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Proinflammatory cytokines are increased in type 2 diabetic women with cardiovascular disease
Journal of Diabetes and Its Complications 18 (2004) 343 – 351
Proinflammatory cytokines are increased in type 2 diabetic women with cardiovascular disease Hillary A. Tuttle a,b, Grace Davis-Gorman a,b, Steve Goldman c, Jack G. Copeland a,b, Paul F. McDonagh a,b,* a
Cardiovascular and Thoracic Surgery Section, University of Arizona Health Sciences Center, Tucson, AZ 85724, USA b Sarver Heart Center, University of Arizona Health Sciences Center, Tucson, AZ 85724, USA c Department of Cardiology, Southern Arizona VA Health Care System, Tucson, AZ 85723, USA Received 8 April 2003; accepted 25 August 2003
Abstract Diabetics have a much greater morbidity and mortality due to cardiovascular disease (CVD) than nondiabetics. Furthermore, diabetic women have a 3.8-fold greater risk for CVD compared to diabetic men. Inflammation is now considered a risk factor for CVD and it has been demonstrated that inflammation also plays a role in diabetes. One component of inflammation that has reported to be increased in patients with diabetes only and CVD only are proinflammatory cytokines, particularly interleukin-6 (IL-6), tumor necrosis factor (TNF-a), and interleukin-1 (IL-1h). This study was performed to test the hypothesis that these proinflammatory cytokines were increased in women with CVD and further increased in diabetic women with CVD compared to nondiabetic women with CVD and healthy age-matched controls. We found that IL-6 was increased in diabetic women with CVD compared to healthy age-matched controls (1.41 = 0.48 to 0.33 F 0.06 pg/ml, P < .05). IL-6 was also increased in diabetic women without CVD compared to healthy age-matched controls, but not significantly (0.96 F 0.27 to 0.33 F 0.06 pg/ml). We found that TNF-a was increased in diabetic women with and without CVD compared to healthy age-matched controls, but not significantly (4.53 F 1.38 to 3.93 F 0.53 to 2.33 F 0.89 pg/ml). IL-1h was not significantly different among any of the four groups of women. These results indicate that both IL-6 and TNF-a are chronically increased in diabetic women with and without CVD compared to nondiabetic women. The additive concentration of cytokines in diabetes and CVD suggests a common inflammatory state in both diabetes and CVD. D 2004 Elsevier Inc. All rights reserved. Keywords: Cytokines; Type 2 diabetes mellitus; Cardiovascular disease; Women
1. Introduction There is a strong link between diabetes and cardiovascular disease (CVD). CVD is the number one killer of patients with diabetes. Men with diabetes are two to three times more likely to die from coronary heart disease (CHD) than those without diabetes, and the risk for women is even higher (Lotufo et al., 2001). Patients with diabetes have increased morbidity and mortality associated with CVD. The precise mechanism as to why patients with diabetes * Corresponding author. Cardiovascular and Thoracic Surgery Section, University of Arizona Health Sciences Center, 1501 North Campbell Avenue, P.O. Box 245071 Tucson, AZ 85724-5071, USA. Tel.: +1-520626-2329; fax: +1-520-626-4042. E-mail address: [email protected] (P.F. McDonagh). 1056-8727/04/$ – see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/S1056-8727(03)00088-6
have such an increased risk of CVD is unclear. It is currently understood that inflammation plays a role in CVD (Libby, 2002; Libby, Ridker, & Maseri, 2002). One hypothesis to explain why diabetic patients have more severe CVD is that diabetic patients have an increase in inflammation compared to the nondiabetic patient. One component of inflammation that has been reported in both diabetes and CVD is the production of proinflammatory cytokines, particularly interleukin-6 (IL-6), tumor necrosis factor (TNF-a), and interleukin-1 (IL-1h). Cytokines are soluble polypeptides released from cells of the immune system, particularly macrophages. Proinflammatory cytokines including, IL-6, TNF-a, and IL-1h, are major mediators in the induction of the acute phase response (Nomura et al., 2000). Previous studies indicate that IL-6, TNF-a, and IL-1h are increased in many different types of
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Table 1 Demographics of the female patients
Patient characteristics Age (years) Body mass index (kg/m2) Duration of Diabetes (years) HbA1c (%) Hypertension (%) Hypercholesteremia (%) Current smoker (%) Prior cardiovascular events (%) Myocardial Infarction Stroke PTCA CABG
ND-CVD (Hokama et al., 1999)
ND + CVD (Yazdani et al., 1998)
D-CVD (American Diabetes Association)
D + CVD (Yudkin et al., 1999)
58.5 F 1.7 26.8 F 1.0* N/A 5.4 F 0.1* 8 25 0
63.1 F 5.0 29.9 F 2.8* N/A 5.2 F 0.2* 86** 57 15
54.5 F 2.4 37.7 F 1.7 8.9 F 1.5 8.2 F 0.4 53 56 11
64.4 F 2.9* 29.5 F 2.0* 11.4 F 3.6 6.9 F 0.5 69** 54 15
0 0 0 0
29 0 29 14
0 0 0 0
62*,** 23 54 15
* P < .05 compared to D-CVD group. ** P < .05 compared to ND-CVD group.
CVD including unstable angina, myocardial dysfunction, chronic heart failure, myocardial infarction, and different cardiac surgeries (Cain et al., 1999; Kanda et al., 2000; Pannitteri et al., 1997; Roig et al., 1998; Tsutamoto et al., 2000; Yazdani et al., 1998). All three cytokines play a key role in the development of atherosclerosis (Huber, Sakkinnen, Conze, Hardin, & Tracy, 1999). It was also demonstrated that IL-6 enhanced the development of atherosclerosis and IL-1h was associated with the development of a lethal arterial inflammation in mice (Dinarello, 2000; Huber et al., 1999). The role of cytokines in diabetes is also under intense investigation. Studies by Lechleitner, Koch, Herold, Dzien, and Hoppichler (2000), McCarty (1999), and Yudkin, Yajnik, Mohamed-Ali, and Bulmer (1999) indicate TNF-a may play a role in the development of diabetes by impairing insulin action. IL-6 and TNF-a have been observed to be increased in diabetic patients (Bastard et al., 2000; Pickup, Chusney, Thomas, & Burt, 2000). In another study, IL-6 was increased in response to acute hyperglycemia (Kato, Morohoshi, Uchimura, & Numano, 1997). There is little investigation of IL-1h in diabetic patients. It is apparent that cytokines play a pathologic role in both diabetes and CVD. It is possible that cytokine concentrations are additive in patients with diabetes and CVD, causing more severe disease. Cytokine concentrations in patients, particularly women, with CVD and diabetes combined have not been reported previously. In this study three proinflammatory cytokines, IL-6, TNF-a, and IL-h were examined via Enzyme Linked-Immuno-Sorbent Assay (ELISA) in four different groups of women: healthy agematched controls, nondiabetic women with cardiovascular disease (ND + CVD), diabetic women without cardiovascular disease (D-CVD), and diabetic women with cardiovascular disease (D + CVD). We found that IL-6 in D + CVD was significantly greater in comparison to healthy agematched controls. There was also a trend for TNF-a to be greater in D + CVD and D-CVD in comparison to healthy
age-matched controls, but no statistical significance. IL-1h was not significantly different among the four groups. This study demonstrates an increase of TNF-a and IL-6 in diabetic women with and without cardiovascular disease in comparison to healthy age-matched controls. The additive increase in cytokines suggests a common inflammatory etiology in cardiovascular disease and diabetes in women.
