We examined the influences of obesity and diabetes on endothelium-dependent and -independent vasodilation, inflammatory cytokines, and growth factors. We included 258 subjects, age 21–80 years in four groups matched for age and gender: 40 healthy nonobese (BMI <30 kg·m−2) nondiabetic subjects, 76 nonobese diabetic patients, 37 obese (BMI >30) nondiabetic subjects, and 105 obese (BMI >30) diabetic patients. The flow-mediated dilation (FMD, endothelium-dependent) and nitroglycerin-induced dilation (NID, endothelium-independent) in the brachial artery, the vascular reactivity at the forearm skin and serum growth factors and inflammatory cytokines were measured. FMD was reduced in the nonobese diabetic patients, obese nondiabetic controls, and obese diabetic patients (P < 0.0001). NID was different among all four groups, being highest in the obese nondiabetic subjects and lowest in the obese diabetic patients (P < 0.0001). The resting skin forearm blood flow was reduced in the obese nondiabetic subjects (P < 0.01). Vascular endothelial growth factor (VEGF) was higher in the obese nondiabetic subjects (P < 0.05), tumor necrosis factor–α was higher in the obese diabetic patients (P < 0.0001) and C-reactive protein was higher in both the obese nondiabetic and diabetic subjects (P < 0.0001). Soluble intercellular adhesion molecule-1 was elevated in the two diabetic groups and the obese nondiabetic subjects (P < 0.05). We conclude that diabetes and obesity affect equally the endothelial cell function but the smooth muscle cell function is affected only by diabetes. In addition, the above findings may be related to differences that were observed in the growth factors and inflammatory cytokines.
Both obesity and diabetes are known to be risk factors for the development of cardiovascular disease (1). Impairment of endothelial function and the induction of a proinflammatory state are currently considered to be two major mechanisms that are responsible for this increased risk (2,3). C-reactive protein and tumor necrosis factor–α are most commonly studied cytokines regarding the development of cardiovascular disease (4,5). However, possible differences between diabetic patients and obese, nondiabetic subjects regarding vascular reactivity and inflammation have not been adequately studied.
In the present study, we have compared the changes in the endothelium-dependent and -independent vasodilation, inflammatory cytokines, and growth factors among nondiabetic nonobese and obese subjects and nonobese and obese diabetic patients. Our study design allowed us to test the hypothesis that obesity and diabetes have differential effects on the vasculature and that the observed abnormalities are mediated by varying circulating factors.
Methods and Procedures
We included 258 subjects, age 21–80 years who were previously enrolled in four clinical trials that run in parallel in our unit and divided them into four groups: 40 healthy nonobese (BMI <30 kg·m−2) nondiabetic control subjects, 76 nonobese diabetic patients, 37 obese (BMI >30 kg·m−2) nondiabetic, control subjects and 105 obese (BMI >30 kg·m−2) diabetic patients. Patients with type 1 or 2 diabetes according to ADA criteria were included (6). Exclusion criteria were: congestive heart failure, cardiac arrhythmias, stroke or transient ischemic attack, end stage renal failure, uncontrolled hypertension, severe dyslipidemia, chronic liver disease or any other severe chronic medical condition requiring active treatment. Active smokers were asked to refrain from smoking the day of the testing. The experimental protocols and experimental design were approved by the institutional review board of the Beth Israel Deaconess Medical Center. All participants gave informed consent for the original trials.
All clinical examinations and evaluations were conducted under fasting conditions. The cytokines and growth factors were measured using a Luminex 200 apparatus (Luminex, Austin, TX) and Millipore multiplex immunoassay panels (Millipore, Chicago, IL). We opted to test a large array of these molecules because there are very limited data available regarding the association of vascular reactivity to cytokines and growth factors. This approach was deemed the most suitable as it could provide us with a better view of possible associations and could lead to additional, specific hypothesis-driven research. Serum specimens were stored in a −80 °C until there were all analyzed together during the first thaw at the end of the study.
In order to test the reliability of the multiplex assay measurements, we measured insulin levels using our standard hospital lab and the multiplex assay. We found a very satisfactory correlation (r = 0.9). Furthermore, using a 39-plex platform, we performed two repeated measurements on the same samples and we noticed very satisfactory correlations. The intra- and interassay coefficient variation of the performed cytokine measurements, as provided by the manufacturer (Millipore) is similar to measurements performed using standard ELISA techniques in our unit (7).
