Abstract
Objective Inadequate vascular remodeling is contributory to increased cardiovascular events in people with type 2 diabetes mellitus (DM) and impaired fasting glucose (IFG). Vascular endothelial growth factor (VEGF) and its regulatory molecule soluble Flt-1(sFlt-1) play important roles in atherogenesis.
Research Design We measured fasting plasma concentrations of VEGF and sFlt-1 in 11 nondiabetic (ND) (aged 46.1 ± 2.1 years; body mass index [BMI], 26.1 ± 0.9 kg/m2; glucose, 5.0 ± 0.1 mM), 15 IFG (aged 52.9 ± 1.8 years; BMI, 32.7 ± 1.3 kg/m2; glucose, 6.4 ± 0.1 mM), and 8 DM (aged 55.8 ± 3.2 years; BMI, 30.0 ± 1.0 kg/m2; glucose, 9.3 ± 0.5 mM) subjects.
Results Plasma VEGF (42.1 ± 4.0 vs 24.2 ± 0.9 vs 29.4 ± 3.8 pg/mL, respectively) and sFlt-1 (119.4 ± 4.9 vs 58.9 ± 3.2 vs 56.7 ± 1.2 pg/mL, respectively) concentrations were higher (P < 0.04) in DM than IFG and ND subjects. Whereas VEGF concentrations were significantly lower (P < 0.05) in IFG than in ND subjects, sFlt-1 concentrations did not differ between the IFG and ND subjects.
Conclusions Although plasma VEGF concentrations were higher (35%) in DM than in ND subjects, VEGF action on vascular remodeling was likely attenuated by higher sFlt-1 concentrations in DM. In contrast, IFG subjects did not have major perturbations in either VEGF or sFlt-1 levels. Further studies defining the roles of these mediators in DM and IFG are necessary to extend these observations.
Patients with type 2 diabetes mellitus (DM) have a cardiac risk equivalent to a 2- to 4-fold greater prevalence of cardiovascular disease than do nondiabetic (ND) individuals. Diabetic individuals without prior myocardial infarction have at least as high a risk of myocardial infarction as ND individuals with a prior history of myocardial infarction.1Furthermore, inadequate vascular remodeling and insufficient collateral formation are critical responses to hypoxia that are likely contributory to increased cardiovascular events in DM and impaired fasting glucose (IFG). A large international contemporary database of established atherothrombotic disease showed a prevalence of DM of 44% and that of IFG of ∼37%.2This staggering statistics showing that almost 80% of those with macrovascular disease have either DM or IFG is a testimony to the burden of the disease process.
Vascular endothelial growth factor (VEGF) is a heparin-binding growth factor that is mostly secreted by vascular endothelial cells and is a crucial determinant of vascular remodeling and collateral formation by binding to its receptor VEGF receptor 1 (fms-like tyrosine kinase or Flt-1). This binding is normally inhibited by a naturally occurring soluble form of VEGFR-1 (sFlt-1). Elevated plasma levels of sFlt-1 have been linked to the development of toxemia of pregnancy likely resulting from impaired placental vascular development.3,4
To our knowledge, there have been no prior simultaneous assessments of plasma VEGF and sFlt-1 in those with IFG or DM. To elucidate this further, we performed simultaneous measurements of VEGF and sFlt-1 in cohorts of ND subjects, patients with IFG, and those with type 2 DM. We report that subjects with DM had plasma VEGF levels that were ∼35% higher than did ND and IFG subjects. In contrast, plasma sFlt-1 levels were 2-fold elevated in DM than in ND and IFG subjects. This could have substantial implications in the pathogenesis of macrovascular disease and collateral formation in those with DM.
MATERIALS AND METHODS
After approval from the Mayo institutional review board, blood samples were collected after an overnight fast in 11 ND, 15 IFG, and 8 DM subjects. None of the subjects had any history of overt cardiovascular, hepatic, or renal disease or hypertension. The ND subjects did not have a history of diabetes in first-degree relatives. All oral antidiabetic agents were discontinued 2 weeks before sampling in DM subjects. None of the DM subjects were on insulin therapy. None of the IFG subjects were on antidiabetic medication. None of the subjects underwent vigorous physical activity for a week before sampling. Fasting plasma glucose and hemoglobin A1c (HbA1c) were measured along with VEGF and sFlt-1 concentrations.
Vascular endothelial growth factor was measured by QuantiGlo Chemiluminescent Human VEGF assay (R&D Systems, Minneapolis, MN). The amount of VEGF bound was measured by a microplate luminometer.5The coefficient of variation for intra-assay was 1.8% at 78.8 pg/mL, 2.2% at 1629 pg/mL, and 3.5% at 11,984 pg/mL and for interassay was 7.9% at 75.2 pg/mL, 4.1% at 1639 pg/mL, and 6.4% at 11,705 pg/mL. This assay measured free VEGF165 in plasma with minimal interference from sFlt-1.
Plasma-soluble VEGF R1/Flt-1 was measured by Quantikine Human Soluble VEGF R1/Flt-1 immunoassay (R&D Systems) that used the quantitative sandwich enzyme immunoassay technique. Proportion of the amount of Flt-1 bound was measured by a microplate luminometer that reads the microplate at 450 nM within 30 minutes and λ correction at 540 or 570 nM. The coefficient of variation for intra-assay was 2.6% at 96.6 pg/mL, 3.8% at 596 pg/mL, and 3.2% at 1213 pg/mL and for interassay was 9.8% at 112 pg/mL, 7.0% at 645 pg/mL, and 5.5% at 1279 pg/mL.
All data are expressed as mean ± SEM. Analysis of variance was used to determine differences between the 3 groups. P < 0.05 was determined as statistically significant, and Student unpaired t test was performed to detect differences between individual groups.
