Relevance of VEGFA in rat livers subjected to partial hepatectomy under ischemia-reperfusion
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We examined the effects of VEGFA on damage and regeneration in steatotic and non-steatotic livers of rats submitted to PH under I/R, and characterized the underlying mechanisms involved. Our results indicated that VEGFA levels were decreased in both steatotic and non-steatotic livers after surgery. The administration of VEGFA increased VEGFA levels in non-steatotic livers, reducing the incidence of post-operative complications following surgery through the VEGFR2-Wnt2 pathway, independently of Id1. Unexpectedly, administration of VEGFA notably reduced VEGFA levels in steatotic livers, exacerbating damage and regenerative failure. After exogenous administration of VEGFA in steatotic animals, circulating VEGFA is sequestered by the high circulating levels of sFlt1 released from adipose tissue. Under such conditions, VEGFA cannot reach the steatotic liver to exert its effects. Consequently, the concomitant administration of VEGFA and an antibody against sFlt1 was required to avoid binding of sFlt1 to VEGFA. This was associated with high VEGFA levels in steatotic livers and protection against damage and regenerative failure, plus improvement in the survival rate via up-regulation of PI3K/Akt independently of the Id1-Wnt2 pathway. The current study highlights the different effects and signaling pathways of VEGFA in liver surgery requiring PH and I/R based in the presence of steatosis.
VEGFA administration improves PH+I/R injury only in non-steatotic livers of Ln animals.
VEGFA benefits are exerted through the VEGFR2-Wnt2 pathway in non-steatotic livers.
In Ob rats, exogenous VEGFA is sequestered by circulating sFlt1, exacerbating liver damage.
Therapeutic combination of VEGFA and anti-sFlt1 is required to protect steatotic livers.
VEGFA+anti-sFlt1 treatment protects steatotic livers through a VEGFR2-PI3K/Akt pathway.
KeywordsIschemia-reperfusion Vascular endothelial growth factor A Soluble vascular endothelial growth factor receptor 1 Liver Steatosis Adipose tissue
In clinical situations, partial hepatectomy (PH) under ischemia/reperfusion (I/R) is a common strategy to control bleeding during parenchymal dissection . More than 20% of patients destined for liver resection present some degree of steatosis, a condition usually related to obesity [1, 2, 3], and the prevalence of steatosis is constantly increasing in society. Importantly, hepatic steatosis represents a major risk factor for liver surgery, being associated with high rates of complications and postoperative mortality after major liver resection [1, 4, 5].
A number of experimental studies on PH without I/R in steatotic and non-steatotic livers have shown that vascular endothelial growth factor A (VEGFA) levels are increased after surgery and that infusion of VEGFA can reduce injury and increase hepatocyte proliferation [6, 7, 8, 9]. Furthermore, protective effects on damage have been reported regarding the role of VEGFA in non-steatotic livers in experimental models of I/R without PH [10, 11].
A number of studies have shown that VEGF receptor-2 (VEGFR2) is the principal mediator of several physiological and pathological effects of VEGFA [12, 13, 14, 15, 16, 17]. It has also been shown that in non-steatotic livers undergoing PH without I/R, VEGFR2 activation is induced, initiating Id1 up-regulation and secretion of Wnt2 angiocrine factor . Recent studies have suggested an important role of Wnt2 signaling in the proliferative response in non-steatotic livers undergoing I/R without PH .
As mentioned above, the role of VEGFA has been evaluated in PH without I/R and in I/R without PH, mainly focusing on non-steatotic livers. Nevertheless, the effect of VEGFA on liver regeneration and damage in conditions of PH under I/R has not been investigated. This scenario is addressed in the present study since PH under I/R is commonly used in the clinical practice to control bleeding during parenchymal dissection. We postulated that the expression and role of VEGFA as well as the mechanisms by which VEGFA might affect damage or regeneration might differ depending on the hepatic surgical conditions as well as the presence or absence of steatosis in the liver submitted to surgery. Consequently, strategies aimed at protecting the liver during surgery might be specific for each surgical procedure and for steatotic and non-steatotic livers.
