Increased Prevalence of Type 2 Diabetes–Related Complications in Combined Type 2 Diabetes and Sickle Cell Trait
Abstract
OBJECTIVE The prevalence of type 2 diabetes (T2D) is rapidly increasing in sub-Saharan Africa, where sickle cell trait (SCT) is also frequent. Although SCT is generally considered a benign condition, evidence suggests that SCT could exaggerate vascular dysfunction in T2D. However, it remains unclear whether SCT could increase the risk of the development of T2D complications. Therefore, this study was conducted to determine whether T2D complications were more prevalent among Senegalese individuals with SCT and T2D than among those with T2D only.
RESEARCH DESIGN AND METHODS Rates of hypertension, retinopathy, peripheral neuropathy, peripheral artery disease, and impaired renal function as well as arterial stiffness, blood rheology, and concentrations of plasma advanced glycation end products (AGEs) and cytokines were compared between groups of Senegalese individuals with combined SCT and T2D (T2D-SCT) (n = 60), T2D (n = 52), SCT (n = 53), and neither T2D nor SCT (control) (n = 56). Human aortic endothelial cell (HAEC) expression of inflammatory and adhesion factors was measured after treatment with tumor necrosis factor-α and subjects’ plasma. Effects of AGE inhibition or tiron on HAEC expression of E-selectin were measured.
RESULTS Retinopathy, hypertension, and reduced renal function were more prevalent, and arterial stiffness, blood viscosity at high shear rates, and thixotropic index were higher, in the T2D-SCT group compared with the other groups. Multivariable analysis showed that plasma AGE concentration was significantly associated with arterial stiffness. E-selectin expression was elevated in HAECs treated with T2D-SCT plasma compared with the other groups, but AGE inhibition reversed this.
CONCLUSIONS SCT could potentially augment the risk of the development of T2D-related complications, including retinopathy, nephropathy, and hypertension.
Introduction
Type 2 diabetes (T2D) is an insidious disease characterized by progressive insulin resistance, resulting in elevated levels of blood glucose (1). Hyperglycemia results in increased levels of advanced glycation end products (AGEs), oxidative stress, chronic low-grade inflammation, and platelet hyperactivity (2). These biological alterations lead to impaired endothelial function, increased vasoconstriction, abnormal blood rheology, and a prothrombotic state, thereby augmenting the risk of microvascular and macrovascular complications (2). An estimated 425 million people around the globe have T2D, and current projections indicate that this number will increase by 48% by 2045 (3). The prevalence of T2D is increasing particularly rapidly in Africa, where urbanization, lifestyle changes, and an aging population contribute to an expected 162% increase in the disease by 2045 (3).
Sickle cell trait (SCT), the heterozygous form of sickle cell anemia, is highly frequent in sub-Saharan African countries, with an estimated prevalence of 6–10% in Senegal (4). Sickle cell anemia is caused by the mutation of the sixth amino acid of the β-globin gene and results in the production of abnormal hemoglobin, called hemoglobin S (HbS) (5). Under deoxygenated conditions, HbS polymerizes and rigidifies, leading to severe acute and chronic complications (5). Unlike sickle cell anemia, SCT is generally considered to be benign (6). However, accumulating evidence shows that individuals with SCT have altered blood rheology and increased coagulation activity (6–8). Moreover, studies show that SCT may increase the risk of stroke, venous thromboembolism, chronic kidney disease, and end-stage renal disease (9).
The high and increasing rates of T2D in sub-Saharan Africa indicate that there is likely a large and growing population of individuals with both SCT and T2D. This could become problematic, as a recent study suggests that SCT could exaggerate vascular dysfunction and arterial stiffness in T2D and also could amplify blood rheological alterations, oxidative stress, AGE, and inflammation (10). However, it remains unclear whether SCT could increase the risk of the development of diabetes-related vascular complications (11–14).
