Endothelial Microparticles and Cardiometabolic Risk
Endothelial Microparticles and Cardiometabolic Risk
In this first study investigating EMPs levels in a community cohort, we found that EMP plasma levels are associated with several cardiovascular risk factors, including higher triglyceride levels, hypertension, and metabolic syndrome. These findings raise the possibility that one consequence of a high burden of metabolic risk factors is increased endothelial activation or injury, in particular in individuals with hypertriglyceridaemic waist.
Microparticles are plasma membrane vesicle fragments that are released from various cell types during activation or apoptosis. In recent years, a growing body of literature has highlighted the role of MPs as mediators of intercellular communication, given that the biological properties of MPs are influenced by both their cellular origin and their composition. Routine MP measures in clinical samples require ongoing efforts to establish standardized protocols for plasma preparation, anticoagulation, and storage conditions in addition to techniques for quantification; these determinants are still under investigation. Nonetheless, in clinical studies performed to date, circulating MPs derived from the endothelium (EMPs) have been shown to demonstrate a special relationship with alterations in endothelial homeostasis. Increased levels of circulating EMPs have been observed in patients with stable or unstable coronary artery disease, stroke, and peripheral artery disease. These changes in plasma EMP levels usually averaged a 2- to 3-fold increase in patients with cardiovascular diseases, when compared with healthy controls. Elevated EMPs levels have also been measured in the presence of cardiovascular risk factors such as uncontrolled systemic hypertension, diabetes, and dyslipidemia. However, these results were observed in highly selected groups of patients and were limited by modest sample sizes. Thus, the influence of different CV risk factors and medications (i.e. cholesterol-lowering therapy, anti-hypertensive medications) on EMP levels has yet to be examined in a larger community-based population.
Our data indicate that elevated triglycerides are strongly associated with higher EMP levels. These results are consistent with observations from previous pilot studies in humans. Ferreira et al. showed that CD31+ EMP levels increase along with triglyceride levels following a high-fat meal in a sample of 18 healthy individuals without known cardiovascular risk factors. Similar findings were also reported for CD144+ EMPs in 27 patients with diabetes. High levels of triglycerides have been shown to impair endothelial function in vivo, which may be related to the pro-inflammatory and pro-apoptotic effects of remnant lipoprotein particles on the endothelium that are potentially mediated by changes in local oxidative stress.
High triglyceride levels are associated with an increased risk for development of cardiovascular disease in certain subgroups such as women and young healthy men. Potential explanations for this association include increased atherogenic cholesterol-enriched remnant lipoprotein particle production, interference with HDL or LDL metabolism, and enhanced recruitment and attachment of monocytes to the vascular wall. Interestingly, the association of EMPs with triglycerides observed in our community-based sample was independent of total cholesterol levels, other cardiovascular risk factors, and the presence of any medication therapies for cardiovascular risk. Thus, our findings offer potentially new insights regarding the deleterious role of triglycerides on vascular homeostasis. As EMPs interfere with coagulation, inflammation, regulation of vascular tone, and angiogenesis, these vesicles might further support the initiation and promotion of vascular disease. We did not observe any independent relationship between HDL or total cholesterol values and EMPs levels. Although oxidized LDL cholesterol has been shown to increase EMP release in vitro, the current data suggest that the previously observed links in vivo between these parameters could have been influenced by the presence of confounding factors (such as associated cardiovascular risk factors, including hypertension and diabetes) or that the presence of lipid-lowering therapy might have affected this relationship.
We also observed independent associations between circulating levels of EMPs and the presence of metabolic profiles associated with high cardiovascular risk. In particular, the present paper demonstrates robust correlations between circulating EMP levels and hypertryglyceridaemic waist, suggesting that the vascular endothelium of these participants at high risk of coronary artery disease may be prematurely injured or inflamed. Metabolic abnormalities are associated with impaired endothelial function and altered vascular structure. The pathophysiology of the deleterious effects on the endothelium has not been fully elucidated but may involve macrophage infiltration within white adipose tissue, release of adipokines by adipocytes and pro-inflammatory cytokines by leukocytes, insulin resistance, and changes in lipid profiles. All of these factors can negatively impact endothelial homeostasis, leading to local inflammation, increased oxidative stress, and cellular vesiculation. Inflammatory cytokines are elevated in the presence of metabolic syndrome and have been shown to stimulate EMP release in vitro. Thus, in turn, EMPs may interfere with coagulation, inflammation, or enhance endothelial damage, leading to increased endothelial vesiculation and MP release.