Herein we examined VEGFA levels in rat steatotic and non-steatotic livers undergoing PH under I/R. We also investigated whether modulating the actions of VEGFA could protect both steatotic and non-steatotic livers against damage and regenerative failure following surgery. Finally, we investigated whether the VEGFR2-Id1-Wnt2 pathway is involved in the underlying action mechanisms of VEGFA in both steatotic and non-steatotic livers in an experimental model of PH under vascular occlusion, a liver surgery setting of potential clinical and scientific interest. In our opinion, the use of experimental surgical models that resemble as much as possible the clinical conditions in which the strategy is intended to be applied will lead to the translation of those strategies to clinical practice in the short term.
Material and methods
Male homozygous obese (Ob) (400–450 g) and heterozygous lean (Ln) Zucker rats (350–400 g) and male Sprague Dawley (SD) and choline-deficient SD (CDD-SD) rats (350–380 g) were used. Ob Zucker and CDD-SD rats showed severe macrovesicular and microvesicular fatty infiltration in hepatocytes (60–70% steatosis) [19, 20].
Protocol 1. VEGFA impact and availability in Ln and Ob Zucker rats undergoing PH+I/R
Sham group (6 Ln and 6 Ob Zucker rats). Hepatic hilar vessels of animals were dissected.
PH+I/R+VEGFA group (6 Ln and 6 Ob Zucker rats). As in group 2, but treated with VEGFA (5 μg/kg i.v.) .
PH+I/R+VEGFA+anti-sFlt1 group (6 Ob Zucker rats). As in group 3, but treated with an antibody against soluble VEGF receptor-1 (sVEGFR1; also known as sFlt1) (0.6 mg/kg i.v.) .
Protocol 2. Role of adipose tissue in the circulating sFlt1 levels in Ln and Ob Zucker rats undergoing PH+I/R
Sham+LPT group (6 Ln and 6 Ob Zucker rats). Same as group 1, but mesenteric, perirenal, retroperitoneal, and epididymal adipose tissue were resected and extracted .
PH+I/R+LPT group (6 Ln and 6 Ob Zucker rats). Same as group 2, but mesenteric, perirenal, retroperitoneal, and epididymal adipose tissue were resected and extracted before starting PH+I/R .
PH+I/R+LPT+VEGFA group (6 Ln and 6 Ob Zucker rats). Same as group 3, but mesenteric, perirenal, retroperitoneal, and epididymal adipose tissue were resected and extracted before starting PH+I/R .
Protocol 3. Underlying mechanisms of VEGFA in Ln and Ob Zucker rats undergoing PH+I/R
PH+I/R+VEGFA+anti-VEGFR2 group (6 Ln Zucker rats). As in group 3, but treated with ZD6474, a potent inhibitor of VEGFR2 (2.5 mg/kg i.v.) .
PH+I/R+Wnt2 group (6 Ln Zucker rats). As in group 2, but treated with Wnt2 (5 ng/kg, i.v.) .
PH+I/R+VEGFA+anti-sFlt1+anti-VEGFR2 group (6 Zucker Ob rats). As in group 4, but treated with ZD6474, a potent inhibitor of VEGFR2 (2.5 mg/kg i.v.) .
PH+I/R+VEGFA+anti-sFlt1+PI3K/Akt-inh group (6 Ob Zucker rats). As in group 4, but treated with LY294002, a potent inhibitor of Akt (0.5 mg/kg i.p.), and Wortmannin, a potent inhibitor of PI3K (1 mg/kg i.p.) .
Protocol 4. VEGFA impact and availability in SD and CDD-SD rats undergoing PH+I/R
Sham group (6 SD and 6 CDD-SD rats). Hepatic hilar vessels of animals were dissected.
PH+I/R+VEGFA group (6 SD and 6 CDD-SD rats). As in group 13, but treated with VEGFA (5 μg/kg i.v.) .
PH+I/R+VEGFA+anti-sFlt1 group (6 CDD-SD rats). As in group 14, but treated with an antibody against soluble VEGF receptor-1 (sVEGFR1; also known as sFlt1) (0.6 mg/kg i.v.) .
The sample collection and the measurements for protocols 1–4 at the corresponding reperfusion times are shown in Online Resource 1.
VEGFA impact in Ln and Ob Zucker rats undergoing PH+I/R
In terms of liver regeneration, the administration of VEGFA (PH+I/R+VEGFA group) increased the percentage of Ki67-positive-hepatocytes in non-steatotic livers (Fig. 1G). The effect of VEGFA on the progression of the cellular cycle was also examined. The levels of cyclin D1, which is necessary for G1 phase progression , and cyclin E, which is induced during G1 and mediates transition into the S phase  were unchanged in non-steatotic livers of the PH+I/R+VEGFA group with respect to those of the PH+I/R group (Fig. 1G). However, hepatic cyclin A expression, which is necessary for S phase progression , was higher in non-steatotic livers of the PH+I/R+VEGFA group when compared with the PH+I/R group (Fig. 1G). The administration of VEGFA (PH+I/R+VEGFA group) reduced lethality in Ln animals when compared with the PH+I/R group (Fig. 1G).
