THE ROLE OF RED BLOOD CELLS IN CARDIAC AND ENDOTHELIAL DYSFUNCTION IN CARDIOMETABOLIC DISEASE

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From Unit of Cardiology, Department of Medicine, Solna Karolinska Institutet, Stockholm, Sweden

THE ROLE OF RED BLOOD CELLS IN CARDIAC AND ENDOTHELIAL

DYSFUNCTION IN CARDIOMETABOLIC DISEASE

Tong Jiao 焦通

Stockholm 2022

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All previously published papers were reproduced with permission from the publisher.

Published by Karolinska Institutet.

Printed by Universitetsservice US-AB, 2022

ISBN 978-91-8016-607-2

Cover illustration: "The building Aula Medica in summer". Photoed by KK in 2020.

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The Role of Red Blood Cells in Cardiac and Endothelial Dysfunction in Cardiometabolic Disease

THESIS FOR DOCTORAL DEGREE (Ph.D.)

By

Tong Jiao

The thesis will be defended in public at Jan Lindsten Lecture Hall A4:04, Karolinska University Hospital, Solna.

Monday, May 9, 2022, at 09:00 a.m.

Principal Supervisor:

Professor John Pernow Karolinska Institutet

Department of Medicine Solna Division of Cardiology

Co-supervisor(s):

Senior Researcher Jiangning Yang Karolinska Institutet

Asst. Prof. Zhichao Zhou Karolinska Institutet

Opponent:

Assoc. Prof. Alexandru Schiopu Lund University

Department of Clinical Sciences Division of Cardiovascular Research Examination Board:

Professor Tomas Nyström Karolinska Institutet

Department of Clinical Research and Education Division of Internal Medicine

Professor Daniel Ketelhuth Karolinska Institutet

Department of Medicine Solna Division of Cardiovascular Medicine Professor Malin Levin

University of Gothenburg Institute of Medicine

Department of Molecular and Clinical Medicine

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To my grey hair grew in my PhD life.

仁心，仁术；坚定，坚持

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ABSTRACT

Background

Cardiometabolic disease, which includes the combination of cardiovascular disease,

especially coronary artery disease and type 2 diabetes (T2D), is a major health problem and cause of mortality worldwide. ST-elevation myocardial infarction (STEMI) occurs when coronary arteries are occluded and is regarded as a life-threatening disorder. In these situations, both cardiac and endothelial function are impaired but specific treatment to alleviate the dysfunction is lacking which is partially due to that the underlying mechanisms remain unclear.

Red blood cells (RBCs) have for long been considered as passive transporters of respiratory gases. Emerging evidence suggests that RBCs are critically involved in physiological cardiovascular regulation by exporting nitric oxide (NO) bioactivity and adenosine

triphosphate, especially under hypoxic and ischemic conditions. It has been further suggested that dysfunctional RBCs may act as mediators of cardiovascular injury under pathological conditions. However, the role of RBCs in patients with cardiovascular disease associated with T2D has not been explored.

Purpose

The purpose of this thesis was to investigate the role of RBCs in the development of cardiac and endothelial dysfunction in cardiometabolic disease.

Methods and results

In Study I, RBCs from both patients and mice with T2D were given to isolated Langendorff- perfused hearts from mice or rats ex vivo at the onset of myocardial ischemia followed by reperfusion. The post-ischemic cardiac recovery was impaired and infarct size was increased via a mechanism depending on upregulated RBC-arginase which led to increased formation of reactive oxygen species by the NO-producing enzyme NO synthase.

In Study II, RBCs from patients with T2D under poor or improved glycemic control were given to Langendorff-perfused hearts or incubated with rat aorta. Improvement in glycemic control attenuated cardiac but not endothelial dysfunction induced the by RBCs. RBC- arginase activity was reduced following improvement in glycemic control. Inhibition of arginase attenuated the negative effect of RBCs on cardiac function irrespective of the glycemic control.

In Study III, stimulation of the NO receptor soluble guanylyl cyclase (sGC) in RBCs from patients with T2D attenuated the impairment in post-ischemic cardiac recovery and the increase in infarct size induced by the RBCs. The supernatant collected from RBCs incubated with the sGC stimulator also improved post-ischemic cardiac recovery and reduced infarct size. sGC stimulation in RBCs increased export of cyclic guanosine monophosphate (cGMP) to the supernatant and phosphorylation of cardiac vasodilator phosphoprotein as a marker of activation of cGMP-dependent protein kinase G. This suggests that cGMP may be the cardioprotective mediator from the RBCs.

In Study IV, RBCs from patients with STEMI protected against post-ischemic cardiac dysfunction and reduced infarct size in isolated hearts subjected to ischemia-reperfusion.

Mechanistic studies indicated that the beneficial effect was mediated via increased NO-sGC signaling in the RBCs mediated by the purinergic P2Y13 receptor.

Conclusions

The present studies importantly increase our understanding of the role of RBC function in cardiometabolic disease. The RBC represents a novel mediator of cardiac and endothelial dysfunction and a potentially important therapeutic target in patients with T2D and STEMI.

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LIST OF SCIENTIFIC PAPERS

I. Yang J, Zheng X, Mahdi A, Zhou Z, Tratsiakovich Y, Jiao T, Kiss A, Kövamees O, Alvarsson M, Catrina SB, Lundberg JO, Brismar K, Pernow J.

Red Blood Cells in Type 2 Diabetes Impair Cardiac Post-Ischemic Recovery Through an Arginase-Dependent Modulation of Nitric Oxide Synthase and Reactive Oxygen Species

JACC Basic Transl Sci. 2018 Jul 18;3(4):450-463

II. Mahdi A, Jiao T, Yang J, Kövamees O, Alvarsson M, von Heijne M, Zhou Z, Pernow J.

The Effect of Glycemic Control on Endothelial and Cardiac Dysfunction Induced by Red Blood Cells in Type 2 Diabetes

Front Pharmacol. 2019 Aug 2;10:861

III. Jiao T, Mahdi A, Zhou Z, Tengbom J, Tratsiakovich Y, Milne G.Todd, Alvarsson M, Yang J, Pernow J.

Stimulation of Soluble Guanylyl Cyclase in Erythrocytes from Patients with Type 2 Diabetes Induces Export of cGMP and Protection Against Myocardial Ischemia-Reperfusion Injury

Manuscript

IV. Jiao T, Mahdi A, Tengbom J, Collado A, Jurga J, Saleh N, Verouhis D, Böhm F, Zhou Z, Yang J, Pernow J.

Erythrocytes from Patients with ST-Elevation Myocardial Infarction Induce Cardioprotection through the Purinergic P2Y13 Receptor and Nitric Oxide Signaling

Manuscript

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Related publications:

Mahdi A, Collado A, Tengbom J, Jiao T, Wodaje T, Johansson N, Farnebo F, Färnert A, Yang J, Lundberg JO, Zhou Z, Pernow J.

Erythrocytes induce vascular dysfunction in COVID-19 JACC Basic Tranl Sci. 2022 Feb 16.

Zhou Z, Collado A, Sun C, Tratsiakovich Y, Mahdi A, Winter H, Chernogubova E, Seime T, Narayanan S, Jiao T, Jin H, Alvarsson M, Zheng X, Yang J, Hedin U, Catrina SB,

Maegdefessel L, Pernow J.

Downregulation of Erythrocyte mir-210 Induces Endothelial Dysfunction in Type 2 Diabetes.

