|Home | About | Journals | Submit | Contact Us | Français|
Atherothrombotic vascular disease is the major cause of death and disability in obese and diabetic subjects with insulin resistance. Although increased systemic risk factors in the setting of insulin resistance contribute to this problem, it is likely exacerbated by direct effects of insulin resistance on the arterial wall cells that participate in atherosclerosis. A critical process in the progression of atherosclerotic lesions to those that cause clinical disease is necrotic breakdown of plaques. Plaque necrosis, which is particularly prominent in the lesions of diabetics, is caused by the combination of macrophage apoptosis and defective clearance, or efferocytosis, of the apoptotic macrophages. One cause of macrophage apoptosis in advanced plaques is activation of a pro-apoptotic branch of the endoplasmic reticulum stress pathway known as the Unfolded Protein Response (UPR). Macrophages have a functional insulin receptor signal transduction pathway, and down regulation of this pathway in the setting insulin resistance enhances UPR-induced apoptosis. Moreover, other aspects of the obesity/insulin-resistance syndrome may adversely affect efferocytosis. These processes may therefore provide an important mechanistic link among insulin resistance, plaque necrosis, and atherothrombotic vascular disease and suggest novel therapeutic approaches to this expanding health problem.
The incidence of insulin resistance, metabolic syndrome, and type 2 diabetes is rising rapidly due to the epidemic of obesity in the industrialized world.1 While a number of disease processes are associated with insulin resistance and type 2 diabetes, the leading cause of morbidity and mortality is cardiovascular disease.2 An important factor in accelerated heart disease in type 2 diabetes is likely to be insulin resistance and hyperinsulinemia. For example, the risk of cardiovascular disease is increased in metabolic syndrome, which is characterized by insulin resistance without overt hyperglycemia.3–5 Moreover, rapid weight gain during childhood leads to hyperinsulinemia and increased coronary artery disease risk in adult life.6 Part of the association between insulin resistance and cardiovascular disease is likely related to associated risk factors, including dyslipidemia (increased VLDL, reduced HDL, and possibly altered LDL), hypertension, and a pro-thrombotic state.3 However, insulin resistance may have direct pro-atherogenic effects at the level of the arterial wall, and an emerging concept that will be explored in this review is that insulin resistance in lesional macrophages promotes a series of cellular events critical for advanced plaque progression. After a brief review of atherogenesis, we will focus on new findings related to plaque progression and the role of macrophage insulin resistance that have appeared in the literature since the last review of this topic in this journal in 2007.7
Atherogenesis begins with the retention of atherogenic lipoproteins in the subendothelium of susceptible areas of the arterial tree.8 In response to these retained lipoproteins, particularly those that undergo atherogenic modifications such as oxidation and aggregation, a series of biological and maladaptive inflammatory responses ensue: (a) monocytes and other inflammatory cells enter the intima; (b) monocytes differentiate into macrophages, which then ingest retained and modified lipoproteins and become cholesteryl ester-loaded foam cells; (c) macrophages and other inflammatory cells contribute to a state of inflammation that fails to properly resolve; and (d) smooth muscle cells populate the intima, leading to collagen synthesis.9–12. At this stage, the plaques are usually asymptomatic due to outward remodeling of the artery to preserve lumenal blood flow and a fibrous cap that protects the lesion from disruption.13, 14 However, some of these plaques, unrelated to plaque size per se, may undergo necrotic breakdown, thinning of the fibrous cap, a heightened state of inflammation, and an accumulation of unesterified cholesterol.13–19. Many of the hallmarks of impaired inflammation resolution are evident in these plaques, including continued entry and poor egress of inflammatory cells, defective clearance of apoptotic cells, and a suppressed fibrotic “scarring” response.12, 20 These so-called “vulnerable plaques” are at risk for plaque disruption through fibrous cap rupture or endothelial erosion, which in turn can trigger acute thrombosis. If the thrombosis is extensive and not quickly resolved, acute vascular occlusion and tissue infarction occurs, leading to acute myocardial infarction, unstable angina, sudden cardiac death, or stroke.
