In a rat model of bypass DuraGraft ameliorates endothelial dysfunction of arterial grafts

Coronary artery bypass surgery can result in endothelial dysfunction due to ischemia/reperfusion (IR) injury. Previous studies have demonstrated that DuraGraft helps maintain endothelial integrity of saphenous vein grafts during ischemic conditions. In this study, we investigated the potential of DuraGraft to mitigate endothelial dysfunction in arterial grafts after IR injury using an aortic transplantation model. Lewis rats (n = 7–9/group) were divided in three groups. Aortic arches from the control group were prepared and rings were immediately placed in organ baths, while the aortic arches of IR and IR + DuraGraft rats were preserved in saline or DuraGraft, respectively, for 1 h before being transplanted heterotopically. After 1 h after reperfusion, the grafts were explanted, rings were prepared, and mounted in organ baths. Our results demonstrated that the maximum endothelium-dependent vasorelaxation to acetylcholine was significantly impaired in the IR group compared to the control group, but DuraGraft improved it (control: 89 ± 2%; IR: 24 ± 1%; IR + DuraGraft: 48 ± 1%, p < 0.05). Immunohistochemical analysis revealed decreased intercellular adhesion molecule-1, 4-hydroxy-2-nonenal, caspase-3 and caspase-8 expression, while endothelial cell adhesion molecule-1 immunoreactivity was increased in the IR + DuraGraft grafts compared to the IR-group. DuraGraft mitigates endothelial dysfunction following IR injury in a rat bypass model. Its protective effect may be attributed, at least in part, to its ability to reduce the inflammatory response, oxidative stress, and apoptosis.

Arterial grafts are considered better conduits than SVGs in CABG 10 .Recently, Aschacher et al. demonstrated that DuraGraft reduces oxidative stress and improves cellular integrity in radial artery grafts used for CABG 11 .These studies have nevertheless examined the effect of DuraGraft during the ischemic preservation phase.However, few studies have investigated the effects of DuraGraft on arterial grafts submitted to cold ischemia followed by warm reperfusion in an in vivo model.
This study analyzes the effect of DuraGraft on endothelial dysfunction induced by cold ischemia followed by blood reperfusion in a rat bypass model.

Effect of intraoperative preservation with DuraGraft on aortic contractile responses
In response to high potassium (K + )-induced depolarization, contraction was significantly reduced in the IR group compared to controls.However, the use of DuraGraft did not show any significant effect on the contractile responses (Fig. 1A).Conversely, the increased contractility to phenylephrine observed in the IR group compared to controls was significantly decreased after preservation of aortic rings with DuraGraft (Fig. 1B).The pD 2 values, which indicate the sensitivity of the aorta to phenylephrine, were significantly lower in the IR group compared to controls but similar between the IR + DuraGraft and the control groups (Table 1).Table 1.Quantitative analysis of vascular function: effect of DuraGraft after cold ischemia followed by blood reperfusion in the aorta.Contractile responses to phenylephrine (control, n = 14 rings from 7 rats; IR, n = 18 rings from 9 rats; IR + Duragraft, n = 17 rings from 9 rats) were expressed as gram and as a percentage of the maximum contraction induced by 80 mM potassium chloride (KCl) (control, n = 14 rings from 7 rats; IR, n = 18 rings from 9 rats; IR + Duragraft, n = 17 rings from 9 rats).Maximum relaxation to acetylcholine (control, n = 14 rings from 7 rats; IR, n = 18 rings from 9 rats; IR + Duragraft, n = 17 rings from 9 rats) and sodium nitroprusside (SNP) (control, n = 14 rings from 7 rats; IR, n = 18 rings from 9 rats; IR + Duragraft®, n = 17 rings from 9 rats) were expressed as a percentage of the contraction induced by phenylephrine.R max indicates maximum relaxation and pD 2 , negative logarithm of the corresponding half-maximal response (EC 50 ).Results are presented as mean ± standard error of the mean (SEM).*p < 0.05 versus control and # p < 0.05 versus IR.All aortic rings that were pre-contracted with phenylephrine showed concentration-dependent relaxation to acetylcholine at concentrations of 10 -9 to 10 -4 M (Fig. 2A).Vasorelaxation induced by acetylcholine was significantly lower in the IR group compared to controls, but it was improved in aortic rings preserved with DuraGraft (Fig. 2A).The sensitivity of the aorta to acetylcholine, indicated by pD 2 values, was significantly decreased in the IR + DuraGraft group compared to the IR aortas (Table 1).

