Objectives: Ischemia-reperfusion injury is correlated with a substantial inflammatory response. Inflam­mation triggers the migration of cells through vessel endothelium and leads to serious tissue injury. Our hypothesis was that an early application of mammalian target of rapamycin inhibitors has an impact on human vessels after ischemia-reperfusion injury.

Materials and Methods: After exposure to ischemia for 5 hours, human vessels (veins and arteries) from 20 patients were reperfused for 120 minutes in an in vitro bioreactor with heparinized human blood after oxygenation and warming to 37 °C. The vessels were treated with mammalian target of rapamycin inhibitor everolimus (5 ng/mL, n = 7) or sirolimus (10 ng/mL, n = 6). As a control group, untreated human vessels were reperfused (n = 7). During the reperfusion period, blood samples were collected continuously (after 0, 15, 30, 60, 120 minutes); vessel biopsies were performed at the end. Oxygen consumption was measured during reperfusion to determine vessel viability. Inflammatory markers (interleukin 6, tumor necrosis factor α, vascular endothelial growth factor) were analyzed in blood samples. To quantify vascular inflammation, we investigated the expression of CD11 and CD31.

Results: Physiological oxygen consumption and pH values verified vessel viability. After reperfusion, interleukin 6 and vascular endothelial growth factor levels were significantly increased in the control group over time, whereas everolimus and sirolimus showed no significant differences. Furthermore, tumor necrosis factor α level increased significantly in the sirolimus group, whereas the everolimus and control groups showed constant values. A significant decrease of expression of CD11b and CD31 in both mammalian target of rapamycin inhibitor cohorts compared with control cohort was investigated.

Conclusions: Early use of mammalian target of rapamycin inhibitors may limit an inflammatory rise of interleukin 6 and vascular endothelial growth factor after ischemia-reperfusion injury and could be associated with a restriction in vascular cell transmigration.

Key words : CD11b, CD31, Interleukin 6

Introduction

Ischemia-reperfusion injury (IRI) refers to an acute inflammation process in tissues caused by ischemia and hypoxia, an accumulation of toxic metabolites, and finally an inflammatory response caused by reperfusion with oxygenated blood. Ischemia-reperfusion injury has a significant role in the context of solid-organ transplant and the recovery of tissues after cerebral and cardiac infarction.¹

In the first step of the IRI cascade, ischemia and hypoxia cause activation of the endothelial cells of the vessels within the injured organ. During reperfusion, white blood cells are activated by chemokines and adhesion molecules released or expressed by the activated endothelial cells. Once activated, white blood cells, especially neutrophils and lymphocytes, transmigrate through the endothelium in a process involving the integrin complex CD11b/18 and adhesion molecules such as CD31 (platelet-endothelial cellular adhesion molecule [PECAM]) and cause the inflammatory IRI.² Activated white blood cells further intensify the IRI by releasing cytokines themselves (eg, interleukin 6 [IL-6] and tumor necrosis factor α [TNF-α]), as well as through a respiratory burst of reactive oxygen species.

It has already been demonstrated that the intracellular mammalian target of rapamycin (mTOR) complex is a key factor with regard to the expression and secretion of inflammatory cytokines and adhesion molecules in the course of IRI through the transcription factor known as nuclear factor κB.3 Inhibition of the mTOR complex was shown to reduce and protect against IRI in animal models.⁴ Previous investigators concluded that mTOR inhibition by sirolimus as well as everolimus decreases interleukin secretion (for example, IL-2, IL-6, IL-7, IL-12) and TNF-α-mediated neutrophil activation.^3,5 Other investigations demonstrated further effects of sirolimus and everolimus causing decreases in the expression of adhesion molecules such as CD11a, CD11b/CD18, VCAM-1, ICAM-1, and E-selectin.⁵ The ability of mTOR inhibitors to interrupt the cell cycle and prevent T- and B-lymphocyte development is the basis for use of sirolimus and everolimus in maintenance immunosuppression.⁶ Finally, mTOR inhibitors were also shown to reduce hypoxia-induced angiogenetic factors like vascular endothelial growth factor (VEGF) in the process of IRI.⁷

Since our study group has already demonstrated pharmacological effects on key mediators of IRI in a primate model and effects of early application of mTOR inhibitors have been shown in murine models, this experimental investigation aimed to compare the effects of an early application of the mTOR inhibitors sirolimus and everolimus upon IRI in a human vessel bioreactor model.^4,8,9

Materials and Methods

Pre-reperfusion stage: vessel procurement, preparation, and treatment groups
After approval by the ethics committee of the University Hospital Frankfurt (AZ 299/14), we used a bioreactor model developed at the Technical University of Munich10 and improved it at the University Hospital of Frankfurt, as previously described.¹¹

Discarded segments of 20 human arteries or veins (3 cm, saphenous vein/internal thoracic artery) from 20 patients, obtained in the course of coronary artery bypass surgery, were surgically installed in a human bioreactor model after exposure to standardized cold ischemia for 5 hours. Following this, each vessel was reperfused with 150 mL blood (AB Rh-negative). For this purpose, the blood was heparinized (25 000 IE/5 mL heparin, Ratiopharm), warmed to a temperature of 37 °C, diluted to a hematocrit of 30% by adding Krebs-Henseleit buffer (Sigma-Aldrich),8 and preoxygenated, for which we used a heart-lung machine for small animals (model 2000, from Martin Humbs, Ingenieurbuero für Feinwerktechnik) to achieve physiological oxygen saturation levels.