Relevance of VEGFA availability in Ln and Ob Zucker rats undergoing PH+I/R
Role of adipose tissue in the circulating sFlt1 levels in Ln and Ob Zucker rats undergoing PH+I/R
Underlying mechanisms of VEGFA in Ln and Ob Zucker rats undergoing PH+I/R
The impact of VEGFA in SD and CDD-SD rats undergoing PH+I/R
As in the genetic obesity model (Zucker rats), in CDD-SD rats, hepatic VEGFA protein levels were lower in the PH+I/R group than in the Sham group (Online Resource 3). In addition, our results revealed an improvement in damage and regenerative process of PH+I/R+VEGFA in non-steatotic livers from SD rats when compared with the PH+I/R group. On the other hand, in steatotic livers from CDD-SD rats, VEGFA administration increased sFlt1 levels in adipose tissue and circulation, and exacerbated hepatic damage and regenerative failure. In CDD-SD rats, the co-administration of VEGFA+anti-sFlt1 was required to confer protection against damage and regenerative failure. The underlying protective mechanisms of VEGFA in an experimental model of steatosis induced by CDD were similar to those described in the genetic obesity model (data not shown).
To the best of our knowledge, no previous studies have reported changes in VEGFA levels in both steatotic and non-steatotic livers submitted to PH under I/R, a procedure commonly applied in surgery to reduce bleeding. Herein, we observed reduced mRNA and protein levels of VEGFA in steatotic and non-steatotic livers. In our study, VEGFA administration exhibited differential effects on damage and regeneration in steatotic and non-steatotic livers. Thus, under PH+I/R conditions, VEGFA administration in Ln Zucker animals increased VEGFA in non-steatotic livers and decreased hepatic injury and regeneration failure. On the other hand, VEGFA administration in Ob Zucker animals undergoing surgery reduced VEGFA levels in steatotic livers and negatively affected damage and regeneration. These effects of VEGFA have also been demonstrated in an experimental model of steatosis induced by CDD.
An attempt was made to discern the role of adipose tissue in the circulating sFlt1 levels in Ln and Ob Zucker rats undergoing PH+I/R, for the following reasons: functional differences between lean and obese adipose tissue have been extensively reported [37, 38, 39], obesity is associated with oxidative stress and inflammatory response in adipose tissue [40, 41, 42], and impairs adipocyte function and secretion of mediators derived from adipose tissue to circulation [34, 35, 36]. Our results in Zucker rats indicate the following: (a) adipose sFlt1 levels were higher in Ob than in Ln animals, (b) the increase in adipose tissue sFlt1 levels as consequence of PH+I/R occurred only in Ob animals, and (c) the adipose tissue was involved as a source of circulating sFlt1 levels. VEGFA treatment increased sFlt1 in adipose tissue only in Ob animals, and this resulted in the release of sFlt1 from adipose tissue to the circulation. Thus, we believe that the high levels of circulating sFlt1 (derived from adipose tissue) in Ob Zucker animals undergoing liver surgery sequestrate exogenous VEGFA, reduce circulating VEGFA bioavailability, and consequently restrict the opportunity for VEGFA to be taken up by the liver and to exert its protective effects [29, 30]. The concomitant administration of VEGFA with an antibody against sFlt1 ensures that the exogenous VEGFA reaches the steatotic liver in order to protect against damage and regenerative failure.