2. Research design and methods 2.1. Subjects Four groups were studied; nondiabetic women without cardiovascular disease (ND-CVD), nondiabetic women with cardiovascular disease (ND + CVD), diabetic women without cardiovascular disease (D-CVD), and diabetic women with cardiovascular disease (D + CVD). These subjects were enrolled from the patients attending the Cardiology Clinic at the Veterans Administration Hospital (Tucson, AZ) and the Diabetes Clinic at the University of Arizona Medical Center (Tucson, AZ). The protocol was approved by the University of Arizona Institutional Review Board. Patient’s characteristics are summarized in Table 1 and their medications are summarized in Table 2. The severity of diabetes was determined by the patient’s hemoglobin A1C. A subject was considered to have CVD if they had a previous cardiovascular event [myocardial infarction, CABG (coronary artery bypass graft), stroke, PTCA (percutaneous transluminal coronary angioplasty), or some combination]. Fifteen milliliters of blood were drawn in a citrate tube from all the patients. 2.2. Cytokine measurement The blood samples were centrifuged (Megafuge 1.0R, Baxter Scientific Products) at 2500 rpm for 30 min at 4 jC. The plasma was pipetted from each sample and then
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Table 2 Distribution of medications in female patients (%) Medication (%)
ND-CVD (Hokama et al., 1999)
ND + CVD (Yazdani et al., 1998)
D-CVD (American Diabetes Association)
D + CVD (Yudkin et al., 1999)
Beta blockers Diuretics Nitrates ACE inhibitors Positive intropes Ca2 + channel blockers Aspirin Anticoagulants Antioxidants Hypoglycemic agents Antihyperlipidemia HRT
0 4 4 4 0 0 21 0 50 0* 13 50
86*,** 29 29 14 14 29 100*,** 0 43 0* 57 71
11 11 0 63** 5 11 21 5 11 69 32 58
38 46 15 38 15 23 69 7 23 31 46 46
* P < .05 compared to D-CVD group. ** P < .05 compared to ND-CVD group.
immediately stored at 70 jC. Once a sufficient number of samples were collected, they were removed from the freezer and thawed to room temperature. ELISA human IL-6, TNFa, and IL-1h Ultrasensitive kits from Biosource International were used to measure the plasma concentrations of IL-6, TNF-a, and IL-1h. The samples were prepared according to the directions provided by the company. First, the standard was reconstituted and serially diluted. Next, the number of eight-well strips needed for the assay was determined and set aside. Standard diluent buffer was added to the zero wells. Standards, samples, and controls were added to the appropriate microtiter wells. The plate was then covered, incubated, and washed according to the specific kits. Biotinylated anti-IL-6, anti-TNF-a, and antiIL-1b solutions were added to respective wells except the chromogen blank wells. The plate was then covered, incubated, and washed according to the specific kits. Streptavidin-HRP was added to each well (except the chromogen blank) and the plate was covered, incubated, and washed. The stabilized chromogen was added to each well and the liquid in the wells turned blue. The wells were incubated for
30 min at room temperature in the dark. After the final incubation, the stop solution was added to each well and the wells turned from blue to yellow. The plates were then read at 450 nm (Vmax, Molecular Devices) having blanked the plate reader against a chromogen blank composed of stabilized chromogen and stop solution. The plate was read within 2 h of adding the stop solution. A standard curve was plotted and all the unknown samples were determined from the standard curve (Fig. 1). 2.3. Data analysis Data were collected in notebooks and transferred to a Computer Spreadsheet Format (Excel for Windows). The summary results are represented as mean F standard error of the mean (S.E.M.). Cytokine concentrations are expressed as picogram per milliliter (pg/ml). Comparisons among the four groups were made with an ANOVA (SigmaStat 3.0). A Dunn’s and Holm-Sidak post hoc test was performed (Sigma-Stat). P < .05 was considered as statistically significant.
3. Results 3.1. Patient characteristics
Fig. 1. Representative standard curve for IL-6 concentration (IL-6 Ultrasensitive kit from Biosource International). The x-axis represents the concentration of IL-6 and the y-axis represents the mean OD value.
Table 1 summarizes the characteristics of the four groups of women studied. There was a significant difference of age in the D + CVD group compared to the D-CVD group ( P < .05). There was also a significant difference in body mass index (BMI) of the D-CVD group compared to three other groups ( P < .01). There was no significant difference of the duration of diabetes between the two diabetic groups. The glycated hemoglobin (HbAIC) was significantly greater in diabetic women without CVD compared to nondiabetic women with and without CVD ( P < .01). There are significantly more patients with hypertension in the ND + CVD and D-CVD group compared to the ND-CVD group. There
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Fig. 2. Summary of ELISA results of IL-6 for all women in the study. Comparison of ND-CVD vs. ND + CVD vs. D-CVD vs. D + CVD. The IL6 production was significantly greater in diabetic women with and without cardiovascular disease in comparison to healthy age-matched controls ( P < .05).
was no significant difference in hypercholesteremia or percent of smokers among any of the groups. The various drug therapies that the patients were receiving are given in Table 2. There was no significant difference in the percentages of women taking HRT among any of the groups. 3.2. IL-6 production Fig. 2 indicates that IL-6 concentration was graded. In comparison to ND-CVD, we found that IL-6 was significantly increased in D + CVD (Fig. 2) ( P < .05). IL-6 in NDCVD was 0.33 F 0.06. IL-6 in ND + CVD was 0.47 F 0.11. IL-6 in D-CVD was 0.96 F 0.27. IL-6 was greatest in D + CVD (1.41 F 0.48). 3.3. TNF-a production Fig. 3 indicates that TNF-a also appeared to be graded. There was a trend for the TNF-a production in D + CVD to be greater than the other groups, but the increase did not reach statistical significance. In comparison to ND-CVD, we found that TNF-a was increased in diabetic women with
Fig. 3. Summary of ELISA results of TNF-a for all women in the study. Comparison of ND-CVD vs. ND + CVD vs. D-CVD vs. D + CVD.
Fig. 4. Summary of ELISA results of IL-lh for all women in the study. Comparison of ND-CVD vs. ND + CVD vs. D-CVD vs. D + CVD.
and D-CVD. TNF-a in ND-CVD was 2.29 F 0.37. TNF-a in ND + CVD was 2.33 F 0.89. TNF-a in D-CVD was 3.93 F 0.53. TNF-a was greatest in D + CVD (4.53 F 1.38). 3.4. IL-1h production Fig. 4 indicates that there was not a graded response in IL-1h for the four groups. There was no significance among any of the groups of women. IL-1h in ND-CVD was 0.15 F 0.05. IL-1h in ND + CVD was 0.08 F 0.08. IL-1h in D-CVD was 0.25 F 0.09. IL-1h in D + CVD was 0.22 F 0.14.