Therefore, the primary objective of this study was to determine whether T2D complications are more prevalent among individuals with combined T2D and SCT (T2D-SCT) than among individuals with T2D alone. The secondary objective was to explore the possible mechanisms implicated in the pathogenesis of vascular complications in individuals with both SCT and T2D.
Research Design and Methods
Setting and Participants
This study was performed at the Cheikh Anta Diop University in Dakar, Senegal. A total of 221 subjects were recruited from the Diabetic Centre at the Hospital Abass Ndao, from the National Center of Blood Transfusion, and from the general population in Dakar. Subjects were screened for T2D and SCT and then assigned to one of the following four groups: subjects with T2D-SCT (n = 60), subjects with T2D (n = 52), subjects with SCT (n = 53), and subjects with neither SCT nor T2D (control group [n = 56]). Calculations conducted prior to subject recruitment demonstrated that a minimum total sample size (all groups combined) of 57 participants would be required to address the main objective of the study (α = 0.05, power = 90%). T2D was defined using the American Diabetes Association Standards of Medical Care in Diabetes guidelines (15). Biological analyses were conducted to confirm whether subjects were carriers of the HbS gene. Briefly, blood was screened using isoelectric focusing. Results were then confirmed using citrate agar electrophoresis, hemoglobin fraction quantification using high-performance liquid chromatography, and a solubility test to confirm the presence of HbS. The high-performance liquid chromatography results were also used to confirm that subjects had no other hemoglobinopathies.
Individuals who smoked as well as women who were pregnant or using oral contraception were excluded from the study. The majority of subjects in the T2D (71.2%) and T2D-SCT (71.7%) groups were treated with metformin, sulfonylureas, insulin, or a combination of these. The other T2D and T2D-SCT subjects were not taking any other medications to treat T2D. All participants were Senegalese. The study protocol was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Cheikh Anta Diop University (reference: 0221/2016/CER/UCAD). All subjects gave informed written consent.
Clinical Examination
Blood Sampling
Subjects arrived at the Laboratory of Medical Physiology (Cheikh Anta Diop University) at 8:00 a.m. after an overnight fast. All of the subjects were instructed to refrain from physical activity for 24 h before the visit. Blood was drawn into heparin tubes for lipid measurement, fluoride tubes for glucose measurement, and EDTA tubes for analyses of hemoglobin A1c (HbA1c), blood rheology, AGEs, and cytokines. The blood samples used to measure AGEs and the cytokines were immediately centrifuged, and the plasma was then stored at −80°C until analyses were conducted.
Blood Pressure and Pulse Wave Velocity
Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured in the left arm using a manual sphygmomanometer (Omron M3; Intellisense, Kyoto, Japan), while the subject remained in a seated position. The measurements were taken three times after a 30-min period of rest. Mean arterial pressure (MAP) was calculated as DBP + (SBP − DBP)/3. The carotid-femoral pulse wave velocity (PWV) was measured using an automated system (Pulse Pen; DiaTecne, Milan, Italy). The carotid and femoral waveforms were measured simultaneously using two pressure-sensitive transducers. The transit time of the pulse wave was calculated by the system software. The distance between the carotid and femoral measurement sites was measured over the body surface using a tape measure. The PWVs were calculated as the distance between the two measurement sites divided by the transit time (in meters per second). In order to cover the entire respiratory cycle, at least 12 readings were performed successively on each subject. The mean of three consecutive measures was calculated and used as the PWV value. The same trained individual performed all PWV measurements.
T2D-Associated Complications
All subjects were screened for hypertension, retinopathy, peripheral neuropathy, peripheral artery disease, and impaired renal function. Hypertension was defined as SBP >130 mmHg or DBP >80 mmHg or the use of antihypertensive drugs (16). Indirect ophthalmoscopy with a noncontact slip lens lamp was used to assess the presence of retinopathy. The subjects were screened for diabetic peripheral neuropathy using the Semmes-Weinstein monofilament examination on three test sites (the great toe, the third metatarsal, and the fifth metatarsal) (17). Ankle brachial pressure was calculated using a Doppler probe to confirm the presence or absence of peripheral artery disease (ankle brachial systolic pressures index <0.90).