Our data also show that hypertension is associated with an increase in CD144 EMPs. Prior studies have reported increased circulating EMPs levels in patients with uncontrolled severe hypertension and an effect of anti-hypertensive drugs on platelet or monocyte-derived, but not endothelial, MPs. Our study highlights differences between the two EMP sub-populations (CD144+ or CD31+CD41− MPs) with regards to sex and hypertension, which could result from the preparation or the conservation of the plasma samples. Alternately, these differences might reflect a different condition or site of endothelial injury or a different affinity of CD144 and CD31 antibodies for their endothelial epitopes.
Notably, we observed no significant association between frank diabetes and EMP traits. Prior small studies of patients referred for coronary angiography have suggested that circulating EMP levels may be elevated in diabetics compared with non-diabetics. However, Sabatier et al. reported higher levels of EMPs compared with healthy controls in Type 1 but not Type 2 diabetics. Notably, 99% of individuals with diabetes in the Framingham Offspring cohort are known to have Type 2 diabetes. Thus, previously observed relations of EMPs with diabetes may be related to concomitant burden of cardiometabolic risk factors, active coronary artery disease, or both. Moreover, the absence of any apparent association between diabetes and EMP traits may also be related to the presence of confounding factors. Notably, we observed that the association between triglycerides and EMP levels was more pronounced in diabetics compared with non-diabetics. This observation suggests the possibility that diabetes enhances potentially deleterious effects of hypertriglyceridemia on endothelium. Alternately, the presence of diabetes in association with hypertriglyceridemia may reflect a more severe grade of metabolic disorder that leads to greater endothelial injury. Further research is needed to specifically investigate these observed relations.
Interestingly, there was no significant association between smoking status and EMP traits in our study sample. In previous work, both active and passive smoking exposure has been associated with a small but significant elevation in circulating EMPs in small samples of healthy adults, with mean ages ranging from 30 to 47 years. By contrast, our larger study sample was older (mean age 66 years) with a presumably larger burden of cardiometabolic risk factors. Thus, it is possible that whereas smoking leads to elevated EMP levels in otherwise healthy younger adults, this relation is attenuated with advancing age and the cumulative burden of cardiometabolic risk that is frequently seen in older adults.
Several limitations of this study merit consideration. The present study was not designed to precisely assess quantity of small-sized MPs, with diameters between 0.1 and 0.5 μm; thus, our conclusions are only valid for large EMPs with diameters ranging from 0.5 to 1.0 μm. The platelet-poor plasma preparation used in the present study might have included residual platelets which could generate artifactual MPs; thus, findings related to platelet-derived MPs or the overall pool of annexinV+ MPs expressing phosphatidylserine would need to be interpreted with extreme caution in this cohort. Because our analyses were cross sectional, we cannot determine whether high-EMPs preceded or followed the development of metabolic syndrome. Additionally, HOMA-IR and HbA1c data were not available as concurrent assessments of insulin resistance or glycaemic exposure. The lack of outcomes data for this ambulatory cohort with low event rates precluded analyses of associations with incident cardiovascular disease, which warrants future investigation. Concurrent data on surrogate measures of subclinical atherosclerosis, such as carotid intima media thickness, were also not available at the time of the EMP assessment. Thus, further research is needed to examine the relations of EMPs and such imaging based markers of subclinical vascular disease in the community. Anti-hypertensive medications including angiotensin receptors blockers, β-blockers or calcium channel blockers have an impact on various subgroups of circulating EMPs. Endothelial microparticle traits have been assessed in very few large samples because they are difficult to measure and require specialized specimen handling. In future prospective cohorts, the ability to obtain samples suitable for EMP and other cell-based phenotyping may help to provide confirmatory data for our findings. Additionally, our sample was predominantly white of European ancestry; therefore, the extent to which our findings are generalizable to other racial/ethnic populations is unknown.
In conclusion, in this large community-based study, higher levels of circulating plasma EMPs were associated with elevated triglycerides, hypertension, and the metabolic syndrome. Our results support the need for further studies exploring the potential mechanisms linking metabolic and lipid abnormalities to the production of EMPs. Further studies may also be warranted to determine whether EMP levels might represent an appropriate biomarker for identification of asymptomatic individuals at risk of developing cardiovascular events.