The exact mechanisms of plaque disruption are not known. Cap thinning per se may be caused by a combination of protease-mediated digestion of extracellular matrix molecules, particularly by matrix metalloproteinases (MMPs), and decreased collagen synthesis, perhaps exacerbated by death of the collagen-synthesizing cells in the intima.13 These processes, as well as coagulation and thrombosis, are likely promoted by inflammatory cytokines, many of which are secreted by lesional macrophages.13 Lesional necrosis of vulnerable plaques, which is caused by the combination of macrophage death and defective clearance, or “efferocytosis,” of dead macrophages,21–23 can promote plaque disruption by a number of mechanisms.15, 22, 24–27. For example, although matrix proteases are secreted by living macrophages in lesions, they may also be released by dead and dying macrophages.28 Moreover, lesional necrosis triggers a heightened state of inflammation, which, as mentioned above, promotes MMP secretion, coagulation, and thrombosis.29 Finally, the necrotic core is rich in lipids and poor in cells and extracellular matrix, and the structural properties resulting from this composition are thought to contribute to mechanical stresses in the overlying cap, which may contribute to cap rupture.30 Thus, macrophage death and defective clearance of the dead cells, leading to lesional necrosis, is an important process in the formation of the vulnerable plaque, and, as described in this review, exacerbations of these processes may help explain accelerated atherothrombotic disease in insulin-resistant states.
To understand how insulin resistance may promote advanced plaque progression in general, and plaque necrosis in particular, it is necessary to review our latest understanding of the mechanisms and consequences of macrophage death in advanced atheromata. A number of hypotheses have been conceived to explain advanced lesional macrophage apoptosis, and undoubtedly more than one mechanism is involved. Examples include growth factor deprivation, toxic cytokines, and oxidized lipids or lipoproteins,31 but there is as yet little proof for these ideas in vivo. Recent mechanistic data in cultured cells and correlative and genetic-causation evidence in vivo support a role for endoplasmic reticulum (ER) stress in advanced lesional macrophage apoptosis and its major consequence, plaque necrosis. As had been previously demonstrated in other models of ER stress-induced apoptosis, macrophages subjected to ER stress undergo apoptosis in a manner that is partially dependent on the CHOP (GADD153) branch of the ER stress pathway known as the Unfolded Protein Response (UPR).32, 33 CHOP-mediated apoptosis can be modeled in cultured macrophages by either potent inducers of ER stress or by the combination of more subtle ER stressors and a “second hit.” An example of an atherosclerosis-relevant inducer of the single-hit ER stress apoptosis is 7-ketocholesterol,33, 34 the most abundant oxysterol in advanced atherosclerotic lesions. Examples of the two-hit model include the combination of low-level ER stressors with pattern recognition receptor (PRR) ligands, such as modified lipoproteins.35, 36 Another example of the two-hit model is incubation of macrophages with atherogenic lipoproteins under conditions of genetic or pharmacologic inhibition of intracellular cholesterol re-esterification.37, 38 In this model, which is often referred to as the “FC” (free cholesterol) model and is designed to mimic FC-loaded macrophages in advanced atheromata,39, 40 the ER stress hit is provided by excess accumulation of unesterified cholesterol in the ER membrane, and the second hit is activation of PRRs by the lipoproteins themselves. The contribution of the second hit to apoptosis involves both amplification of pro-apoptotic pathways and suppression of cell-survival pathways that are activated in ER-stressed cells.36 The tendency of macrophages to undergo apoptosis when subjected to ER stress in combination with PRR activation may have evolved as a host defense mechanism against intracellular organisms that require living macrophages to survive.