Diabetes. 2022 Feb 1;71(2):285-297.

Kang X*, Jiao T*, Wang H, Pernow J, Wirdefeldt K.

Mendelian Randomization Study on the Causal Effects of Tumor Necrosis Factor Inhibition on Coronary Artery Disease and Ischemic Stroke Among the General Population

EBioMedicine. 2022 Feb;76:103824.

Mahdi A*, Jiao T*, Tratsiakovich Y, Wernly B, Yang J, Östenson CG, Danser AHJ, Pernow J, Zhou Z.

Therapeutic Potential of Sunitinib in Ameliorating Endothelial Dysfunction in Type 2 Diabetic Rats.

Pharmacology. 2021 Dec 20:1-7.

Mahdi A, Tratsiakovich Y, Tengbom J, Jiao T, Garib L, Alvarsson M, Yang J, Pernow J, Zhou Z.

Erythrocytes Induce Endothelial Injury in Type 2 Diabetes Through Alteration of Vascular Purinergic Signaling.

Front Pharmacol. 2020 Nov 30;11:603226.

Sun C, Jiao T, Merkus D, DunckerDJ, Mustafa SJ, Zhou Z.

Activation of Adenosine A2A but Not A2B Receptors is Involved in Uridine Adenosine Tetraphosphate-Induced Porcine Coronary Smooth Muscle Relaxation.

J Pharmacol Sci. 2019 Sep;141(1):64-69.

Mahdi A, Jiao T, Tratsiakovich Y, Yang J, Östenson CG, Pernow J, Zhou Z.

Altered Purinergic Receptor Sensitivity in Type 2 Diabetes-Associated Endothelial Dysfunction and Up4A-Mediated Vascular Contraction.

Int J Mol Sci. 2018 Dec 7;19(12):3942

*Contributed equally

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Cardiovascular disease (CVD) is a major health problem and constitutes the most common cause of death world-wide (1-4). The causes of CVD are multiple, but is mainly driven by the common risk factors of physical inactivity, overweight, smoking, hypercholesterolemia, hypertension and diabetes (5, 6). Despite observations that the prevalence of coronary artery disease (CAD) is declining due to the recent improvement in diagnostics and treatments, a large portion of patients are still suffering from cardiovascular events. Thus, although improvement in the management and introduction of novel therapeutic strategies in CVD may reduce the burden and risk of CVD in the general population (1, 7, 8), there are still much more that needs to be done to tackle risks of CVD in the community.

1.1.1 Coronary artery disease and myocardial infarction

CAD, the most common type of CVD and the leading cause of death both in men and women, develops when coronary arteries develop atherosclerotic lesions that obstruct blood flow and thereby oxygen delivery to the heart (9). The atherosclerotic process is believed to be initiated by activated endothelium with expression of adhesion molecules that promotes mononuclear leukocytes to attach to the endothelium and then entering the intima (Figure 1) (10). The differentiation of monocytes to macrophages occurs after internalization of

modified lipoprotein which leads to the formation of foam cell. The process results in an inflammatory response and further activation of the endothelium and vascular smooth muscle cells (VSMC) to form fibromuscular plaque. Previously considered as a cholesterol storage disease, atherosclerosis is currently viewed as an inflammatory disease based on the finding that immune competent cells are abundant in atherosclerosis lesions with over-production of inflammatory cytokines (11). This continuous process results in reduction in lumen diameter and impaired coronary blood flow. The fibrous cap covering the plaque may become

vulnerable and prone to rupture, which promotes local thrombus formation and further inhibition or complete blockage of blood flow (12, 13). The impairment of coronary blood flow results in myocardial ischemia and cardiomyocyte necrosis if not relieved, a situation referred to as myocardial infarction (MI). MI is included in the spectrum of acute coronary syndromes which includes unstable angina and MI with or without ST-segment elevation, which occur when there is thrombus formation at the site of a ruptured atherosclerotic plaque in the coronary artery and consequently a reduction of myocardial perfusion (14, 15). ST- segment elevation myocardial infarction (STEMI) occurs when a coronary artery is occluded following plaque rupture and thrombus formation that prevents supply of oxygen and

nutrient-rich blood to the heart with subsequent transmural myocardial ischemia and necrosis

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(16). An extended period of myocardial ischemia, for more than 20 min, causes a “wave front” of cardiomyocyte injury from the subendocardium transmurally toward the epicardium (17). This explains why the most effective and crucial therapy to date for patients who suffer from STEMI is timely reperfusion of the myocardium by thrombolytic treatment or primary percutaneous coronary intervention (PCI).

Figure 1. Schematic illustration of acute myocardial infarction (MI) caused by the development of coronary artery disease (CAD). Created with BioRender.com and inspired by Dr. Ali Mahdi.

The annual incidence of hospitalization for MI is 1900 per million with an incidence of STEMI of approximately 800 per million (18). Primary PCI and thrombolysis, which represent two alternative strategies to restore myocardial perfusion, are essential approaches to reduce mortality of STEMI patients. The most recent European Society of Cardiology guidelines recommend primary PCI performed within 12 h from symptom onset (19).

Although timely reperfusion is essential to salvage ischemic myocardium from the infarction, the reperfusion can itself induce injury to the cardiomyocytes, a process referred to as

ischemia-reperfusion (IR) injury (20-22) and typically arises in patients with STEMI after PCI or bypass surgery. Experimental studies have shown that nearly 50% of the final infarct size can be attributable to IR injury (23). The multitude of factors contributing to IR injury include endothelial dysfunction, microvascular obstruction, inflammation, and formation of reactive oxygen species (ROS) (23, 24). Although therapeutic approaches targeting the individual components of the IR injury such as oxidative stress, calcium overload and inflammation to attenuate the lethal myocardial IR injury have been proven effective in experimental models, they have remained ineffective in the clinical setting in patients with STEMI (25-27). Therefore, the mechanism behind the IR injury needs to be further

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investigated and novel therapeutic strategies to limit the extent of the myocardial infarction needs to be explored.

1.1.2 Type 2 diabetes and cardiovascular disease

Diabetes mellitus is a chronic disorder that develops either when the pancreas produces insufficient insulin or the insulin produced cannot be used effectively by body tissue to facilitate glucose update. Such defect production of insulin or resistance to the cellular actions of insulin subsequently results in hyperglycemia, which is the key feature of uncontrolled diabetes. The prevalence of diabetes has been rising rapidly globally and has become a major healthy concern with increasing mortality. Type 2 diabetes (T2D), formerly named non- insulin dependent diabetes, is the most common form that accounts for 90-95% of all causes of diabetes and is characterized by chronic hyperglycemia, insulin resistance and impaired carbohydrate, lipid, and protein metabolism (28). Obesity, physical inactivity, sedentary life style, smoking and excessive alcohol consumption are usually the risk factors that contribute to T2D (29-32). The global prevalence of T2D among adults, who constitute the majority of patients with T2D, has increased twofold in three decades. The number of pediatric patients with T2D has also increased dramatically in recent years which needs more attention (33).

