Search

The CANTOS (Canakinumab Anti-inflammatory Thrombosis Outcome Study) and colchicine trials suggest an important role of inflammasomes and their major product IL-1β (interleukin 1β) in human atherosclerotic cardiovascular disease. Moreover, studies in mouse models indicate a causal role of inflammasomes and IL-1β in atherosclerosis. However, recent studies have led to a more granular view of the role of inflammasomes in atherosclerosis. Studies in hyperlipidemic mouse models suggest that prominent activation of the NLRP3 inflammasome requires a second hit such as defective cholesterol efflux, defective DNA repair, clonal hematopoiesis or diabetes. Similarly in humans some mutations promoting clonal hematopoiesis increase coronary artery disease risk in part by promoting inflammasome activation. Recent studies in mice and humans point to a wider role of the AIM2 (absent in melanoma 2) inflammasome in promoting cardiovascular disease including in some forms of clonal hematopoiesis and diabetes. These developments suggest a precision medicine approach in which treatments targeting inflammasomes or IL-1β might be best employed in clinical settings involving increased inflammasome activation.

This study provides evidence that hypothalamic gliosis is a key early event through which androgen deficiency in combination with a Western-style diet might lead to cardiometabolic dysregulation in males. Furthermore, this work provides the first evidence in humans of a positive association between hypothalamic gliosis and LDL-cholesterol, advancing our knowledge of CNS influences on CVD risk progression.

Both type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM) are associated with an increased risk of atherosclerotic cardiovascular disease. Research based on human-first or bedside-to-bench approaches has provided new insights into likely mechanisms behind this increased risk. Although both forms of diabetes are associated with hyperglycemia, it is becoming increasingly clear that altered lipoprotein metabolism also plays a critical role in predicting cardiovascular disease risk in people with diabetes. This review examines recent findings indicating that increased levels of circulating remnant lipoproteins could be a missing link between diabetes and cardiovascular disease. Although cardiovascular disease risk associated with diabetes is clearly multifactorial in nature, these findings suggest that we should increase efforts in evaluating whether remnant lipoproteins or the proteins that govern their metabolism are biomarkers of incident cardiovascular disease in people living with diabetes, and whether reducing remnant lipoproteins will prevent the increased cardiovascular disease risk associated with diabetes.

APOA1 and APOB are the structural proteins of high-density lipoprotein and APOB-containing lipoproteins, such as low-density lipoprotein and very low-density lipoprotein, respectively. The 4 smaller APOCs (APOC1, APOC2, APOC3, and APOC4) are exchangeable apolipoproteins; they are readily transferred among high-density lipoproteins and APOB-containing lipoproteins. The APOCs regulate plasma triglyceride and cholesterol levels by modulating substrate availability and activities of enzymes interacting with lipoproteins and by interfering with APOB-containing lipoprotein uptake through hepatic receptors. Of the 4 APOCs, APOC3 has been best studied in relation to diabetes. Elevated serum APOC3 levels predict incident cardiovascular disease and progression of kidney disease in people with type 1 diabetes. Insulin suppresses APOC3 levels, and accordingly, elevated APOC3 levels associated with insulin deficiency and insulin resistance. Mechanistic studies in a mouse model of type 1 diabetes have demonstrated that APOC3 acts in the causal pathway of diabetes-accelerated atherosclerosis. The mechanism is likely due to the ability of APOC3 to slow the clearance of triglyceride-rich lipoproteins and their remnants, thereby causing an increased accumulation of atherogenic lipoprotein remnants in lesions of atherosclerosis. Less is known about the roles of APOC1, APOC2, and APOC4 in diabetes.

Serum apolipoprotein C3 (APOC3) predicts incident cardiovascular events in people with type 1 diabetes and silencing of APOC3 prevents both lesion initiation and advanced lesion necrotic core expansion in a mouse model of type 1 diabetes. APOC3 acts by slowing the clearance of triglyceride-rich lipoproteins, but lipid-free APOC3 has recently been reported to activate an inflammasome pathway in monocytes. We therefore investigated the contribution of hematopoietic inflammasome pathways to atherosclerosis in mouse models of type 1 diabetes. LDL receptor-deficient diabetes mouse models were transplanted with bone marrow from donors deficient in NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3), absent in melanoma 2 (AIM2), or gasdermin D (GSDMD), an inflammasome-induced executor of pyroptotic cell death. Mice with diabetes exhibited inflammasome activation, and consistently, increased plasma interleukin-1b (IL-1b) and IL-18. Hematopoietic deletions of NLRP3, AIM2 or GSDMD caused smaller atherosclerotic lesions in diabetic mice. The increased lesion necrotic core size in diabetic mice was independent of macrophage pyroptosis because hematopoietic GSDMD-deficiency failed to prevent necrotic core expansion in advanced lesions. Our findings demonstrate that AIM2 and NLRP3 inflammasomes contribute to atherogenesis in diabetes and suggest that necrotic core expansion is independent of macrophage pyroptosis.