While it has been known that activation of the CHOP pathway of the UPR can cause apoptosis, the molecular mechanisms linking CHOP to death execution pathways is poorly understood. Recent work in our laboratories has provided evidence for a calcium-dependent mechanism in ER stress-induced macrophage apoptosis. ER stress in macrophages leads to the release of calcium from the ER lumen into the cytosol.32 The cytosolic calcium chelator BAPTA-AM can block ER stress-induced apoptosis in macrophages, and recent work has shown that a key integrator of cytosolic calcium and death execution in these cells is a calcium-responsive kinase called calcium/calmodulin-dependent protein kinase II (CaMKII).36, 41, 42 Activation of CaMKII leads to multiple death pathways, including induction of the cell-surface death receptor Fas; stimulation of mitochondrial calcium uptake and release of pro-apoptotic cytochrome c from the mitochondria; activation of pro-apoptotic STAT-1; and accumulation of reactive oxygen species through activation of NADPH oxidase.42 CHOP amplifies this calcium-death pathway by leading to activation of IP3 receptors, which are calcium-release channels in the ER membrane.43 The mechanism involves oxidative activation of IP3R by the downstream CHOP transcriptional target, ER oxidase-1α (ERO1α). Net calcium release can also be promoted through inhibition of sarco/endoplasmic reticulum calcium-dependent ATPase (SERCA), which pumps calcium back into the ER lumen. SERCA is inhibited by alterations in the ER membrane by certain ER stressors, such as unesterified cholesterol or saturated fatty acids,44 and SERCA is down-regulated in the setting of insulin resistance, as will be summarized below.
Macrophage apoptosis by itself would not be expected to be detrimental, because apoptotic cells are normally cleared rapidly by phagocytosis (“efferocytosis”) in a manner that prevents post-apoptotic cellular necrosis and that promotes anti-inflammatory processes.27 Indeed, manipulations that accelerate early lesional macrophage apoptosis decrease lesion cellularity and plaque progression, and vice versa,23, 45 suggesting that efferocytosis is very efficient in the early stages of atherogenesis. This principle has been applied recently to a mouse model of type 2 diabetes and early atherosclerosis.46 However, in the later stages of atherosclerosis, macrophage apoptosis is associated with plaque necrosis,23 and there is evidence in humans that efferocytosis is defective in advanced plaques.47 The mechanisms of defective efferocytosis in advanced lesions are not known, but several interesting ideas have been advanced based on in vitro and in vivo observations. For example, oxidized lipids and proteins exist in these plaques, and some of these molecules can competitively inhibit efferocytosis by binding to efferocytosis receptors.48 Thus, to the extent that these oxidized molecules accumulate as lesions progress,10 they may reach a high enough level in advanced atheromata to take on this competitive inhibitory role. In another scenario, the efferocytosis receptor Mertk has been shown to play a role in efferocytosis and plaque necrosis in mouse lesions,49, 50 and inflammation-induced cleavage of this receptor by membrane sheddases51 may contribute to defective clearance of apoptotic cells in advanced plaques. The fact that inflammation increases as lesions progress may offer an explanation as to why this anti-efferocytic process occurs only in advanced plaques.
The concept that ER stress-induced macrophage apoptosis in combination with defective efferocytosis in advanced lesions promotes plaque necrosis is supported by a number of genetic-causation studies in mice and by correlative studies in humans. In fat-fed Apoe−/− or Ldlr−/− mice, ER stress markers are induced as lesions progress.32, 52–56 Most importantly, genetic targeting of CHOP and STAT-1, the pro-apoptotic signaling transducer activated by the CHOP calcium-CaMKII pathway (above), as well as prevention of cholesterol-induced ER damage, inhibit advanced lesional macrophage apoptosis and plaque necrosis.33, 42, 52 Moreover, deletion of two “second-hit” PRRs—SRA and CD36—decreases macrophage apoptosis and plaque necrosis in the lesions of fat-fed Apoe−/− mice.57 In humans, there are close correlations among markers of ER stress, apoptosis, and plaque vulnerability in coronary arteries.55 In terms of efferocytosis, studies have shown an increase in plaque necrosis that correlates with a worsening of lesional efferocytosis in several mouse models in which efferocytosis effectors have been targeted, including Mertk, MFG-E8, transglutaminase-2, and complement factor C1q.49, 58 In summary, in vitro and in vivo evidence support a model in which macrophage apoptosis in advanced lesions, induced in part by a pro-apoptotic ER stress-calcium pathway, plus defective efferocytosis promote plaque necrosis (Figure 1). Because plaque necrosis is strongly associated with disrupted plaques and acute lumenal thrombosis,59 and because plaque necrosis is particularly prominent in atherosclerotic lesions from diabetic subjects, as described in the following section, these insights should be useful in our understanding of and therapeutic approaches to accelerated plaque progression in the setting of insulin resistance.