Herein, we report for the first time that the benefits of VEGFA in non-steatotic livers in the event of PH were maintained when implementing vascular occlusion, which increases our knowledge about the applicability of this growth factor [6, 7, 9]. However, it is important to denote that the effects of VEGFA on the VEGFR2-Id1-Wnt2 pathway in non-steatotic livers subjected to PH were dependent on the presence or absence of vascular occlusion. In fact, in previous studies [7, 43], Id1 was required to induce Wnt2 activation in non-steatotic livers submitted to PH whereas in our hands, Id1 seemed to play a minor role in damage and regeneration in non-steatotic livers under PH+I/R conditions. The pharmacological regulation of VEGFA or its receptor, VEGFR2, affected the degree of damage and regeneration response in non-steatotic livers under PH+I/R conditions independently of changes in Id1 expression. Indeed, VEGFA administration in Ln animals (without the induction of changes in Id1 expression) increased Wnt2 in non-steatotic livers and this was associated with improvements in the levels of damage and regeneration. In addition, antibodies against VEGFR2 abolished the beneficial effects of VEGFA on Wnt2, damage, and the regenerative process. Furthermore, similar to that occurring for VEGFA treatment, the administration of Wnt2 decreased hepatic injury and regeneration failure under PH+I/R conditions, resulting in reduced damage and inflammation in non-steatotic livers and improved liver functionality, regeneration, and survival rate. Taken together, our results demonstrate for the first time that the beneficial effects of VEGFA against damage and regenerative failure in non-steatotic livers under PH+I/R conditions are exerted through the VEGFR2-Wnt2 pathway, independently of Id1.
A previous study indicated that the administration of VEGFA protected diet-induced steatotic livers under PH without I/R . On the other hand, our results indicated that VEGFA administration should not be considered as an effective strategy to reduce damage and improve liver regeneration in surgical conditions requiring both liver regeneration and vascular occlusion. Previous results in obese mice with NAFLD indicated that the down-regulation of Wnt2 signaling was associated with the inhibition of hepatocyte proliferation [44, 45]. However, in our study, Wnt2 seemed to play a minor role in steatotic livers under PH+I/R conditions. Indeed, the concomitant administration of VEGFA and anti-sFlt1 (which increased VEGFA availability in the steatotic liver) did not induce changes in either Id1 or Wnt2, whereas the PI3K/Akt pathway was up-regulated. This increase in PI3K/Akt expression was abolished when we inhibited VEGFA using antibodies against VEGFR2, whereas the levels of both Id1 and Wnt2 were unchanged. Indeed, the inhibition of PI3K/Akt pathway abolished the benefits resulting from VEGFA+anti-sFlt1 treatment on damage and regeneration in steatotic livers. Thus, if sFlt1 action is blocked, VEGFA might exert its effects on steatotic livers by activating the PI3K/Akt rather than the Id1-Wnt2 pathway.
Given our experimental results, circulating sFlt1 may determine VEGFA availability and its effects on target organs. Obviously, intensive investigations (which were not part of the present study) will be necessary to determine whether all these experimental results could be extrapolated to clinical practice in liver surgery or in other pathologies. Yilmaz et al.  showed that patients with NAFLD have significantly lower serum sFlt1 concentrations than matched controls. On the other hand, Coulon et al.  found that serum levels of sFlt1 were significantly higher in NASH and/or simple steatosis patients compared to controls. The authors indicate that the reason for these different results may lie in the diversity of the patient populations and their clinical characteristics (differences in BMI and inclusion or exclusion of diabetes mellitus patients) or in the differences in laboratory techniques. Indeed, the results reported by Coulon et al.  are in line with other papers indicating increases in circulating sFlt1 levels in patients with liver cirrhosis as well as in patients with chronic kidney disease [32, 33, 48].
MECP has received a Sara Borrell postdoctoral contract from ISCIII, Madrid, Spain, and CGAL is the recipient of a fellowship from CONACYT, México. J Gracia-Sancho received continuous funding from the Instituto de Salud Carlos III (currently FIS PI17/00012) & the CIBEREHD, from Ministerio de Ciencia, Innovación y Universidades. MBJC also has a contract from EMPLEA Program-MINECO, Madrid, Spain.
EB and MECP performed experimental groups, participated in data collection and in biochemical and histological analyses. JG, FR, CAL, and ENS contributed in biochemical and histological analyses and data acquisition. JGS contributed to data analysis/interpretation and critical revision of this article. AN contributed in critical revision of this article. MBJC and CP contributed to the study concept/design, acquisition of experimental data, data analysis/interpretation, and drafting and critical revision of this article. All authors have approved the final manuscript.
This research was supported by the Ministerio de Ciencia, Innovación y Universidades (MCIU) (RTI2018-095114-B-I00) Madrid, Spain, by the European Union (Fondos Feder, ‘una manera de hacer Europa’), by CERCA Program/Generalitat de Catalunya, and by the Secretaria d’Universitats i Recerca (2017SGR-551) Barcelona, Spain.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
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