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Endothelial dysfunction mediated by interleukin-18 in patients with ischemic heart disease undergoing coronary artery bypass grafting surgery
Accepted Manuscript Endothelial dysfunction mediated by interleukin-18 in patients with ischemic heart disease undergoing coronary artery bypass grafting surgery Sokolic Jadranko, Vlatka Sotosek Tokmadzic, Knezevic Danijel, Medved Igor, Vukelic Damjani Nada, Balen Sanja, Rakic Marijana, Lanca Bastiancic Ana, Laskarin Gordana PII: DOI: Reference:
S0306-9877(17)30175-5 http://dx.doi.org/10.1016/j.mehy.2017.05.009 YMEHY 8553
To appear in:
Medical Hypotheses
Received Date: Accepted Date:
16 February 2017 6 May 2017
Please cite this article as: S. Jadranko, V.S. Tokmadzic, K. Danijel, M. Igor, V.D. Nada, B. Sanja, R. Marijana, L.B. Ana, L. Gordana, Endothelial dysfunction mediated by interleukin-18 in patients with ischemic heart disease undergoing coronary artery bypass grafting surgery, Medical Hypotheses (2017), doi: http://dx.doi.org/10.1016/ j.mehy.2017.05.009
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Endothelial dysfunction mediated by interleukin-18 in patients with ischemic heart disease undergoing coronary artery bypass grafting surgery
Sokolic Jadranko1, Vlatka Sotosek Tokmadzic1,2, Knezevic Danijel2, Medved Igor3, Vukelic Damjani Nada4, Balen Sanja4, Rakic Marijana5, Lanca Bastiancic Ana5, Laskarin Gordana5,6
1
Clinic of Anesthesiology and Intensive Care Medicine, Clinical Hospital Center Rijeka, 51 000
Rijeka, Kresimirova 42, Croatia 2
Department of Anesthesiology, Reanimatology and Intensive Care, Faculty of Medicine,
University of Rijeka, 51000 Rijeka, B. Branchetta 20, Croatia 3
Department of Surgery, Faculty of Medicine, University of Rijeka, 51000 Rijeka, Tome Strizica
3, Croatia 4
Department of Transfusion Medicine, Clinical Hospital Center Rijeka, 51 000 Rijeka,
Kresimirova 42, Croatia 5
Division of Cardiology, Hospital for Medical Rehabilitation of the Hearth and Lung Diseases
and Rheumatism “Thalassotherapia” Opatija, 51410 Opatija, M. Tita 188, Croatia 6
Department of Physiology and Immunology, Medical Faculty, University of Rijeka, 51000
Rijeka, B. Branchetta 20, Croatia
Corresponding author: Vlatka Sotosek Tokmadzic, MD, PhD Department of Anesthesiology, Reanimatology and Intensive Care, Faculty of Medicine, University of Rijeka, Medical Faculty, University of Rijeka B. Branchetta 20 1
51000 Rijeka, Croatia Tel. ++ 385 51 65 11 85 Fax number: ++ 385 51 67 56 99 E-mail: [email protected]
Running head: IL-18 mediated endothelial dysfunction
2
Abstract When medication management or percutaneous coronary intervention is not successful in patients with advanced ischaemic heart disease, surgical revascularisation—predominantly coronary artery bypass grafting (CABG)—is considered the gold standard. However, CABG surgery can lead to ischaemia/reperfusion injury, which is characterized by a strong inflammatory response. Interleukin (IL)-18, is a strong inflammatory mediator, that is released from cardiomyocytes and can be found in the systemic circulation of patients during and immediately after CABG surgery. The existing damage of endothelial glycocalyx in patients with ischaemic heart disease is further impaired concurrently during the surgery due to the anaesthesia-surgical technique used and intravascular fluid loading. This results in the increased incidence of adverse events, including myocardial infarction. IL-18 leads to the activation of lymphocyte cytotoxicity via cytotoxic mediators (Fas ligand, Tumor necrosis factor (TNF)-related apoptosis-inducing ligand, perforin, and granulysin). We hypothesize that IL-18 is released locally in the heart and the systemic circulation in patients undergoing CABG surgery and may be correlated with the level of activity of circulating lymphocytes. In turn, this may lead to lymphocyte-mediated cytotoxicity directed toward damaged and activated endothelial cells. Shear stress glycocalyx, as well as damaged and activated endothelial cells then become the main the source of pro-inflammatory cytokines, chemokines, and adhesion molecules. These attract activated lymphocytes to adhere to the endothelium or enter the subintimal layer, increasing existing or initiating the formation of new plaques, which leads to the development of myocardial infarction during or shortly after surgery. To evaluate our hypothesis, we will measure the local concentration of IL-18 in the sinus coronarius and systemic circulation. These values will then be correlated with immunological and biochemical parameters, predominantly with the concentration of degradation products of glycocalyx and cytotoxic mediators in activated lymphocytes. If our hypothesis is correct, 3
measuring the IL-18 concentration that is responsible for glycocalyx deterioration, may become a useful tool for predicting myocardial infarction occurrence in patients undergoing CABG surgery.