4. Discussion There are 17 million people in the United States who have diabetes and 95% of those have type 2 diabetes (American Diabetes Association). The relative excess risk for cardiovascular disease (CVD) is two- to eightfold higher in people with diabetes than in nondiabetic individuals of similar sex, age, and ethnicity (Howard et al., 2002). Diabetes mellitus is a much stronger risk factor for coronary heart disease (CHD) in women than in men. Diabetes is associated with a three- to sevenfold elevation in CHD risk among women compared with a two- to threefold elevation among men (Mosca et al., 1997). Howard et al. (1995) reported that the prevalence rate ratio for CHD in individuals with type 2 diabetes was 4.6 in women compared to 1.8 in men. According to the American Heart Association (2003), cardiovascular disease was the number one cause of death in women in the year 2000. There were over 945,000 deaths from CVD in the United States, accounting for one out of five deaths. Fifty-three percent of these deaths due to CVD were in women. It is apparent that there is a factor or combination of factors that causes women with diabetes to have more severe cardiovascular disease. It is known that inflammation mediates the formation of atherosclerotic plaques, which contributes to cardiovascular disease. Inflammation is now considered a cardiovascular risk factor and a recent study of predictors of
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cardiovascular risk ranks markers of inflammation as comparable to markers of cholesterol (Libby, 2002). The association of inflammation and diabetes is a topic of active research. Our laboratory has demonstrated that neutrophil activation and platelet-neutrophil interactions are increased in type 2 diabetic men (Hokama et al., 1999). Hokama et al. (2000) reported diabetic rat hearts suffer excessive reperfusion injury following ischemia. An enhanced inflammatory response may contribute to the greater recurrence of myocardial infarction and ischemic heart failure in diabetes. In this study, we tested the hypothesis that a component of the inflammatory response, proinflammatory cytokines, was increased in CVD and further increased in diabetic women with CVD. There appears to be an additive effect of CVD and diabetes on the chronic concentrations of proinflammatory cytokines in women. We examined interleukin-6 (IL-6), interleukin-1 (IL-1h), and tumor necrosis factor (TNF-a) in women with only CVD, women with only diabetes, and women with both diabetes and CVD, and then compared them to healthy age-matched controls. These proinflammatory cytokines appear throughout the literature to play an important role in the pathology of both diabetes and CVD. Cytokines aggravate the development of atherosclerosis in CVD, while cytokines potentially cause insulin insensitivity in diabetes. We found trends towards increasing plasma cytokines and the incidence of CVD and diabetes. The association of plasma IL-6 was the most consistent and dramatic finding. Women with diabetes, with and without CVD, demonstrated a significant increase in plasma IL-6. This study also revealed an interesting pattern of IL-6 and TNF-a cytokine. Women with both diabetes and CVD had the highest concentration of these cytokines. Lower concentrations were observed in diabetic women without CVD, and women with only CVD, respectively. Finally, healthy agematched controls had the lowest concentrations of IL-6 and TNF-a. This trend suggests that as women develop diabetes and cardiovascular disease, the inflammatory stimuli in the blood increases, which could then lead to more severe cardiovascular disease, including atherosclerosis. Alternatively, these data suggest the chronic inflammation underlies both CVD and diabetes. In this study, IL-1h did not differ significantly among any of the groups, suggesting that this cytokine may not be a key component in the cardiovascular disease process observed in diabetic women. 4.1. Inflammation in diabetes and cardiovascular disease The metabolic events that occur during an inflammatory response to a pathogen also occur in certain disease processes, i.e., atherosclerosis and diabetes mellitus. These changes do not appear to have a beneficial purpose but rather contribute to the pathology of the disease. Hayden and Tyagi (2003) hypothesized that type 2 diabetes mellitus is a vascular disease (atherosclerosis) and that NOS, NO, redox stress, and components of inflammation, play a causative role in type 2 diabetes, in turn causing atheroscle-
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rosis. Inflammation, in particular cytokines, plays a key role in atherosclerosis (Ross, 1999). In atherogenesis, endothelial dysfunction is an early step. Injury to the endothelium increases adhesion of leukocytes and platelets. The inflammatory response stimulates migration and proliferation of smooth-muscle cells that become intermixed with the area of inflammation to form an intermediate lesion. Chronic inflammation results in increased numbers of macrophages and lymphocytes. Activation of these cells leads to release of hydrolytic enzymes, cytokines, chemokines, and growth factors that cause more damage and eventually lead to focal necrosis. The lesion is then covered by a fibrous cap that overlies a core of lipid and necrotic tissue (Ross, 1999; Selzman, Miller, & Harken, 2001). Inflammation has also been reported to be increased in type 2 diabetes. Esposito et al. (2002) reported that hyperglycemia acutely increased circulating IL-6 and TNF-a. Pradhan, Manson, Rifai, Buring, and Ridker (2001) reported baseline IL-6 was increased in women who later developed type 2 diabetes. The relative risk of future type 2 diabetes in women between the highest and lowest quartiles was 7.5 for interleukin-6. The results of this study support a role for inflammation in the development of diabetes (Pradhan et al., 2001). These acute phase reactants may explain the clinical and biochemical features of type 2 diabetes and its complications. The increased cytokines in diabetes appear to originate from noncirculating cells (Pickup et al., 2000) and the likely candidates are adipocytes and endothelial cells. If cytokines chronically circulate in diabetic patients, then there could be continuous aggravation of atherosclerosis causing more sites of atherosclerosis and acceleration of the progression of the lesions. Despite the apparent links between diabetes and cardiovascular disease, there have been no reports focusing on the concentrations of cytokines in women with both diabetes and cardiovascular disease. 4.2. Cytokines and cardiovascular disease Cytokine activity has been implicated in various aspects of the pathogenesis in cardiovascular disease including atherosclerosis, acute myocardial infarction, congestive heart failure, myocarditis, and allograft rejection (Oyama, Shimokawa, Morita, Yasui, & Takeshita, 2001). Cardiovascular disease is characterized by a chronic low-level inflammatory process with increased circulating concentrations of proinflammatory cytokines (such as IL-6, TNF-a, IL-1h) (Huber et al., 1999). 4.2.1. Atherosclerosis and cytokines Our data suggests that patients with diabetes and CVD have increased IL-6 and TNF-a. The pathogenesis of atherosclerosis involves many different cytokines, particularly the proinflammatory cytokines, IL-6, IL-1h, and TNF-a,b. Evidence that IL-6 plays an active role in cardiovascular disease is demonstrated in a study by Huber et al. (1999). This investigation injected Apo-E-deficient mice
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with IL-6 or saline. Fatty streak lesions were 1.9- to 5.1-fold larger in the IL-6 treated mice compared to the saline-treated mice, demonstrating that exogenously administered IL-6 significantly enhanced fatty lesion development in the atherosclerosis-prone, but not in the atherosclerosis-resistant animals. This suggests that IL-6 likely has an active role in the atherosclerosis disease process (Huber et al., 1999). It has also been determined that there were large quantities of IL-6 found in human atherosclerotic plaques, typically in macrophage rich area (Rus, Vlaicu, & Niculescu, 1996). IL-1h has also been observed in atherosclerotic lesions (Munro & Cotran, 1988; Ross, 1993). In a study by Nicklin, Hughes, Barton, Ure, and Duff (2000) mice in which the gene for IL-1 receptor antagonist was knocked out were examined. It was observed that there was a lethal arterial inflammation characterized by transmural infiltration of neutrophils, macrophages, and T lymphocytes. The inflammatory nature of the lesions was consistent with the biologic effects of IL-1 on endothelial cells and smooth-muscle cells. IL-1 receptor antagonist produced in healthy mice protects them from the effects of interleukin-1 (Nicklin et al., 2000). 4.2.2. Interleukin-6 In several studies, IL-6 was increased after acute myocardial infarctions and unstable angina (Kanda et al., 2000; Pannitteri et al., 1997; Yazdani et al., 1998). Studies by Roig et al. (1998) and Tsutamoto et al. (2000) demonstrated that IL-6 was increased to 4.1 F 0.6 and 73 F 4 pg/ ml, respectively, in patients with congestive heart failure compared with normal subjects. In the study by Roig et al., IL-6 was associated with lower ejection fraction and worse prognosis (Kanda et al., 2000; Roig et al., 1998). Suzuki, Ishiwata, Hassegawa, Yamamoto, and Yamazaki (2000) reported that patients with stable angina pectoris undergoing elective coronary angioplasty IL-6 was significantly increased 1 and 6 h after the procedure (3.6- and 4.4-fold, respectively). These patients with increased IL-6 later presented with restenosis (Suzuki et al., 2000). IL-6 was also reported to be increased preoperatively (Nomura et al., 2000) and after various cardiac surgeries including CABG and valvular surgery (Berendes et al., 1997; Corbi et al., 2000; Kawahito, Adachi, & Ino, 2000; Liebold, Langhammer, Brunger, & Birnbaum, 1999; Whitten, Hill, Ivy, Greilich, & Lipton, 1998; Yamada et al., 1998). These studies demonstrate clear evidence that IL-6 is increased in response to various different cardiac diseases. These studies agree with our findings of increased IL-6 in patients with different forms of CVD. In this study, we found that IL-6 was significantly increased in diabetic women with CVD in comparison to healthy age-matched controls. We also found women with CVD only had an increase in IL-6 production compared to healthy age-matched controls, but the data were not significant (Fig. 2). This increased concentration of IL-6 observed in diabetic women with and without CVD could reflect the diabetogenesis. The