T2D, which is a heterogeneous and complex disorder with multiple associated complications, requires a multifaceted and individualized therapeutic approach (34). Exposure to prolonged hyperglycemia is responsible for the reversible and irreversible changes in tissue metabolism and structure, which may potentially result in multiple diabetic complications. The impaired regulation system of glucose transport in T2D constitutes part of the metabolic syndrome phenotype which also includes hypertension, obesity, dyslipidemia and abnormal levels of free fatty acids (35-37). T2D is associated with both microvacular and macrovascular complications. The microvascular complications include retinopathy, nephropathy and neuropathy and macrovascular complications include CAD, peripheral artery disease and stroke. Among the multisystem complications caused by T2D, CVD is the most prevalent complication and the major cause of mortality and morbidity in T2D despite significant progress in the treatments during recent years (38). Patients with T2D have two to four times higher risk of developing cardiovascular events than the general population (39). CAD usually develops earlier in patients with T2D and increases the mortality from cardiovascular causes two to six times compared to healthy subjects (40-43). As a result, nearly 75% of T2D patients die as a consequence of cardiovascular complication (44). MI may often be the primary presentation in individuals with T2D or impaired glucose intolerance (45).

Interestingly, the risk of occurrence of MI in patients with T2D without prior MI is comparable to that of patients with a previous MI but without T2D, a phenomenon called

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“cardiovascular disease risk equivalent” (46). This illustrates the high risk of CAD in patients with T2D. Early diagnosis of T2D may provide a timely and intensive management of

treatment, resulting in improved prognosis and quality of life for patients with T2D (47). The term “cardiometabolic disease” in this thesis is used to stress the clear association between CVD and T2D.

Glycation is clearly defined as a deleterious process in diabetic complications (48). Advanced glycation end-products (AGEs) and related oxidative end products are reactive products of proteins, lipids, and nucleic acids under chronic hyperglycemia conditions (49-51). Increased AGEs lead to oxidative stress, which facilitates further AGE formation (52). Moreover, vascular inflammation, vasoconstriction, thrombosis and atherogenesis develop as consequences of increased AGEs formation, thereby contributing to cardiovascular complications in diabetes (53).

Based on the important role of hyperglycemia for cardiovascular complications in T2D patients, it would be assumed that glucose lowering therapy would be beneficial to prevent such complications (54-56). However, evidence from large clinical trials investigating the efficacy of glycemic control in T2D to reduce cardiovascular events is not overall

convincing (57). Early large-scale study reported that intensive hypoglycemic treatment in T2D significantly reduces the incidence of myocardial infarction, but negatively affects stroke and cardiovascular mortality (58). Recently, tight glucose control therapy with novel classes of drugs such as sodium-glucose cotransporter-2 (SGLT-2) inhibitors and glucagon- like peptide-1 (GLP-1) agonists effectively protect against CVD and improve

cardiovascular outcomes among patients with T2D (54, 55). The mechanisms behind the cardioprotection induced by these hypoglycemic agents not only involve lowering of glucose, but also several effects beyond glucose lowering such as reduction in body weight and blood pressure, decrease in uric acid and oxidative stress, improvement in endothelial function and cardiomyocyte protection (59, 60). β-cell function and insulin sensitivity are improved as a consequence of reduced glucotoxicity (61). Of note, studies have shown that these classes of drugs produce a ≥10% risk reduction in major cardiovascular events (53).

However, further research is still essential to develop treatments beyond glucose lowering drugs that specifically target the cardiovascular complications occurring in T2D.

1.2 Endothelial dysfunction in type 2 diabetes

The vascular endothelium is the monolayer of cells that separates blood cells from the vascular wall and was for long considered as a simple vascular barrier. The endothelium has been recognized as a key regulator of vascular homeostasis and an essential modulator of

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several biological function in the vasculature under physiological and pathological conditions including vascular tone, inflammation, hemostasis and redox balance (62, 63).

Endothelial function may be altered and impaired by cardiovascular risk factors including T2D that are associated with overproduction of ROS causing increased oxidative stress (64, 65). Endothelial dysfunction reflects the vascular phenotype prone to atherosclerosis and precedes the development of morphological atherosclerosis changes, thus contributing to atherosclerotic lesion and subsequent development of clinical complications (66).

Endothelial dysfunction is identified to play a key role in the development of cardiovascular complications in T2D. The substantially impaired vasodilating properties of the

endothelium in T2D is regarded as an early stage of vascular injury in T2D and the first step in the progression of microvascular and macrovascular complications (67, 68). The endothelial dysfunction is characterized by reduced bioavailability of the major vasodilator molecule nitric oxide (NO) and elevated production of ROS, which results in altered redox balance and imbalance between endothelium-derived relaxing and contracting factors, promoting vascular injury in T2D (69). The cause of endothelial dysfunction in T2D is not clearly defined but hyperglycemia is suggested as an important factor (67). Hyperglycemia and insulin resistance are important factors that increase the formation of ROS, in particular superoxide, which scavenges NO (70). The process of ROS production contributes to incidence of CVD by triggering the activation of protein kinase C (PKC), which has been shown to affect vascular cell growth and apoptosis, permeability and extracellular matrix synthesis (71, 72), thus resulting in alteration of vascular homeostasis. Meanwhile, expression and activity of the NO-producing enzyme endothelial NO synthase (eNOS) is attenuated by PKC via increased production of ROS (73). It is also of importance that eNOS can be a source of ROS formation under conditions of oxidative stress which is discussed further below (74).

The understanding of the complete biochemical mechanism of the vascular functional changes by hyperglycemia is still incompletely understood and the cellular links between hyperglycemia and the relevant vascular lesions remains unknown (67). A recent study demonstrated that upregulation of arginase activity and overproduction of NOS-derived ROS in red blood cells are involved in endothelial dysfunction in T2D as novel

mechanisms (75).

Appreciation of the central role of the endothelium throughout the atherosclerotic disease process has led to the development of a range of methods to test different aspects of its function, which include measures of both endothelium dependent and -independent

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relaxations (76). These provide the opportunities to detect novel mechanisms and therapeutic strategies to target endothelial dysfunction.

1.3 Nitric oxide signaling in type 2 diabetes 1.3.1 Nitric oxide

NO is a short-lived gaseous free radical secreted from the endothelium that plays a key role in multiple biological processes including regulation of cardiovascular, metabolic and cellular effects (77-80). As a cardiovascular regulator, NO mediates vasodilation, inhibition of

platelet aggregation and inhibition of inflammation under both physiological and pathological conditions (81).

NO is generated in a variety of cell types by the three NOS isoforms of inducible NOS (iNOS), neuronal NOS (nNOS) and eNOS. iNOS is mainly expressed in macrophages in response to cytokine release and inflammation and nNOS is expressed in neurons and is responsible for central regulation of blood pressure (82). eNOS, which is constitutively expressed in endothelial cells, is important for the regulation of vascular tone by producing NO with amino acid L-arginine and molecular oxygen as substrates together with cofactors including NADPH and tetrahydrobiopterin (BH4) (82, 83). The activity of eNOS is primarily dependent on intracellular calcium (83). iNOS and nNOS are of less importance for the regulation of vascular tone, although it should be mentioned that nNOS is present in vessels and regulates vascular resistance independently of eNOS (82, 84, 85).