4
Introduction Endothelial dysfunction is caused by chronic endogenous inflammation and altered metabolic conditions on the endothelium. It develops into a chronic process of varying intensity in different organs and manifests itself in a range of clinical entities (1). Recently, it has become evident that identifying the unique roles of inflammation and metabolism share in patient populations with atherosclerotic disease. This relationship is important to identify potentially anti-inflammatory and/or metabolic therapeutic approaches to prevent and stabilize patients with atherosclerotic disease (2). Ischemic heart disease occurs from endothelial dysfunction of large or small coronary arteries and usually presents as acute or chronic coronary syndrome (3). Surgical revascularization is the gold standard to treat patients with coronary artery disease, in cases where complementary treatment, including proper nutrition, moderate physical activity, medication, and percutaneous coronary intervention, have become insufficiently effective (4). However, there is an increased risk of creating oxidative stress associated with ischemia/reperfusion injury via on-pump coronary artery bypass grafting surgery (CABG) (5). CABG is associated with systemic inflammatory response (SIRS), which can be measured using the systemic concentration of tumour necrosis factor alpha (TNF-α) (6). In an in vitro model, TNF-α and H2O2 induced the expression of interleukin (IL)-18 mRNA and precursor protein in cardiomyocytes; IL-18 was also released into the culture supernatants (6). In an experimental animal model, researchers have shown that isolated cardiomyocytes enhanced oxidative stress and biologically active IL-18 expression
via
the
IKK-dependent
nuclear
factor
(NF)-kappa
B
pathway
after
ischemia/reperfusion (5). In humans, IL-18 is a key pro-inflammatory mediator in the pathogenesis and deterioration of patients with heart and vascular disease (5). It mediates plaque
5
rupture and the development of acute coronary syndrome, which is a predictive factor of myocardial infarction and heart failure in hospitalized patients three to seven months after myocardial infarction (7), potentially by increasing cytotoxicity of activated T and NK cells recruited in subintimal layer. Patients who underwent CABG surgery and valvular replacement had similar increases in plasma levels of circulating IL-18 protein. However, in the epicardial adipose tissue of patients who underwent myocardial revascularization, gene expression of the components of IL-18 system, such as IL-18, activating IL-18 receptor (IL-18R) 1, and IL-18R accessory protein, were higher than those in patients who underwent valvular replacement (8). These data indicate that local production of pro-inflammatory protein IL-18, and its autocrine or paracrine effects could provide more information about endothelial dysfunction in the coronary arteries than IL-18 concentration in peripheral blood (8). With the use of off-pump CABG, researchers observed lower SIRS, faster postoperative recovery, and shorter hospital stay; however, local inflammatory events in the myocardium (4) were not attenuated. Even with the adjustment of surgical techniques, perioperative myocardial infarction remains the most common adverse complications, probably because of thinning of the glycocalyx during specific conditions of shear stress (3). Glycocalyx represents a highly fragile and unstable endothelial surface layer, which is primarily composed of syndecan-1, hyaluronic acid, heparan sulfate, and chondroitin sulfate (9). Integral glycocalyx assumes the mechanical load in the form of local torque, disperses it, and transduces the signal through proteoglycan chains to the anchored membrane proteins (10). The main result is endothelial nitric oxide synthase (eNOS) activation, which leads to the synthesis of endogenous nitric oxide (NO). NO has a vasodilatory effect and causes the reorganization of the actin cytoskeleton, providing the adaptation of intercellular contacts to the mechanical load (10). Shedding or total absence of a glycocalyx layer 6
leads to the direct mechanical load on the apical endothelial cell membrane and suppresses their NO production, resulting in high blood pressure, impairment of intercellular contacts with increased endometrial permeability, and higher endothelial activation status (10). Therefore, recently the perturbation of the endothelial glycocalyx is shown to be involved in critically ill patients with septic shock or acute respiratory distress syndrome (11), severely injured trauma patients in haemorrhagic shock or low plasma colloid osmotic pressure (12). It is also observed in patients with chronic kidney disease (13), type II diabetes (14), tumour angiogenesis (15), acute coronary syndrome (16), and in patients during CABG in characteristic patterns of released glycocalyx fragments (17). However, the relationship of pro-inflammatory cytokines and glycocalyx damage is still widely unexplored.
Hypothesis We hypothesize that the serum concentration of IL-18 in patients with ischemic heart disease measured immediately before CABG surgery is correlated with the level of activity of circulating lymphocytes. The activity of circulating lymphocytes was estimated using the expression of activated IL-18R1, adhesion receptors (L-selectin [CD62L], lymphocyte function associated antigen (LFA-1) [CD11b/CD18], LFA-2 [CD2]), and membrane expression or intracellular localization of cytotoxic mediators (FasL, TNF-related apoptosis-inducing ligand (TRAIL), perforin and granulysin) in their granule. Therefore, IL-18 could lead and predict lymphocyte– mediated cytotoxicity in dysfunctional and activated endothelial cells associated with glycocalyx shedding, as well as cause adverse events during and immediately after CABG surgery. Analysing these immunological parameters in the blood samples of patients taken from the sinus coronarius could be a valuable tool to shed more light in the series of events in the coronary
7
circulation leading to ischemic heart disease. In addition, analysing blood samples taken from the systemic circulation of these patients may be useful to evaluate endothelial dysfunction. Endothelial dysfunction occurs in various arteries of patients with ischemic heart disease beside coronary circulation, due to the systemic impact of unhealthy habits, inflammatory-metabolic, and genetic factors (1, 2), as well as seismic endothelial strain caused by intravascular fluid overload during surgeries that damage the glycocalyx (18). Furthermore, CABG surgery (19) and ischemia/reperfusion injury (3) may cause additional damage to the endothelial glycocalyx and lead to the activation of uncovered endothelial cells, primarily in the coronary circulation. IL-18 is a marker of endothelial dysfunction, that is expressed in human endothelial cells and covered by atheroma and mediate plaque rupture (7). In adult mice cardiomyocytes, IL-18 has been shown to induce ischemia/reperfusion injury (5). Unfortunately, the mechanism of action for IL-18 action is not well-understood in humans, nor is its correlation with cytotoxic mediators in circulating lymphocytes during CABG surgery. IL-18 may be more intensively expressed on the surface of activated endothelium in patients who underwent CABG surgery, after the shedding of glycocalyx components when the activation of dysfunctional endothelium becomes the main source of pro-inflammatory cytokines (IL-1β, IL-6, IL-15, TNF-α), chemokines (fractalkine, monocyte chemoattractant protein-1 (MCP1), granulocyte-colony stimulating protein [G-CSF]), and adhesion molecules (CXC/CC chemokine receptors, intercellular adhesion molecule [ICAM] -1 [CD54], ICAM-3 [CD50]) (20). We also hypothesise that the shedding or total absence of the glycocalyx layer during CABG surgery, not only worsens endothelial mechanical stress (17), but may also be the first step of atherosclerosis enhancing IL-18-mediated tethering and adhesion of IL-18R1 expressing CD2+CD62L+ T lymphocytes and NK cells for endothelial layer (20). This is supported by
8
previously published research demonstrating that: (I) hyaluronic acid cleaving during seismic tear expose receptors (CD44) for hyaluronic acid on the surface of endothelial cells (21); (II) uncoupled CD44 play a crucial role in adhesion of L-selectin expressing leukocytes under flow, but not under static condition (22); (III) damage of the glycocalyx causes up-regulation of ICAM1 combined with de-regulation of NFkB signalling in response to flow (23); and (IV) significant positive correlation between hyaluronic acid release and monocyte-endothelial cell adhesion occurs in vitro (24).