even higher IL-6 concentrations observed in diabetic women with CVD could then represent an added risk factor of atherosclerosis. 4.2.3. Tumor necrosis factor-alpha The literature on TNF-a found in various different cardiac disease states is conflicting. Both proinflammatory cytokines TNF-a and IL-1h have been implicated in the pathogenesis of myocardial dysfunction in ischemia – reperfusion injury and chronic heart failure. In a study by Cain et al. (1999), it was demonstrated that TNF-a and IL-1h separately and synergistically decreased human myocardial function in a dose-dependent fashion, affecting systolic more than diastolic function. Concentrations as low as 1.25 pg/ml of TNF-a decreased the force of myocardial development (Cain et al., 1999). Roig et al. (1998) and Tsutamoto et al. (2000) observed TNF-a to be increased in patients with congestive heart failure compared to control subjects. In contrast, Pannitteri et al. (1997) observed no significant rise of TNF-a in patients following an acute myocardial infarction and Nomura et al. (2000) found TNFa did not increase preoperatively in a group of patients prior to vascular surgery. Regarding TNF-a we found this cytokine to be increased in patients with CVD only, diabetes only, and both CVD and diabetes in comparison to normal controls, but not significantly (Fig. 3). This increase in TNFa, agrees with some reports in the literature on TNF-a (Roig et al., 1998; Tsutamoto et al., 2000). The increase in TNF-a observed in diabetic women with CVD could be one of the mediators of atherogenesis. It could also be one of the contributors to the pathogenesis of diabetes itself. 4.2.4. Interleukin-1h The literature on IL-1h found in various cardiac disease states is conflicting. It was demonstrated that IL-1h decreased the function of human myocardial trabeculae in a dose-dependent fashion, affecting systolic more than diastolic function (Cain et al., 1999). Nomura et al. (2000) found that IL-1 was increased preoperatively in a group of patients prior to vascular surgery, but Yazdani et al. (1998) found that IL-1 was not significantly different between stable and unstable angina. The result of IL-1h in this study demonstrated that there was no significant difference among the four different groups that were studied; CVD only, diabetes only, CVD and diabetes, and healthy age-matched controls (Fig. 4). The data on the role of IL-1h in diabetes and cardiovascular disease is less convincing. IL-1h may not be one of the cytokines that is chronically increased in patients with diabetes and CVD, therefore not contributing to the increased severity of disease observed in diabetic women with CVD. 4.3. Diabetes and cytokines It is understood that cytokines play a significant role in diabetes. There is evidence that inflammation is an initiating
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factor in insulin insensitivity and proinflammatory cytokines are the key linkage between inflammation and insulin insensitivity (Feinstein, Kanety, Papa, Lunenfeld, & Karasik, 1993; Hotamisligil, 1999; McCarty, 1999; Yudkin et al., 1999). Our data demonstrated that type 2 diabetic women with and without CVD had increased concentrations of IL-6 and TNF-a. Several studies reported an increase in circulating cytokines in patients with diabetes (Bastard et al., 2000; Pickup et al., 2000). This increase in cytokine production could then lead to inappropriate metabolic effects causing arteriosclerosis. IL-6 and TNF-a were significantly increased in type 2 diabetic patients compared to normal subjects in a study by Pickup et al. (2000). The diabetic patients were observed to have higher body mass indices, glycated hemoglobin percentage (HbAIC), and plasma total cholesterol and triglyceride concentration. Five of the 20 diabetic patients in this study had microvascular (including retinopathy and nephropathy) and/or macrovascular disease (including myocardial infarction, angina, peripheral vascular disease, or stroke) and there was no significant difference in comparison to diabetic patients without vascular disease (Pickup et al., 2000). In contrast, Mooradian, Reed, Meredith, and Scuderi (1991) reported that diabetes mellitus was not associated with significant changes in serum TNF-a or IL-1h. Bastard et al. (2000) observed obese nondiabetic and diabetic patients with high insulin resistance had increased IL-6 and TNF-a in comparison to lean controls. IL-6, not TNF-a, was found to be proportional to insulin resistance and blood glucose. Thus, severe diabetes is associated with increased IL-6. When the obese patients were placed on a very low-calorie diet (VLCD) for a 3-week period, the IL-6 decreased significantly in both adipose tissue and serum. TNF-a however was unchanged. They found serum concentration of IL-6 and TNF-a were significantly correlated with BMI. The authors concluded that the circulating IL-6 possibly reflected adipose tissue production and insulin resistance. Vozarova et al. (2002) also reported that plasma IL-6 relates positively to adiposity and negatively to insulin sensitivity. A study by Kato et al. (1997) determined that IL-6 production was positively regulated by hyperglycemia (Kato et al., 1997). TNF-a has a complex relationship with diabetes. TNFa exerts its action on insulin signaling and lipid metabolism by suppressing expression of the insulin sensitive glucose transporter GLUT-4, insulin receptor substrate-1 (IRS-1), and insulin receptor (Feinstein et al., 1993; Long & Pekala, 1996; Stephens, Lee, & Pilch, 1997). This causes an excessive free fatty acids (FFA) flux from hypertrophied insulin-resistant adipocytes (Feinstein et al., 1993; Long & Pekala, 1996; Stephens et al., 1997). This mechanism demonstrates a possible link between TNF-a, obesity, insulin resistance, and type 2 diabetes (Feinstein et al., 1993; Hotamisligil, 1999; McCarty, 1999; Yudkin et al., 1999). It has also been demonstrated that incubating rat skeletal muscle cells with TNF-a results in
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significant impairment of insulin action (Begum & Ragolia, 1996). 4.4. Conclusion TNF-a and IL-6 are produced by cells of the immune system and adipocytes and this may provide the link between inflammation and insulin insensitivity (Grimble, 2002; Mohamed-Ali et al., 1997; Yudkin et al., 1999). Since the majority of patients with diabetes and cardiovascular disease are overweight, this is possibly one of the mechanisms by which diabetic patients have an increased production of these two cytokines. It has been reported that TNF-a and IL-6 correlate with BMI (Bastard et al., 2000). Increased concentrations of TNF-a and IL-6 may affect the development of atherosclerosis, creating a more severe lesion. The increased circulating concentrations of both TNF-a and IL-6 could also negatively affect myocardial function. Therefore, a patient with these increased cytokines may have a worse prognosis after a myocardial infarction, angina, or congestive heart failure. Diabetic patients also have more severe atherosclerosis and a higher degree of morbidity/mortality associated with myocardial infarction, congestive heart failure, and other cardiovascular events (Abbott, Donahue, Kannel, & Wilson, 1988; American Diabetes Association). In conclusion, the findings of this study indicate that (1) D + CVD have significantly increased IL-6 in comparison to age-matched controls, which indicates the potential additive effects of inflammation in these two disease processes; (2) diabetic women with and without cardiovascular disease have increased TNF-a in comparison to age-matched controls, which also indicates the potential additive effects of inflammation in these two disease processes; and (3) there was no correlation between diabetes or CVD of IL-1h. Thus, the increased concentrations of IL-6 and TNF-a in women as a result of diabetes and as a result of cardiovascular disease could work synergistically in comparison to women with cardiovascular disease only or diabetes only and could be a contributor to the more severe cardiovascular disease in this population.
