hepatocytes entering the replicative process to form these islands, decreasing the amount of functional liver tissue available even as gross mass is restored . This helps explain why multiple studies, including our own, have reported that functional regeneration does not correlate with volumetric regeneration during the early stages of liver regrowth [4, 5, 51-54] and that strategies that decelerate regeneration can actually lead to better preservation of normal hepatic microarchitecture and improved survival during the regenerative process .
Based on the results of this study, we conclude that the perioperative administration of somatostatin in SFS liver transplantation offers a reversible means to reduce portal inflow and protect the cells lining the hepatic sinusoid in the critical postreperfusion period, thereby significantly improving posttransplantation outcomes. Additionally, somatostatin exerts a direct and immediate cytoprotective effect on HSC, independent of the reduction in PVF. Taken together, these findings indicate that somatostatin may play an important role in alleviating SFSS and increasing the applicability of partial liver transplantation in the clinical setting.