Effect of intraoperative preservation with DuraGraft on aortic smooth muscle relaxation
In all phenylephrine pre-contracted aortic rings, concentration-dependent relaxation was elicited by sodium nitroprusside (10 -10 -10 -5 M) (Fig. 2B).There were no significant changes observed among the experimental groups in terms of maximum relaxation responses or pD 2 values to sodium nitroprusside (Table 1; Fig. 2B).Our findings showed a significant increase in the immunoreactivity of caspase-3 and caspase-8, essential regulators of the apoptotic response, in the IR group compared to the control group (Fig. 3).However, preservation of aortas with DuraGraft solution led to a decrease in caspase3 and caspase-8 immunoreactivity (Fig. 3).Furthermore, the increased expression of ICAM-1, which is responsible for regulating leukocytes recruitment from circulation to sites of inflammation, and 4-hydroxy-2-nonenal, a marker of oxidative stress, observed in the IR aortas compared to controls, was significantly reduced after preservation with DuraGraft (Fig. 4A and 4C).Additionally, a significant decrease in endothelial PECAM-1 immunoreactivity was observed in the IR aortas compared to controls, which was increased following preservation with DuraGraft (Fig. 4B).

Discussion
Our findings suggest that the preservation of arterial grafts using DuraGraft, a novel inhibitor of endothelial damage, mitigates endothelial dysfunction and reduces enhanced smooth muscle contraction induced by agonists after cold ischemia and warm reperfusion injury.The mechanism underlying the positive effect of DuraGraft preservation on endothelial function and smooth muscle contraction involves a decrease in ICAM-1 expression, an increase in PECAM-1 expression, a reduction in oxidative stress, and a decrease in caspasemediated apoptosis.
CABG is a surgical technique that involves using autologous arteries or SVGs to bypass occlusion and restore blood supply to the heart muscle 12,13 .Unfortunately, conventional storage solutions used during CABG surgery do not adequately protect the endothelium.Consequently, post-procedural lumen loss can occur following CABG due to functional and structural changes in endothelial cells, among other factors 12,14 .In our study, we confirmed that cold ischemia followed by reperfusion with blood leads to endothelial dysfunction and impairs contractile responses in arterial grafts.These impairments are attributed, in part, to inflammation, oxidative stress, and cell apoptosis, which are crucial factors in IR-induced vascular damage 12,14,15 .Moreover, our findings revealed significant changes in the levels of intercellular adhesion molecules, especially ICAM-1 and PECAM-1, known to contribute to the inflammatory mechanisms in blood vessels.Additionally, we observed modifications in the levels of 4-hydroxy-2-nonenal, a protein biomarker of oxidative stress, as well as caspase-3 and caspase-8, important regulators of apoptosis.Therefore, novel treatments are required to protect vascular grafts against IR injury, potentially improving long-term outcomes following CABG 6 .In this context, DuraGraft, an endothelial damage inhibitor, has shown promising results in preclinical and clinical investigations in protecting the integrity and function of SVG's endothelium during ischemic storage 16 .A retrospective study found that treatment of SVGs with DuraGraft during the CABG procedure was linked to lower rates of major adverse cardiac events and repeat revascularization compared to the use of heparinized saline in a cohort of 2436 patients 9 .In a recent study, we demonstrated that DuraGraft improves both endothelial and contractile dysfunction resulting from in vitro vascular injury caused by IR, without the involvement of leukocytes 17 .Building upon the promising findings from our in vitro investigation 17 , we conducted an experimental study using a clinically relevant in vivo rat model to compare the impact of DuraGraft and saline on arterial vascular damage.Acetylcholine binds to muscarinic receptors on endothelial cells to induce NO release, which exhibits a crucial role in vascular tone, local blood flow, leukocyte-endothelial cell interactions, and platelet aggregation 18 .Our study showed that DuraGraft protects against endothelial dysfunction.This is demonstrated by the enhancement of endothelium-dependent vasorelaxation to acetylcholine and attenuation of exaggerated agonist-induced smooth muscle contraction following IR injury.To evaluate the loss of endothelial cells within the grafts, we performed PECAM-1 immunoreactivity.Our results demonstrated that the decreased immunoreactivity of PECAM-1, a marker indicating the presence of endothelial cells, caused by IR injury, was increased by DuraGraft treatment.These observations are in line with previous studies that have shown DuraGraft to preserve the functionality and integrity of endothelial and intimal cells, prevent DNA damage, and reduce cell death in radial artery grafts used for CABG 11 .The authors demonstrated in their study that DuraGraft decreases ROS production and inhibits progressive neo-intimal