Acknowledgments This research was supported by NIH HLB 58859.
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Proinflammatory cytokines are increased in type 2 diabetic women with cardiovascular disease Hillary A. Tuttle a,b, Grace Davis-Gorman a,b, Steve Goldman c, Jack G. Copeland a,b, Paul F. McDonagh a,b,* a
Cardiovascular and Thoracic Surgery Section, University of Arizona Health Sciences Center, Tucson, AZ 85724, USA b Sarver Heart Center, University of Arizona Health Sciences Center, Tucson, AZ 85724, USA c Department of Cardiology, Southern Arizona VA Health Care System, Tucson, AZ 85723, USA Received 8 April 2003; accepted 25 August 2003
Abstract Diabetics have a much greater morbidity and mortality due to cardiovascular disease (CVD) than nondiabetics. Furthermore, diabetic women have a 3.8-fold greater risk for CVD compared to diabetic men. Inflammation is now considered a risk factor for CVD and it has been demonstrated that inflammation also plays a role in diabetes. One component of inflammation that has reported to be increased in patients with diabetes only and CVD only are proinflammatory cytokines, particularly interleukin-6 (IL-6), tumor necrosis factor (TNF-a), and interleukin-1 (IL-1h). This study was performed to test the hypothesis that these proinflammatory cytokines were increased in women with CVD and further increased in diabetic women with CVD compared to nondiabetic women with CVD and healthy age-matched controls. We found that IL-6 was increased in diabetic women with CVD compared to healthy age-matched controls (1.41 = 0.48 to 0.33 F 0.06 pg/ml, P < .05). IL-6 was also increased in diabetic women without CVD compared to healthy age-matched controls, but not significantly (0.96 F 0.27 to 0.33 F 0.06 pg/ml). We found that TNF-a was increased in diabetic women with and without CVD compared to healthy age-matched controls, but not significantly (4.53 F 1.38 to 3.93 F 0.53 to 2.33 F 0.89 pg/ml). IL-1h was not significantly different among any of the four groups of women. These results indicate that both IL-6 and TNF-a are chronically increased in diabetic women with and without CVD compared to nondiabetic women. The additive concentration of cytokines in diabetes and CVD suggests a common inflammatory state in both diabetes and CVD. D 2004 Elsevier Inc. All rights reserved. Keywords: Cytokines; Type 2 diabetes mellitus; Cardiovascular disease; Women
1. Introduction There is a strong link between diabetes and cardiovascular disease (CVD). CVD is the number one killer of patients with diabetes. Men with diabetes are two to three times more likely to die from coronary heart disease (CHD) than those without diabetes, and the risk for women is even higher (Lotufo et al., 2001). Patients with diabetes have increased morbidity and mortality associated with CVD. The precise mechanism as to why patients with diabetes * Corresponding author. Cardiovascular and Thoracic Surgery Section, University of Arizona Health Sciences Center, 1501 North Campbell Avenue, P.O. Box 245071 Tucson, AZ 85724-5071, USA. Tel.: +1-520626-2329; fax: +1-520-626-4042. E-mail address: [email protected] (P.F. McDonagh). 1056-8727/04/$ – see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/S1056-8727(03)00088-6
have such an increased risk of CVD is unclear. It is currently understood that inflammation plays a role in CVD (Libby, 2002; Libby, Ridker, & Maseri, 2002). One hypothesis to explain why diabetic patients have more severe CVD is that diabetic patients have an increase in inflammation compared to the nondiabetic patient. One component of inflammation that has been reported in both diabetes and CVD is the production of proinflammatory cytokines, particularly interleukin-6 (IL-6), tumor necrosis factor (TNF-a), and interleukin-1 (IL-1h). Cytokines are soluble polypeptides released from cells of the immune system, particularly macrophages. Proinflammatory cytokines including, IL-6, TNF-a, and IL-1h, are major mediators in the induction of the acute phase response (Nomura et al., 2000). Previous studies indicate that IL-6, TNF-a, and IL-1h are increased in many different types of
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Table 1 Demographics of the female patients
Patient characteristics Age (years) Body mass index (kg/m2) Duration of Diabetes (years) HbA1c (%) Hypertension (%) Hypercholesteremia (%) Current smoker (%) Prior cardiovascular events (%) Myocardial Infarction Stroke PTCA CABG
ND-CVD (Hokama et al., 1999)
ND + CVD (Yazdani et al., 1998)
D-CVD (American Diabetes Association)
D + CVD (Yudkin et al., 1999)
58.5 F 1.7 26.8 F 1.0* N/A 5.4 F 0.1* 8 25 0
63.1 F 5.0 29.9 F 2.8* N/A 5.2 F 0.2* 86** 57 15
54.5 F 2.4 37.7 F 1.7 8.9 F 1.5 8.2 F 0.4 53 56 11
64.4 F 2.9* 29.5 F 2.0* 11.4 F 3.6 6.9 F 0.5 69** 54 15
0 0 0 0
29 0 29 14
0 0 0 0
62*,** 23 54 15
* P < .05 compared to D-CVD group. ** P < .05 compared to ND-CVD group.
CVD including unstable angina, myocardial dysfunction, chronic heart failure, myocardial infarction, and different cardiac surgeries (Cain et al., 1999; Kanda et al., 2000; Pannitteri et al., 1997; Roig et al., 1998; Tsutamoto et al., 2000; Yazdani et al., 1998). All three cytokines play a key role in the development of atherosclerosis (Huber, Sakkinnen, Conze, Hardin, & Tracy, 1999). It was also demonstrated that IL-6 enhanced the development of atherosclerosis and IL-1h was associated with the development of a lethal arterial inflammation in mice (Dinarello, 2000; Huber et al., 1999). The role of cytokines in diabetes is also under intense investigation. Studies by Lechleitner, Koch, Herold, Dzien, and Hoppichler (2000), McCarty (1999), and Yudkin, Yajnik, Mohamed-Ali, and Bulmer (1999) indicate TNF-a may play a role in the development of diabetes by impairing insulin action. IL-6 and TNF-a have been observed to be increased in diabetic patients (Bastard et al., 2000; Pickup, Chusney, Thomas, & Burt, 2000). In another study, IL-6 was increased in response to acute hyperglycemia (Kato, Morohoshi, Uchimura, & Numano, 1997). There is little investigation of IL-1h in diabetic patients. It is apparent that cytokines play a pathologic role in both diabetes and CVD. It is possible that cytokine concentrations are additive in patients with diabetes and CVD, causing more severe disease. Cytokine concentrations in patients, particularly women, with CVD and diabetes combined have not been reported previously. In this study three proinflammatory cytokines, IL-6, TNF-a, and IL-h were examined via Enzyme Linked-Immuno-Sorbent Assay (ELISA) in four different groups of women: healthy agematched controls, nondiabetic women with cardiovascular disease (ND + CVD), diabetic women without cardiovascular disease (D-CVD), and diabetic women with cardiovascular disease (D + CVD). We found that IL-6 in D + CVD was significantly greater in comparison to healthy agematched controls. There was also a trend for TNF-a to be greater in D + CVD and D-CVD in comparison to healthy
age-matched controls, but no statistical significance. IL-1h was not significantly different among the four groups. This study demonstrates an increase of TNF-a and IL-6 in diabetic women with and without cardiovascular disease in comparison to healthy age-matched controls. The additive increase in cytokines suggests a common inflammatory etiology in cardiovascular disease and diabetes in women.
