Vascular Biology of Atherosclerosis in Patients with Diabetes: Dyslipidemia, Hypercoagulability, Endothelial Dysfunction, and Inflammation

Overview

The interaction between diabetes and atherosclerosis is complex and multifactorial. Despite unequivocal evidence for increased cardiovascular disease (CVD) risk in patients with diabetes; a well-documented epidemic of obesity and diabetes; intensive research efforts that include major preclinical scientific progress using unbiased “-omic” approaches; large cardiovascular (CV) outcome studies in diabetes; and new glucose-lowering therapies, the mechanisms that link diabetes to atherosclerosis remain murky. Indeed, challenges in this area begin with simple issues regarding definitions and expand quickly into problems of epistemology. Type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM) differ fundamentally in their root causes, but share increased risk of micro-CVD and macro-CVD as compared with nondiabetic patients. Although these diseases are defined clinically by hyperglycemia, the pathologic picture of T2DM extends beyond glucose. Indeed, recent clinical trial data raise questions regarding whether glucose should be the primary therapeutic target for improving CVD outcomes. Such issues force consideration of other factors in the vascular biology of diabetic atherosclerosis that are outside the glucose-insulin axis discussed in Chapter 1, Chapter 2, Chapter 3 .

Although it remains unlikely that one single pathway accounts for how diabetes promotes atherogenesis, atherosclerosis, and atherothrombotic complications, various mediators and pathogenic forces have been uncovered that help explain how the diabetic and even the prediabetic state modulate vascular biology, including specific responses in different cell types ( Fig. 10-1 ). Aside from changes in glucose, diabetes is typically characterized by a dyslipidemia involving elevated triglycerides (TGs), lower high-density lipoprotein (HDL) levels, and a low-density lipoprotein (LDL) particle that is more atherogenic. Diabetic atherosclerosis involves a prothrombotic state, suggesting basic changes in the coagulation system and its players. Although all cellular components of the arterial wall and the inflammatory system appear involved in diabetic atherosclerosis, the endothelium and its functional roles have been especially implicated in the natural history of T2DM. Inflammation has arisen as a potential central driver in the pathogenesis of diabetes, atherosclerosis, and their intersection. The breadth of abnormalities, whether molecular or clinical, proposed to play a part in T2DM and atherosclerosis independent of glucose is impressive and beyond the scope of any one summary, especially given ongoing rapid evolution in this area. Here we review key concepts regarding how dyslipidemia, hypercoagulability, endothelial dysfunction, and inflammation alter cellular responses that promote atherosclerosis in the setting of diabetes, with an emphasis on emerging concepts, novel targets, and clinical relevance.

Diabetic Dyslipidemia

Type 2 diabetes is characterized by a distinct lipid profile involving LDL cholesterol (LDL-C) levels that are often not particularly elevated, higher TG values, and lower HDL cholesterol (HDL-C) concentrations. Also associated with diabetic dyslipidemia are elevated levels of circulating free fatty acids (FFAs). Often this constellation of lipid abnormalities arises early in T2DM including in prediabetic states, drawing further attention to diabetic dyslipidemia as a contributor to the pathogenesis of diabetic atherosclerosis and its complications. Multiple inputs appear to foster diabetic dyslipidemia. Central adiposity may promote dyslipidemia, including the development of secondary factors such as increased inflammation within the fat, systemically, as well as through higher levels of FFAs. The hypertriglyceridemia of diabetes involves changes in both production and combustion: the hepatic secretion of TG-rich lipoproteins such as very low-density lipoproteins (VLDLs) and altered hydrolysis of these and other TG-rich lipoproteins. ^, Yet another potential component of hypertriglyceridemia may be postprandial excursions in TG levels, which may be more predictive of CV risk than the fasting levels usually obtained in the clinic.

Lipoprotein lipase (LPL), a key enzyme involved in hydrolyzing fatty acids from TGs and delivering these fatty acids to tissues, may be defective in T2DM. It is interesting to note that LPL-mediated hydrolysis of TGs has been shown to be a mechanism for generating natural ligands for the nuclear receptor known as peroxisome proliferator-activated receptor alpha (PPAR-α), which, when activated by ligands, controls the expression of multiple genes involved in lipid metabolism, inflammation, and fatty acid oxidation. Fibrates, lipid-lowering agents used to treat hypertriglyceridemia, are thought to work as PPAR-α agonists. ^, Of note, other endogenous lipolytic pathways including adipose tissue TG lipase (ATGL) and hepatic lipase as well as fatty acid synthase can generate PPAR ligands in different physiologic contexts as well. These lines of evidence suggest that in diabetes, loss of endogenous LPL action decreases activation of the PPAR-α–regulated gene cassette, which would be predicted to result in decreased expression of apolipoprotein (apo) A-I, which is involved in HDL function, and increased endothelial inflammation. It is important to note that fibrates, as synthetic PPAR-α agonists, may not faithfully replicate cellular responses to natural PPAR-α ligands. Of interest, the potential role of LPL has expanded to include other proteins involved in LPL action. For example, C-III is an endogenous inhibitor of LPL activity. Recent studies implicate apo C-III in promoting proatherogenic, proinflammatory responses, which may occur through various mechanisms, including potential modulation of endogenous PPAR responses as outlined previously as well as other means.