Urinary albumin concentration (UAC) was measured using early morning spot urine (HemoCue Albumin 201 System). Serum creatinine level was measured using the standard Jaffe method. The estimated glomerular filtration rate (eGFR) was calculated using the Chronic Kidney Disease Epidemiological Equation, and the appropriate corrections for race and sex were applied (18). Subjects with an eGFR <60 mL/min/1.73 m2 were classified as having reduced renal function (moderately or severely decreased eGFR) (18).
Laboratory Analyses
Biochemical Parameters
Fasting glucose was measured using an enzymatic glucosidase-peroxidase method (Urit Medical Electronic Co., Guilin, People’s Republic of China). HbA1c was measured using capillary electrophoresis on a Capillary 3 Tera device (Sebia, Lisses, France). Plasma lipids (triglycerides, total cholesterol, HDL cholesterol, and LDL cholesterol) were evaluated using standard enzymatic methods.
Hemorheological Parameters
Hematocrit was measured after blood microcentrifugation. Plasma viscosity was measured at 37°C using a cone/plate viscometer at a shear rate of 375 s−1. Oxygenated whole blood at native hematocrit was used to measure whole-blood viscosity at varying shear rates (5.62, 11.25, 22.5, 45, 90, 225, and 375 s−1), at 37°C, using a cone/plate viscometer (Pro DV-II+, with CPE40 spindle; Brookfield, Middleboro, MA). Furthermore, a “loop protocol” was used to characterize the blood thixotropic index, a measure that reflects the impact of altered red blood cell aggregation, and deformability (to a lesser extent), on blood viscosity (19). For this method, a cone/plate viscometer is used to measure blood viscosity because the shear rate is increased every 30 s from an initial shear rate of 22.5 s−1 to a maximum shear rate of 225 s−1 (curve 1). The shear rate is then decreased every 30 s progressively back to the original shear rate (curve 2). The difference between the blood viscosities at each of the shear rates on curve 1 and curve 2 was calculated for each subject and then plotted versus shear rate. The area under the curve for this graph was calculated for each subject and corresponded to the thixotropic index (19,20).
AGEs and Cytokines
ELISA kits were used to measure plasma concentrations of AGEs (Cell Biolabs, Inc., San Diego, CA) and interleukin (IL)-1β (Genetex, Irvine, CA). The ELISA assays were carried out according to the manufacturer instructions. The Bio-Plex Pro Human Cytokine 8-Plex Immunoassay Kit (Bio-Rad) was used to measure concentrations of IL-2, IL-4, IL-6, IL-8, IL-10, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon (IFN)-γ, and tumor necrosis factor-α (TNF-α) in subjects’ plasma. The assays were performed following the instructions with the kit, and cytokine concentrations were measured using the MAGPIX xPONENT 4.2 System (Luminex Corporation, Austin, TX).
Human Aortic Endothelial Cells Culture and Treatment
A subset of 25 plasma samples (control n = 7, T2D n = 7, SCT n = 5, and T2D-SCT n = 6) was randomly selected from the whole cohort for use in cell culture experiments. Human aortic endothelial cells (HAECs) from PromoCell were grown in Endothelial Cell Medium MV2 (PromoCell) and treated with TNF-α (0.3 ng/mL), either alone or together with plasma (5%), for 4 h.
Flow Cytometry: E-Selectin and Vascular Cell Adhesion Molecule-1
HAECs were detached using accutase (Thermo Fisher Scientific), labeled with FITC-conjugated antibodies against CD106 (vascular cell adhesion molecule-1 [VCAM-1], clone STA; Thermo Fisher Scientific) and phycoerythrin-conjugated antibody against CD62E (E-selectin, clone P2H3; eBioscience), and then analyzed using an Accuri C6 Flow Cytometer (Becton Dickinson).