It is now well-established that type 2 diabetes and insulin resistance are major risk factors for atherothrombotic vascular disease.3–5 While many theories have arisen to explain this relationship,60, 61 a common endpoint of plaque progression associated with atherothrombotic vascular disease, as mentioned in the previous section, is plaque necrosis. In this context, a number of independent studies have found that advanced atherosclerotic lesions in diabetic subjects are characterized by particularly large necrotic cores when compared to similarly sized lesions from non-diabetic individuals.62–67 For example, Burke et al.62 found that necrotic core size in the coronary arteries of subjects who died suddenly was positively correlated with the presence of diabetes independently of other factors. Similar results were found when coronary atherectomy specimens of diabetics and non-diabetics were compared.63 Nasu et al.66 used virtual histology based on intravascular ultrasound (IVUS) data to assess coronary arterial necrotic cores in non-diabetic and diabetic patients with stable angina and found an approximate 50% increase in the percent area covered by necrotic cores in the diabetic group. Almost identical findings were reported in similar studies conducted by Hong et al.65 in Korea and Pundziute et al.66 in the Netherlands. A prospective study of subjects with coronary artery disease in which radiofrequency data from IVUS was used to assess necrotic core size in coronary arteries found that only diabetes and age were associated positively with necrotic core size in logistic regression analysis.67 These collective data raise the issue as to whether the cellular events described in the previous sections, particularly advanced lesional macrophage apoptosis and/or defective efferocytosis, are enhanced in the setting of diabetes, leading to increased plaque necrosis and, ultimately, accelerated atherothrombotic vascular disease.
In view of the role of insulin resistance in diabetic heart disease and the larger necrotic cores in the coronary arteries of diabetic subjects, we and others have examined how insulin resistance at the level of the macrophage affects mechanisms and consequences of macrophage death in vitro and in vivo. Macrophages have insulin receptors, and acute exposure of the cells to insulin in vitro results in phosphorylation of the insulin receptor, insulin receptor substrate-2 (IRS-2), and Akt, leading, among other responses, to nuclear exclusion and inactivation of FoxO transcription factors.7, 68 Moreover, pre-treatment of macrophages in vitro with high-dose insulin leads to down-regulation of their insulin receptors and suppression of insulin receptor signaling, which is also observed in freshly isolated macrophages from insulin-resistant mice, such as the hyperinsulinemic leptin-deficient ob/ob mouse.68 Thus, macrophages show the hallmarks of “insulin resistance” at a cellular level in the setting of high insulin concentrations.