Reduced bioavailability of eNOS-derived NO induced by a series of pathological conditions including oxidative stress, inflammation, insulin resistance and hyperglycemia is the central phenotype in atherosclerosis and cardiovascular complications associated with T2D. The reduction in NO is caused by diminished biosynthesis of NO and increased scavenging of NO by superoxide, which create a situation referred to as reduced “NO bioactivity” and is a critical mechanism of endothelial dysfunction (86). eNOS uncoupling is a phenomenon that occurring when the function of the enzyme is altered in a situation of deficiency of the substrate of L-arginine or the co-factor BH4, whereby eNOS produces superoxide instead of NO (87). A previous study have implicated that NO inactivity and abnormal response of NO- mediated vasodilation exist in patients with T2D (88), which may account for the key

mechanism of endothelial dysfunction. The decreased production of NO in these pathological states causes disturbances to the endothelial equilibrium and that is the reason why numerous therapies have been investigated to assess the possibility of reversing endothelial dysfunction by enhancing endothelium-derived NO bioactivity (89). Improved understanding of these mechanisms by which vascular function is impaired in T2D may lead to the identification of novel therapeutic strategies to reduce CVD risk.

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1.3.2 Soluble guanylyl cyclase

Soluble guanylyl cyclase (sGC) is known as the intracellular receptor of NO. Activation of sGC by NO requires binding of NO with the ferrous heme of sGC (90). This triggers the catalytic center and facilitates the conversion of guanosine triphosphate into cyclic guanosine monophosphate (cGMP), that in turn transduces signaling to downstream targets including cGMP-dependent protein kinases G (PKG) and cGMP-gated cation channels (91, 92).

Depending on the cell type, cGMP thereby elicits different biological responses. PKG- mediated activity modulates several cardiovascular functions including vascular smooth muscle relaxation, endothelial permeability and cardiac contractility (93, 94). Vasodilator- stimulated phosphoprotein (VASP) is a well-known and widely-accepted substrate that is phosphorylated by PKG. cGMP is a well-known vasodilator that relaxes VSMC and increases blood flow. Collectively, sGC acts as a crucial mediator to activate a series of beneficial cardiovascular effects elicited by NO-sGC-cGMP signaling.

The vasorelaxing response of sGC stimulation by exogenous NO was found to be blunted in a rat model of T2D (95), which was initially explained by decreased sGC bioactivity. However, vascular sGC activity did not differ between patients with T2D and healthy subjects under basal conditions or following activation by exogenous NO (96). Pharmacological compounds that act on sGC have been developed with the purpose to induce vasorelaxation. These compounds are divided into 2 categories: sGC stimulators and sGC activators based on their pattern of action (97). Stimulators sensitize sGC in an NO-independent manner but are dependent on a reduced (ferrous) prosthetic heme (98, 99). By contrast, activators activate sGC function via its oxidized and heme-free state in the presence of NO (98, 100, 101). These compounds are currently approved for the treatment of pulmonary hypertension and heart failure (102-104). Since NO donors and organic nitrate vasodilators are associated with problematic adverse effects such as hypotension and development of drug tolerance (105, 106), the strategy of enhancing NO bioactivity by sGC stimulators seems to be an attractive therapeutic opinion in T2D to prevent cardiovascular complication (107, 108).

1.3.3 Arginase

Arginase, which is an enzyme hydrolyzing L-arginine to ornithine and urea, is considered as a critical regulator of NO production by competing with eNOS for their common substrate L- arginine (109, 110), thus leading to reduction of NO production. The two distinct genetic isoforms arginase I and II have been identified and share 60% amino acid homology (111, 112). Arginase I is a cytosolic enzyme expressed abundantly in the liver and contributes a major part of total arginase activity. By contrast, arginase II is present in the mitochondria and is abundant in the kidney and prostate (112). Both isoforms are expressed in endothelial

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cells and VSMC (113). A previous study has shown that arginase expression was upregulated in the mammary artery from patients with T2D and arginase inhibition attenuated endothelial dysfunction existing in T2D (114). Moreover, it has been showed that rodent and human RBCs contain large amounts of functional arginase I but not arginase II (115). Increased arginase activity induced by ROS, pro-inflammatory cytokines and hypoxia may reduce the availability of substrate L-arginine for eNOS, therefore, decreasing NO production. Arginase also increases ROS formation from uncoupled eNOS due to substrate deficiency (109, 110).

Hypoxia may accelerate the activity of arginase and reduce NO production. Several studies have demonstrated a key pathological role of increased arginase activity during IR and previous findings have shown that pharmacological arginase inhibition increases coronary flow and limits myocardial infarction following IR in rats and pigs (116-118). In addition, it has been established that arginase inhibition protects against endothelial dysfunction caused by IR in patients with CAD and T2D (119).

Collectively, upregulation of arginase is considered a key factor driving endothelial impairment in T2D. Arginase inhibition may therefore provide a promising therapeutic strategy against IR injury and endothelial dysfunction in T2D.

1.3.4 Oxidative stress and reactive oxygen species

Oxidative stress reflects a state of imbalance between production of oxidative species and antioxidant defenses, which leads to vascular damage in cardiometabolic disease and atherogenesis (120, 121). Cardiometabolic pathological conditions are associated with

overproduction of ROS in combination with reduced antioxidant response. Oxidative stress is thought to be implicated in the development of multiple pathophysiological processes.

Oxidative stress plays a pivotal role in mediating the generation and secretion of cytokines (122-124), and overproduction of ROS accelerates the chronic inflammatory progress

underlying cardiometabolic disease (125, 126). The alteration of vascular function is initiated by the imbalance between NO bioavailability and accumulation of ROS, leading to

endothelial dysfunction (70).

ROS are considered to be important as reactive intermediates of molecular oxygen in cell signaling and homeostasis and act as a second messenger during cellular interaction.

Although characterized as byproducts of the normal metabolism of oxygen, ROS have been considered primarily detrimental because of their highly damaging entity to cells and tissues and therefore of pathological importance in a wide range of CVD and endothelial dysfunction (127, 128). The generation of superoxide from uncoupled eNOS and the activation of oxidase enzymes that subsequently react with NO to form peroxynitrite (ONOO^-), which is

considered as an important mode of NO inactivation (82, 129-131). In turn, peroxynitrite

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contributes to endothelial dysfunction and promotes protein nitration (132-134) and may further disrupts NO signaling leading to cardiovascular complication.

1.4 Red blood cells

1.4.1 Red blood cells and cardiovascular function

RBCs are well known to transport the respiratory gases oxygen and carbon dioxide as well as nutrients. During recent years, RBCs are identified to be involved in several additional biological processes of central importance for cardiovascular function (135). It has become increasingly evident that RBCs are involved in the regulation of cardiovascular function (136-138) by exporting NO bioactivity and adenosine triphosphate (ATP) especially under hypoxic conditions (139). Previous studies have demonstrated that RBCs via these effects are of importance to mediate hypoxic vasodilation and thereby are crucial for physiological regulation of blood flow (140, 141). RBCs have also been suggested to express a functional eNOS and release NO-like bioactivity which has led to the suggestion to consider NO as the third gas of the RBC (115, 142). It has been known that RBCs actively regulate vascular tone and cardiovascular homeostasis by regulating ATP export and redox balance via its

antioxidant system (143, 144). The release of ATP from RBCs is suggested to elicit vascular relaxation. The involvement of purinergic signaling in the biology of RBCs was recognized earlier and the expression of several purinergic receptors in RBCs have been identified (145- 148).

Emerging evidence indicates that RBC-derived NO bioactivity plays a key role in protecting heart against IR injury (115). This finding unravels a new role of RBC in ischemic heart disease. Intriguingly, this protective role of RBC can be turned to be detrimental in patients with cardiometabolic disease and when exposed to cardiovascular risks. This is supported by the observations that RBCs from patients with T2D induce endothelial dysfunction (75, 135).