Flow Cytometry: the Effect of an Antioxidant (Tiron) or AGE Inhibition on HAEC E-Selectin Expression
The effects of reactive oxygen species and AGEs on HAEC E-selectin expression were tested in control subjects (n = 5) and individuals with T2D-SCT (n = 6) using tiron (ab146234; Abcam), a cell-permeable superoxide scavenger and antioxidant, or aminoguanidine hydrochloride (ab120123; Abcam), an AGE inhibitor. HAECs from PromoCell were grown in Endothelial Cell Medium MV2 (PromoCell) and treated for 4 h with TNF-α (0.3 ng/mL) together with plasma (5%) from control subjects or subjects with T2D-SCT, with plasma (5%) from control subjects or subjects with T2D-SCT and tiron (5 mmol/L), or with plasma (5%) from control subjects or subjects with T2D-SCT and aminoguanidine hydrochloride (5 mmol/L).
HAECs were detached using accutase (Thermo Fisher Scientific), labeled with a phycoerythrin-conjugated antibody against CD62E (E-selectin, clone P2H3; eBioscience), and then analyzed using an Accuri C6 Flow Cytometer (Becton Dickinson).
Real-Time Quantitative PCR: Zonula Occludens-1 and Nuclear Factor-κB
Analysis of HAECs was performed using established protocols. Total RNA was isolated from cells using Tri-Reagent (Sigma-Aldrich, St. Louis, MO). Real-time quantitative PCR was performed using diluted cDNAs and LightCycler 480 SYBR Green I Master Mix (Roche). Triplicate assays were performed for both genes (nuclear factor-κB [NF-κB] and zonula occludens-1 [ZO-1]). Gene expression was normalized with TATA-binding protein (TBP).
Western Blotting
HAEC lysate preparation and Western blotting were performed as previously described (21). Semiquantitative assessment of band density was performed using ImageJ software. Band density for the protein of interest was normalized to β-actin. Antibodies against phosphorylated (phospho-) and total p38 mitogen-activated protein kinase (MAPK) were purchased through Cell Signaling Technology (Danvers, MA). β-Actin antibody was purchased from Sigma-Aldrich.
Statistical Analysis
Results are expressed as the mean ± SD. One-way ANOVA tests were performed with Tukey post hoc tests (to answer the primary objective) and with the Holm-Bonferroni correction (to answer the secondary objective, which is more exploratory) to compare differences among groups for mean age; SBP; DBP; MAP; PWV; anthropometric, biochemical, and biological parameters; microalbumin; serum creatinine; and eGFR. Differences between groups for PWV were also determined by ANCOVA, using MAP as a covariate. χ2 tests were used to compare the sex distribution and the frequency of hypertension, retinopathy, peripheral neuropathy, peripheral artery disease, and impaired renal function between groups. A multivariate linear regression model was used to predict the independent associations between PWV and several parameters of interest. Statistical significance was defined as P < 0.05. Analyses were conducted using SPSS Statistics software (version 24; IBM, Chicago, IL).
Results
Anthropometrics, Blood Pressures, and Biochemical Parameters
There were no significant differences in sex distribution or age among the four groups (Table 1). The duration of diabetes did not differ between the T2D (6.7 ± 5.7 years) and T2D-SCT (5.9 ± 4.6 years) groups. Furthermore, there was no significant difference between the proportion of subjects receiving metformin (T2D group 63.4%; T2D-SCT group 50.0%), sulfonylurea (T2D group 21.2%; T2D-SCT group 16.7%), or insulin (T2D group 5.7%; T2D-SCT group 10.0%) treatment between the T2D and T2D-SCT groups.
Participant characteristics and biochemical parameters
BMI, SBP, MAP, HbA1c, and fasting glucose levels were higher in subjects with T2D compared with levels in those without T2D (Table 1). DBP was significantly higher in the T2D group compared with the SCT group and was significantly higher in the T2D-SCT group compared with the control and SCT groups. LDL cholesterol was elevated in the two groups with T2D compared with the control group. Total cholesterol level was higher in the T2D group compared with the control and SCT groups and was elevated in the T2D-SCT group compared with the control group (Table 1). Triglyceride levels were higher in the T2D-SCT group compared with the three other groups (Table 1). HDL cholesterol was lower in the two groups with T2D compared with the control group (Table 1).