Macrophages rendered insulin resistant through pre-incubation with insulin, genetic deletion of the insulin receptor, or pharmacologic inhibition of insulin signaling, and macrophages freshly isolated from hyperinsulinemic mice, show an increase in the levels of the scavenger receptor SRA.7, 68 As mentioned in the previous section, SRA can serve as a “second-hit” PRR in ER stress-induced macrophage apoptosis both in vitro and in advanced lesional macrophage death and plaque necrosis in vivo. In this regard, insulin-resistant macrophages show markedly enhanced apoptosis in vitro when exposed to ER stress conditions plus an SRA-mediated second hit, as is the case with macrophages loaded with lipoprotein-derived unesterified cholesterol.56, 69, 70
ER stress in macrophages triggers compensatory cell-survival pathways, notably those activated by Akt and NF-κB, and apoptosis is temporally correlated with a down-regulation of these pathways and can be accelerated by their inhibition.36, 71, 72 Moreover, Akt deficiency in Apoe−/− mice was shown to enhance lesional macrophage apoptosis and inflammation and plaque progression.73 In this context, an important observation was that phosphorylation of Akt is suppressed in ER-stressed, insulin-resistant macrophages.69, 71 Consistent with a decrease in Akt phosphorylation, Senokuchi et al.71 found an increase in nuclear FoxO1 in insulin-resistant, ER-stressed macrophages, which normally translocates to the cytoplasm in response to Akt-dependent phosphorylation.74 Moreover, macrophages genetically lacking FoxO1, 3 and 4, were resistant to ER stress-induced apoptosis.71 However, FoxO-overexpression experiments indicated that nuclear localization of these transcription factors was not by itself sufficient for macrophage apoptosis but rather led to an enhancement of apoptosis in the setting of ER stress. The apoptosis-enhancing mechanism of FoxO1 is directly related to the role of another compensatory cell-survival factor in ER-stressed macrophages, namely NF-κB.71, 72, 75, 76 In ER-stressed macrophages, FoxO1 induces the expression of the NF-κB inhibitor IκBε and thereby enhances apoptosis.71
Importantly, insulin resistance potentiates the ER stress response itself.77 ER stress in macrophages leads to activation of the mitogen-activated protein kinase ERK,76 and Liang et al.77 found that this response was blunted in insulin-resistant macrophages. Additional studies revealed that the MEK-ERK pathway induces SERCA,77 which, as explained above, can abrogate ER stress by replenishing ER lumenal calcium stores and can protect macrophages from ER stress-induced apoptosis by lowering cytosolic calcium levels. Thus, the blunted MEK-ERK-SERCA pathway in insulin-resistant macrophages exacerbates the ER stress response and the calcium-mediated apoptosis pathway described above, and restoration of MEK1 in these cells is protective against both ER stress and apoptosis.77
In summary, mechanistic studies using various cell culture models of insulin-sensitive and insulin-resistant macrophages, including primary macrophages freshly harvested from ob/ob mice, have revealed an integrated pathway of cell signaling events responsible for the increased apoptotic response to ER stress in the setting of insulin resistance. Key among these events are those related to the compromise of compensatory cell survival pathways and the exacerbation of pro-apoptotic calcium signaling pathways (Figure 2).
To test relevance of enhanced ER stress-induced apoptosis in insulin-resistant macrophages in vivo, irradiated Ldlr−/− mice were transplanted with bone marrow from Insr+/+ or Insr−/− mice.69 It should be noted that this proof-of-concept model represents the most extreme form of “insulin resistance.” After recovery of the graft, the mice were fed a high-fat diet, and lesions were analyzed for overall area and, most importantly, plaque morphology. Consistent with the in vitro data, the advanced lesions of the Insr−/−→ Ldlr−/− mice fed the diet for 12 weeks had more apoptotic cells, particularly in macrophage-rich regions of the plaque, and more plaque necrosis than those of the Insr+/+ → Ldlr−/− control mice. Overall lesion area, the less important endpoint for the hypothesis being tested, showed no change after 8 weeks of diet and only a modest increase after 12 weeks. Baumgartl et al.78 used the cre-lox system to create Apoe−/− with macrophage-targeted deficiency of insulin receptors. After 4 months on a high-fat diet, these mice had a modest decrease in lesion area compared with control Apoe−/− mice. Apoptotic cells and necrotic areas were not quantified. Immortalized macrophages derived from these mice had a marked reduction in LPS-induced interleukin-6 (IL-6) secretion. The authors also tested the effects of global and bone marrow-derived IRS-2 deficiency in