Given that HDL cholesterol levels are inversely associated with coronary heart disease (CHD) risk, significant effort has focused on the mechanisms underlying the low HDL commonly observed in patients with diabetes. Both abnormal production of HDL and remodeling of this lipid by plasma enzymes may contribute to the low level of circulating HDL cholesterol observed in T2DM. Expression and activity of endothelial lipase (EL), a phospholipase that is synthesized in and expressed on the surface of vascular endothelium, catabolizes HDL, resulting in decreased levels of this putatively antiatherogenic lipoprotein. Elevated concentrations of EL protein are significantly correlated with coronary artery calcification score as well as other features of metabolic syndrome including waist circumference, blood pressure, TGs, HDL levels, and fasting glucose in individuals with a family history of premature CHD. In addition, direct correlations have been observed between EL levels and circulating markers of inflammation including high-sensitivity C-reactive protein (hsCRP), interleukin 6 (IL-6), and soluble intercellular adhesion molecule. Low-dose endotoxemia in 20 subjects increased EL concentrations 12 to 16 hours after injection, and this increase in EL correlated with reductions in plasma HDL. Collectively these data suggest that low-intensity inflammation, a common feature of T2DM, controls HDL through effects on EL, providing a possible mechanism for the low HDL in T2DM and the exaggerated CV risk associated with insulin-resistant states including metabolic syndrome and diabetes mellitus. Despite the clear epidemiologic inverse association between HDL and CV risk, the hypothesis that raising HDL can reduce CV events has not yet been proven. The recent failure of large randomized, placebo-controlled trials designed to test this hypothesis using cholesteryl ester transfer protein (CETP) inhibitors and niacin, which both raise HDL cholesterol levels, suggests that the biology of HDL’s atheroprotective effects are likely very complex and cannot be ascribed exclusively to a single parameter such as HDL cholesterol quantity—the current lipid parameter measured in the clinic. ^,

Another input into diabetic dyslipidemia is hepatic dysregulation, itself a consequence of fatty liver, hyperinsulinemia, and hyperglycemia. Hyperglycemia per se can alter the carefully controlled system of lipid metabolism, as, for example, through the glycation of proteins and lipoproteins. In addition to altering the normal function of these entities, the breakdown of glycated proteins and lipoproteins, known as advanced glycation endproducts (AGEs), activates specific receptors for AGEs (RAGEs), resulting in responses closely linked to atherosclerotic complications, such as increases in matrix metalloproteinases (MMPs) thought to promote plaque destabilization and rupture.

Although total LDL-C levels are often average in patients with T2DM, LDL continues to appear as a significant predictor of CV risk in this patient population. As is usually seen with higher TG values, LDL particles in T2DM are considered more pathogenic as a result of their being smaller, more dense, and hence more prone to entry, oxidation, and retention in the arterial wall. The notion that lipoprotein retention in the subendothelial space may contribute to atherosclerosis may be especially relevant in diabetes. Extensive evidence implicates the oxidation of LDL as a major player in atherosclerosis. Given that hyperglycemia and other aspects of diabetes may promote altered redox balance and increased oxidative stress, increased LDL oxidation in diabetes may be an additional factor in diabetic atherosclerosis. An intriguing newer direction for this field has been evidence that autoantibodies to oxidized LDL (oxLDL) may be involved in atherosclerosis and coronary calcification, which may extend to diabetes, including T1DM.

Placing lipid metabolism into a broader context, lipoprotein particles can be reconsidered as circulating, biologically active entities whose very nature and function afford systemic pathologic effects. Lipoproteins in various forms exit the liver and interact with the vasculature. In their transit through the circulation, lipoproteins also encounter other factors in addition their interactions with vessel walls, including circulating cells and many other proteins. In this regard, one functional unit with which lipoproteins interact is the coagulation system, including both the relevant procoagulant and anticoagulant proteins as well as platelets. Consistent with this concept, studies have reported increased platelet reactivity and thrombogenicity in response to VLDL and TG-rich lipoproteins. Such interactions connect dysregulated lipid metabolism in diabetes to a potent force in atherosclerosis strongly suggested as being altered in the diabetic milieu, namely the coagulation system.

This brief preceding overview underscores the extent to which pathogenesis in diabetes, including alterations in lipid and cholesterol metabolism, are influenced by diverse, often overlapping issues.