T2D-Associated Complications
Retinopathy, hypertension, and reduced renal function were significantly more frequent in the T2D-SCT group compared with the three other groups (Table 2). The prevalence of neuropathy was elevated in the two T2D groups compared with the control and SCT groups (Table 2). Finally, the prevalence of peripheral artery disease did not vary significantly among the four groups (Table 2). The average values for UAC, serum creatinine concentration, and eGFR for each group are displayed in Table 1. UAC was higher in the T2D and T2D-SCT groups compared with the control group. Serum creatinine concentration was significantly higher in the T2D-SCT group compared with the other three groups. The eGFR was higher in the control group compared with the other three groups and was lower in the T2D-SCT group compared with the other three groups. Three subjects in the SCT group presented with retinal hemorrhages, which occur in the early stages of diabetic and nondiabetic retinopathy.
Prevalence of hypertension, retinopathy, neuropathy, peripheral artery disease, and reduced renal function in the control, T2D, SCT, and T2D-SCT groups
PWV
PWV was higher in the T2D group compared with the control group only. Additionally, PWV was significantly higher in the T2D-SCT group compared with the other three groups, even when MAP was fixed as a covariate (Fig. 1).
PWV values of patients with T2D with SCT (T2D-SCT) or without SCT (T2D) and of individuals without T2D with SCT or without SCT (Control). The two extremities of the horizontal bars indicate the difference between two given groups (*P < 0.05; **P < 0.01; ***P < 0.001). Results are presented as the mean ± SD.
Multivariate Analysis
Five variables were included in the model to predict PWV: plasma AGE concentration, blood viscosity at 225 s−1, age, triglyceride level, and HbA1c. The other variables, which were significantly different between groups by univariate analyses, were not included in the model because of the high risk of colinearity effect (variance inflation factor >3) with one of the five variables included. The overall model was statistically significant (R2 = 0.09; df = 5; P < 0.05). AGE concentration was significantly associated with PWV (β = 0.16; P = 0.04), and blood viscosity was marginally associated with PWV (β = 0.15; P = 0.08).
Hemorheological Parameters
No significant differences in hematocrit or plasma viscosity were observed between groups (Table 1). Whole blood viscosity was higher in the two T2D groups compared with the control and SCT groups at low shear rates ranging from 5.62 to 45 s with the following exceptions: at 22.5 s−1 blood viscosity was higher in the T2D-SCT group compared with the control group only, and at 5.62 and 45 s−1 blood viscosity was higher in the T2D group compared with the control group only (Table 1). At higher shear rates (90–375 s−1), blood viscosity in the T2D group was elevated only in comparison with the control group, whereas blood viscosity was higher in the T2D-SCT group compared with the other three groups (Table 1). Furthermore, blood viscosity was higher in the SCT group compared with the control group at the two highest shear rates (225–375 s−1) (Table 1). The thixotropic index was significantly greater in the T2D-SCT group compared with the other three groups (Table 1).
Plasma AGEs and Cytokines
Plasma concentrations of AGEs were significantly increased in the T2D-SCT group compared with the control and SCT groups, and there was a trend for elevated AGE levels in the T2D-SCT group compared with the T2D group after application of the Holm-Bonferroni correction (P = 0.03) (Table 1). Plasma concentrations of IL-1β did not vary among groups (Table 1). No statistical differences in plasma concentrations of IL-6, IL-8, and TNF-α were found among groups (Table 1). The plasma concentrations of IL-2, IL-4, IL-10, GM-CSF, and IFN-γ were very low and below the limits of detection of the kits used.
FACS: E-Selectin and VCAM-1
After incubation with plasma, HAEC expression of E-selectin was significantly higher in the T2D-SCT group compared with the other three groups. No differences in the expression of VCAM-1 were observed among the groups (Fig. 2A).