To be qualified for this study, patients had to be over 18 years old. Exclusion criteria were infections, insulin-dependent diabetes mellitus, a history of vein thrombosis, varicose veins, and blood storage exceeding 48 hours.

Vessels reperfused without addition of drugs served as the control group (n = 7). The sirolimus group (n = 6) consisted of vessels reperfused with blood containing sirolimus at a concentration of 10 ng/mL (Rapamune, Pfizer),12 and the everolimus group (n = 7) consisted of vessels reperfused with blood containing everolimus at a concentration of 5 ng/mL (Certican, Novartis).13 Both concentrations were used in accordance with the manufacturers’ directions.

Reperfusion and blood parameters
The reperfusion time was standardized to 2 hours. The flow through the vessels was adjusted to 70 to 80 mL/min with a roller pump, which corresponds to the physiological coronary flow. For blood quality and bioreactor function monitoring purposes, oxygen saturation, pH, and CO2 partial pressure were repeatedly determined by means of a blood-gas analyzer (Radiometer model A/S ABL 5) from 2-mL blood samples taken at the beginning of reperfusion (t-0), and after 15 (t-1), 30 (t-2), 60 (t-3), and 120 (t-4) minutes. At the same time points, 5-mL samples were obtained for quantification of the inflammatory markers IL-6, TNF-α, and VEGF.

Postreperfusion stage: enzyme-linked immuno­sorbent assay and immunohistochemical analyses
The 5-mL blood samples were drawn into EDTA tubes and centrifuged at 4 °C (3000 rpm for 10 minutes). The blood serum was stored at -80 °C for subsequent treatment. We used commercially available enzyme-linked immunosorbent assay kits (QuantiGlo ELISA, R&D Systems Bio-Techne) for immunohistochemical testing for IL-6, TNF-α, and VEGF, according to the manufacturer’s instructions, by inserting into each well 100 μL of serum for the analysis of IL-6 and TNF-α and 50 μL for VEGF. We measured the results with an absorbance microplate reader (Infinite M200, Tecan).

After the reperfusion period, biopsies of the vessels were obtained for immunohistological investigation of the inflammatory process through quantification of the adhesion molecule CD31 and integrin CD11b. The biopsies were stored for 24 hours in diluted (1:5) formalin at 24 °C and then transferred to 70% ethanol where they remained at 4 °C until fixation in paraffin. From the paraffin-embedded tissue, 5-µm sections were cut (cryostat section) at a temperature of -15 °C. These were subsequently dewaxed with xylol and ethanol.

We used primary monoclonal rabbit antibodies (Abcam) to identify CD31 and CD11b. For this purpose, the air-dried and fixed sections were blocked by a 1:19 dilution of goat serum in phosphate-buffered saline (1 hour), followed by blocking of the endogenous peroxidase for 10 minutes. Thus prepared, the sections were incubated with the primary antibody (CD31, dilution 1:500; CD11b, dilution 1:4000) overnight at 4 °C. After a number of washing steps, the secondary antibody (horseradish peroxidase rabbit antibodies; Nichirei) was added, and the sections were stored for 40 minutes in a wet and dark incubator. After that, diaminobenzidine substratum was added, and incubation continued under microscopic monitoring until the required color intensity was achieved. This was followed by hematoxylin staining, a number of washing steps, and fixation on microscope slides.

Statistical analyses and immunohistochemical evaluation
Results are presented as the mean of the measurement ± SEM. We used 1-way or 2-way analysis of variance to analyze statistical differences. CD31 was considered positive as an endoluminal CD31 expression in vessels. The semiquantitative evaluation was done as described before.8,9 The number of CD31-positive vessels per slide was counted and described as an expression ratio. Three randomly chosen optical fields per vessel were analyzed. Expression of CD11b was considered positive if CD11b-positive cells were identified. The number of CD11b-positive cells was counted in 5 randomly chosen optical fields per vessel. Graphics and diagrams were produced by GraphPad Prism 7 software. P < .05 was considered statistically significant.