Diabetes: A Prothrombotic State

T2DM is characterized by a prothrombotic and hypercoagulable state that is a significant contributor to the pathogenesis and progression of diabetic vascular complications. Multiple factors have been implicated in promoting the prothrombotic state in diabetes, including platelet hyperreactivity, increased coagulation, and impaired fibrinolysis. Although hyperglycemia itself may be a major factor in these pathways, as noted, other components of the clinical picture in diabetes, such as lipid abnormalities, obesity, and inflammation, as well as more specific pathogenic mechanisms such as oxidative stress may also contribute to the prothrombotic, procoagulant state found in those with diabetes, including changes in platelet function, changes in coagulation factors, and shifts in the fibrinolytic balance, as are considered here.

Altered Platelet Function

Platelets of patients with T2DM are characterized by dysregulation of several signaling pathways, leading to hyperreactive platelets with enhanced adhesion, aggregation, and activation ( Fig. 10-2 ). Processes that define the diabetic state—hyperglycemia, insulin resistance, dyslipidemia, inflammation, and increased oxidation— are all implicated in platelet hyperactivity in diabetes. Hyperglycemia increases platelet reactivity by altering different biochemical pathways, including protein kinase C (PKC) activation, with subsequent increased platelet granule release and aggregation. ^, Glucose also has direct osmotic effects that can increase platelet reactivity. In addition, by inducing nonenzymatic glycation of proteins on the surface of platelets, hyperglycemic states may decrease membrane fluidity while increasing adhesion and activation. Consistent with these findings, acute hyperglycemia has been shown to increase markers of platelet activation such as P-selectin and CD40 ligand, whereas improved glycemic control may decrease platelet reactivity. ^,

Platelet aggregation is mediated by platelet surface receptors and adhesive proteins such as glycoproteins GPIIb/IIIa, GPIb, and P2Y12, each of which is altered in T2DM. Platelet turnover in patients with diabetes appears accelerated. Hyperglycemia increases the release of reticulated, larger, and thus more reactive platelets, including a higher capability of forming thromboxane—a potent vasoconstrictor and proaggregant. Diabetic platelets may also have altered signaling through the P2Y12 pathway, a key player in adhesion, aggregation, and procoagulant activity. Increased levels of circulating microparticles, derived from platelets and various stimulated cell types, may also underlie the procoagulant potential in diabetes. Microparticle size is larger in those with T2DM than in normal controls, and increases in microparticle number have been associated with an increased incidence of diabetic complications.

Intracellular calcium is a central mechanism for regulating platelet function. Platelets in patients with diabetes contain lower cyclic adenosine monophosphate (cAMP) levels and higher intracellular calcium levels than in normal patients, which may contribute to hyperreactivity, increased aggregation and activation, and stimulation of thromboxane synthesis. Altered calcium homeostasis may be in part attributable to changes in the activity of calcium ATPases, which are highly sensitive to oxidative damage. ^, Recent research suggests that activity of calcium-activated proteases (calpains) is increased in platelets from diabetic patients, contributing to dysregulation of platelet calcium signaling and hyperreactivity of platelets.

Insulin resistance and insulin deficiency can both alter platelet reactivity. Insulin opposes the effects of platelet agonists through activation of an inhibitory G protein by insulin receptor substrate 1 (IRS-1). During insulin resistance, impaired insulin receptor signaling attenuates insulin-mediated antagonism of platelet activation, thus increasing platelet reactivity. Insulin-like growth factor 1 (IGF-1), which is present in granules of platelets with IGF-1 receptors present on the platelet surface, stimulates tyrosine phosphorylation of IRS, potentiating platelet activation. ^, Reduced insulin sensitivity in platelets lowers cAMP levels and increases intracellular calcium levels, enhancing platelets degranulation and aggregation. In addition, platelets from insulin-resistant patients display diminished sensitivity to the actions of nitric oxide (NO) and prostacyclin while also manifesting significantly lower platelet NO-synthase activity.

As noted, some of the systemic abnormalities often concomitant with diabetes can also alter platelet biology. Hypertriglyceridemia increases platelet reactivity, perhaps in part through apo E. Glycation of LDL particles may also lead to impaired NO production and increased intraplatelet calcium concentration, with subsequent increased platelet hyperreactivity and microparticle formation in diabetic patients. Central obesity appears to promote platelet dysfunction, with reduced platelet sensitivity to insulin, impaired platelet responses to nitrates and prostacyclin, elevated platelet count and volume, increased cytosolic calcium concentration, and evidence for increased oxidative stress. Furthermore, weight loss reverses some of these changes, reducing platelet activation. Increased platelet reactivity has been tied to increased oxidative stress found in T2DM. ^, Superoxide and reactive oxygen species (ROSs) may increase platelet reactivity by enhancing postactivation intraplatelet activation calcium. In addition, lipid peroxidation and protein glycation may affect platelet activation. Inflammation may foster platelet reactivity by increasing expression of mediators of platelet activation, such as CD40 ligand, whose plasma-soluble levels are increased in T2DM. CD40L, found in activated platelets, has proinflammatory properties.