Collectively, these observations may give us a hint that the pathological events that lead to cardiovascular dysfunction in patients with CVD may in fact start in the RBCs.

1.4.2 Nitric oxide signaling in red blood cells

The biology of NO in RBCs has been a matter of discussion for long. The export of

biological functional NO from RBCs remains controversial due to the fact that NO is rapidly and effectively inactivated and scavenged by hemoglobin leading to production of

methemoglobin and nitrate (149, 150). eNOS, which is one of the three identified NOS isoforms, is present and functionally active in RBCs (142). The fundamental role of RBC eNOS for maintaining cardiovascular homeostasis was recently demonstrated using a RBC- specific eNOS knockout mouse model (151). In this study, it was shown that selective deletion of eNOS in RBCs caused elevation of blood pressure. Conversely, knock-in of

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eNOS in RBCs of global eNOS knockout mice led to reduction in blood pressure. This finding actually led to the suggestion to redefine eNOS as “erythrocytic and endothelial NOS” (152). The role of eNOS in RBCs in exporting NO bioactivity has been supported by the finding that eNOS inhibition significantly reduce the production of NO metabolites and the NOS product citrulline (142, 153, 154). The molecular form of NO that may be released remains unclear. One theory first brought forward by the Stamler group is that the NO binds to oxygenated hemoglobin in the hemoglobin β-chain (β-93 cysteine), which results in formation of S-nitrosohemoglobin (155). When deoxygenated, S-nitrosohemoglobin exports NO bioactivity from the RBCs, resulting in hypoxic vasorelaxation. This theory has recently been challenged as hemoglobin β-93 cysteine was demonstrated not to be essential for RBCs to export NO bioactivity (156). Another theory is that NO is transported in the form of nitrate that is reduced to nitrite by deoxygenated hemoglobin (157). This theory has been questioned by the proposed role of nitrite-methemoglobin complexes which challenges the involvement of deoxygenated hemoglobin-dependent generation of NO (158). Recently, extracellular ATP has been proposed to induce NO production by activating eNOS in RBCs through

intercellular Ca²⁺ and the PI3K/Akt pathway (159). Above all, controversy remains in the field of NO generation from RBCs and further investigations are still needed.

Arginase I is abundantly expressed in human RBCs (115). Previous studies demonstrated endothelial cell arginase as a key regulator of NO production by competing with eNOS for their shared substrate L-arginine (109, 110). The beneficial effect of RBC-arginase inhibition was determined using an isolated ischemic heart model and administration of RBCs (115).

Production of NO metabolites including nitrate and nitrite was increased and cardiac functional recovery after ischemia was improved following RBC-arginase inhibition (115).

Moreover, the cardioprotection was dependent on the present of eNOS in RBCs (115).

Therefore, arginase inhibition in RBCs represents a novel therapeutic target against myocardial IR injury (116).

It is also reported that a catalytically active sGC exist in RBCs and that activation of sGC in RBCs results in formation of cGMP (92). Thus, sGC is characterized as an important regulator of intracellular cGMP. It has been suggested that vascular responses to sGC is reduced in T2D animal models (160). However, vascular sGC activity and sensitivity to NO- dependent and –independent sGC stimulation is preserved in T2D patients (96). Multiple experimental studies indicate that treatment with sGC stimulator exerts beneficial

cardiovascular effects in animal models of diabetes (161-163). An intact sGC-cGMP

signaling is present in RBCs and remains fully responsive to sGC stimulation in patients with

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CAD (92). This signaling pathway in RBCs therefore appears very attractive to improve cardiac performance after ischemia in patients with T2D.

1.4.3 Purinergic signaling in red blood cells

The importance of purinergic signaling in cardiovascular pathophysiology and its therapeutic potential had been well discussed (164) and the involvement of purinergic signaling in RBCs was recognized early (145). The substantial release of ATP from RBCs in hypoxic conditions is considered as crucial for the regulation of tissue perfusion (135, 165). Once released, ATP is degraded to ADP and adenosine by various nucleotidases, after which they act on their corresponding P2 receptors on endothelial cells to activate eNOS and enhance NO production (165-168). P2 receptors are present on all blood cells including RBCs (145). Activation of P2 receptors in RBCs stimulates signaling pathway mediating ATP release from cells. The P2Y13, a member in the P2Y purinergic receptor family, has been suggested to be a key target to regulate the ATP release from human RBCs (148). The role of this pathway and its

interaction with export of NO bioactivity from RBCs in the setting of ischemia in STEMI is of potential interest but remains unexplored.

1.4.4 Red blood cells in type 2 diabetes

It has been suggested that RBCs undergo functional changes which result in increased adhesion to the endothelium and enhanced oxidative stress in pathological progress of diabetes, thereby potentially affecting cardiovascular function (169). Increased adhesion of RBCs from diabetic patients to the endothelium is considered to be associated with the severity of vascular injury (170). Of interest, the degree of endothelial adhesion correlates with levels of glucose and glycated hemoglobin in patients with type 1 diabetes (171). The cell free zone, the space between RBCs and the endothelium, is disrupted as an important consequence of increased adhesion. Thereby, the interaction between RBCs and the vascular endothelium is further enhanced. The interaction between glycated RBC Band 3 protein and the receptor for AGEs (RAGE) on the endothelium leads to endothelial cell oxidative stress and this effect was blocked directly by antibodies against AGEs on RBCs and antibodies against RAGE on the endothelium (172). Moreover, hepatic oxidative stress was induced by transfusing RBCs from T2D rats to healthy controls which could be

partially prevented by a RAGE antibody (172). Collectively, AGE-RAGE signaling plays a key role in the induction of endothelial oxidative stress by RBCs. However, it remains unknown whether endothelial dysfunction or other cardiovascular complications in diabetes are directly caused by endothelial oxidative stress induced by RBCs.

Growing evidence suggest that dysfunctional RBCs are associated with multiple CVD processes in T2D patients (135). As mentioned above, it has been demonstrated that RBCs

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exert cardioprotective effects by exporting NO bioactivity during IR and that this effect is tightly regulated by arginase (115). Of importance, we have recently reported the arginase expression and activity are upregulated in RBCs from patients with T2D, which results in impairment in endothelial function (75). Furthermore, either arginase inhibition or NOS inhibition significantly reduced arginase and ROS production, which resulted in improved endothelial function. These data suggest that the deteriorating phenomenon induced by RBCs from patients with T2D are mediated by increased arginase activity and eNOS-derived ROS production. Extracellular accumulation of nitrate and nitrite was also observed following arginase inhibition (115), supporting stimulation of NO production, which may explain the beneficial cardiovascular effects of arginase inhibition in RBCs. In addition, as mentioned earlier, the release of ATP from RBCs is diminished in pathological condition including T2D, which also contribute to attenuated vascular relaxation (167). The adverse effects caused by dysfunctional RBCs, which is referred to as ‘erythropathy’ (135), lead to endothelial and cardiomyocyte injury.