Testing of the hypothesis To evaluate our hypothesis we need to identify the plasma level of components in the IL-18 system. In addition, we need to evaluate possible effects of serum IL-18 due to the presence of its functional antagonists, including IL-18 receptor antagonist and IL-18 binding protein in patients undergoing CABG surgery. Both of these parameters would be evaluated using enzyme-linked immunosorbent assay (ELISA). Peripheral blood can be sampled immediately pre-CABG (time point 1), at the end of surgical revascularisation (time point 2), and 24 (time point 3) and 72 hours (time point 4) postoperatively. This would allow us to actively monitor the parameters of interest. Blood samples from the sinus coronarius, vena cava superior, vena cava inferior, and vena femoralis dextra or sinistra can also be also sampled (3-4 mL) at time point 1 and time point 2. Sampling blood this way will allow us to measure the parameters predominantly associated with coronary artery circulation (sinus coronarius), circulation in the head, neck and upper limbs (vena cava superior), chest, abdomen and lower limbs (vena cava inferior), and right or left lower limb (vena femoralis dextra or sinistra, respectively). We are currently conducting a study analysing the activation of IL-18R1 expression using triple CD3/CD56/IL-18R1 flow cytometry in the blood samples of patients who underwent CABG 9
surgery at time point 1. Our preliminary data shows an increase in IL-18R1 expression on the membrane of CD3+CD56-, CD3-CD56+, and CD3+CD56+ lymphocyte subsets in the coronary artery circulation measured in blood samples from the sinus coronarius, compared with those from peripheral blood in healthy study participants. These data suggest an increase in the sensitivity of lymphocytes associated with IL-18 stimulation in patients who underwent CABG. Therefore, it could be valuable to conduct a follow-up analysis to determine whether and how IL18 is correlated with cytotoxic molecule expression, including perforin, FasL, TRAIL, and granulysin at the mRNA and protein level in patients who underwent CABG surgery, using different methods (real time polymerase chain reaction, Western blotting, flow cytometry). Particular attention should be paid to the intracellular setting of cytotoxic molecules based on their relation with the cell surface membrane and co-localization with lysosomal-associated membrane protein 1 (LAMP-1), a marker of exocytotic granules, and perforin, as the prototype of the cytotoxic pore forming protein (25) using confocal microscopy. Different cellular positioning of cytotoxic mediators can signify their current role in on-going immune reaction during CABG surgery and possible killing of endothelial cells by silent, unscheduled apoptosis, which may be measured by the detection of circulating von Willebrand expressing endothelial cells in blood samples in vitro using flow cytometry. If our hypothesis is correct, the concentration of IL-18 in the coronary circulation before revascularization (time point 1) may be positively correlated with the preoperative Sintax score— a lesion-based angiographic grading tool to determine the complexity of coronary artery disease—based on advanced endothelial dysfunction. Systemic IL-18 levels could also be correlated with broad spectrum laboratory markers, imaging grading tools, and clinical parameters of endothelial dysfunction, which is manifested in different organs in patients with coronary artery disease before the CABG surgery. In addition, further analysis may be useful to 10
evaluate the relationship between IL-18 and N-terminal proBrain Natriuremic Peptide (NTproBNP), c troponin (TN)-T, ST segment depression, ejection fraction, heart wall motility, Agatston score (26), known blood pressure parameters of heart condition and function, percentage of von Willebrand factor positive cells, MCP-1, IL-15 levels, intima/media thickness of carotid artery, flow velocity and the degree of the stenosis of carotid and renal artery, resistance index of renal artery to determine vessel condition and glomerular filtration rate, creatinine clearance, albumin/creatinine ratio, albuminuria to determine kidney function as well as with metabolic parameters by measuring the levels of fasting glucose, HbA1c, uric acid, homocistein, total cholesterol, low-density lipoproteins, high-density lipoproteins, triglycerides), Framingham score (27) and EuroScore (28), as the algorithms used to estimate the 10-year cardiovascular risk of an individual. The relationship between IL-18 and the degradation products of the glycosaminoglycan layer, including levels of syndecan 1, hyaluronic acid, chondroitin sulfate, heparin sulfate, and soluble von Willebrand factor would be very important in the light of anesthesiology-surgical procedure of myocardial revascularisation, because they have been shown to be important factors of glycocalyx shedding in different pathophysiological settings (11,12). The glycocalyx-uncovered endothelium can express and secrete more IL-18, among other activation and chemotactic markers (ICAM-1, IL-15, IL-17A, TNF-a, MCP-1, fractalkine, matrix metalloproteinase) (9), and can enhance the devastating circle of an inflammatory reaction. Correlation of IL-18 with particular marker of glycocalyx shedding could clarify the role of glycocalyx in the expression of IL-18. In patients who underwent on-pump CABG surgery, gradual decreases of proliferation and interferon gamma secretion in CD4+ peripheral blood T cells, after the phorbol myristate acetate/ionomycin stimulation in vitro, was observed in early postoperative period (26). It could suggest down-regulation of IL-18–mediated inflammatory
11
reaction and acceleration of the healing process, which going ahead with rapid overall turnover rate of glycocalyx component (a half a day to several days) (8). Altogether, dynamic monitoring of the aforementioned parameters represents a reliable time window during recovery of patients undergoing CABG surgery.