This manuscript was sent to Joyce Bischoff, Guest Editor, for review by expert referees, editorial decision, and final disposition. Final decisions were approved by Jane Leopold, Guest Editor-in-Chief.

Objective: Niacin therapy fails to reduce cardiovascular events in statin-treated subjects even though it increases plasma HDL-C (HDL [high-density lipoprotein] cholesterol) and decreases LDL-C (LDL [low-density lipoprotein] cholesterol) and triglyceride levels. To investigate potential mechanisms for this lack of cardioprotection, we quantified the HDL proteome of subjects in 2 niacin clinical trials: the CPC study (Carotid Plaque Composition) and the HDL Proteomics substudy of the AIM-HIGH trial (Atherothrombosis Intervention in Metabolic Syndrome with Low HDL/High Triglycerides). Approach and Results: Using targeted proteomics, we quantified levels of 31 HDL proteins from 124 CPC subjects and 120 AIM-HIGH subjects. The samples were obtained at baseline and after 1 year of statin monotherapy or niacin-statin combination therapy. Compared with statin monotherapy, niacin-statin combination therapy did not reduce HDL-associated apolipoproteins APOC1, APOC2, APOC3, and APOC4, despite significantly lowering triglycerides. In contrast, niacin markedly elevated HDL-associated PLTP (phospholipid transfer protein), CLU (clusterin), and HP/HPR (haptoglobin/haptoglobin-related proteins; P ≤0.0001 for each) in both the CPC and AIM-HIGH cohorts. Conclusions: The addition of niacin to statin therapy resulted in elevated levels of multiple HDL proteins linked to increased atherosclerotic risk, which might have compromised the cardioprotective effects associated with higher HDL-C levels and lower levels of LDL-C and triglycerides. Registration: URL: https://www.clinicaltrials.gov ; Unique identifier: NCT00715273; NCT00880178; NCT00120289.

Objective: HDL (high-density lipoprotein) can exert both anti-inflammatory and proinflammatory effects in macrophages due to its ability to induce cholesterol depletion. Because cholesterol depletion also increases sheddase activity of the membrane protease ADAM17 (ADAM metallopeptidase domain 17) in other cells, we examined if ADAM17 plays a role in HDL’s effects on inflammatory processes in macrophages in vitro and in vivo. Approach and Results: Sorted peritoneal macrophages from human APOA1 (apolipoprotein A1) transgenic LDL (low-density lipoprotein) receptor-deficient ( APOA1 Tg ; Ldlr −/− ) mice with and without myeloid cell-targeted ADAM17-deficiency were studied in parallel with wildtype and ADAM17-deficient bone marrow-derived macrophages stimulated with HDL in vitro. HDL increased ADAM17 expression and activity in macrophages. Furthermore, ADAM17-deficient macrophages exhibited reduced expression of ABCA1 (ATP-binding cassette A1) and reduced cholesterol efflux and were cholesterol loaded. This was caused by the absence of shedding of TNFα, a major ADAM17 substrate. Sorted thioglycollate-elicited peritoneal macrophages freshly isolated from APOA1 Tg ; Ldlr −/− mice, which have higher HDL levels than Ldlr −/− controls, showed reduced expression of interferon-inducible genes in response to lipopolysaccharide or interferon β, but exacerbated proinflammatory responses to lipopolysaccharide for Tnfa , Cxcl1 , Ccl2 , and Il1b , phenocopying cells stimulated with HDL in vitro. These effects were all prevented in ADAM17-deficient macrophages and associated with lower concentrations of large HDL particles in APOA1 Tg ; Ldlr −/− mice with myeloid cell-targeted ADAM17-deficiency. Conclusions: The increased cholesterol loading of ADAM17-deficient macrophages prevents both anti-inflammatory and proinflammatory responses of HDL. Our findings demonstrate a novel role for ADAM17 in maintaining cholesterol efflux in macrophages, thereby regulating the immune functions of these cells.