fat-fed Apoe−/− mice. In the holo-knockout model, lesion area was modestly increased, and in the bone marrow transplant model, lesion area was modestly decreased. Plaque morphology was not quantified. The authors interpreted these data as showing that myeloid-derived insulin receptors suppress atherosclerosis by blunting the inflammatory response.78 Senokuchi et al.71 also observed decreased inflammatory responses during ER stress in insulin-resistant macrophages. In that study, reduced NF-κB responses led to both increased apoptosis, as noted in the previous section, and decreased expression of some inflammatory genes. In summary, a careful comparison of Han et al.69 and Baumgartl et al.78 reveal a common finding of relatively modest effects of macrophage insulin resistance on overall lesion size, with subtle differences between the two studies perhaps arising from differences in genetic background (mixed vs. inbred C67Bl/6J), diets used (Western-type diet vs. the pro-inflammatory high-cholesterol/bile salt diet), and stage of lesion development. As noted, those specific features of atherosclerotic lesions related to the novel concept that insulin-resistant macrophages are more susceptible to apoptosis, i.e., advanced lesional macrophage apoptosis and plaque necrotic area, were assessed in only one of the two studies, and the data supported that concept.69
Our laboratories have been working with another model of advanced lesional apoptosis and plaque necrosis in mice that may relate to the findings above. During certain types of ER stress, macrophages respond with activation of a compensatory cell-survival pathway in which the MAP kinase p38α enhances phosphorylation/activation of Akt, a potent survival signal in these cells (see previous section).79 In essence, this ER stress-activated pathway delays or suppresses apoptosis, but eventually the survival pathway gets overwhelmed, and apoptosis ensues. As predicted by this concept, we found that gene-targeting of macrophage p38α partially impedes Akt activation and promotes ER stress-induced apoptosis both in vitro and in advanced plaques in fat-fed Ldlr−/− mice. Because insulin resistance also partially impedes Akt signaling, we reasoned that the two pathways might be additive. Indeed, treatment of macrophages from Insr−/−;Ldlr−/− mice with an ER stressor plus a p38 inhibitor enhanced apoptosis to a very high level, i.e., above the high level already seen when these macrophages are subjected to ER stress alone79 (see previous section). These findings further demonstrate the importance of defective Akt signaling, a critical component of intact insulin receptor signaling, in ER stress-induced macrophage apoptosis and raise caution about the use of p38 inhibitors, currently under development as anti-inflammatory agents in a number of diseases including type 2 diabetes,80 in insulin-resistance subjects.
Two models of global insulin resistance have also shown an effect on plaque necrosis. A recent study examining Western diet-fed ob/ob;Ldlr−/− mice, which have obesity and insulin resistant secondary to leptin deficiency, showed an increase in necrotic core size compared to similarly fed Ldlr−/− mice.81 As explained below, the mechanism not only involves increased susceptibility to apoptosis but also defective efferocytosis in the macrophages of these mice. Hsueh and colleagues82 compared 3 mo/old and 12 mo/old Ldlr−/− mice fed a high-fat diet for 3 months. The older mice developed worse insulin resistance and worse atherosclerosis than the younger mice, and the lesions in the older mice appeared to be associated with a marked increase in plaque necrosis. The insulin-resistant older mice had a blunted anti-oxidant response that might be caused by a defective DJ-1—Nrf2 anti-oxidant pathway,83 and a higher lesional expression of the NADPH oxidase subunit, p47. Atherosclerosis and plaque morphology were improved by treating the mice with the NADPH oxidase inhibitor and anti-oxidant, apocynin. One implication of these findings is that aging, a major risk factor for cardiovascular disease in humans,84 may interact with insulin resistance to promote plaque necrosis, and in this regard it is interesting to note that aging is associated with both enhanced ER stress and defective efferocytosis.85, 86 Second, a critical downstream pro-apoptotic effector of ER stress and ER calcium release is activation of NADPH oxidase, and, given the pathways described in Figure 2, this response may be further enhanced in the setting of insulin resistance. Although vitamin E has not been shown to be effective in decreasing cardiovascular risk in humans,87 more targeted anti-oxidants, such as NADPH oxidase inhibitors, in the specific setting of insulin resistance and possibly aging, may be more mechanistically justified and have more promise.