2. Research design and methods 2.1. Subjects Four groups were studied; nondiabetic women without cardiovascular disease (ND-CVD), nondiabetic women with cardiovascular disease (ND + CVD), diabetic women without cardiovascular disease (D-CVD), and diabetic women with cardiovascular disease (D + CVD). These subjects were enrolled from the patients attending the Cardiology Clinic at the Veterans Administration Hospital (Tucson, AZ) and the Diabetes Clinic at the University of Arizona Medical Center (Tucson, AZ). The protocol was approved by the University of Arizona Institutional Review Board. Patient’s characteristics are summarized in Table 1 and their medications are summarized in Table 2. The severity of diabetes was determined by the patient’s hemoglobin A1C. A subject was considered to have CVD if they had a previous cardiovascular event [myocardial infarction, CABG (coronary artery bypass graft), stroke, PTCA (percutaneous transluminal coronary angioplasty), or some combination]. Fifteen milliliters of blood were drawn in a citrate tube from all the patients. 2.2. Cytokine measurement The blood samples were centrifuged (Megafuge 1.0R, Baxter Scientific Products) at 2500 rpm for 30 min at 4 jC. The plasma was pipetted from each sample and then
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Table 2 Distribution of medications in female patients (%) Medication (%)
ND-CVD (Hokama et al., 1999)
ND + CVD (Yazdani et al., 1998)
D-CVD (American Diabetes Association)
D + CVD (Yudkin et al., 1999)
Beta blockers Diuretics Nitrates ACE inhibitors Positive intropes Ca2 + channel blockers Aspirin Anticoagulants Antioxidants Hypoglycemic agents Antihyperlipidemia HRT
0 4 4 4 0 0 21 0 50 0* 13 50
86*,** 29 29 14 14 29 100*,** 0 43 0* 57 71
11 11 0 63** 5 11 21 5 11 69 32 58
38 46 15 38 15 23 69 7 23 31 46 46
* P < .05 compared to D-CVD group. ** P < .05 compared to ND-CVD group.
immediately stored at 70 jC. Once a sufficient number of samples were collected, they were removed from the freezer and thawed to room temperature. ELISA human IL-6, TNFa, and IL-1h Ultrasensitive kits from Biosource International were used to measure the plasma concentrations of IL-6, TNF-a, and IL-1h. The samples were prepared according to the directions provided by the company. First, the standard was reconstituted and serially diluted. Next, the number of eight-well strips needed for the assay was determined and set aside. Standard diluent buffer was added to the zero wells. Standards, samples, and controls were added to the appropriate microtiter wells. The plate was then covered, incubated, and washed according to the specific kits. Biotinylated anti-IL-6, anti-TNF-a, and antiIL-1b solutions were added to respective wells except the chromogen blank wells. The plate was then covered, incubated, and washed according to the specific kits. Streptavidin-HRP was added to each well (except the chromogen blank) and the plate was covered, incubated, and washed. The stabilized chromogen was added to each well and the liquid in the wells turned blue. The wells were incubated for
30 min at room temperature in the dark. After the final incubation, the stop solution was added to each well and the wells turned from blue to yellow. The plates were then read at 450 nm (Vmax, Molecular Devices) having blanked the plate reader against a chromogen blank composed of stabilized chromogen and stop solution. The plate was read within 2 h of adding the stop solution. A standard curve was plotted and all the unknown samples were determined from the standard curve (Fig. 1). 2.3. Data analysis Data were collected in notebooks and transferred to a Computer Spreadsheet Format (Excel for Windows). The summary results are represented as mean F standard error of the mean (S.E.M.). Cytokine concentrations are expressed as picogram per milliliter (pg/ml). Comparisons among the four groups were made with an ANOVA (SigmaStat 3.0). A Dunn’s and Holm-Sidak post hoc test was performed (Sigma-Stat). P < .05 was considered as statistically significant.
3. Results 3.1. Patient characteristics
Fig. 1. Representative standard curve for IL-6 concentration (IL-6 Ultrasensitive kit from Biosource International). The x-axis represents the concentration of IL-6 and the y-axis represents the mean OD value.
Table 1 summarizes the characteristics of the four groups of women studied. There was a significant difference of age in the D + CVD group compared to the D-CVD group ( P < .05). There was also a significant difference in body mass index (BMI) of the D-CVD group compared to three other groups ( P < .01). There was no significant difference of the duration of diabetes between the two diabetic groups. The glycated hemoglobin (HbAIC) was significantly greater in diabetic women without CVD compared to nondiabetic women with and without CVD ( P < .01). There are significantly more patients with hypertension in the ND + CVD and D-CVD group compared to the ND-CVD group. There
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Fig. 2. Summary of ELISA results of IL-6 for all women in the study. Comparison of ND-CVD vs. ND + CVD vs. D-CVD vs. D + CVD. The IL6 production was significantly greater in diabetic women with and without cardiovascular disease in comparison to healthy age-matched controls ( P < .05).
was no significant difference in hypercholesteremia or percent of smokers among any of the groups. The various drug therapies that the patients were receiving are given in Table 2. There was no significant difference in the percentages of women taking HRT among any of the groups. 3.2. IL-6 production Fig. 2 indicates that IL-6 concentration was graded. In comparison to ND-CVD, we found that IL-6 was significantly increased in D + CVD (Fig. 2) ( P < .05). IL-6 in NDCVD was 0.33 F 0.06. IL-6 in ND + CVD was 0.47 F 0.11. IL-6 in D-CVD was 0.96 F 0.27. IL-6 was greatest in D + CVD (1.41 F 0.48). 3.3. TNF-a production Fig. 3 indicates that TNF-a also appeared to be graded. There was a trend for the TNF-a production in D + CVD to be greater than the other groups, but the increase did not reach statistical significance. In comparison to ND-CVD, we found that TNF-a was increased in diabetic women with
Fig. 3. Summary of ELISA results of TNF-a for all women in the study. Comparison of ND-CVD vs. ND + CVD vs. D-CVD vs. D + CVD.
Fig. 4. Summary of ELISA results of IL-lh for all women in the study. Comparison of ND-CVD vs. ND + CVD vs. D-CVD vs. D + CVD.
and D-CVD. TNF-a in ND-CVD was 2.29 F 0.37. TNF-a in ND + CVD was 2.33 F 0.89. TNF-a in D-CVD was 3.93 F 0.53. TNF-a was greatest in D + CVD (4.53 F 1.38). 3.4. IL-1h production Fig. 4 indicates that there was not a graded response in IL-1h for the four groups. There was no significance among any of the groups of women. IL-1h in ND-CVD was 0.15 F 0.05. IL-1h in ND + CVD was 0.08 F 0.08. IL-1h in D-CVD was 0.25 F 0.09. IL-1h in D + CVD was 0.22 F 0.14.