The mechanism of cardiovascular complications including ischemic heart disease and endothelial dysfunction in T2D still remains largely unknown despite considerable studies that have been conducted. As illustrated from the results of our group and other researchers, RBCs have been identified as a novel trigger and mediator of CVD as exemplified by the cause of endothelial injury in T2D. These findings have led to a change in the focus of fundamental disease mechanisms from the vascular wall to the RBC as a driver of cardiovascular injury. Further efforts still are needed to comprehensively understand the mechanisms driving RBC dysfunction in order to identify novel therapies to target cardiovascular dysfunction in T2D and especially therapeutic strategies that target RBC dysfunction. The present studies in this thesis are therefore designed to investigate in detail the role of RBCs in development of CVD with special focus on targeting molecular signaling in the RBC to improve cardiovascular function. The identification of the intact NO-sGC- cGMP signaling pathway in RBCs opens up several attractive therapeutic strategies to enhance RBC-derived NO signaling in CVD.

1.4.5 Red blood cells and ST-Elevation Myocardial Infarction

Experimental procedures to limit IR injury and infarct size in patients with STEMI tend to focus on the cardiomyocyte due to its critical role for cardiac function. Recently, the importance of circulating blood cells had been well discussed in this pathology (173).

Interaction between blood components including platelets, inflammatory cells, and red blood cells (RBCs) with endothelium and cardiomyocytes is considered to be a critical factor that contributes to thrombosis (174). Moreover, the formation of erythrocyte-rich thrombi is

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associated with inflammation and oxidative stress (175). Platelets are known to influence the development of IR injury by secreting cytokines and platelet activating factor and

contributing to increased oxidative stress (173, 176). Emerging evidence suggests RBCs as cardioprotective, supported by the observation that production of NO and its metabolites are elevated in blood under hypoxic/ischemic conditions (141, 177). In addition, our previous experimental data demonstrate that RBCs are able to protect the heart from IR injury via export of NO bioactivity (115). This opens up the fascinating possibility that RBCs may play a role as an endogenous protector in the setting of IR. In addition, it has been suggested that RBC-derived NOS activation and NO bioactivity are involved in the cardioprotection mediated by remote ischemia conditioning (178).

As mentioned above, ATP released by multiple cell types also exerts important

cardiovascular effects. Endothelial cells release ATP in response to blood flow change or hypoxia to act on P2 receptors on endothelial cells to produce NO (168). Apart from endothelial cells, ATP is also released from RBCs in response to hypoxia and purinergic receptors are expressed by these cells (164, 179). Moreover, P2Y13 has been suggested to act as crucial target to regulate ATP release from RBCs (148). Collectively, RBCs can be a novel target to affect cardiac function in STEMI patients by releasing ATP and exporting NO bioactivity.

1.5 Motivation of this thesis

The Nobel Assembly at Karolinska Institutet awarded the Nobel Prize in Physiology or Medicine for 1998 to Robert F Furchgott, Louis J Ignarro and Ferid Murad for their

discoveries of NO as a signaling molecular in the cardiovascular system. Since then, the door to NO discoveries is widely open to all researchers. Remarkable attention and significant efforts have been drawn to this small gaseous molecule with abundant biological effects.

Emerging evidence have shown that NO, as an intra- and extracellular messenger, mediates signaling in target cells and evokes protective effects in multiple physiological responses including cardiovascular function (74, 180, 181). Abnormalities in NO signaling have been linked to several pathological conditions including atherosclerosis, diabetes mellitus, thrombosis and other CVDs (78, 182, 183). Therefore, this field has attracted tremendous interest in the identification of ways to enhance NO signaling under pathological conditions.

Clinical applications of NO donor has so far not been satisfying, and although organic nitrate are effective as anti-angina medication, their use is otherwise of limited benefit due to

development of drug tolerance (83, 184, 185). Further, clinical trials using inhaled NO or administration of nitrite in the treatment of patients with STEMI did not reduce infarct size

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(186, 187). Hence, therapeutic modulation of NO signaling is challenging and novel strategies for preserving NO signaling is needed.

The theory that RBCs export NO bioactivity has attracted considerable additional interest to this field (155). Recent studies suggest RBCs as important mediators of endothelial

dysfunction in T2D (75, 188). It has become evident that the NOS pathway is impaired in T2D (189). However, it remains unknown how RBCs regulate and affect the cardiac function in the clinical situation of patients with STEMI and T2D. In this thesis, Study I showed that RBCs from mice and patients with T2D aggravate myocardial IR injury. The mechanism behind is through increased RBC arginase I activity and elevated ROS production. Study II clearly showed that improved glycemic control in patients with T2D attenuated the

impairment of post-ischemic cardiac function induced by RBCs but failed to attenuate the negative effects of RBCs on endothelium-dependent relaxation. In addition, arginase I activity from RBCs is decreased following intensive glycemic control in T2D. Study III demonstrate that stimulation of sGC in RBCs from patients with T2D markedly attenuated the cardiac IR injury. This protective effect was associated with increased release of cGMP from the RBCs and activation of cardiac PKG. Study IV showed that RBCs from STEMI patients induce a cardioprotective effect, which is mediated via the NO-sGC signaling pathway and P2Y13 receptor activation.

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2. RESEARCH AIMS

The overall aim of the current thesis is to investigate the role of the RBCs as key regulator in cardiac and endothelial function in patients with T2D and STEMI. A particular focus is to explore the importance of the arginase-NO-sGC and purinergic signaling in RBCs for the regulation of cardiac and endothelial function.

The specific aims for each individual study are:

Ⅰ. To investigate the importance of RBCs in T2D for cardiac dysfunction following IR and the regulatory role of arginase and ROS.

Ⅱ. To determine the effect of glycemic control on RBC-induced cardiac and endothelial dysfunction in patients with T2D.

Ⅲ. To investigate the benefit of stimulation of sGC in RBCs from patients with T2D as a novel therapeutic strategy to improve cardiac functional recovery following IR and to explore the mechanism behind this effect.

Ⅳ. To explore the potential protective role of RBCs from patients with STEMI on cardiac functional recovery and the involvement of purinergic and NO signaling in RBCs.

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3. MATERIALS AND MAIN METHODS 3.1 Overview

The general description of each included study is illustrated in Table 1.

Table 1.

Study Title Studied

population n Intervention(s) Primary endpoint(s)

Secondary

endpoint(s) Methods

I

Red blood cells in type 2 diabetes impair cardiac post-ischemic recovery through an arginase-dependent modulation of nitric oxide synthase and reactive oxygen species

Healthy vs.

Type 2 diabetes 50

RBCs +/- nor-NOHA, ABH, L-NAME, 1400W, NAC, L- arginine

Post-ischemic cardiac function ex vivo

RBC-arginase expression and activity, ROS level

Langendorff, Western blot, enzyme activity assay, flow cytometry, EPR

II

The effect of glycemic control on endothelial and cardiac dysfunction induced by red blood cells in type 2 diabetes

Healthy vs.

Type 2 diabetes 25 RBCs and glycemic control

Post-ischemic cardiac function and endothelial function ex vivo

Arginase activity

Langendorff, Myography, enzyme activity assay

III

Stimulation of soluble guanylyl cyclase in erythrocytes from patients with type 2 diabetes induces export of cGMP and protection against myocardial ischemia – reperfusion injury

Healthy vs.

Type 2 diabetes 51

RBCs +/- DMSO, CYR715, ODQ, DEA- NO, MK-571, 8-bromo- cGMP

Cardiac pVASP expression, cGMP level

Langendorff, Western blot, ELISA

IV

Erythrocytes from patients with ST-elevation myocardial infarction induce

cardioprotection through the purinergic P2Y13 receptor and nitric oxide signaling

Healthy vs.