Results

Quality of the bioreactor model
To evaluate the quality of the bioreactor model, blood samples were obtained at time points t-0 to t-4 to quantify oxygen saturation, pH, and CO₂ partial pressure. Analysis of these blood samples demonstrated a constant high quality of the bioreactor model during the reperfusion process and showed no significant differences between the 3 groups (Figure 1). Also, no differences were seen in the cold ischemia time between the compared groups.

Influence of sirolimus and everolimus on blood parameters
The IL-6 levels determined in the control group were found to have a significant increase after 60 and 120 minutes of the reperfusion process (Figure 2). The treatment groups, in contrast, showed no significant increase in IL-6 levels over the reperfusion period. Everolimus as well as sirolimus showed a slight increase of the IL-6 concentration within the last 60 minutes of reperfusion, which was lower compared with the control group. The everolimus concentration at the end of the reperfusion was lower than at the beginning.

Measurement of TNF-α yielded a statistically insignificant increase in the control group (Figure 3). In the sirolimus group, a significant increase in TNF-α was noted at t-4. Tumor necrosis factor α in the everolimus group showed an increase that was not statistically significant.

The proangiogenic marker VEGF showed a significant increase over the reperfusion period in the control group (Figure 4). The increases in the everolimus and sirolimus groups, in contrast, were not statistically significant.

Influence of sirolimus and everolimus on the expression of CD31 and CD11b
The expression of the adhesion molecules CD31 and CD11b was significantly decreased by mTOR inhibitors after reperfusion versus that shown in the control group (Figures 5 through 7). The expression ratio of CD31-positive vessels per microscopic slide as well as CD11b-positive cells per microscopic slide was significantly increased in the control group compared with the everolimus and sirolimus groups. There were no significant differences in the comparison between sirolimus und everolimus regarding CD31 and CD11b.

Discussion

Ischemia-reperfusion injury is an acute inflammatory and multifactorial process caused by a period of ischemia and hypoxia, the accumulation of toxic metabolites, and inflammation caused by reperfusion with oxygenated blood. This sequence of events results in organ damage starting early after ischemia and following reperfusion and contributes, in the context of transplantation, to primary as well as chronic graft dysfunction.^1,2 A variety of substances including tacrolimus, cyclosporine, monoclonal antibodies against the CD11b/CD118 complex, and antithymocyte globulin have been proved capable of modifying the IRI cascade.^9,14 Furthermore, it has already been shown that the time component of pharmacological intervention also plays an essential role in IRI.¹⁵ In this context, Beiras-Fernandez and colleagues demonstrated, in a nonhuman primate model, that antithymocyte globulin reduces the production of inflammatory cytokines and adhesion molecules in IRI.^8,9

The bioreactor model is an established and verified model for the investigation of IRI, hypoxia, and cardiovascular research.¹⁰ This study aimed to investigate the effects of IRI on human vessels perfused with oxygenated blood supplemented with sirolimus or everolimus, respectively, versus a control group. The quality of the bioreactor model was monitored throughout the experiments. Constant oxygen saturation during the reperfusion period represents one of the parameters for the quality of a bioreactor. The lower mean oxygen saturation at the beginning of the experiments in the sirolimus group could be explained by the limited sample size. Additionally, pH measurement at regular intervals proved the valid technical set-up of our model. Increases in pH to levels near the physiological range occurred equally in all groups and were attributed to the washout of acid metabolites from cells damaged by IRI. This phenomenon is well known in the literature as the pH paradox.16 Although the decrease of the pH during the ischemia period appears to be a protection mechanism against apoptosis and cell death, the pH returns to the physiological range in the course of reperfusion.

To date, sirolimus and everolimus are mainly used in combination with other immunosup­pressants to prevent primary and chronic graft rejection after transplantation.^17,18 Beyond this, it is known that mTOR inhibitors also provide protection against coronary artery disease and myocardial infarction.¹⁹ Therefore, sirolimus and everolimus are commonly used in drug-eluting stents for the treatment of coronary artery disease, and lower restenosis rates and reduced neointimal proliferation have been repeatedly demonstrated for everolimus- and sirolimus-eluting stents.^20,21 The optimal dose for everolimus and sirolimus for daily clinical use is unknown. Therefore, we used the dose as directed by the manufacturer’s declaration.²² Previous inves­tigations suggested that everolimus is a more potent inhibitor of endothelial cell signaling, nuclear factor κB activity, migration, and proliferation than is sirolimus, which may result in a higher efficacy of everolimus in the treatment of coronary artery stenosis.^4,23

The significant increase in IL-6 that we observed in the control group during the reperfusion period (Figure 2) is consistent with the well-established relationship between IL-6 levels and myocardial ischemia and dysfunction.24 Sirolimus and everolimus had positive effects in that they limited a significant increase in IL-6 over the reperfusion period and showed a protection against IRI, as IL-6 is a main chemokine that triggers the neutrophil activation and white blood cells transmigration through endothelial cells into injured tissue.²⁵ Furthermore, the early increase of IL-6 may emphasize the potential importance of an early application to reduce IRI.