4. Discussion There are 17 million people in the United States who have diabetes and 95% of those have type 2 diabetes (American Diabetes Association). The relative excess risk for cardiovascular disease (CVD) is two- to eightfold higher in people with diabetes than in nondiabetic individuals of similar sex, age, and ethnicity (Howard et al., 2002). Diabetes mellitus is a much stronger risk factor for coronary heart disease (CHD) in women than in men. Diabetes is associated with a three- to sevenfold elevation in CHD risk among women compared with a two- to threefold elevation among men (Mosca et al., 1997). Howard et al. (1995) reported that the prevalence rate ratio for CHD in individuals with type 2 diabetes was 4.6 in women compared to 1.8 in men. According to the American Heart Association (2003), cardiovascular disease was the number one cause of death in women in the year 2000. There were over 945,000 deaths from CVD in the United States, accounting for one out of five deaths. Fifty-three percent of these deaths due to CVD were in women. It is apparent that there is a factor or combination of factors that causes women with diabetes to have more severe cardiovascular disease. It is known that inflammation mediates the formation of atherosclerotic plaques, which contributes to cardiovascular disease. Inflammation is now considered a cardiovascular risk factor and a recent study of predictors of
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cardiovascular risk ranks markers of inflammation as comparable to markers of cholesterol (Libby, 2002). The association of inflammation and diabetes is a topic of active research. Our laboratory has demonstrated that neutrophil activation and platelet-neutrophil interactions are increased in type 2 diabetic men (Hokama et al., 1999). Hokama et al. (2000) reported diabetic rat hearts suffer excessive reperfusion injury following ischemia. An enhanced inflammatory response may contribute to the greater recurrence of myocardial infarction and ischemic heart failure in diabetes. In this study, we tested the hypothesis that a component of the inflammatory response, proinflammatory cytokines, was increased in CVD and further increased in diabetic women with CVD. There appears to be an additive effect of CVD and diabetes on the chronic concentrations of proinflammatory cytokines in women. We examined interleukin-6 (IL-6), interleukin-1 (IL-1h), and tumor necrosis factor (TNF-a) in women with only CVD, women with only diabetes, and women with both diabetes and CVD, and then compared them to healthy age-matched controls. These proinflammatory cytokines appear throughout the literature to play an important role in the pathology of both diabetes and CVD. Cytokines aggravate the development of atherosclerosis in CVD, while cytokines potentially cause insulin insensitivity in diabetes. We found trends towards increasing plasma cytokines and the incidence of CVD and diabetes. The association of plasma IL-6 was the most consistent and dramatic finding. Women with diabetes, with and without CVD, demonstrated a significant increase in plasma IL-6. This study also revealed an interesting pattern of IL-6 and TNF-a cytokine. Women with both diabetes and CVD had the highest concentration of these cytokines. Lower concentrations were observed in diabetic women without CVD, and women with only CVD, respectively. Finally, healthy agematched controls had the lowest concentrations of IL-6 and TNF-a. This trend suggests that as women develop diabetes and cardiovascular disease, the inflammatory stimuli in the blood increases, which could then lead to more severe cardiovascular disease, including atherosclerosis. Alternatively, these data suggest the chronic inflammation underlies both CVD and diabetes. In this study, IL-1h did not differ significantly among any of the groups, suggesting that this cytokine may not be a key component in the cardiovascular disease process observed in diabetic women. 4.1. Inflammation in diabetes and cardiovascular disease The metabolic events that occur during an inflammatory response to a pathogen also occur in certain disease processes, i.e., atherosclerosis and diabetes mellitus. These changes do not appear to have a beneficial purpose but rather contribute to the pathology of the disease. Hayden and Tyagi (2003) hypothesized that type 2 diabetes mellitus is a vascular disease (atherosclerosis) and that NOS, NO, redox stress, and components of inflammation, play a causative role in type 2 diabetes, in turn causing atheroscle-
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rosis. Inflammation, in particular cytokines, plays a key role in atherosclerosis (Ross, 1999). In atherogenesis, endothelial dysfunction is an early step. Injury to the endothelium increases adhesion of leukocytes and platelets. The inflammatory response stimulates migration and proliferation of smooth-muscle cells that become intermixed with the area of inflammation to form an intermediate lesion. Chronic inflammation results in increased numbers of macrophages and lymphocytes. Activation of these cells leads to release of hydrolytic enzymes, cytokines, chemokines, and growth factors that cause more damage and eventually lead to focal necrosis. The lesion is then covered by a fibrous cap that overlies a core of lipid and necrotic tissue (Ross, 1999; Selzman, Miller, & Harken, 2001). Inflammation has also been reported to be increased in type 2 diabetes. Esposito et al. (2002) reported that hyperglycemia acutely increased circulating IL-6 and TNF-a. Pradhan, Manson, Rifai, Buring, and Ridker (2001) reported baseline IL-6 was increased in women who later developed type 2 diabetes. The relative risk of future type 2 diabetes in women between the highest and lowest quartiles was 7.5 for interleukin-6. The results of this study support a role for inflammation in the development of diabetes (Pradhan et al., 2001). These acute phase reactants may explain the clinical and biochemical features of type 2 diabetes and its complications. The increased cytokines in diabetes appear to originate from noncirculating cells (Pickup et al., 2000) and the likely candidates are adipocytes and endothelial cells. If cytokines chronically circulate in diabetic patients, then there could be continuous aggravation of atherosclerosis causing more sites of atherosclerosis and acceleration of the progression of the lesions. Despite the apparent links between diabetes and cardiovascular disease, there have been no reports focusing on the concentrations of cytokines in women with both diabetes and cardiovascular disease. 4.2. Cytokines and cardiovascular disease Cytokine activity has been implicated in various aspects of the pathogenesis in cardiovascular disease including atherosclerosis, acute myocardial infarction, congestive heart failure, myocarditis, and allograft rejection (Oyama, Shimokawa, Morita, Yasui, & Takeshita, 2001). Cardiovascular disease is characterized by a chronic low-level inflammatory process with increased circulating concentrations of proinflammatory cytokines (such as IL-6, TNF-a, IL-1h) (Huber et al., 1999). 4.2.1. Atherosclerosis and cytokines Our data suggests that patients with diabetes and CVD have increased IL-6 and TNF-a. The pathogenesis of atherosclerosis involves many different cytokines, particularly the proinflammatory cytokines, IL-6, IL-1h, and TNF-a,b. Evidence that IL-6 plays an active role in cardiovascular disease is demonstrated in a study by Huber et al. (1999). This investigation injected Apo-E-deficient mice
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with IL-6 or saline. Fatty streak lesions were 1.9- to 5.1-fold larger in the IL-6 treated mice compared to the saline-treated mice, demonstrating that exogenously administered IL-6 significantly enhanced fatty lesion development in the atherosclerosis-prone, but not in the atherosclerosis-resistant animals. This suggests that IL-6 likely has an active role in the atherosclerosis disease process (Huber et al., 1999). It has also been determined that there were large quantities of IL-6 found in human atherosclerotic plaques, typically in macrophage rich area (Rus, Vlaicu, & Niculescu, 1996). IL-1h has also been observed in atherosclerotic lesions (Munro & Cotran, 1988; Ross, 1993). In a study by Nicklin, Hughes, Barton, Ure, and Duff (2000) mice in which the gene for IL-1 receptor antagonist was knocked out were examined. It was observed that there was a lethal arterial inflammation characterized by transmural infiltration of neutrophils, macrophages, and T lymphocytes. The inflammatory nature of the lesions was consistent with the biologic effects of IL-1 on endothelial cells and smooth-muscle cells. IL-1 receptor antagonist produced in healthy mice protects them from the effects of interleukin-1 (Nicklin et al., 2000). 4.2.2. Interleukin-6 In several studies, IL-6 was increased after acute myocardial infarctions and unstable angina (Kanda et al., 2000; Pannitteri et al., 1997; Yazdani et al., 1998). Studies by Roig et al. (1998) and Tsutamoto et al. (2000) demonstrated that IL-6 was increased to 4.1 F 0.6 and 73 F 4 pg/ ml, respectively, in patients with congestive heart failure compared with normal subjects. In the study by Roig et al., IL-6 was associated with lower ejection fraction and worse prognosis (Kanda et al., 2000; Roig et al., 1998). Suzuki, Ishiwata, Hassegawa, Yamamoto, and Yamazaki (2000) reported that patients with stable angina pectoris undergoing elective coronary angioplasty IL-6 was significantly increased 1 and 6 h after the procedure (3.6- and 4.4-fold, respectively). These patients with increased IL-6 later presented with restenosis (Suzuki et al., 2000). IL-6 was also reported to be increased preoperatively (Nomura et al., 2000) and after various cardiac surgeries including CABG and valvular surgery (Berendes et al., 1997; Corbi et al., 2000; Kawahito, Adachi, & Ino, 2000; Liebold, Langhammer, Brunger, & Birnbaum, 1999; Whitten, Hill, Ivy, Greilich, & Lipton, 1998; Yamada et al., 1998). These studies demonstrate clear evidence that IL-6 is increased in response to various different cardiac diseases. These studies agree with our findings of increased IL-6 in patients with different forms of CVD. In this study, we found that IL-6 was significantly increased in diabetic women with CVD in comparison to healthy age-matched controls. We also found women with CVD only had an increase in IL-6 production compared to healthy age-matched controls, but the data were not significant (Fig. 2). This increased concentration of IL-6 observed in diabetic women with and without CVD could reflect the diabetogenesis. The