Rationale: Hypertriglyceridemia and low HDL-C (high-density lipoprotein cholesterol), both of which are regulated by LpL (lipoprotein lipase) activity, associate with increased cardiovascular disease. Genetic regulators of LpL actions track with cardiovascular disease risk in humans. Whether this is due to changes in HDL-C or function or circulating triglyceride levels is unresolved. Objective: We created hypertriglyceridemia and HDL-C reduction in atherosclerotic mice to allow the assessment of how hypertriglyceridemia and reduced HDL-C affect regression of atherosclerosis and the phenotype of plaque macrophages. Methods and Results: Atherosclerosis regression was studied in control LpL floxed ( Lpl fl/fl ) mice and tamoxifen-inducible whole-body LpL knockout ( iLpl −/− ) mice with hypertriglyceridemia (≈500 mg/dL) and reduced HDL-C (≈50% reduction). Atherosclerosis regression was studied using 2 models in which advanced plaques resulting from hypercholesterolemia are exposed to normal LDL-C (low-density lipoprotein cholesterol) levels using aortic transplantation or treatments with oligonucleotides. In a subset of mice, we expressed hCETP (human cholesterol ester transfer protein) to humanize the relationship between apoB-lipoproteins and HDL. HDL particle number, cholesterol efflux capacity, and HDL proteome were measured in hypertriglyceridemia mice and humans. Surprisingly, hypertriglyceridemia and reduced HDL-C levels due to loss of LpL did not affect atherosclerosis lesion size or macrophage content (CD68+cells) in either model. Expression of hCETP and further reduction of HDL-C did not alter lesions. Sera from iLpl −/− mice had a decrease in total cholesterol efflux capacity, but not ABCA1 (ATP-binding cassette transporter A1)-mediated cholesterol efflux capacity. Hypertriglyceridemic humans, including those with LpL deficiency, had greater ABCA1-mediated cholesterol efflux capacity and total cholesterol efflux capacity per HDL particle number. Conclusions: Atherosclerosis regression in mice is driven by LDL-C reduction and is not affected by hypertriglyceridemia and plasma HDL-C levels.

In recent years, highly sensitive mass spectrometry–based phosphoproteomic analysis is beginning to be applied to identification of protein kinase substrates altered downstream of increased cAMP. Such studies identify a very large number of phosphorylation sites regulated in response to increased cAMP. Therefore, we now are tasked with the challenge of determining how many of these altered phosphorylation sites are relevant to regulation of function in the cell. This minireview describes the use of phosphoproteomic analysis to monitor the effects of cyclic nucleotide phosphodiesterase (PDE) inhibitors on cAMP-dependent phosphorylation events. More specifically, it describes two examples of this approach carried out in the authors’ laboratories using the selective PDE inhibitor approach. After a short discussion of several likely conclusions suggested by these analyses of cAMP function in steroid hormone–producing cells and also in T-cells, it expands into a discussion about some newer and more speculative interpretations of the data. These include the idea that multiple phosphorylation sites and not a single rate-limiting step likely regulate these and, by analogy, many other cAMP-dependent pathways. In addition, the idea that meaningful regulation requires a high stoichiometry of phosphorylation to be important is discussed and suggested to be untrue in many instances. These new interpretations have important implications for drug design, especially for targeting pathway agonists. SIGNIFICANCE STATEMENT: Phosphoproteomic analyses identify thousands of altered phosphorylation sites upon drug treatment, providing many possible regulatory targets but also highlighting questions about which phosphosites are functionally important. These data imply that multistep processes are regulated by phosphorylation at not one but rather many sites. Most previous studies assumed a single step or very few rate-limiting steps were changed by phosphorylation. This concept should be changed. Previous interpretations also assumed substoichiometric phosphorylation was not of regulatory importance. This assumption also should be changed.

Clinical effort in lipidology focuses largely on mitigating effects of atherosclerosis, a pathologic process localized to the intimal layer of larger arteries. This JCL Roundtable brings together 3 leading researchers to discuss the current understanding of pathogenesis in atherosclerosis. We begin by recognizing that low density lipoprotein concentrations in arterial intima far exceed concentrations in other connective tissues, consistent with the response-to-retention hypothesis of atherogenesis. High density lipoproteins facilitate reverse cholesterol transport and also have antioxidant and anti-inflammatory roles. New evidence points to remnants of triglyceride-rich lipoproteins as promoters of atherogenesis, highlighted by deleterious effects of apolipoprotein C-III. The multifaceted role of inflammation is becoming clearer through discoveries related to leukocyte recruitment, efferocytosis, resolution of inflammation, and crystal formation. MicroRNAs represent a new, complex mode of gene regulation bearing on lipoprotein and inflammation biology. Progress in understanding atherosclerosis portends a future in which residual risk related to obesity, diabetes, and other factors will yield to new targeted therapies.