Vascular reactivity measurements
All tests were performed in the same lab. Vascular reactivity of the macrocirculation was measured in the brachial artery using a high-resolution ultrasound with a 10.0 MHz linear array transducer and an HDI Ultramark 9 system (Advanced Technology Laboratories, Bothell, WA). To measure endothelial-dependent vasodilation, the brachial artery diameter was measured before and after flow-mediated dilation (FMD) during reactive hyperemia. Reactive hyperemia was assessed by inflating a pneumatic tourniquet distal to the brachial artery to 50 mm Hg above systolic blood pressure for 5 min and then deflating it (7). All techniques were performed according to published guidelines (8). The skin blood flow was evaluated by employing a LASER Doppler Perfusion Imager (LDPI Lisca 2.0, Lisca Development AB, Linkoping, Sweden), as previously described (9). The above methods, including information regarding their coefficient of variation, have been previously described (7,8,9). Vascular reactivity was measured in 165 subjects (22 healthy nonobese nondiabetic control subjects, 40 nonobese diabetic patients, 31 obese nondiabetic, control subjects, and 72 obese diabetic patients). There were no major differences between the subjects who underwent these tests vs. those who did not.
Statistical analysis was performed in collaboration with a biostatistician (C.G.). The analysis was undertaken by univariate techniques and modeling the data through multiple linear regression using the SPSS statistical package (version 17.0; SPSS, Chicago, IL). For normally distributed data, the ANOVA was used followed by the Fisher's multiple comparison tests to identify differences between groups. For nonparametrically distributed data, the Kruskal-Wallis test was used. Multiple regression analysis was used to compare the reactivity variables after adjusting for age, gender, treatment with angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor blockers (ARBs), statins, oral antidiabetic agents, insulin, and diabetes type. The contribution of cytokines and growth factors in the variation of vascular reactivity measurements was assessed by multivariate stepwise regression analysis. Significant predictors were included in the models with a stepwise procedure, after adjusting for age and gender. Two sided P values were assessed for all models.
The comparisons of the demographics among the three groups are shown in Table 1. All groups were matched for age and gender. The systolic blood pressure was slightly higher in the two diabetic groups (P < 0.001) whereas total cholesterol was lower (P < 0.0001). As expected, a higher number of diabetic patients were treated with antihypertensives that can affect endothelial function such as ACE inhibitors and ARBs and statins when compared to the two nondiabetic groups.
Table 1. Clinical characteristics of the studied groups
The vascular reactivity results are shown in Table 2. The resting brachial diameter was higher in the nonobese diabetic patients, obese nondiabetic subjects, and obese diabetic patients when compared to the nonobese healthy subjects (P < 0.001). The flow-mediated vasodilation (FMD) was lower the nonobese diabetic patients, obese nondiabetic subjects and obese diabetic patients when compared to the nonobese healthy subjects (P < 0.0001) (Figure 1a). The nitroglycerin-induced vasodilation (NID) was different among all four groups, being highest in the obese nondiabetic subjects and lowest in the obese diabetic patients (P < 0.0001) (Figure 1b). The resting forearm skin blood flow was lower in the obese nondiabetic subjects when compared all other three groups (P < 0.01) (Figure 1c). The maximal vasodilation after the iontophoresis of acetylcholine (endothelium-dependent) and sodium nitroprusside (endothelium-independent) was lower in the obese nondiabetic patients when compared to all of the other three groups (P < 0.05 and P < 0.0001, respectively). Similar results were observed in multiple regression analysis after controlling for age, gender, treatment with ACE inhibitors, ARBs, statins, oral antidiabetic agents, insulin, and the type of diabetes (Table 3). Gender and treatment with ACE inhibitors and ARBs, influenced the FMD and NID results whereas age and the type of diabetes did not have any effect in any of the study parameters.