RESULTS
The subject characteristics are provided in Table 1. By design, fasting plasma glucose was higher (P < 0.0001) in DM and IFG subjects than in ND individuals; fasting glucose was also higher (P < 0.01) in DM than in IFG subjects and between IFG and ND subjects. Hemoglobin A1c concentrations were also higher in DM than in IFG and ND subjects but were no different between IFG and ND subjects. Both age and body mass index (BMI) differed (P < 0.04) between groups, with differences in both these parameters occurring between the ND subjects and each of the 2 other groups. There were no differences in age or BMI between the DM and IFG subjects.
Plasma VEGF Concentrations
Plasma VEGF concentrations were higher (P < 0.001) in DM than in IFG and ND subjects (42.1 ± 4.0 vs 24.2 ± 0.9 vs 29.4 ± 3.8 pg/mL, respectively) (Fig. 1, upper panel). Further analyses confirmed that DM subjects had higher VEGF levels than did both IFG subjects (P < 0.001) and ND subjects (P < 0.04). There was also a slight but significant (P < 0.02) difference in the VEGF levels between IFG and ND subjects.
Plasma sFlt-1 Concentrations
Plasma sFlt-1 concentrations were also higher (P < 0.0001) in DM than in IFG and ND subjects (119.4 ± 4.9 vs 58.9 ± 3.2 vs 56.7 ± 1.2 pg/mL, respectively) (Fig. 1, lower panel). Further analyses confirmed that DM subjects had higher sFlt-1 levels than did both IFG (P < 0.0001) and ND (P < 0.0001) subjects. In contrast to VEGF levels, however, there were no differences (P = 0.6) in sFlt-1 levels between IFG and ND subjects.
Correlation Between Plasma Glucose, VEGF, and sFlt-1 Concentrations
There were significant correlations (P < 0.01) between fasting glucose concentrations and VEGF levels (r 2 = 0.46) (Fig. 2, upper panel) and also between fasting glucose and sFlt-1 levels (r 2 = 0.7) (Fig. 2, lower panel) in the entire cohort. However, within the IFG and ND groups, taken together or separately, there were no correlations between fasting glucose, VEGF, or sFlt-1 concentrations. There were no correlations between HbA1c, VEGF, and sFlt-1 concentrations in the cohorts studied.
DISCUSSION
Our results show that DM subjects without overt macrovascular disease had higher plasma VEGF and sFlt-1 concentrations than did individuals with IFG or ND. Furthermore, individuals with IFG had slightly but significantly lower plasma VEGF concentrations, whereas their sFlt-1 concentrations did not differ when compared with those of ND individuals. Contrary to prior report,6our data clearly reveal dramatic increases in sFlt-1 concentrations that are up to 2-fold higher in DM than in IFG and ND subjects. These initial observations suggest intriguing possibilities regarding vascular remodeling issues in both DM and IFG subjects.
The activity of VEGF-mediated angiogenesis is limited by the extent of its binding to circulating soluble receptor Flt-1.7Our results suggest the possibility that, in those with diabetes, although VEGF levels are increased when compared with those of IFG and ND subjects, the dramatic doubling of sFlt-1 concentrations likely limits the angiogenic potential of VEGF, which could lead to impaired vascular remodeling and collateral formation. That said, in the absence of measuring VEGF bioactivity in plasma as well as exploring the stoichiometric relationship between VEGF and sFlt-1, our line of reasoning remains speculative and needs to be further evaluated in future studies. In contrast, in IFG subjects, although the sFlt-1 levels were no different when compared with those with ND, the slight but significant fall in VEGF levels could potentially be a factor in impaired angiogenesis and vascular remodeling in these individuals.
However, use of VEGF and/or sFlt-1 as biomarkers for cardiovascular disease in individuals with type 2 diabetes will need to be further explored and tested. Further population-based studies will need to be performed to compare the predictive power of these novel biomarkers with conventional cardiovascular risk markers.
Elevated levels of VEGF have been found in the vitreous humor8and plasma9of those with diabetic retinopathy, in the urine of those with diabetic nephropathy,10and in plasma of individuals with DM with or without vascular disease.6,11In contrast, although reduced expression of VEGF has been found in myocardium of subjects with DM with acute coronary syndrome,12other studies have found higher myocardial VEGF mRNA and protein levels but lower Flt-1 mRNA and protein levels in diabetic subjects with chronic congestive cardiac failure than in their ND counterparts.13
Few existing studies have examined plasma sFlt-1 levels in individuals with DM. Although sFlt-1 levels have been shown to be increased in the urine in those with diabetic nephropathy,10there were no changes in plasma sFlt-1 levels in those with diabetic retinopathy.9In the only other study that measured VEGF and sFlt-1 levels in subjects with DM,6VEGF concentrations were increased in those with DM and vascular disease (but not in those without vascular disease), whereas there were no alterations in sFlt-1 levels when compared with ND subjects.
The positive correlations between fasting plasma glucose and both VEGF and sFlt-1 concentrations for the entire cohort are intriguing, suggesting a mechanistic link between rising glucose concentrations and regulation of angiogenesis. Although a relationship between glycemic control as measured by HbA1c and VEGF levels has been reported earlier,11,14there has been no prior information of correlations between glycemic status and sFlt-1 levels. Additionally, there have been studies that have shown reduction in plasma VEGF concentrations with improved glycemic control.11,15,16Whether that translates into improved vascular outcomes is uncertain. Large clinical trials either have17,18or have not affected19major cardiovascular outcomes with improved glycemic control in diabetes. However, it remains to be seen whether a combined approach of improvement in glycemic control linked with modulation of angiogenic factors could pave the way for improving the considerable cardiovascular morbidity in these high-risk individuals.