Tumor necrosis factor α also plays a major role in the inflammatory process associated with IRI by activating neutrophils among other cells.²⁶ Tumor necrosis factor α has been shown to induce apoptosis, especially of endothelial cells, after periods of hypoxia and ischemia.²⁷ In our study, the level of TNF-α increased, although not significantly, in the control group during the reperfusion period (Figure 3). This is consistent with the fact that TNF-α-induced apoptosis is enhanced by reoxygenation through reperfusion.²⁸ In our control group, the level of TNF-α decreased after an initial peak at t-1. In the sirolimus group, this peak was not present, but the TNF-α level rose significantly during the last hour of the reperfusion time. This may be explained by the initial inhibitory effect by sirolimus on the apoptosis of endothelial cells induced by hypoxia and TNF-α, followed by apoptosis caused by a different mechanism such as mTOR-induced suppression of the nuclear factor κB signaling pathway.²⁹ In the everolimus group, no significant differences in TNF-α levels were observed over the reperfusion period. There was, however, the same tendency as in the sirolimus group, ie, lower TNF-α than in the control group in the beginning, followed by a slight increase toward the end of the reperfusion period. Nevertheless, no significant difference in the concentration of TNF-α was observed between both mTOR groups after the reperfusion. These data are consistent with the hypothesis that everolimus causes a decrease in expression of proapoptotic proteins.³⁰

With regard to VEGF, there is evidence that it is mainly expressed through the hypoxia-inducible transcription factor HIF-1α during periods of hypoxia.³¹ Previous investigators showed that inhibition of VEGF is capable of suppressing and limiting IRI.³² Our data are consistent with previous reports that suggest that sirolimus and everolimus decrease and limit IRI based on VEGF expression.³³ Our data showed a significant abrupt increase in VEGF in the control group after 60 minutes of reperfusion (Figure 4). Sirolimus and everolimus, in contrast, were able to control an abrupt significant increase in VEGF levels and kept VEGF nearly constant. Since it is known that the expression and secretion of VEGF in the context of IRI are mainly caused by neutrophils, we assume that the inhibition of a significant increase in VEGF could be related to the significant reduced activation of the CD31 expression ratio and transmigration of CD11b-positive cells compared with the control group.³⁴

Analysis of adhesion molecule expression showed a significantly higher expression ratio of CD31 (PECAM) in the control group (Figure 5) in comparison with the sirolimus and everolimus groups. Some authors suggest that reduction of CD31 expression is associated with less IRI as well as with better regeneration after IRI,⁸ as leukocyte activation, endothelial dysfunction, and leukocyte transmigration through tissues damaged by IRI are not possible without CD31.³⁵

Our data showed a significantly higher CD11b expression in the control group (Figure 5) than in the sirolimus and everolimus groups. CD11b is mainly expressed by leukocytes, especially neutrophils, and is a major contributor to the process of leukocyte transmigration in the framework of IRI.³⁶ Previous studies showed connections between the expression of CD11b and the effect of drugs on IRI.^9,37

In animal models, sirolimus was shown to reduce acute rejection, prevent coronary artery disease after heart transplantation, and protect against cardiac IRI.^19,38 Our present in vitro study, in contrast, investigated the influence of sirolimus in a human vessel model. Our data suggest that sirolimus can reduce and limit IRI by control of the increases in inflammatory chemokines and expression of adhesion molecules, which reduces leukocyte transmigration. Some studies suggest that everolimus can prevent allograft rejection and vasculopathy in cardiac transplant recipients,¹⁸ but its effects on IRI have not been widely investigated, and few investigators have provided information on beneficial effects such as those reported from a study on renal IRI in rats.³⁹ Our study is one of the first to provide data on the potential positive effects of everolimus on IRI of human vessels. Our results suggest that an early usage of everolimus substantially reduces and limits IRI because it prevents increases of chemokine levels and inhibits leukocyte transmigration because it reduces the expression of adhesion molecules.

The present study has several limitations. First, our investigations were performed with a small sample size without knowledge about patients’ preexisting conditions, except from exclusion criteria. This fact could have led to nonsignificant differences between investigated parameters. Moreover, arteries and veins were used for our investigations, which have well-known functional differences. Furthermore, because of structural conditions, the assumption was that 5 hours of cold ischemia time and 2 hours of reperfusion would fully induce the IRI, even though cold ischemia time during kidney or liver transplant procedures could be up to 48 or 12 hours, respectively. As reported, the IRI cascade is affected by more than the investigated cytokines. To fully understand the effect of mTOR inhibitors on IRI, future studies should investigate different doses within the usual clinically used ranges as well as additional markers, especially through in vivo findings.

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