Discussion Surgical revascularization impacts immune parameters, primarily cell–mediated immunity (5). In their study, Akbas et al. (29) showed a significant decrease in the number of T lymphocytes (CD4+ and CD8+), B lymphocytes, and NK cells in the peripheral blood of patients who underwent myocardial revascularisation, particularly in those who underwent on-pump CABG surgery. The down-regulation of cell immunity could be responsible for the occurrence of infections (pneumonia, urinary infection, sepsis and multiorgan failure), which complicate recovery early in the postoperative period (30). In particular, perioperative myocardial infarction is the most common complication after myocardial revascularisation. A high level of troponin T is usually found in patients who have not been properly diagnosed with myocardial infarction based on European Society of Cardiology guidelines (31). To our knowledge, whether the decrease in lymphocytes in the peripheral blood is due to their recruitment in the myocardium or lymphocyte infiltration is unknown. In one study, researchers observed T and NK cells in patients who died immediately after an acute myocardial infarction, shortly afterward, or even longer (32). This is supported by previously published data indicating that peripheral blood NK cells have increased activity (27) and they could travel toward myocardial chemotactic factors, which are overexpressed during ischemia/reperfusion injury (1). Peripheral blood NK cells have also been shown to move toward sites with damaged endothelial cells all over the body. Together with shear stress (18) and surgical procedures (19), ischemia/reperfusion injury (3) have been shown 12
to promptly cause disorganisation of endothelial glycocalyx at the local (coronary) and systemic level. Glycocalyx physiologically protects the endothelium from mechanical stress and superabundant expression of activating receptors, preventing the access of leukocytes and soluble plasma components to the surface of the endothelium, and preventing inflammation (10). In a 3dimensional cell culture model, investigators demonstrated that endothelial cells developed a proinflammatory phenotype after the enzymatic degradation of the glycocalyx and subsequent exposure to shear stress, leading to an increase in leukocyte adhesion (23). Therefore, the shedding of degradable components or total absence of the glycocalyx layer during this pathophysiological process, in addition to mechanically inducing endothelial dysfunction (10), may fine-tune endothelial functions, including activation of immune response. It was recently suggested that fragile glycocalyx release endogenous damage associated molecular patterns, which trigger the pro-inflammatory cascade by engaging toll like receptor 4 (TLR4) (16). Activation of TLR4 is mediated by NFkB, which cause cytokine production, such as IL-6 (16). IL-6 in combination with IL-1β, IL-8, and TNFα, induced by IL-18, contribute to plaque instability, causing pain (33). These cytokines can worsen inflammatory response during unstable angina and acute myocardial infarction. Significant increases in the levels of these cytokines as well as C-reactive protein (CRP) have been observed in the serum of patients with coronary artery disease (33). Thinned, disorganized glycocalyx increases adhesion capacity of activated leukocytes from the blood stream of the endothelium in rats (24). This was also shown in a 3-dimensional cell culture model (23) that was mediated by chemoattractant and adhesion molecules, including fractalkine, MCP-1, IL-15, and IL-18.
13
MCP-1 (34) and IL-15 (35) are expressed on the plaque surface and seem to mediate the homing of circulating T and NK cells at the site of inflammation, such as the locally thickened subendothelial layer during the formation of early atherosclerotic lesion (36). Similarly, we demonstrated earlier the IL-15 abundant expression in viable cardiomyocytes surrounding necrotic infarction zone (37). IL-15 may increase the cytotoxic potential of immune cells attached to the endothelium. In particular, IL-15 has been shown to increase perforin mRNA and/or protein expression and Fas ligand (38); granulysin (39), even in tolerogenic; and uterine NK cells in early pregnancy. However, it increased their cytotoxicity only against NK sensitive targets (40). This is similar to our recent results showing that IL-15 stimulation of DL in vitro decreased colocalization of cytotoxic 9 kDa ganulysin form and LAMP-1, potentially decreasing their release from the cell (41). The regulatory 15 kDa granulysin was stored in LAMP-1 expressing granules prepared for exocytosis (41). However, in the peripheral blood lymphocytes of the pregnant women, IL-15 had the opposite effect, significantly increased 9 KDa granulysin, and diminished 15 kDa granulysin in colocalization with LAMP-1 (41), suggesting that the effects of IL-15 is dependent on the entire cellular microenvironment. High IL 18/IL-10 ratio is an independent predictor of adverse cardiovascular events in hospitalized patients (42). Antagonist of IL-18 function, IL-18BP reduces intensity of inflammation and protects myocardium after ischemia and acute myocardial infarction (43). IL-18 is considered a strong activator of cytotoxic action, which may have complementary function with IL-15 in cell–mediated cytotoxicity in patients who underwent revascularisation. Specifically, IL-18 is a member of the broad IL-1 family that acts as a strong pro-inflammatory cytokine associated with Th1 immune response (40). The main promoter of Th1 immune response, IL-12 encourages expression of activated IL-18R1 on NK cells and T lymphocytes (41). IL-18 can induce leukocyte extravasation through upregulation of endothelial cell adhesion 14
molecules, and mediated recruitment of monocytes, lymphocytes, and neutrophil at the site of plaque formation; this was previously reported in the synovial membrane of patients with rheumatoid arthritis (44). Moreover, IL-18 enhances mouse (42) and human (43) T and NK cell IFN-γ production after lypopolisaccharide treatment and increases cytotoxic activity of NK cells indispensably of IFN-γ. IL-18 strongly augments perforin and Fas ligand–mediated killing of murine NK cell clones (45), but does not enhance TRAIL expression in animal models (46). In humans however, locally activated T cells are thought to kill smooth muscle cells by TRAILmediated apoptosis, which greatly determine atherosclerotic plaque rupture (46). The mechanisms of secretory cytotoxicity mediated by perforin and granulysin have not been broadly investigated in patients who underwent CABG surgery. In an in vitro model of internalisation of blood particles, endothelial cells with disrupted glycocalyx increased the influx of polystiren nanoparticles (47). Based on these data, we believe that cytotoxic granules fill with cytotoxic mediators (perforin, granulysin) that are released in the synaptic gap between an activated NK cell and the endothelium, and could easily target endothelial cells. This observation is supported by data showing increased plasma concentration of IL-18 in ischemic rather than necrotic myocardium (48), and variation of IL-18 genes represents increased susceptibility to acute myocardial infarction (49). IL-18 correlates with NT pro-BNP and hsCRP, and cause cardiomyocytes hypertrophy, dysfunction of contractility, and unwanted myocardial recasting (48). In conclusion, IL-18 activated lymphocytes from the circulation of patients with coronary artery disease adhere to the endothelium with disturbed glycocalyx, and damage or more likely, enters the subintimal space faster, thereby increasing existing or starting to form new plaque during or immediately after CABG surgery. Results obtained from this study will contribute to general
15
understanding of endothelial dysfunction, and potentially lead to the change of exiting anaesthesia-surgical techniques to prevent damage of endothelia during anaesthesia and surgery.
