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
Post-ischemic cardiac function ex vivo
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
Post-ischemic cardiac function ex vivo
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 - NG-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 - Nω -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.
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
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
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 Nω-hydroxy-nor-L-arginine (nor-NOHA, 1 and 3 mM) and 2 (S)-amino-6-boronohexanoic acid (ABH, 1 mM), the NOS inhibitor Nω-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).
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
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 10-5 M) 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
mixture was incubated for 60 min at 100 °C and centrifuged for 5 min at 5,000 g. The concentration of urea was determined in a spectrophotometer at 540 nm.
Figure 3. Illustration of Langendorff-perfused heart model and protocol. Hearts from rats or mice were isolated and the aorta was cannulated to a Langendorff apparatus and perfused with gassed (5%
CO2 in O2) Krebs-Henseleit buffer at 37 °C. A balloon-tipped catheter connected to a pressure transducer was inserted into the left ventricle to monitor cardiac functional parameters. Hearts were stabilized for 30 min and followed by 25 min (for rat hearts) or 40 min (for mouse hearts) of ischemia.
Red blood cells (RBCs) collected from db/db or wild-type (WT) mice, patients with type 2 diabetes (T2D), ST-elevation myocardial infarction (STEMI) or age- and sex- matched healthy controls were isolated and administered to hearts at the onset of ischemia. Hearts were reperfused for 60 min, after which the hearts were frozen and stained for infarct size measurement. Adapted from Yang et al.
JACC BTS 2018 (Study I of this thesis). LVDP: left ventricular developed pressure. LVEDP: left ventricular end diastolic pressure.
3.10 Measurement of reactive oxygen species
In Study Ⅰ, ROS production was measure with two different methods. For flow cytometry, the RBC suspension (hematocrit, 1%) was after incubated with different pharmacological compounds for 30 min at 37 °C mixed with the fluorescent probe 5-(and-6)-
chloromethyl-2’,7’-dichlorodihydrofluorescein diacetate (10 μM) for 1 h in the dark room at room temperature. The fluorescence (fluorescein isothiocyanate) intensity was analyzed using Beckman Coulter CyAn ADP Flow Cytometer. Autofluorescence induced by RBCs from wild-type and db/db mice was excluded by determining fluorescence in the absence of fluorescent probe. For ROS measurement using electron spin resonance, washed RBCs were diluted with Krebs/N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid buffer (hematocrit, 1%). The suspension was mixed with 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (200 μM) for 30 min at 37 °C in the presence of different
pharmacological agents followed by subsequent detection with electron spin resonance (Bruker EPR technology).
3.11 Protein expression
In Study Ⅰ, arginase protein expression was determined in lysed RBCs from mice. Protein was separated by 10% sodium dodecylsulfate–polyacrylamide gel electrophoresis
(SD/PAGE) and transferring to nitrocellulose membrane. Following an overnight incubation at 4 °C using rabbit anti-arginase I antibody (#HPA003595, Atlas Prestige Antibody, Sigma-Aldrich, MI, USA), membranes were then incubated with goat anti-rabbit immunoglobulin G (#926-32211, IR Dye 800CW, LI-COR, NE, USA) for 1 h at room temperature. Immunoreactive bands were visualized using infrared fluorescence.
In Study Ⅲ, Langendorff-perfused rat hearts were subjected to 10 min ischemia following 30 min stabilization. RBCs from patients with T2D were given to the isolated heart at the onset of ischemia. Following ischemia, the hearts were reperfused for 1 min, after which they were snap frozen in liquid nitrogen and stored in -80°. The hearts were lysed by using
radioimmunoprecipitation assay buffer containing protease inhibitors. The protocol of Western blotting was described elsewhere (118). Briefly, proteins were separated by 12%
SDS/PAGE, transferred to nitrocellulose membrane, and blocked in 5% nonfat milk for 1 h at room temperature. Membranes were incubated overnight at 4 °C with rabbit
anti-phosphorylated VASP (pVASP) (#3114, Cell Signaling Technology, MA, USA) following stripping of the membrane with rabbit anti-GAPDH (#G9545, Sigma, MI, USA). Separate membranes were incubated with rabbit anti-VASP (#3112, Cell Signaling Technology, MA, USA) and following stripping with the same anti-GAPDH antibody. The reason for this was that incubation with pVASP/VASP antibodies resulted in unspecific staining when used on
stripped membranes. Membranes were washed by Tris-buffered saline with 0.1% Tween 20 and incubated with goat anti-rabbit IgG (#AP132, Sigma, MI, USA). Immunoreactive bands were visualized by using infrared fluorescence densities and analyzed with Image Studio Lite Version 3.1. Expression of pVASP/VASP was normalized to GAPDH.
3.12 Quantification of extracellular cGMP level
In Study Ⅲ, cGMP levels in the supernatant were determined by ELISA using the cyclic GMP complete kit (ADI-900-013, Enzo life science, NY, USA) according to the
manufacturer’s instructions. The 96 microplate was read by a Victor Multilabel Plate Reader to measure the absorbance at 405 nm. Each sample was run in duplicates and the mean values are presented.
3.13 Statistical analysis
LVDP and dP/dt are presented as percentage recovery during reperfusion from baseline level. LVEDP is expressed as absolute value during reperfusion. Infarct size is presented as percentage of the heart. EDR is presented as percentage relaxation from precontriction induced by U46619 during application of cumulative concentrations of ACh. Normal distribution of data was analyzed using D’Agostino Pearson’s test or Shapiro-Wilk test, depending on the number of observations in each group. Differences between 2 groups were analyzed by Student’s t-test, Mann-Whitney test or Wilcoxon signed-rank test, depending on the matching and distribution. Cardiac performance during the reperfusion period and vessel relaxation after pre-contraction were compared using 2-way analysis of variance including both treatment and time or concentration followed by Tukey’s or
Dunnett’s post hoc test for multiple comparisons. n in all analyses denotes to the number of subjects included. All statistical analysis was calculated by using GraphPad Prism version 7.04 (GraphPad, San Diego, California). Data are unless otherwise stated presented as mean
± standard deviation (SD) in Study Ⅰ-Ⅳ. A probability p <0.05 was considered as statistical significance.