The increase in plaque necrosis in diabetic lesions raises the important issue as to whether efferocytosis is defective in these lesions and, if so, how this is mechanistically linked to insulin resistance. For example, defective phosphatidylinositol 3-kinase signaling in the setting of insulin resistance could, in theory, lead to a defect in efferocytosis in general and a specific defect in Mertk-mediated efferocytosis in particular.88, 89 Using an in situ assay that quantifies the percentage of apoptotic cells that have been engulfed by phagocytic macrophages vs. not associated with phagocytes,49 Li et al.81 found that the aortic root lesions of Western diet-fed ob/ob;Ldlr−/− mice had evidence of defective efferocytosis and, as predicted, increased plaque necrosis compared with lesions of Western diet-fed Ldlr−/− mice. In vitro studies showed that primary macrophages isolated from ob/ob mice have a defect in efferocytosis that was associated with defective PI3 kinase activity, but those from Insr−/− mice do not. Further studies revealed that the key defect in ob/ob macrophages was an increase in the saturated fatty acid:unsaturated fatty acid ratio in the macrophage membranes, perhaps through “stiffening” the plasma membrane to the point where phagocytosis is compromised.90 The efferocytosis defect on ob/ob macrophage could be corrected by treating the cells with the omega-3 polyunsaturated fatty acid eicosapentanoic acid (EPA), and similar results were found when macrophages were harvested from EPA-fed ob/ob mice. Most importantly, lesional efferocytosis was improved in ob/ob;Ldlr−/− mice by EPA feeding, which interestingly has also been associated with protection from heart disease in humans.91 The precise mechanism of how saturated fatty acid impair efferocytosis and how EPA improves it is still under investigation, as are other possible links between insulin resistance and clearance of apoptotic cells. Nonetheless, we can begin to imagine an integrated model in which direct effects of insulin resistance on advanced lesional macrophage apoptosis, combined with defective efferocytosis caused by systemic fatty acid defects in the setting of insulin resistance, can at least partially explain the large neurotic cores and accelerated thrombotic vascular disease in diabetics (Figure 2).
This review focused on one key feature of type 2 diabetes, insulin resistance; one type of lesional cell, the macrophage; and one overall context of atherosclerosis, advanced plaque progression. Even within this focused area of research, more work is needed to further define mechanisms whereby insulin resistance affects specific signaling pathways involved in the panoply of atherosclerosis-relevant macrophage activities, including, interaction with lipoproteins and intracellular metabolism of lipoprotein-derived lipids; inflammation and the resolution thereof; stress responses, including oxidative, heat shock, and ER stress; secretion of proteases, pro-coagulant molecules, and other factors involved in plaque progression; phagocytosis, efferocytosis, and antigen presentation; apoptosis-cell survival balance; and interaction with other cells and extracellular matrix. Moreover, it is likely that insulin resistance affects these processes differently in different subsets of macrophages and in other types of myeloid cells, notably dendritic cells, mast cells, and neutrophils. A limitation of our in vivo studies has been the lack of a mouse model that fully recapitulates features of human plaque disruption and athero-thrombosis,92 and so further developments to improve mouse models of diabetic atherothrombotic vascular disease is an important goal. Nonetheless, it is becoming clear that key morphologic features of such plaques are worsened by ER stress33 and insulin resistance in macrophages.69