16
Acknowledgements The preliminary experiments were financed by the financial support of University of Rijeka, grant No. 13.06.1.1.12.
Conflicts of interest statement Authors declare no conflicts of interest.
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S0306-9877(17)30175-5 http://dx.doi.org/10.1016/j.mehy.2017.05.009 YMEHY 8553
To appear in:
Medical Hypotheses
Received Date: Accepted Date:
16 February 2017 6 May 2017
Please cite this article as: S. Jadranko, V.S. Tokmadzic, K. Danijel, M. Igor, V.D. Nada, B. Sanja, R. Marijana, L.B. Ana, L. Gordana, Endothelial dysfunction mediated by interleukin-18 in patients with ischemic heart disease undergoing coronary artery bypass grafting surgery, Medical Hypotheses (2017), doi: http://dx.doi.org/10.1016/ j.mehy.2017.05.009
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Endothelial dysfunction mediated by interleukin-18 in patients with ischemic heart disease undergoing coronary artery bypass grafting surgery
Sokolic Jadranko1, Vlatka Sotosek Tokmadzic1,2, Knezevic Danijel2, Medved Igor3, Vukelic Damjani Nada4, Balen Sanja4, Rakic Marijana5, Lanca Bastiancic Ana5, Laskarin Gordana5,6
1
Clinic of Anesthesiology and Intensive Care Medicine, Clinical Hospital Center Rijeka, 51 000
Rijeka, Kresimirova 42, Croatia 2
Department of Anesthesiology, Reanimatology and Intensive Care, Faculty of Medicine,
University of Rijeka, 51000 Rijeka, B. Branchetta 20, Croatia 3
Department of Surgery, Faculty of Medicine, University of Rijeka, 51000 Rijeka, Tome Strizica
3, Croatia 4
Department of Transfusion Medicine, Clinical Hospital Center Rijeka, 51 000 Rijeka,
Kresimirova 42, Croatia 5
Division of Cardiology, Hospital for Medical Rehabilitation of the Hearth and Lung Diseases
and Rheumatism “Thalassotherapia” Opatija, 51410 Opatija, M. Tita 188, Croatia 6
Department of Physiology and Immunology, Medical Faculty, University of Rijeka, 51000
Rijeka, B. Branchetta 20, Croatia
Corresponding author: Vlatka Sotosek Tokmadzic, MD, PhD Department of Anesthesiology, Reanimatology and Intensive Care, Faculty of Medicine, University of Rijeka, Medical Faculty, University of Rijeka B. Branchetta 20 1
51000 Rijeka, Croatia Tel. ++ 385 51 65 11 85 Fax number: ++ 385 51 67 56 99 E-mail: [email protected]
Running head: IL-18 mediated endothelial dysfunction
2
Abstract When medication management or percutaneous coronary intervention is not successful in patients with advanced ischaemic heart disease, surgical revascularisation—predominantly coronary artery bypass grafting (CABG)—is considered the gold standard. However, CABG surgery can lead to ischaemia/reperfusion injury, which is characterized by a strong inflammatory response. Interleukin (IL)-18, is a strong inflammatory mediator, that is released from cardiomyocytes and can be found in the systemic circulation of patients during and immediately after CABG surgery. The existing damage of endothelial glycocalyx in patients with ischaemic heart disease is further impaired concurrently during the surgery due to the anaesthesia-surgical technique used and intravascular fluid loading. This results in the increased incidence of adverse events, including myocardial infarction. IL-18 leads to the activation of lymphocyte cytotoxicity via cytotoxic mediators (Fas ligand, Tumor necrosis factor (TNF)-related apoptosis-inducing ligand, perforin, and granulysin). We hypothesize that IL-18 is released locally in the heart and the systemic circulation in patients undergoing CABG surgery and may be correlated with the level of activity of circulating lymphocytes. In turn, this may lead to lymphocyte-mediated cytotoxicity directed toward damaged and activated endothelial cells. Shear stress glycocalyx, as well as damaged and activated endothelial cells then become the main the source of pro-inflammatory cytokines, chemokines, and adhesion molecules. These attract activated lymphocytes to adhere to the endothelium or enter the subintimal layer, increasing existing or initiating the formation of new plaques, which leads to the development of myocardial infarction during or shortly after surgery. To evaluate our hypothesis, we will measure the local concentration of IL-18 in the sinus coronarius and systemic circulation. These values will then be correlated with immunological and biochemical parameters, predominantly with the concentration of degradation products of glycocalyx and cytotoxic mediators in activated lymphocytes. If our hypothesis is correct, 3
measuring the IL-18 concentration that is responsible for glycocalyx deterioration, may become a useful tool for predicting myocardial infarction occurrence in patients undergoing CABG surgery.