STEMI 53

RBCs +/- DMSO, ODQ, 8-PT, PPADS, L-NAME, mATP, MRS2211

Purinergic and NO signaling

Langendorff

n denotes to the total number of participants included in each study. ABH - 2(S)-amino-6-boronohexanoic acid, cGMP – cyclic guanosine monophosphate, DEA-NO – diethylamine NONOate diethylammonium, DMSO - dimethyl sulfoxide, EPR - electron paramagnetic resonance, L-NAME - N^G-nitro-L-arginine methyl ester, mATP - α-β-methylene ATP, MK- 571 - inhibitor (E)-3-[[[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl][[3-(dimethylamino)-3-oxopropyl]thio]methyl]thio]-propanoic acid, NAC - N-acetyl-cysteine, nor-NOHA - N^ω- hydroxy-nor-L-arginine, ODQ - 1H- [1,2,4] oxadiazolo [4,3,-a] quinoxalin-1-one, PPADS - pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid, pVASP - phosphorylated vasodilator-stimulated phosphoprotein, RBCs - red blood cells, ROS - reactive oxygen species, STEMI - ST-elevation myocardial infarction, 8-bromo-cGMP - 8-bromo-guanosine cyclic 3´,5´-hydrogen phosphate, 8-PT - 8-phenyltheophylline. n denotes to the total number of participants included in each study.

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3.2 Workflow

Figure 2. Overall workflow of the research works in Study I-IV. Red blood cells (RBCs) were isolated from patients with type 2 diabetes (T2D) or ST-elevation myocardial infarction (STEMI), and age- and sex-matched healthy controls. The RBCs were subjected to functional and molecular

investigations including administration in isolated Langendorff-perfused rodent heart, incubation with rat aortic segments for endothelium-dependent relaxation (EDR), protein expression of RBC-arginase and cardiac vasodilator-stimulated phosphoprotein, RBC-arginase activity and RBC-reactive oxygen species (ROS). Created with BioRender.com.

3.3 Human subjects

All procedures involving humans were conducted according to the declaration of Helsinki and the protocol was approved by the Swedish Ethical Review Authority. All participants were informed of the purpose of the study and possible risks associated with the

participation and gave their oral and written informed consent.

3.3.1 Type 2 diabetic patients

Patients with T2D for Study Ⅰ, Ⅱ and Ⅲ were recruited from the Department of Diabetology and Endocrinology, Karolinska University Hospital and Center for Diabetes, Academic Specialist Center, Health Care Services Stockholm County. T2D was defined according to the World Health Organization criteria. For Study Ⅰ and Ⅲ, patients with T2D were recruited regardless of glucose level as long as they meet the clinical diagnosis standard of T2D. For Study II, patients were scheduled to visit twice: Visit 1 when they were referred to the Center for Diabetes with poor glycemic control and Visit 2 following optimization of

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glycemic control. Inclusion criteria for Visit 1 were mean daily blood glucose of >12 mM or glycated haemoglobin (HbA1c) of >70 mmol/mol. The improvement in glycemic control was achieved by an educational program including optimized medication, lifestyle

modification, dietary interventions and monitoring devices according to clinical routine.

The mean duration of the program until follow-up at Visit 2 was 17 weeks. The aim before enrollment was to reach <9 mM in mean daily blood glucose at Visit 2.

3.3.2 ST-elevation myocardial infarction

For Study Ⅳ, patients with STEMI (chest pain and ST-elevation of >1 mV in two contiguous leads in electrocardiogram) and planned for primary percutaneous coronary intervention (PCI) at Karolinska University Hospital (Solna and Huddinge) were eligible for inclusion. Patients received double antiplatelet therapy with aspirin (300-500 mg) and any of ticagrelor (180 mg) or clopidogrel (600 mg) in the ambulance or immediately on arrival to the catheterization laboratory. PCI was performed according to local clinical routine.

3.3.3 Healthy controls

For Study Ⅰ-Ⅳ, age- and sex-matched healthy control subjects without diabetes or history of CVD were included. T2D was excluded in the control group by fasting blood glucose levels

<6.0 mM or an oral glucose tolerance test and HbA1c <42 mmol/mol.

3.4 Animals

All animal experiments were approved by the Ethical committee and conform to the Guide for Care and Use of Laboratory Animals published by the U.S National Institute of Health (NIH publication NO.85-23, revised 1996). Male db/db and wild-type (C57BL/6J) mice at age of 8-10 weeks were purchased from Janvier. Male Wistar rats at age of 7-9 weeks were purchased from Charles River. All animals were housed in the animal facility of Karolinska University Hospital (L5) or Karolinska Institutet (Komparativ Medicin Biomedicum, KMB) until 10-15 weeks of age for experiments. db/db mice were only included for experiments if they had a tail vein blood glucose level ˃15 mM. In separate experiments of Study I, db/db and wild-type mice were treated orally with ordinary chow and the anti- oxidant N-acetyl-cysteine NAC (1 mM) in water or normal water for 4 weeks. All animals were kept in a 12:12-hour light-dark cycle with free access to standard chow and water.

3.5 RBCs preparation and supernatant collection 3.5.1 Blood sampling

In patients with T2D and healthy control subjects, whole blood was collected in heparinized tubes from the antecubital vein. Blood from patients with T2D and healthy volunteers was

(33)

collected in the morning following an overnight fasting to minimize the possible influence of circadian or dietary factors. In patients with STEMI, whole blood was collected in pre- chilled heparinized tubes from the radial or femoral artery following the catheterization as part of the coronary angiography procedure. The blood samples were stored either on ice or at +4 °C in a refrigerator before RBCs were isolated. Extra blood samples were sent to routine chemistry lab at Karolinska University Hospital.

3.5.2 RBCs isolation

The isolation of RBCs was established previously (115). Briefly, following separation of blood components by centrifugation at 1,000 g and +4 °C for 10 min, RBCs were isolated by discarding the plasma and buffy coat including the top part of RBCs. Following three

washing cycles with oxygenated (5% CO2 in O2) Krebs-Henseleit (KH) buffer (118.5 mM NaCl, 25.0 mM NaHCO3, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 11.1 mM glucose, and 2.4 mM CaCl2), RBCs were diluted with KH buffer. This procedure results in removal of 99% of white blood cells and 98% of platelet (115). The dilution with oxygenated KH buffer increase blood oxygen saturation level to ˃99%. In separate experiments, blood collected from healthy subjects was placed for 3 h, 6 h or 24 h in a refridgerator at +4 °C before being washed as above. The RBC-KH buffer suspension was used in the functional cardiac and vessel experiment and molecular analyses described below. Samples with hemolysis were discarded.

3.5.3 RBCs incubation

In Study Ⅰ, the RBC-KH buffer suspension (hematocrit, ~45%) was pre-incubated with vehicle, the arginase inhibitors N^ω-hydroxy-nor-L-arginine (nor-NOHA, 1 and 3 mM) and 2 (S)-amino-6-boronohexanoic acid (ABH, 1 mM), the NOS inhibitor N^ω-nitro-L-arginine methyl ester (L-NAME, 0.1 mM) or the combination of nor-NOHA and L-NAME, the specific iNOS inhibitor 1400W (0.1 mM) for 20 min at 37 °C before being administered to the hearts. When NOS and arginase inhibition were combined, L-NAME was added to the RBC suspension 5 min prior to nor-NOHA. Nor-NOHA and ABH are two types of arginase inhibitors with different structure whereby nor-NOHA has a guanidinium chain and ABH binds as a tetrahedral boronate anion (190). In separate experiments, the diluted RBCs (hematocrit, 5%) were incubated with either 5 mM or 25 mM glucose for 24 h. For ROS measurement, the diluted RBCs (hematocrit, 1%) were incubated with the anti-oxidant N- acetyl-cysteine (NAC, 1 mM), ABH (0.1 mM), L-NAME (0.1 mM) and L-arginine (3 mM) for 30 min.