Flow cytometry, real-time quantitative PCR, and Western blot results for subjects with T2D and SCT (T2D-SCT) or without SCT (T2D) and subjects without T2D with SCT (SCT) or without SCT (Control). A: Flow cytometry results for E-selectin and VCAM-1 expression in HAECs incubated with TNF-α and plasma from subjects; *P < 0.05. B: Flow cytometry results for E-selectin expression in HAECs incubated with TNF-α and either control or T2D-SCT plasma (Control or T2D-SCT, respectively), control or T2D-SCT plasma and the antioxidant tiron (Control+Tiron or T2D-SCT+Tiron), and control or T2D-SCT plasma with the AGE inhibitor aminoguanidine hydrochloride (Control+AG or T2D-SCT+AG); *P < 0.05, **P < 0.01. C: Real-time quantitative PCR for results showing relative mRNA expression of NF-κB and ZO-1 in HAECs incubated with TNF-α and plasma from subjects, *P < 0.05. D: Western blot results showing the relative expression of phospho- and total p38 MAPK in HAECs incubated with TNF-α and plasma from subjects. Results are presented as the mean ± SD.
Incubation of HAECs with plasma and aminoguanidine hydrochloride significantly reduced the HAEC expression of E-selectin in the T2D-SCT group only, whereas incubation of HAECs with plasma and tiron resulted in reduced HAEC expression of E-selectin in both the control and T2D-SCT groups (Fig. 2B).
mRNA Expression
Real-time quantitative PCR revealed no significant difference in the expression of ZO-1 mRNA. NF-κB mRNA expression was significantly higher in the T2D group compared with the control group (Fig. 2C).
Western Blot
There were no significant differences in expression of phospho- and total p38 MAPK in HAECs among groups (Fig. 2D).
Conclusions
The primary finding of the current study was that retinopathy, hypertension, and reduced renal function were more prevalent in subjects with SCT and T2D than in those with T2D alone. Our study also found increased arterial stiffness, blood viscosity, thixotropic index, and plasma AGE concentrations in the T2D-SCT group compared with the other three groups. Our results corroborate the findings of a previous study by Diaw et al. (10), which observed amplified vascular dysfunction in individuals with T2D and SCT compared with subjects with T2D, subjects with SCT, and control subjects. However, few previous studies have evaluated whether SCT could increase the risk of the development of T2D-related complications, and the results of these studies have been contradictory. Indeed, several studies (11,13,14,22) have reported that SCT does not aggravate microvascular or macrovascular complications, whereas several other studies (10,12,23) indicate that SCT could potentially increase the risk of vascular complications in male patients or in all patients with T2D. These conflicting results could be attributed to the fact that the ethnic and racial backgrounds of the study populations varied widely between studies. Furthermore, most of the previous studies had small overall sample sizes and/or a small number of subjects with T2D-SCT. In the current study, however, all groups were matched for age, ethnicity, and sex.
Interestingly, our results showed no significant differences in urine albumin concentration among the T2D, SCT, and T2D-SCT groups, although eGFR was significantly lower in the T2D-SCT group compared with the other three groups. However, several studies have shown that renal disease in individuals with T2D is heterogeneous and that renal insufficiency can occur in the absence of albuminuria in individuals with T2D (24).
A large body of evidence suggests that arterial stiffness plays an important role in the pathogenesis of T2D and its complications (25). Indeed, previous studies (25,26) have shown that increased arterial stiffness is associated with the development and progression of hypertension, nephropathy, and retinopathy in individuals with diabetes. Our findings are in accordance with these studies, as arterial stiffness was highest in the T2D-SCT group, which also had the highest rates of these complications. PWV, which is considered one of the most reliable measures of arterial stiffness, is strongly associated with blood pressure (27). However, in our study, PWV remained elevated in the T2D-SCT group even when MAP was fixed as a covariate, indicating that differences in arterial pressure are probably not completely responsible for differences in PWV among the groups. In T2D, accelerated arterial stiffening is partially attributed to the formation of AGEs in the arterial wall, which leads to cross-linking of collagen molecules, resulting in decreased collagen elasticity (26). In our study, plasma AGE concentrations were elevated in the T2D-SCT group. Additionally, the results of our multivariate analysis suggest that the plasma concentrations of AGEs could be the most important factor contributing to the elevated arterial stiffness in the T2D-SCT group. These results suggest that SCT could potentially exaggerate the nonenzymatic glycation cross-linking of collagen, thereby contributing to increased arterial rigidity in these individuals.