4
Introduction Endothelial dysfunction is caused by chronic endogenous inflammation and altered metabolic conditions on the endothelium. It develops into a chronic process of varying intensity in different organs and manifests itself in a range of clinical entities (1). Recently, it has become evident that identifying the unique roles of inflammation and metabolism share in patient populations with atherosclerotic disease. This relationship is important to identify potentially anti-inflammatory and/or metabolic therapeutic approaches to prevent and stabilize patients with atherosclerotic disease (2). Ischemic heart disease occurs from endothelial dysfunction of large or small coronary arteries and usually presents as acute or chronic coronary syndrome (3). Surgical revascularization is the gold standard to treat patients with coronary artery disease, in cases where complementary treatment, including proper nutrition, moderate physical activity, medication, and percutaneous coronary intervention, have become insufficiently effective (4). However, there is an increased risk of creating oxidative stress associated with ischemia/reperfusion injury via on-pump coronary artery bypass grafting surgery (CABG) (5). CABG is associated with systemic inflammatory response (SIRS), which can be measured using the systemic concentration of tumour necrosis factor alpha (TNF-α) (6). In an in vitro model, TNF-α and H2O2 induced the expression of interleukin (IL)-18 mRNA and precursor protein in cardiomyocytes; IL-18 was also released into the culture supernatants (6). In an experimental animal model, researchers have shown that isolated cardiomyocytes enhanced oxidative stress and biologically active IL-18 expression
via
the
IKK-dependent
nuclear
factor
(NF)-kappa
B
pathway
after
ischemia/reperfusion (5). In humans, IL-18 is a key pro-inflammatory mediator in the pathogenesis and deterioration of patients with heart and vascular disease (5). It mediates plaque
5
rupture and the development of acute coronary syndrome, which is a predictive factor of myocardial infarction and heart failure in hospitalized patients three to seven months after myocardial infarction (7), potentially by increasing cytotoxicity of activated T and NK cells recruited in subintimal layer. Patients who underwent CABG surgery and valvular replacement had similar increases in plasma levels of circulating IL-18 protein. However, in the epicardial adipose tissue of patients who underwent myocardial revascularization, gene expression of the components of IL-18 system, such as IL-18, activating IL-18 receptor (IL-18R) 1, and IL-18R accessory protein, were higher than those in patients who underwent valvular replacement (8). These data indicate that local production of pro-inflammatory protein IL-18, and its autocrine or paracrine effects could provide more information about endothelial dysfunction in the coronary arteries than IL-18 concentration in peripheral blood (8). With the use of off-pump CABG, researchers observed lower SIRS, faster postoperative recovery, and shorter hospital stay; however, local inflammatory events in the myocardium (4) were not attenuated. Even with the adjustment of surgical techniques, perioperative myocardial infarction remains the most common adverse complications, probably because of thinning of the glycocalyx during specific conditions of shear stress (3). Glycocalyx represents a highly fragile and unstable endothelial surface layer, which is primarily composed of syndecan-1, hyaluronic acid, heparan sulfate, and chondroitin sulfate (9). Integral glycocalyx assumes the mechanical load in the form of local torque, disperses it, and transduces the signal through proteoglycan chains to the anchored membrane proteins (10). The main result is endothelial nitric oxide synthase (eNOS) activation, which leads to the synthesis of endogenous nitric oxide (NO). NO has a vasodilatory effect and causes the reorganization of the actin cytoskeleton, providing the adaptation of intercellular contacts to the mechanical load (10). Shedding or total absence of a glycocalyx layer 6
leads to the direct mechanical load on the apical endothelial cell membrane and suppresses their NO production, resulting in high blood pressure, impairment of intercellular contacts with increased endometrial permeability, and higher endothelial activation status (10). Therefore, recently the perturbation of the endothelial glycocalyx is shown to be involved in critically ill patients with septic shock or acute respiratory distress syndrome (11), severely injured trauma patients in haemorrhagic shock or low plasma colloid osmotic pressure (12). It is also observed in patients with chronic kidney disease (13), type II diabetes (14), tumour angiogenesis (15), acute coronary syndrome (16), and in patients during CABG in characteristic patterns of released glycocalyx fragments (17). However, the relationship of pro-inflammatory cytokines and glycocalyx damage is still widely unexplored.
Hypothesis We hypothesize that the serum concentration of IL-18 in patients with ischemic heart disease measured immediately before CABG surgery is correlated with the level of activity of circulating lymphocytes. The activity of circulating lymphocytes was estimated using the expression of activated IL-18R1, adhesion receptors (L-selectin [CD62L], lymphocyte function associated antigen (LFA-1) [CD11b/CD18], LFA-2 [CD2]), and membrane expression or intracellular localization of cytotoxic mediators (FasL, TNF-related apoptosis-inducing ligand (TRAIL), perforin and granulysin) in their granule. Therefore, IL-18 could lead and predict lymphocyte– mediated cytotoxicity in dysfunctional and activated endothelial cells associated with glycocalyx shedding, as well as cause adverse events during and immediately after CABG surgery. Analysing these immunological parameters in the blood samples of patients taken from the sinus coronarius could be a valuable tool to shed more light in the series of events in the coronary
7
circulation leading to ischemic heart disease. In addition, analysing blood samples taken from the systemic circulation of these patients may be useful to evaluate endothelial dysfunction. Endothelial dysfunction occurs in various arteries of patients with ischemic heart disease beside coronary circulation, due to the systemic impact of unhealthy habits, inflammatory-metabolic, and genetic factors (1, 2), as well as seismic endothelial strain caused by intravascular fluid overload during surgeries that damage the glycocalyx (18). Furthermore, CABG surgery (19) and ischemia/reperfusion injury (3) may cause additional damage to the endothelial glycocalyx and lead to the activation of uncovered endothelial cells, primarily in the coronary circulation. IL-18 is a marker of endothelial dysfunction, that is expressed in human endothelial cells and covered by atheroma and mediate plaque rupture (7). In adult mice cardiomyocytes, IL-18 has been shown to induce ischemia/reperfusion injury (5). Unfortunately, the mechanism of action for IL-18 action is not well-understood in humans, nor is its correlation with cytotoxic mediators in circulating lymphocytes during CABG surgery. IL-18 may be more intensively expressed on the surface of activated endothelium in patients who underwent CABG surgery, after the shedding of glycocalyx components when the activation of dysfunctional endothelium becomes the main source of pro-inflammatory cytokines (IL-1β, IL-6, IL-15, TNF-α), chemokines (fractalkine, monocyte chemoattractant protein-1 (MCP1), granulocyte-colony stimulating protein [G-CSF]), and adhesion molecules (CXC/CC chemokine receptors, intercellular adhesion molecule [ICAM] -1 [CD54], ICAM-3 [CD50]) (20). We also hypothesise that the shedding or total absence of the glycocalyx layer during CABG surgery, not only worsens endothelial mechanical stress (17), but may also be the first step of atherosclerosis enhancing IL-18-mediated tethering and adhesion of IL-18R1 expressing CD2+CD62L+ T lymphocytes and NK cells for endothelial layer (20). This is supported by
8
previously published research demonstrating that: (I) hyaluronic acid cleaving during seismic tear expose receptors (CD44) for hyaluronic acid on the surface of endothelial cells (21); (II) uncoupled CD44 play a crucial role in adhesion of L-selectin expressing leukocytes under flow, but not under static condition (22); (III) damage of the glycocalyx causes up-regulation of ICAM1 combined with de-regulation of NFkB signalling in response to flow (23); and (IV) significant positive correlation between hyaluronic acid release and monocyte-endothelial cell adhesion occurs in vitro (24).