ADAM17 (A Disintegrin-like And Metalloproteinase 17) is a transmembrane protease that cleaves its substrates at the cell surface. Cell type-specific substrates cleaved by ADAM17, such as TNFα, can function locally and systemically. When fed a diet enriched in saturated fat, sucrose, and added cholesterol (DDC), Ldlr -/- mice develop prediabetes characterized by elevated triglyceride-rich lipoproteins (TRLs), obesity, glucose intolerance and atherosclerosis. Our results demonstrate that when ADAM17 is deleted from hematopoietic cells, DDC-fed Adam17 H-/- Ldlr -/- bone marrow transplant chimeras (n=15-30) are less obese and more insulin sensitive than wildtype Ldlr -/- chimeras. Adam17 H-/- chimeras show decreased plasma cholesterol and triglycerides, primarily due to a reduction in plasma VLDL assayed by FPLC fractionation. Atherosclerotic lesions are significantly (p Adam17 M-/- Ldlr -/- bone marrow transplant chimeras recapitulate the phenotypes of Adam17 H-/- mice, suggesting that the phenotypes are mediated by myeloid cells. Mechanistic studies indicate that the reduced plasma VLDL levels in ADAM17-deficient chimeras are not due to reduced VLDL production or reduced intestinal uptake of triglycerides. Furthermore, analysis of pre- and post-heparin plasma demonstrates that lipoprotein lipase activity is not increased in the ADAM17-/- chimeras. Instead, characterization of the TRLs by proteomics and differential ion mobility analysis revealed that ADAM17-deficient chimeras have reduced levels of 25-30 nm TRL remnant particles (e.g., p=0.027 for 25 nm particles; n=8-10) and reduced levels of APOC1, APOC2 and APOC3 per particle. Together, these results are consistent with the proposal that deletion of ADAM17 in myeloid cells results in increased hepatic clearance of TRLs and their remnants. Our work suggests that myeloid cells play important roles in regulating hepatic TRL remnant clearance and associated atherosclerosis in a model of prediabetes.

Cardiovascular disease (CVD) risk is strongly and inversely associated with plasma levels of high-density lipoprotein cholesterol (HDL-C). Recent clinical studies suggest that the association between HDL-C and CVD risk is indirect and that HDL’s cardioprotective function may be attributed to its ability to promote cholesterol efflux from macrophages. Apolipoprotein A-I (APOA1), the major protein in HDL, serves as a structural scaffold to facilitate protein binding and enzyme activation. Our previous work demonstrated that two or three molecules of APOA1 form antiparallel dimer structures (rotamers) that wrap around discoidal and spherical HDL. Human HDL contains both LL5/5 and LL5/4 rotamers. Because little is known about the impact of different rotamers on HDL function, we generated human APOA1 mutants with single cysteine mutations that produced reconstituted HDL (rHDL) locked in specific rotamer (K133C for LL5/5 and L122C for LL5/4). The LL5/5 rotamer of rHDL demonstrated four-fold higher LCAT activation efficiency than the LL5/4 rotamer. To investigate the roles of APOA1 rotamers in vivo , we used a liver-targeted adeno-associated virus (AAV) to express WT hAPOA1 and the two cysteine mutants in APOA1-deficient ( Apoa1 -/- ) mice. HDL particle concentration (HDL-P) in WT and LL5/5 mice were similar but were 250% higher (n=8; P

Diabetes accelerates atherosclerosis progression in part by increasing lesional necrotic core expansion, but the underlying mechanisms remain elusive. Previous studies have shown that systemic inhibition of the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome prevents atherosclerosis progression in a mouse model of diabetes. Gasdermin D (GSDMD), which acts downstream of inflammasome activation, contributes to pyroptosis and IL-1β and IL-18 release. To explore if hematopoietic inflammasome activation mediates diabetes-related necrotic core expansion, we first generated hematopoietic GSDMD-deficient chimeras in an LDL receptor-deficient ( Ldlr -/- ) virally-induced mouse model of type 1 diabetes (T1D)-accelerated atherosclerosis. Mice with T1D demonstrated elevated plasma glucose, triglycerides, cholesterol, and plasma IL-18 (non-diabetes [ND]=180.4 pg/ml, diabetes [D]=316.6, N=17-18 per group). However, diabetic mice with hematopoietic GSDMD-deficiency had no reduction in plasma IL-18 (D-KO=437.5 pg/ml, p=0.29, N=17). Moreover, we found little evidence of inflammasome activation in peritoneal and splenic macrophages from diabetic mice. A 4-week duration of diabetes significantly increased necrotic core area measured at three different levels in the aortic sinus (p Ldlr -/- hematopoietic NLRP3 and absent in melanoma 2 (AIM2) inflammasome double knockout (DKO) chimeras in a streptozotocin mouse model of diabetes. Mice with diabetes demonstrated hyperglycemia and hypercholesterolemia. However, diabetic hematopoietic DKO chimeras did not exhibit reduced aortic sinus necrotic core area, as compared with diabetic wildtype controls. Our findings suggest that the increased plasma IL-18 levels in diabetic mice are not dependent on hematopoietic GSDMD, and that diabetes-mediated necrotic core expansion is independent of hematopoietic NLRP3 and AIM2 inflammasome and pyroptosis pathways.