formation by downregulating transforming growth factor (TGF)-β-induced vascular endothelial growth factor (VEGF) cellular over-proliferation 11 .Additionally, they showed a direct positive effect on protective markers, such as heme oxygenase-1 and AKT/endothelial NO synthase pathway 11 .Ischemia followed by reperfusion is known to elicit a widespread inflammatory response, which is typically marked by the upregulation of adhesion molecules.The expression of ICAM-1 can be induced by various cytokines (such as interleukin-1β, tumor necrosis factor-α, and interferon (IFN)-γ) 19 , as well as by cytokine-independent stimuli such as free radicals and hypoxia, including reactive and nitrogen species 20 .Our study demonstrated that DuraGraft resulted in a significant decrease in ICAM-1 immunoreactivity in aortic rings subjected to cold ischemia followed by warm reperfusion injury.Furthermore, we performed immunohistochemistry for HNE, an indicator of oxidative stress, based on the role of oxidative stress during IR injury.Our results showed that DuraGraft lowered the increased 4-hydroxy-2-nonenal expression in aortic rings.Additionally, DuraGraft reduced high caspase-3 and caspase-8 immunoreactivity in the arterial grafts, which are key regulators of the apoptotic response, after IR injury.
In a previous study, we have also shown that the storage of grafts with saline or heparinized blood solution (maximum relaxation to acetylcholine: 34 ± 6% or 29 ± 4%) is unable to protect the endothelium against cold ischemia and warm reperfusion injury in rats 21 .In another study, our findings support that the N-acetyl-histidine containing iron chelators-enriched and amino-acid fortified TiProtec solution appears to be superior to saline and custodiol (maximum relaxation to acetylcholine: 46 ± 7% vs. 26 ± 5% and 24 ± 5%) as a preservation solution for arterial storage in a rat model of bypass 22 .Consistent with the results of the present study, the maximum relaxation to acetylcholine in the saline group was 24 ± 1%, whereas with DuraGraft it was 48 ± 1%, indicating similar protective effect of TiProtect and DuraGraft on arterial grafts in a rodent model of revascularization.Additionally, the reduced immunoreactivity of PECAM-1 (CD31), a marker indicating the presence of endothelial cells, resulting from ischemia/reperfusion injury, was ameliorated by TriProtect 22 , as observed in the present study.
The present study has some limitations.First, it should be noted that the vessel wall structure of the aortic tissue differs from that of human internal mammary or radial artery grafts.Therefore, caution should be exercised when interpreting our results in the context of those grafts.Second, although vascular leakage is an important feature in several diseases, including IR injury, endothelial permeability was not measured in this study.
In summary, our study presents novel experimental evidence demonstrating that preservation of arterial grafts with DuraGraft alleviates endothelial dysfunction and attenuates agonist-induced increased smooth muscle contraction following cold ischemia and blood reperfusion in rats.Our findings suggest that the protective effect of DuraGraft can be attributed, at least in part, to its ability to reduce oxidative stress and apoptosis, decrease ICAM-1 expression, and increase PECAM-1 expression.However, additional studies are necessary to fully elucidate the mechanisms underlying the protective effects of DuraGraft.

Animals and humane care
Isogenic male Lewis rats weighing between 250 and 300 g were obtained from Janvier Labs, Saint Berthevin, France, and housed in the Division of Laboratory Animal Resources at Heidelberg University, Germany.Before experiments, the rats were acclimatized for at least 7 days and given standard rodent fodder and water ad libitum.The animal procedures in this study followed the "Principles of Laboratory Animal Care" published by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" developed by the Institute of Laboratory Animal Resources and published by the National Institutes of Health in 2011 (8th edition).Ethical approval for this study was obtained from the Ethical Committee of the Regional Council of Karlsruhe, Germany (G280/20).The animal experiments were conducted in accordance with the ARRIVE (Animals in Research: Reporting in Vivo Experiments) guidelines.

Experimental group
The animals were randomly assigned to three groups: control (n = 7 rats), IR (n = 9 rats), and IR + DuraGraft (n = 9 rats).In the control group, the aortic arches were explanted, and the aortic rings were prepared and immediately www.nature.com/scientificreports/placed in organ baths.In the IR-and IR + DuraGraft groups, the donor aortic arches were explanted, stored in cold physiological saline or DuraGraft solution (Somahlution, Jupiter, FL, United States), respectively, for 1 h before heterotopic aorta transplantation.Following 1 h of reperfusion, the aortic arches were removed and placed in chambers of organ bath for ex vivo measurement of vascular function in the grafts.The experimental protocol is shown in Fig. 5.