Testing of the hypothesis To evaluate our hypothesis we need to identify the plasma level of components in the IL-18 system. In addition, we need to evaluate possible effects of serum IL-18 due to the presence of its functional antagonists, including IL-18 receptor antagonist and IL-18 binding protein in patients undergoing CABG surgery. Both of these parameters would be evaluated using enzyme-linked immunosorbent assay (ELISA). Peripheral blood can be sampled immediately pre-CABG (time point 1), at the end of surgical revascularisation (time point 2), and 24 (time point 3) and 72 hours (time point 4) postoperatively. This would allow us to actively monitor the parameters of interest. Blood samples from the sinus coronarius, vena cava superior, vena cava inferior, and vena femoralis dextra or sinistra can also be also sampled (3-4 mL) at time point 1 and time point 2. Sampling blood this way will allow us to measure the parameters predominantly associated with coronary artery circulation (sinus coronarius), circulation in the head, neck and upper limbs (vena cava superior), chest, abdomen and lower limbs (vena cava inferior), and right or left lower limb (vena femoralis dextra or sinistra, respectively). We are currently conducting a study analysing the activation of IL-18R1 expression using triple CD3/CD56/IL-18R1 flow cytometry in the blood samples of patients who underwent CABG 9
surgery at time point 1. Our preliminary data shows an increase in IL-18R1 expression on the membrane of CD3+CD56-, CD3-CD56+, and CD3+CD56+ lymphocyte subsets in the coronary artery circulation measured in blood samples from the sinus coronarius, compared with those from peripheral blood in healthy study participants. These data suggest an increase in the sensitivity of lymphocytes associated with IL-18 stimulation in patients who underwent CABG. Therefore, it could be valuable to conduct a follow-up analysis to determine whether and how IL18 is correlated with cytotoxic molecule expression, including perforin, FasL, TRAIL, and granulysin at the mRNA and protein level in patients who underwent CABG surgery, using different methods (real time polymerase chain reaction, Western blotting, flow cytometry). Particular attention should be paid to the intracellular setting of cytotoxic molecules based on their relation with the cell surface membrane and co-localization with lysosomal-associated membrane protein 1 (LAMP-1), a marker of exocytotic granules, and perforin, as the prototype of the cytotoxic pore forming protein (25) using confocal microscopy. Different cellular positioning of cytotoxic mediators can signify their current role in on-going immune reaction during CABG surgery and possible killing of endothelial cells by silent, unscheduled apoptosis, which may be measured by the detection of circulating von Willebrand expressing endothelial cells in blood samples in vitro using flow cytometry. If our hypothesis is correct, the concentration of IL-18 in the coronary circulation before revascularization (time point 1) may be positively correlated with the preoperative Sintax score— a lesion-based angiographic grading tool to determine the complexity of coronary artery disease—based on advanced endothelial dysfunction. Systemic IL-18 levels could also be correlated with broad spectrum laboratory markers, imaging grading tools, and clinical parameters of endothelial dysfunction, which is manifested in different organs in patients with coronary artery disease before the CABG surgery. In addition, further analysis may be useful to 10
evaluate the relationship between IL-18 and N-terminal proBrain Natriuremic Peptide (NTproBNP), c troponin (TN)-T, ST segment depression, ejection fraction, heart wall motility, Agatston score (26), known blood pressure parameters of heart condition and function, percentage of von Willebrand factor positive cells, MCP-1, IL-15 levels, intima/media thickness of carotid artery, flow velocity and the degree of the stenosis of carotid and renal artery, resistance index of renal artery to determine vessel condition and glomerular filtration rate, creatinine clearance, albumin/creatinine ratio, albuminuria to determine kidney function as well as with metabolic parameters by measuring the levels of fasting glucose, HbA1c, uric acid, homocistein, total cholesterol, low-density lipoproteins, high-density lipoproteins, triglycerides), Framingham score (27) and EuroScore (28), as the algorithms used to estimate the 10-year cardiovascular risk of an individual. The relationship between IL-18 and the degradation products of the glycosaminoglycan layer, including levels of syndecan 1, hyaluronic acid, chondroitin sulfate, heparin sulfate, and soluble von Willebrand factor would be very important in the light of anesthesiology-surgical procedure of myocardial revascularisation, because they have been shown to be important factors of glycocalyx shedding in different pathophysiological settings (11,12). The glycocalyx-uncovered endothelium can express and secrete more IL-18, among other activation and chemotactic markers (ICAM-1, IL-15, IL-17A, TNF-a, MCP-1, fractalkine, matrix metalloproteinase) (9), and can enhance the devastating circle of an inflammatory reaction. Correlation of IL-18 with particular marker of glycocalyx shedding could clarify the role of glycocalyx in the expression of IL-18. In patients who underwent on-pump CABG surgery, gradual decreases of proliferation and interferon gamma secretion in CD4+ peripheral blood T cells, after the phorbol myristate acetate/ionomycin stimulation in vitro, was observed in early postoperative period (26). It could suggest down-regulation of IL-18–mediated inflammatory
11
reaction and acceleration of the healing process, which going ahead with rapid overall turnover rate of glycocalyx component (a half a day to several days) (8). Altogether, dynamic monitoring of the aforementioned parameters represents a reliable time window during recovery of patients undergoing CABG surgery.