Hyperglycemia is one of the primary factors contributing to AGE formation (28). However, in the current study we found no significant differences in HbA1c or fasting glucose levels between the T2D-SCT and the T2D groups, although concentrations of AGEs were significantly higher in the T2D-SCT group. In addition to glucose concentration, AGE concentrations can also be enhanced by oxidative stress (29). Therefore, the elevated AGE concentrations found in the T2D-SCT group could be attributed to the elevated levels of oxidative stress that have been previously observed in individuals with T2D-SCT (10).
Evidence suggests that AGE accumulation promotes the progression of diabetes complications, including nephropathy, retinopathy, and macroangiopathy, both through the formation of cross-links in the basement membrane of the extracellular matrix and through interaction with the receptor for AGEs (RAGE) (30). AGE-RAGE interaction activates nuclear transcription factors, such as NF-κB, which can upregulate the transcription of inflammatory proteins, such as VCAM-1 and E-selectin (30). E-selectin is expressed by endothelial cells during inflammation and plays an essential role in mediating leukocyte adhesion to the endothelium (31). Furthermore, increased E-selectin expression may contribute to the pathogenesis of vascular disease in T2D (32). We found that E-selectin expression was upregulated in HAECs treated with plasma from individuals with T2D-SCT compared with HAECs treated with plasma from the other three groups. Furthermore, when the HAECs were treated with plasma from the individuals with T2D-SCT and an inhibitor of AGE formation, the expression of E-selectin significantly decreased; however, there was no significant effect of AGE inhibition on HAEC E-selectin expression in the control group. These results confirm that AGEs likely contribute to the upregulation of E-selectin expression in the T2D-SCT group.
Our results showed that NF-κB mRNA expression was not elevated in the T2D-SCT group and that there were no differences in VCAM-1 expression among groups. These findings suggest that a pathway other than AGE-RAGE activation of NF-κB could cause the elevated expression of E-selectin found in the T2D-SCT group. The p38 MAPK signaling pathway is another mechanism that modulates the expression of E-selectin (33,34). However, our findings showed no differences in p38 MAPK expression among the groups, suggesting that a different mechanism is most likely responsible for the increased E-selectin protein expression observed in the T2D-SCT group.
Elevated concentrations of proinflammatory cytokines can contribute to the development of vascular dysfunction and microvascular and macrovascular complications in T2D (35). However, in this study, no differences in plasma concentrations of IL-1β, IL-6, IL-8, or TNF-α were found among groups, indicating that differences in these proinflammatory cytokines likely do not explain the increased arterial stiffness or the increased rates of vascular complications observed in the T2D-SCT group.