Heterotopic aorta transplantation
As we previously described 23 , anesthesia was initially induced by placing the donor male Lewis rats in a chamber filled with 3% isoflurane gas and was maintained by inhaling 1.75-2.5% in oxygen through a connected tube.Heparin (400 IU/kg, Ratiopharm GmbH, Ulm, Germany) was administered through the inferior vena cava, and the brachiocephalic, left common carotid artery, and left subclavian artery were ligated.While the aortic arches obtained from the control group were explanted, the aortic rigs were prepared, cut into rings, and immediately placed in organ bath chambers, those from the IR and IR + DuraGraft groups were preserved in saline or DuraGraft, respectively, for a duration of 1 h.The recipient rats were anesthetized and then heparinized following the same procedure as describe before.Two ends to side anastomoses were used to transplant the donor aortic arch into the recipient aorta.

Ex vivo organ bath experiments
As we previously described 17,24 , the aortic arch was explanted for the control group, while for the IR and IR + DuraGraft groups, the implanted graft was harvested after 1 h of in vivo blood reperfusion.The harvested graft was then placed in Krebs-Henseleit solution (KHL) containing the following components: 118 mM NaCl, 4.7 mM KCl, 1.2 mM KH 2 PO 4 , 1.2 mM MgSO 4 , 1.77 mM CaCl 2 , 25 mM NaHCO 3 and 11.4 mM glucose).Subsequently, the aortic arch was carefully dissected and cut into three rings, each measuring 3-4 mm in width.Two rings were then mounted on the hooks of isolated organ baths containing 30 ml KHL at 37 °C while the remaining ring was used for immunohistochemical staining.The baths were continuously supplied with a gas mixture of 95% O 2 and 5% CO 2 .To maintain a passive tension of 2 g, the rings were stretched and allowed to equilibrate for a duration of 1 h, with KHL being replaced every 30 min to prevent the presence of interfering metabolites.Throughout this period, adjustments were periodically made to the tension, maintaining it at 2 g.Following equilibration, to verify tissue viability, 80 mM potassium chloride (KCl) was used.Then, the rings were pre-contracted using an α-adrenergic receptor agonist, phenylephrine (10 -9 -10 -5 M).Subsequently, relaxation responses were assessed by adding increasing concentrations of acetylcholine (10 -9 -10 -4 M) and sodium nitroprusside (10 -10 -10 -5 M).The relaxation responses were expressed as a percentage of the contraction induced by phenylephrine.The half-maximal response values (EC 50 ) were obtained from sigmoidal equation fitting of individual concentration-response curves to phenylephrine, acetylcholine, or sodium nitroprusside, and the vascular sensitivity was expressed as pD 2 (− logEC 50 ).

Immunohistochemistry for caspase-3, caspase-8, ICAM-1, PECAM-1, and 4-hydroxy-2-nonenal
As we previously reported 17,24 , immunohistochemical analysis was performed on aortic segments that were fixed in a 4% buffered paraformaldehyde solution and embedded in paraffin.Thin sections of 4 µm thickness were cut from the tissue blocks and subjected to various treatments to unmask the antigenic epitopes.Primary antibodies against ICAM-1 (1:50, ab171123, host species: mouse, monoclonal, Thermo Sientific, UK), PECAM-1 (1:5000, ab182981, host species: rabbit, monoclonal, Abcam, Berlin, Germany), HNE (1:1000, ab46545, host species: rabbit, polyclonal, Abcam, Berlin, Germany), caspase-3 (1:1000, #9662, host species: rabbit, polyclonal, . Sequential outline of the experimental procedure.After anesthesia, the donor aortic arch was harvested for all experimental groups.In the control group, after preparation, two rings were mounted in organ bath chambers, while one ring was stored as a paraffin block.For the IR and IR + Duragraft groups, the aortic arch was preserved for 1 h at 4 °C either in saline or Duragraft, respectively.Then, the donor aortic arch was implanted into the recipient.After 1 h of blood reperfusion, the donor aortic arch was harvested, and two prepared rings were mounted in organ bath chambers, while one ring was stored as a paraffin block.The rings were excluded from the organ bath experiments analysis if there was a technical problem, and they were considered damaged.