even higher IL-6 concentrations observed in diabetic women with CVD could then represent an added risk factor of atherosclerosis. 4.2.3. Tumor necrosis factor-alpha The literature on TNF-a found in various different cardiac disease states is conflicting. Both proinflammatory cytokines TNF-a and IL-1h have been implicated in the pathogenesis of myocardial dysfunction in ischemia – reperfusion injury and chronic heart failure. In a study by Cain et al. (1999), it was demonstrated that TNF-a and IL-1h separately and synergistically decreased human myocardial function in a dose-dependent fashion, affecting systolic more than diastolic function. Concentrations as low as 1.25 pg/ml of TNF-a decreased the force of myocardial development (Cain et al., 1999). Roig et al. (1998) and Tsutamoto et al. (2000) observed TNF-a to be increased in patients with congestive heart failure compared to control subjects. In contrast, Pannitteri et al. (1997) observed no significant rise of TNF-a in patients following an acute myocardial infarction and Nomura et al. (2000) found TNFa did not increase preoperatively in a group of patients prior to vascular surgery. Regarding TNF-a we found this cytokine to be increased in patients with CVD only, diabetes only, and both CVD and diabetes in comparison to normal controls, but not significantly (Fig. 3). This increase in TNFa, agrees with some reports in the literature on TNF-a (Roig et al., 1998; Tsutamoto et al., 2000). The increase in TNF-a observed in diabetic women with CVD could be one of the mediators of atherogenesis. It could also be one of the contributors to the pathogenesis of diabetes itself. 4.2.4. Interleukin-1h The literature on IL-1h found in various cardiac disease states is conflicting. It was demonstrated that IL-1h decreased the function of human myocardial trabeculae in a dose-dependent fashion, affecting systolic more than diastolic function (Cain et al., 1999). Nomura et al. (2000) found that IL-1 was increased preoperatively in a group of patients prior to vascular surgery, but Yazdani et al. (1998) found that IL-1 was not significantly different between stable and unstable angina. The result of IL-1h in this study demonstrated that there was no significant difference among the four different groups that were studied; CVD only, diabetes only, CVD and diabetes, and healthy age-matched controls (Fig. 4). The data on the role of IL-1h in diabetes and cardiovascular disease is less convincing. IL-1h may not be one of the cytokines that is chronically increased in patients with diabetes and CVD, therefore not contributing to the increased severity of disease observed in diabetic women with CVD. 4.3. Diabetes and cytokines It is understood that cytokines play a significant role in diabetes. There is evidence that inflammation is an initiating
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factor in insulin insensitivity and proinflammatory cytokines are the key linkage between inflammation and insulin insensitivity (Feinstein, Kanety, Papa, Lunenfeld, & Karasik, 1993; Hotamisligil, 1999; McCarty, 1999; Yudkin et al., 1999). Our data demonstrated that type 2 diabetic women with and without CVD had increased concentrations of IL-6 and TNF-a. Several studies reported an increase in circulating cytokines in patients with diabetes (Bastard et al., 2000; Pickup et al., 2000). This increase in cytokine production could then lead to inappropriate metabolic effects causing arteriosclerosis. IL-6 and TNF-a were significantly increased in type 2 diabetic patients compared to normal subjects in a study by Pickup et al. (2000). The diabetic patients were observed to have higher body mass indices, glycated hemoglobin percentage (HbAIC), and plasma total cholesterol and triglyceride concentration. Five of the 20 diabetic patients in this study had microvascular (including retinopathy and nephropathy) and/or macrovascular disease (including myocardial infarction, angina, peripheral vascular disease, or stroke) and there was no significant difference in comparison to diabetic patients without vascular disease (Pickup et al., 2000). In contrast, Mooradian, Reed, Meredith, and Scuderi (1991) reported that diabetes mellitus was not associated with significant changes in serum TNF-a or IL-1h. Bastard et al. (2000) observed obese nondiabetic and diabetic patients with high insulin resistance had increased IL-6 and TNF-a in comparison to lean controls. IL-6, not TNF-a, was found to be proportional to insulin resistance and blood glucose. Thus, severe diabetes is associated with increased IL-6. When the obese patients were placed on a very low-calorie diet (VLCD) for a 3-week period, the IL-6 decreased significantly in both adipose tissue and serum. TNF-a however was unchanged. They found serum concentration of IL-6 and TNF-a were significantly correlated with BMI. The authors concluded that the circulating IL-6 possibly reflected adipose tissue production and insulin resistance. Vozarova et al. (2002) also reported that plasma IL-6 relates positively to adiposity and negatively to insulin sensitivity. A study by Kato et al. (1997) determined that IL-6 production was positively regulated by hyperglycemia (Kato et al., 1997). TNF-a has a complex relationship with diabetes. TNFa exerts its action on insulin signaling and lipid metabolism by suppressing expression of the insulin sensitive glucose transporter GLUT-4, insulin receptor substrate-1 (IRS-1), and insulin receptor (Feinstein et al., 1993; Long & Pekala, 1996; Stephens, Lee, & Pilch, 1997). This causes an excessive free fatty acids (FFA) flux from hypertrophied insulin-resistant adipocytes (Feinstein et al., 1993; Long & Pekala, 1996; Stephens et al., 1997). This mechanism demonstrates a possible link between TNF-a, obesity, insulin resistance, and type 2 diabetes (Feinstein et al., 1993; Hotamisligil, 1999; McCarty, 1999; Yudkin et al., 1999). It has also been demonstrated that incubating rat skeletal muscle cells with TNF-a results in
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significant impairment of insulin action (Begum & Ragolia, 1996). 4.4. Conclusion TNF-a and IL-6 are produced by cells of the immune system and adipocytes and this may provide the link between inflammation and insulin insensitivity (Grimble, 2002; Mohamed-Ali et al., 1997; Yudkin et al., 1999). Since the majority of patients with diabetes and cardiovascular disease are overweight, this is possibly one of the mechanisms by which diabetic patients have an increased production of these two cytokines. It has been reported that TNF-a and IL-6 correlate with BMI (Bastard et al., 2000). Increased concentrations of TNF-a and IL-6 may affect the development of atherosclerosis, creating a more severe lesion. The increased circulating concentrations of both TNF-a and IL-6 could also negatively affect myocardial function. Therefore, a patient with these increased cytokines may have a worse prognosis after a myocardial infarction, angina, or congestive heart failure. Diabetic patients also have more severe atherosclerosis and a higher degree of morbidity/mortality associated with myocardial infarction, congestive heart failure, and other cardiovascular events (Abbott, Donahue, Kannel, & Wilson, 1988; American Diabetes Association). In conclusion, the findings of this study indicate that (1) D + CVD have significantly increased IL-6 in comparison to age-matched controls, which indicates the potential additive effects of inflammation in these two disease processes; (2) diabetic women with and without cardiovascular disease have increased TNF-a in comparison to age-matched controls, which also indicates the potential additive effects of inflammation in these two disease processes; and (3) there was no correlation between diabetes or CVD of IL-1h. Thus, the increased concentrations of IL-6 and TNF-a in women as a result of diabetes and as a result of cardiovascular disease could work synergistically in comparison to women with cardiovascular disease only or diabetes only and could be a contributor to the more severe cardiovascular disease in this population.
Acknowledgments This research was supported by NIH HLB 58859.
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