Discussion
In this first study investigating EMPs levels in a community cohort, we found that EMP plasma levels are associated with several cardiovascular risk factors, including higher triglyceride levels, hypertension, and metabolic syndrome. These findings raise the possibility that one consequence of a high burden of metabolic risk factors is increased endothelial activation or injury, in particular in individuals with hypertriglyceridaemic waist.
Microparticles are plasma membrane vesicle fragments that are released from various cell types during activation or apoptosis. In recent years, a growing body of literature has highlighted the role of MPs as mediators of intercellular communication, given that the biological properties of MPs are influenced by both their cellular origin and their composition. Routine MP measures in clinical samples require ongoing efforts to establish standardized protocols for plasma preparation, anticoagulation, and storage conditions in addition to techniques for quantification; these determinants are still under investigation. Nonetheless, in clinical studies performed to date, circulating MPs derived from the endothelium (EMPs) have been shown to demonstrate a special relationship with alterations in endothelial homeostasis. Increased levels of circulating EMPs have been observed in patients with stable or unstable coronary artery disease, stroke, and peripheral artery disease. These changes in plasma EMP levels usually averaged a 2- to 3-fold increase in patients with cardiovascular diseases, when compared with healthy controls. Elevated EMPs levels have also been measured in the presence of cardiovascular risk factors such as uncontrolled systemic hypertension, diabetes, and dyslipidemia. However, these results were observed in highly selected groups of patients and were limited by modest sample sizes. Thus, the influence of different CV risk factors and medications (i.e. cholesterol-lowering therapy, anti-hypertensive medications) on EMP levels has yet to be examined in a larger community-based population.
Our data indicate that elevated triglycerides are strongly associated with higher EMP levels. These results are consistent with observations from previous pilot studies in humans. Ferreira et al. showed that CD31+ EMP levels increase along with triglyceride levels following a high-fat meal in a sample of 18 healthy individuals without known cardiovascular risk factors. Similar findings were also reported for CD144+ EMPs in 27 patients with diabetes. High levels of triglycerides have been shown to impair endothelial function in vivo, which may be related to the pro-inflammatory and pro-apoptotic effects of remnant lipoprotein particles on the endothelium that are potentially mediated by changes in local oxidative stress.
High triglyceride levels are associated with an increased risk for development of cardiovascular disease in certain subgroups such as women and young healthy men. Potential explanations for this association include increased atherogenic cholesterol-enriched remnant lipoprotein particle production, interference with HDL or LDL metabolism, and enhanced recruitment and attachment of monocytes to the vascular wall. Interestingly, the association of EMPs with triglycerides observed in our community-based sample was independent of total cholesterol levels, other cardiovascular risk factors, and the presence of any medication therapies for cardiovascular risk. Thus, our findings offer potentially new insights regarding the deleterious role of triglycerides on vascular homeostasis. As EMPs interfere with coagulation, inflammation, regulation of vascular tone, and angiogenesis, these vesicles might further support the initiation and promotion of vascular disease. We did not observe any independent relationship between HDL or total cholesterol values and EMPs levels. Although oxidized LDL cholesterol has been shown to increase EMP release in vitro, the current data suggest that the previously observed links in vivo between these parameters could have been influenced by the presence of confounding factors (such as associated cardiovascular risk factors, including hypertension and diabetes) or that the presence of lipid-lowering therapy might have affected this relationship.
We also observed independent associations between circulating levels of EMPs and the presence of metabolic profiles associated with high cardiovascular risk. In particular, the present paper demonstrates robust correlations between circulating EMP levels and hypertryglyceridaemic waist, suggesting that the vascular endothelium of these participants at high risk of coronary artery disease may be prematurely injured or inflamed. Metabolic abnormalities are associated with impaired endothelial function and altered vascular structure. The pathophysiology of the deleterious effects on the endothelium has not been fully elucidated but may involve macrophage infiltration within white adipose tissue, release of adipokines by adipocytes and pro-inflammatory cytokines by leukocytes, insulin resistance, and changes in lipid profiles. All of these factors can negatively impact endothelial homeostasis, leading to local inflammation, increased oxidative stress, and cellular vesiculation. Inflammatory cytokines are elevated in the presence of metabolic syndrome and have been shown to stimulate EMP release in vitro. Thus, in turn, EMPs may interfere with coagulation, inflammation, or enhance endothelial damage, leading to increased endothelial vesiculation and MP release.