In Study Ⅱ, the diluted RBCs (hematocrit, ~45%) were pre-incubated with or without nor- NOHA (1 mM).

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In Study Ⅲ, the RBC suspension (hematocrit, ~45%) was incubated with one of the

following for 20 min at 37 °C: dimethyl sulfoxide (DMSO as vehicle for the sGC stimulator, 10 μM), the sGC stimulator CYR715 (a ferrous-dependent stimulator of sGC (191), 10 μM in DMSO) provided by Cyclerion (MA, USA), the sGC inhibitor 1H- [1,2,4] oxadiazolo [4,3,-a]

quinoxalin-1-one (ODQ, 5 μM), the NO donor diethylamine NONOate diethylammonium salt (DEA-NO, 200 μM), the cyclic GMP transporter inhibitor (E)-3-[[[3-[2-(7-chloro-2- quinolinyl)ethenyl]phenyl][[3-(dimethylamino)-3-oxopropyl]thio]methyl]thio]-propanoic acid (MK-571, 10 μM), or the combination of CYR715 and ODQ, CYR715 and DEA-NO or CYR715 and MK-571 before being administered to the heart (see below). When the RBCs were incubated in combination with ODQ, MK-571 or DEA-NO, they were added 5 min before addition of CYR715 or vehicle. In other experiments, CYR715 or the cell-permeable analogue of cGMP 8-bromo-guanosine cyclic 3´,5´-hydrogen phosphate (8-bromo cGMP, 1 μM) were given to the heart in KH buffer only (without RBCs).

In Study Ⅳ, the RBC-KH suspension (hematocrit, ~45%) was incubated with one of the following for 20 min at 37°C: dimethyl sulfoxide (DMSO, 5 μM), the sGC inhibitor (ODQ, 5 μM), the non-selective purinergic P1 receptor antagonist 8-phenyltheophylline (8PT, 10 μM), the non-selective purinergic P2 receptor antagonist pyridoxal phosphate-6-azo(benzene-2,4- disulfonic acid) tetrasodium salt hydrate (PPADS, 10 μM), the NOS inhibitor (L-NAME, 100 μM), the cell- permeable analog of ATP α-β-methylene ATP (mATP, 100 μM), the P2Y13 receptor antagonist MRS2211 (10 μM) or the combination of mATP and ODQ, mATP and PPADS or mATP and MRS2211. When co-incubated, ODQ, PPADS and MRS2211 were added 5 min before addition of mATP.

3.5.4 Supernatant collection

In Study Ⅲ, washed RBC-KH buffer suspension was incubated with pharmacological compounds/vehicle described above for 20 min at 37 °C, and the supernatant was collected after one additional centrifugation. Samples with hemolysis were discarded.

3.6 Isolated Langendorff-perfused hearts

In Study Ⅰ-Ⅳ, wild-type mice, db/db mice and Wistar rats were anesthetized with pentobarbital sodium (50 mg/kg, i.p.) and heparinized (100 IU/kg, i.v.). The hearts were excised and placed in ice-cold KH buffer before being mounted to a Langendorff apparatus (Figure 3). The ascending aorta was quickly cannulated and perfused with gassed (5% CO2

in O2) KH buffer in a retrograde manner at a constant pressure (55 mmHg and 80 mmHg for mouse and rat hearts, respectively) at 37 °C. A balloon-tipped catheter connected to a pressure transducer was inserted into the left ventricle to monitor cardiac functional parameters, including left ventricular developed pressure (LVDP), and its positive first

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derivative dP/dt and left ventricular end-diastolic pressure (LVEDP). During the

stabilization period of 30 min, the balloon was given a baseline LVEDP of 4-10 mmHg, after which baseline parameters were registered. Any heart that did not reach a LVDP of

>60 mmHg and heart rate of >250 beats/min during the stabilization period was excluded.

Global ischemia was induced by clamping the inflow tube, and 3 ml of RBC-KH suspension, supernatant or KH buffer only was administered to the heart via a sidearm connected to the ascending aorta at the onset of ischemia. The duration of global ischemia was 25 min for rat hearts and 40 min for mouse hearts, and during this period, the pre- incubated RBCs, supernatant or KH buffer only was present in the coronary circulation.

Reperfusion, which rinsed away the incubation medium, was initiated by releasing the clamp and was maintained for 60 min.

3.7 Determination of heart infarct size

In Study Ⅰ, Ⅲ and Ⅳ, the hearts were collected at the end of reperfusion and frozen at -20 °C and sectioned into 1 mm thick slices from the apex to the base, stained with

triphenyltetrazolium chloride for 15 min, and fixed in 4% formaldehyde for 18 h. The area of necrotic negatively stained myocardium was measured using Adobe Photoshop Elements 2019 Edition and present as percentage of the heart.

3.8 Evaluation of ex vivo vascular reactivity

In Study Ⅱ, rats were anesthetized as described above followed by thoracotomy and isolation of the aorta. Washed RBCs collected from patients with T2D and healthy subjects were diluted with KH buffer to a hematocrit of ~45% and incubated with isolated aortic rings in a cell culture incubator at 37° with 5% CO2 for 18 h. After incubation, aortic segments were thoroughly washed with KH buffer and mounted on wire myographs in organ chamber to measure isometric tension as previously described (75). Briefly, following 30 min equilibration, all vessels were exposed to KCl twice (50 mM and 100 mM, respectively). Vessel segments were pre-constricted with 9,11-dideoxy-9α,11α-methanoepoxy PGF2α (U46619, 30 nM). EDR was determined by administration of accumulatively increasing concentration (10^-9 to 10^-5M) of acetylcholine (ACh) to the pre-constricted vessels.

3.9 Arginase expression and activity assay

In Study Ⅰ and Ⅱ, arginase activity was determined as previously described (75, 115). In brief, following incubation with MnCl2 for 10 min at 56 °C, lysed RBCs were subsequently incubated with L-arginine for 60 min at 37 °C. After addition of 400 μl stop solution

(H2SO4:H3PO4:H2O, 1:3:7) and then 25 μl of α-isonitrosopropiophenone (9% in ethanol), the

THE ROLE OF RED BLOOD CELLS IN CARDIAC AND ENDOTHELIAL DYSFUNCTION IN CARDIOMETABOLIC DISEASE

From Unit of Cardiology, Department of Medicine, Solna Karolinska Institutet, Stockholm, Sweden

THE ROLE OF RED BLOOD CELLS IN CARDIAC AND ENDOTHELIAL

DYSFUNCTION IN CARDIOMETABOLIC DISEASE

The Role of Red Blood Cells in Cardiac and Endothelial Dysfunction in Cardiometabolic Disease

THESIS FOR DOCTORAL DEGREE (Ph.D.)

Tong Jiao

ABSTRACT

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