Beyond the specific areas of plaque macrophages, insulin resistance, and advanced plaque progression, other areas of focus may offer additional clues as to why heart disease is enhanced in type 2 diabetes.61 For example, decreased insulin signaling in endothelial cells, through impaired Akt signaling, is also likely to have important pro-atherogenic consequences through decreased eNOS activity and increased expression of inflammatory genes and VCAM-1.73 In the liver, hyperinsulinemia and insulin signaling may increase VLDL secretion while having the opposite effects on LDL receptor expression.93 The other major feature of type 2 diabetes, hyperglycemia, may promote plaque instability by enhancing the inflammatory response in macrophages through effects on plasma triglyceride-rich lipoproteins and free fatty acids.94 Hyperglycemia may also cause endothelial cell abnormalities, including oxidative stress and RAGE-induced inflammation, that promote the earlier stages of atherogenesis.95, 96 Interestingly, there are recent data suggesting that hyperglycemia may exert some of its pro-atherogenic effects in endothelial cells through FoxO1 and also through the induction of ER stress.97, 98 These hyperglycemia-endothelial cell studies, together with the insulin resistance-macrophage studies described in this review, raise the interesting possibility that hyperglycemia may affect mostly the earlier stages of atherogenesis, while insulin resistance has its greatest effect on promoting advanced plaque progression. In this context, a recent analysis of the Veterans Affairs Diabetes Trial found that intensive glucose lowering reduced cardiovascular events in diabetics with a coronary artery calcium store < 100 (multivariable hazard ratio [HR] = 0.08, p=0.03), but not in those with a calcium score >100 (HR = 0.74, p=0.21).99 Smooth muscle cells, a key cell type in the generation of the “protective” fibrous cap in advanced lesions, and platelets, the final effector of acute vascular occlusion, may be affected by insulin resistance, hyperglycemia, or fatty acid abnormalities, which provide additional opportunities for investigation.61 Continued progress in these areas will provide a more complete understanding of how multiple features of diabetes promotes heart disease.
The ultimate goal of these studies is to complement our current efforts at identifying and treating systemic risk factors that promote cardiovascular disease in diabetics. Despite the relative success of this strategy, risk is still very high,100, and the tremendous scale of this epidemic is such that overall risk will still be high even if compliance is improved and the experimental modalities prove useful. Further understanding of the specific mechanisms of increased vascular disease in diabetics, particularly at the molecular level in arterial wall cells, may be a promising approach for further eradication in the future—and one that should be additive or even synergistic with reduction of lipid and other systemic risk factors. One approach is to increase insulin sensitivity in diabetic macrophages, such has been demonstrated recently using a PPARγ activator in vivo68 and 1,25(OH)2 vitamin D in vitro.101 Another approach is to develop agents to prevent ER stress or downstream pro-apoptotic processes in macrophages by pharmacologic means, e.g., through the use of chemical chaperones102 or inhibitors of the calcium-mediated pro-apoptotic pathway.103, 104 Moreover, in view of the importance of defective efferocytosis in the generation of plaque necrosis and the ob/ob efferocytosis study described above, experimental therapeutic modalities designed to enhance efferocytosis58, 105 may be particularly useful in diabetics. Delivery of such drugs to plaques might be facilitated by specific vehicles targeted to plaques,106 while clinical assessment in phase 2 and phase 3 studies could be assisted by imaging techniques such as carotid MRI, that have the capacity to measure important plaque features such as necrotic core area and cap thickness.107 Studies in these areas occurring in parallel with ongoing efforts at systemic risk reduction offer the best chance to curb the growing epidemic of diabetes-associated atherothrombotic vascular disease.
The authors gratefully acknowledge the outstanding members of our laboratories who contributed to the studies described herein, including Seongah Han, Chien-Ping Liang, Takafumi Senokuchi, Suzhao Li, Tracie Seimon, Gang Li, Jenelle Timmins, Lale Ozcan, Edward Thorp, Rebecca Haeusler, Jun Tanaka, and Li Qiang.
Sources of Funding
This work was supported by National Institutes of Health Grants HL087123, HL075662, and HL054591 and US Army Medical Research and Materiel Command (USAMRMC) grant W81XWH-06-1-0212.