Our data also show that hypertension is associated with an increase in CD144 EMPs. Prior studies have reported increased circulating EMPs levels in patients with uncontrolled severe hypertension and an effect of anti-hypertensive drugs on platelet or monocyte-derived, but not endothelial, MPs. Our study highlights differences between the two EMP sub-populations (CD144+ or CD31+CD41− MPs) with regards to sex and hypertension, which could result from the preparation or the conservation of the plasma samples. Alternately, these differences might reflect a different condition or site of endothelial injury or a different affinity of CD144 and CD31 antibodies for their endothelial epitopes.
Notably, we observed no significant association between frank diabetes and EMP traits. Prior small studies of patients referred for coronary angiography have suggested that circulating EMP levels may be elevated in diabetics compared with non-diabetics. However, Sabatier et al. reported higher levels of EMPs compared with healthy controls in Type 1 but not Type 2 diabetics. Notably, 99% of individuals with diabetes in the Framingham Offspring cohort are known to have Type 2 diabetes. Thus, previously observed relations of EMPs with diabetes may be related to concomitant burden of cardiometabolic risk factors, active coronary artery disease, or both. Moreover, the absence of any apparent association between diabetes and EMP traits may also be related to the presence of confounding factors. Notably, we observed that the association between triglycerides and EMP levels was more pronounced in diabetics compared with non-diabetics. This observation suggests the possibility that diabetes enhances potentially deleterious effects of hypertriglyceridemia on endothelium. Alternately, the presence of diabetes in association with hypertriglyceridemia may reflect a more severe grade of metabolic disorder that leads to greater endothelial injury. Further research is needed to specifically investigate these observed relations.
Interestingly, there was no significant association between smoking status and EMP traits in our study sample. In previous work, both active and passive smoking exposure has been associated with a small but significant elevation in circulating EMPs in small samples of healthy adults, with mean ages ranging from 30 to 47 years. By contrast, our larger study sample was older (mean age 66 years) with a presumably larger burden of cardiometabolic risk factors. Thus, it is possible that whereas smoking leads to elevated EMP levels in otherwise healthy younger adults, this relation is attenuated with advancing age and the cumulative burden of cardiometabolic risk that is frequently seen in older adults.
Several limitations of this study merit consideration. The present study was not designed to precisely assess quantity of small-sized MPs, with diameters between 0.1 and 0.5 μm; thus, our conclusions are only valid for large EMPs with diameters ranging from 0.5 to 1.0 μm. The platelet-poor plasma preparation used in the present study might have included residual platelets which could generate artifactual MPs; thus, findings related to platelet-derived MPs or the overall pool of annexinV+ MPs expressing phosphatidylserine would need to be interpreted with extreme caution in this cohort. Because our analyses were cross sectional, we cannot determine whether high-EMPs preceded or followed the development of metabolic syndrome. Additionally, HOMA-IR and HbA1c data were not available as concurrent assessments of insulin resistance or glycaemic exposure. The lack of outcomes data for this ambulatory cohort with low event rates precluded analyses of associations with incident cardiovascular disease, which warrants future investigation. Concurrent data on surrogate measures of subclinical atherosclerosis, such as carotid intima media thickness, were also not available at the time of the EMP assessment. Thus, further research is needed to examine the relations of EMPs and such imaging based markers of subclinical vascular disease in the community. Anti-hypertensive medications including angiotensin receptors blockers, β-blockers or calcium channel blockers have an impact on various subgroups of circulating EMPs. Endothelial microparticle traits have been assessed in very few large samples because they are difficult to measure and require specialized specimen handling. In future prospective cohorts, the ability to obtain samples suitable for EMP and other cell-based phenotyping may help to provide confirmatory data for our findings. Additionally, our sample was predominantly white of European ancestry; therefore, the extent to which our findings are generalizable to other racial/ethnic populations is unknown.
In conclusion, in this large community-based study, higher levels of circulating plasma EMPs were associated with elevated triglycerides, hypertension, and the metabolic syndrome. Our results support the need for further studies exploring the potential mechanisms linking metabolic and lipid abnormalities to the production of EMPs. Further studies may also be warranted to determine whether EMP levels might represent an appropriate biomarker for identification of asymptomatic individuals at risk of developing cardiovascular events.