Elevated whole blood viscosity has been shown to play an important role in the pathogenesis of hypertension (36), diabetic retinopathy (37), and chronic kidney disease (38). In our study, whole blood viscosity was significantly increased at high shear rates (90–375 s−1) in the T2D-SCT group compared with the other three groups. Whole blood viscosity is dependent on plasma viscosity, hematocrit, and red blood cell rheological properties, including red blood cell aggregation and deformability (39). However, hematocrit and plasma viscosity did not vary among groups. Therefore, the increased blood viscosity observed in the T2D-SCT group is likely due to alterations in red blood cell rheological properties (39). The impact of red blood cell rheological properties on blood viscosity is highly dependent on shear rate (39). At low-to-moderate shear rates, red blood cell aggregation is the main determinant of blood viscosity, whereas red blood cell deformability plays a more important role at high shear rates. Decreased red blood cell deformability contributes to increased blood viscosity in both T2D and SCT (8). Indeed, SCT red blood cells contain HbS and have been shown to have increased lactate transporter activity, which could increase lactate and hydrogen ion accumulation in the red blood cells, thereby decreasing red blood cell deformability (8). Furthermore, in T2D, hyperglycemia leads to increased nonenzymatic glycation of hemoglobin and membrane proteins in red blood cells, and these alterations are associated with decreased membrane fluidity (39). Evidently, the combination of these factors could potentially amplify decreased red blood cell deformability, and thus increase whole blood viscosity, in individuals with T2D-SCT. Additionally, the elevated plasma AGE concentrations observed in the T2D-SCT group suggest that nonenzymatic glycation of red blood cell membrane proteins could be increased in these individuals, thereby contributing to the increased red blood cell rigidity (40).
We also observed a higher thixotropic index in the T2D-SCT group compared with the other three groups. Blood is considered a thixotropic fluid because its viscosity depends not only on shear rate but also on the previous history of motion of the fluid (19). Indeed, at low shear rates, red blood cells form large, reversible aggregates, thereby increasing blood viscosity (19,38). However, as the shear rate rises, viscosity gradually decreases as the aggregates separate into smaller aggregates and eventually into individual red blood cells (19). If the shear rate is then reduced, red blood cell aggregates will gradually reform, and blood viscosity will increase again (19). Therefore, the increased thixotropic index observed in the T2D-SCT group may reflect the amplified red blood cell alterations present in the T2D-SCT group. Furthermore, the high thixotropic index in the T2D-SCT group could potentially increase vascular disease risk in these individuals because an increased thixotropic index has been linked to the occurrence of microvascular and macrovascular disorders in sickle cell anemia and coronary artery disease (20, 41).
In conclusion, our study found that hypertension, retinopathy, and decreased renal function were more prevalent in individuals with both SCT and T2D than in those with T2D only. Furthermore, our results suggest that SCT could exacerbate multiple factors that increase the risk of development of T2D-related vascular complications, including arterial stiffness, blood hyperviscosity, AGE accumulation, and increased E-selectin expression. Overall, our results indicate that individuals with T2D-SCT should be monitored more frequently for diabetic retinopathy, diabetic nephropathy, and hypertension. Additionally, SCT is prevalent not only in sub-Saharan Africa but also in the Middle East, India, and populations of African ancestry in Europe and the Americas; the prevalence of T2D is projected to increase in all of these populations (3,4). Therefore, additional studies are needed to confirm whether or not SCT increases the risk of development of T2D-related vascular complications across all populations (4).
Article Information
Acknowledgments. The authors thank the personnel of Laboratoire de Biochimie et Biologie Moléculaire du Lyon-Est and of Laboratoire de Biochimie du Centre Lyon-Sud for their help with measurements of HbA1c.
Funding. This work has been partly funded by Campus France.
Duality of Interest. No potential conflicts of interest relevant to this article were reported.
Author Contributions. S.C.S. contributed to, implemented, and designed the study; wrote the first draft of the manuscript; and carefully read and approved the final version of the manuscript. M.D. contributed to, implemented, and designed the study and carefully read and approved the final version of the manuscript. V.P., P.M., and D.S. contributed to the study and carefully read and approved the final version of the manuscript. M.N.M., P.L., D.B., F.G., D.D., P.J., C.R., S.D., and A.S. contributed to and implemented the study and carefully read and approved the final version of the manuscript. B.R. and A.V. contributed to and designed the study and carefully read and approved the final version of the manuscript. N.G. contributed to, implemented, and designed the study and carefully read and approved the final version of the manuscript. P.C. contributed to and designed the study, reviewed and revised the manuscript, and carefully read and approved the final version of the manuscript. P.C. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
- Received June 15, 2018.
- Accepted September 24, 2018.
- © 2018 by the American Diabetes Association.
Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. More information is available at http://www.diabetesjournals.org/content/license.
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