Discussion Surgical revascularization impacts immune parameters, primarily cell–mediated immunity (5). In their study, Akbas et al. (29) showed a significant decrease in the number of T lymphocytes (CD4+ and CD8+), B lymphocytes, and NK cells in the peripheral blood of patients who underwent myocardial revascularisation, particularly in those who underwent on-pump CABG surgery. The down-regulation of cell immunity could be responsible for the occurrence of infections (pneumonia, urinary infection, sepsis and multiorgan failure), which complicate recovery early in the postoperative period (30). In particular, perioperative myocardial infarction is the most common complication after myocardial revascularisation. A high level of troponin T is usually found in patients who have not been properly diagnosed with myocardial infarction based on European Society of Cardiology guidelines (31). To our knowledge, whether the decrease in lymphocytes in the peripheral blood is due to their recruitment in the myocardium or lymphocyte infiltration is unknown. In one study, researchers observed T and NK cells in patients who died immediately after an acute myocardial infarction, shortly afterward, or even longer (32). This is supported by previously published data indicating that peripheral blood NK cells have increased activity (27) and they could travel toward myocardial chemotactic factors, which are overexpressed during ischemia/reperfusion injury (1). Peripheral blood NK cells have also been shown to move toward sites with damaged endothelial cells all over the body. Together with shear stress (18) and surgical procedures (19), ischemia/reperfusion injury (3) have been shown 12
to promptly cause disorganisation of endothelial glycocalyx at the local (coronary) and systemic level. Glycocalyx physiologically protects the endothelium from mechanical stress and superabundant expression of activating receptors, preventing the access of leukocytes and soluble plasma components to the surface of the endothelium, and preventing inflammation (10). In a 3dimensional cell culture model, investigators demonstrated that endothelial cells developed a proinflammatory phenotype after the enzymatic degradation of the glycocalyx and subsequent exposure to shear stress, leading to an increase in leukocyte adhesion (23). Therefore, the shedding of degradable components or total absence of the glycocalyx layer during this pathophysiological process, in addition to mechanically inducing endothelial dysfunction (10), may fine-tune endothelial functions, including activation of immune response. It was recently suggested that fragile glycocalyx release endogenous damage associated molecular patterns, which trigger the pro-inflammatory cascade by engaging toll like receptor 4 (TLR4) (16). Activation of TLR4 is mediated by NFkB, which cause cytokine production, such as IL-6 (16). IL-6 in combination with IL-1β, IL-8, and TNFα, induced by IL-18, contribute to plaque instability, causing pain (33). These cytokines can worsen inflammatory response during unstable angina and acute myocardial infarction. Significant increases in the levels of these cytokines as well as C-reactive protein (CRP) have been observed in the serum of patients with coronary artery disease (33). Thinned, disorganized glycocalyx increases adhesion capacity of activated leukocytes from the blood stream of the endothelium in rats (24). This was also shown in a 3-dimensional cell culture model (23) that was mediated by chemoattractant and adhesion molecules, including fractalkine, MCP-1, IL-15, and IL-18.
13
MCP-1 (34) and IL-15 (35) are expressed on the plaque surface and seem to mediate the homing of circulating T and NK cells at the site of inflammation, such as the locally thickened subendothelial layer during the formation of early atherosclerotic lesion (36). Similarly, we demonstrated earlier the IL-15 abundant expression in viable cardiomyocytes surrounding necrotic infarction zone (37). IL-15 may increase the cytotoxic potential of immune cells attached to the endothelium. In particular, IL-15 has been shown to increase perforin mRNA and/or protein expression and Fas ligand (38); granulysin (39), even in tolerogenic; and uterine NK cells in early pregnancy. However, it increased their cytotoxicity only against NK sensitive targets (40). This is similar to our recent results showing that IL-15 stimulation of DL in vitro decreased colocalization of cytotoxic 9 kDa ganulysin form and LAMP-1, potentially decreasing their release from the cell (41). The regulatory 15 kDa granulysin was stored in LAMP-1 expressing granules prepared for exocytosis (41). However, in the peripheral blood lymphocytes of the pregnant women, IL-15 had the opposite effect, significantly increased 9 KDa granulysin, and diminished 15 kDa granulysin in colocalization with LAMP-1 (41), suggesting that the effects of IL-15 is dependent on the entire cellular microenvironment. High IL 18/IL-10 ratio is an independent predictor of adverse cardiovascular events in hospitalized patients (42). Antagonist of IL-18 function, IL-18BP reduces intensity of inflammation and protects myocardium after ischemia and acute myocardial infarction (43). IL-18 is considered a strong activator of cytotoxic action, which may have complementary function with IL-15 in cell–mediated cytotoxicity in patients who underwent revascularisation. Specifically, IL-18 is a member of the broad IL-1 family that acts as a strong pro-inflammatory cytokine associated with Th1 immune response (40). The main promoter of Th1 immune response, IL-12 encourages expression of activated IL-18R1 on NK cells and T lymphocytes (41). IL-18 can induce leukocyte extravasation through upregulation of endothelial cell adhesion 14
molecules, and mediated recruitment of monocytes, lymphocytes, and neutrophil at the site of plaque formation; this was previously reported in the synovial membrane of patients with rheumatoid arthritis (44). Moreover, IL-18 enhances mouse (42) and human (43) T and NK cell IFN-γ production after lypopolisaccharide treatment and increases cytotoxic activity of NK cells indispensably of IFN-γ. IL-18 strongly augments perforin and Fas ligand–mediated killing of murine NK cell clones (45), but does not enhance TRAIL expression in animal models (46). In humans however, locally activated T cells are thought to kill smooth muscle cells by TRAILmediated apoptosis, which greatly determine atherosclerotic plaque rupture (46). The mechanisms of secretory cytotoxicity mediated by perforin and granulysin have not been broadly investigated in patients who underwent CABG surgery. In an in vitro model of internalisation of blood particles, endothelial cells with disrupted glycocalyx increased the influx of polystiren nanoparticles (47). Based on these data, we believe that cytotoxic granules fill with cytotoxic mediators (perforin, granulysin) that are released in the synaptic gap between an activated NK cell and the endothelium, and could easily target endothelial cells. This observation is supported by data showing increased plasma concentration of IL-18 in ischemic rather than necrotic myocardium (48), and variation of IL-18 genes represents increased susceptibility to acute myocardial infarction (49). IL-18 correlates with NT pro-BNP and hsCRP, and cause cardiomyocytes hypertrophy, dysfunction of contractility, and unwanted myocardial recasting (48). In conclusion, IL-18 activated lymphocytes from the circulation of patients with coronary artery disease adhere to the endothelium with disturbed glycocalyx, and damage or more likely, enters the subintimal space faster, thereby increasing existing or starting to form new plaque during or immediately after CABG surgery. Results obtained from this study will contribute to general
15
understanding of endothelial dysfunction, and potentially lead to the change of exiting anaesthesia-surgical techniques to prevent damage of endothelia during anaesthesia and surgery.
16
Acknowledgements The preliminary experiments were financed by the financial support of University of Rijeka, grant No. 13.06.1.1.12.
Conflicts of interest statement Authors declare no conflicts of interest.
17
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