Table 2. Vascular reactivity results
Table 3. Multiple regression derived coefficients (b) and P values for vascular reactivity results
The results from the measurements of the various growth factors, cytokines and cellular adhesion molecules are shown in Table 4. The platelet-derived growth factor AA was lower in the nonobese diabetic patients and the obese nondiabetic subjects when compared to the nonobese subjects (P < 0.01) (Table 2). The vascular endothelial growth factor (VEGF) was higher in the obese nondiabetic subjects when compared to the remaining three groups (P < 0.05). The osteoprotegerin (OPG) tended to be higher in the obese diabetic patients when compared to the nonobese and obese control subjects but marginally failed to reach statistical significance (P = 0.053). The osteopontin was higher in the obese nondiabetic subjects when compared to all other three groups (P < 0.05) whereas the growth-regulated oncogene was lower in the obese nondiabetic subjects when compared to the nonobese subjects and the obese diabetic patients (P < 0.05). The chemokine (C-X-C motif) ligand 10 (interferon-inducible protein-10) was lower in both diabetic groups (P < 0.0001), the monocyte chemotactic protein-1 was higher in the obese nondiabetic subjects when compared to the nonobese subjects and the nonobese diabetic patients (P < 0.01) whereas the tumor necrosis factor–α was higher in the obese diabetic patients when comparing to all other three groups (P < 0.0001). The eSelectin tended to be lower in the nonobese subjects comparing to the other three groups but failed to reach statistical significance (P = 0.076) whereas the soluble intercellular adhesion molecule-1 was lower in the nonobese subjects comparing to the other three groups (P < 0.05). The C-reactive protein was higher in the obese subjects and obese diabetic patients when compared to the nonobese subjects and nonobese diabetic patients (P < 0.0001).
Table 4. Results of growth factors and cytokines
A stepwise regression analysis was also undertaken to assess the contribution of the tested cytokines and growth factors in the variation of FMD, NID and the three microvascular measurements (see Supplementary Table S1 online). Regarding the variation of FMD, the main contributing factors were gender, platelet-derived growth factor-AA, insulin, human osteopontin, and interferon-inducible protein-10 whereas for NID were gender and human osteoprotegerin. Macrophage-derived chemokine was the common contributing factor for all microvascular measurements whereas adiponectin additionally contributed in the variation of the forearm resting skin blood flow.
In the present study, we have shown that obesity primarily affects the endothelial cell function in the macrocirculation whereas diabetes affects the function of both the endothelial and vascular smooth muscle cells. In addition, under resting conditions, the baseline forearm skin blood flow and the response to the iontophoresis of acetylcholine (endothelium-dependent) and sodium nitroprusside (endothelium-independent) are reduced in obese nondiabetic subjects. We have also found that obesity and diabetes had different effects on various inflammatory cytokines and VEGF. These results indicate that although diabetes and obesity share common mechanisms, there are also considerable differences in the pathways that lead to the development of a proinflammatory state and impaired vascular function.
Previous studies have shown that obesity affects the flow-mediated vasodilation (FMD, endothelium-dependent) but has no effect on the NID (endothelium-independent (10,11)). In contrast, the majority of large studies in diabetic patients have shown that both FMD and NID are impaired (12,13). Regarding the forearm skin microcirculation, previous studies have shown that both endothelium-dependent and endothelium-independent vasodilation are reduced in both type 1 and 2 diabetic patients (14,15). Our data are also in agreement with these findings as no differences were observed between the type 1 and 2 diabetic patients who in this study were of similar age and did not have severe cardiovascular disease or severe diabetic complications such as end-stage diabetic neuropathy. However, no information is available regarding possible impairment of the microvascular reactivity in obese, nondiabetic subjects.
In the present study, we have shown that FMD was similarly reduced in obese nondiabetic subjects and both obese and nonobese diabetic patients. These results indicate that both obesity and diabetes independently affect the function of the endothelial cell. In contrast, the function of the vascular smooth muscle, as indicated by NID measurements, was affected only by diabetes whereas the obese, nondiabetic had higher levels when compared to the nonobese controls. The reasons for the observed increase in the nondiabetic obese subjects are not clear and will need further exploration. These results indicate that although obesity and diabetes have different effects on different vascular cells that can affect the vascular function. Finally, in agreement with previous studies, we have shown that the resting brachial artery diameter was increased in the diabetic patients and in the obese nondiabetic subjects (16).
At the forearm skin microcirculation, the resting blood flow was reduced in the obese, nondiabetic subject when compared to all other three groups. In addition, the maximal vasodilation after the iontophoresis of acetylcholine (endothelium-dependent) and sodium nitroprusside (endothelium-independent) were also reduced in this group, obviously a result of the lower baseline levels. The reasons for these observed results are not clear but are compatible with sympathoexcitation and increased catecholamines levels that have been reported in obesity (17,18). The fact that diabetic obese patients, in whom a certain degree of autonomic dysfunction should be expected, did not have a similar reduction, further supports this hypothesis. However, further investigations will be required before final conclusions are reached.
Previous studies have reported an increase in serum VEGF in obese subjects and have hypothesized that adipocytes produce angiogenic factors that stimulate neovascularization that plays an important role in allowing fat mass expansion (19,20,21). Our results have confirmed these findings and have shown increased serum VEGF levels in the obese, nondiabetic patients. However, it is of interest that the obese diabetic group did not have a similar increase, suggesting that diabetes can affect the circulating VEGF levels. The platelet-derived growth factor-AA serum levels were lower in the nonobese diabetic patients and the obese nondiabetic subjects whereas no differences were observed in the epidermal growth factor, fibroblast growth factor and transforming growth factor–α levels. VEGF and platelet-derived growth factor play a major role in angiogenesis and wound healing but their possible role in adipogenesis in diabetes has not been explored (22).
The insulin, leptin, and adiponectin levels were similar in nonobese nondiabetic subjects and diabetic patients whereas the same was also true for the obese nondiabetic subjects and the diabetic patients. OPG was marginally elevated in the obese diabetic patients whereas osteopontin was higher in the obese nondiabetic subjects. OPG levels have been reported to be higher in diabetes and are associated with impaired endothelial function, vascular calcification, and the development of cardiovascular disease (23,24). Our results indicate that the combination of diabetes and obesity increase the OPG levels and this may explain the particularly high incidence of cardiovascular disease in this group. Previous studies have also shown that osteopontin levels are higher in obese subjects and that these high levels are related to increased expression by macrophages that are recruited into adipose tissue and play a major role in the development of inflammation and insulin resistance (25,26). In the present study, we have confirmed that OPN or osteopontin levels are high in obese subject but it is of interest that obese diabetic patients did not have high levels. The same results were also observed with macrophage-derived chemokine. In contrast, growth-regulated oncogene levels where reduced in the obese nondiabetic subjects when compared to the nonobese controls and the obese diabetic patients. To the best of our knowledge, there are no other data regarding growth-regulated oncogene in obesity or in diabetes but in animal studies growth-regulated oncogene has been shown to enhance endothelial recovery (27).
Tumor necrosis factor–α levels were increased only in the diabetic obese patients. Previous studies have reported that in obese subjects the serum levels are not increased despite increased expression in adipose tissue (28). However, our results indicate that diabetes and obesity have a synergistic effect that leads to increased levels of this cytokine. It is also of interest that C-reactive protein was increased in both nondiabetic and diabetic obese subjects but was normal in the nonobese diabetic patients. Further studies will be required to examine the exact contribution of obesity and diabetes in the expression of these two major inflammatory cytokines. Finally, soluble intercellular adhesion molecule-1 levels were increased in both diabetic groups and the obese nondiabetic subjects. These results are in agreement with the observed endothelial dysfunction, assessed by FMD measurements, in the same groups.
In agreement with previous studies, we have observed that gender can affect vascular reactivity in the macrocirculation (15,29). Age and the type of diabetes did not affect our results. Treatment with medications that can positively affect endothelial function, such as ACE inhibitors and ARBs was found to be related with a deterioration of vascular reactivity rather than improvement in the present study, whereas treatment with statins did not have any effect. The main reason for these results is that treatment with statins, ACE inhibitors, and ARBs was mainly restricted to diabetic patients and acted mainly as surrogate measurements of the existence of diabetes-related vascular reactivity impairment. In any case, as the observed differences in the vascular reactivity measurements among the four groups were still present after adjusting for the above variables, strongly suggesting that obesity and diabetes were the main responsible factors. Finally, stepwise analysis showed that some of the tested cytokines and growth factors had a significant, although small, contribution to the observed variation in all vascular reactivity measurements. These results indicate that additional identified factors may considerably contribute to the observed variation and further investigations will be needed to identify them.
Taken all together, our results indicate that obesity and diabetes share common mechanisms that affect vascular function and there is synergy in some of these mechanisms between these two conditions in augmenting these mechanisms. However, obesity and diabetes also diverge regarding their effect on other pathways. Additional investigation of these similarities and differences may prove of particular help in understanding the causal pathways that are involved in the development of cardiovascular disease.
The present study has its limitations. The main one is that this is a cross-sectional study and changes over time cannot be assessed. As a result, the inclusion/exclusion criteria may have resulted in the selection of diabetic patients with long duration of diabetes who were spared of serious complications and this may be reflected in their cytokine levels. Further prospective studies will be required to clarify this issue. In addition, due to the nature of human research, there are likely to be unrecognized variables leading to residual confounding. For example, we have not systematically assessed obstructive sleep apnea in all of our participants which could also have effects on vascular function and circulating markers.
In summary, we have shown that diabetes and obesity affect equally the endothelial cell function but the smooth muscle cell function is affected only by diabetes. In addition, the above findings may be related to differences that were observed in the growth factors and inflammatory cytokines serum levels.