We tested whether accumulation of chylomicrons as an addition to LDL would enhance atherosclerosis. To do this, whole body LpL deficiency was combined with LDL receptor (LDLR) deficiency. Induced global LpL deficiency (i Lpl -/- ) was created by tamoxifen injections to activate beta actin-driven cre recombinase in mice. To knock down hepatic LDLR we utilized anti-sense oligonucleotides (ASOs) or a PCSK9 expressing AAV. i Lpl -/- mice on chow diet have elevated plasma TGs, increased VLDL cholesterol, and lower LDL-C compared to control Lpl fl/fl mice. In contrast, when hepatic LDLR was knocked down, the mice displayed elevated LDL-C (~150 mg/dl), similar to the levels seen in control mice with LDLR knockdown. i Lpl -/- mice fed a Western diet for 12 weeks developed severe hypertriglyceridemia (1400-7000 mg/dl) and increased total cholesterol reaching ~1500 mg/dl. LDL-C levels were lower in iLpL -/- mice (160 mg/dl vs 359 mg/dl) and the increase in cholesterol was primarily in chylomicrons compared to the control mice with LDLR knockdown (1064 mg/dl vs 238 mg/dl). i Lpl -/- mice had bigger lesions in the brachiocephalic artery (46000 um 2 vs 13000 um 2 ) but macrophage content was lower than the control LDLR-deficient mice (24% vs 69%). Aortic root lesion size was larger (60000 um 2 vs 26000 um 2 ), due to a greater the percentage of collagen (34% vs 23%), but not macrophages (60% vs 58%) or neutral lipid content (34% vs 35%). The lack of differences in macrophage accumulation and neutral lipid content and the increase in the collagen content indicates an advanced lesion in the iLpl -/- mice. Thus, our data suggest that intact chylomicrons contribute to atherosclerosis.

Despite the benefits of LDL-cholesterol-reducing medications, patients with type 1 diabetes (T1D) continue to have an increased risk of incident CVD. Preclinical and clinical observations indicate that triglyceride-rich lipoproteins (TRLs) and their remnants may contribute significantly to CVD risk in T1D. Recently, we have shown that diabetes increases levels of atherogenic TRL remnants and that improvement of remnant clearance halts atherosclerosis in a mouse model of T1D. Cholesteryl ester transfer protein (CETP), which is normally absent in mice, links metabolism of TRLs to that of HDL by mediating the transfer of cholesteryl ester from HDL to VLDL and other APOB-containing lipoproteins in exchange for triglycerides. We hypothesized that CETP impairs the atheroprotective effects of APOA1 by altering TRL and HDL metabolism in diabetes. To test this hypothesis, we developed a virally-induced T1D mouse model expressing the human APOA1 transgene ( APOA1 Tg , Ldlr –/– ). Cohorts of these mice were injected with a liver-targeted adeno-associated virus to express human CETP, and the effects on lipoproteins, plasma apolipoproteins, HDL particle concentrations (HDL-P), and atherosclerosis in the presence and absence of diabetes were determined. This mouse model exhibits an HDL profile similar to humans, with small, medium and large HDL-P, whereas wildtype ( Ldlr -/- ) littermates have only one sized HDL-P. Expression of the APOA1 transgene significantly suppressed diabetes-accelerated necrotic core expansion and accumulation of APOB and APOE in the aortic sinus (compared to wildtype Ldlr -/- mice), while CETP impaired the protective effect of APOA1 on these parameters in diabetic APOA1 Tg , Ldlr –/– mice (n= 22-30, p < 0.05). Further characterization of plasma lipoproteins and apolipoproteins by targeted mass spectrometry, FPLC and differential ion mobility analysis indicated that CETP leads to smaller TRL remnant particles, increases triglyceride content in HDL, lower levels of plasma APOA1 and reduction in large HDL-P under diabetic conditions (n= 8-10, p < 0.05). Our results are consistent with the proposal that inhibition of CETP might be cardioprotective in the setting of T1D and highlight the close links between TRL and HDL metabolism in diabetes.

Atherosclerotic cardiovascular disease (CVD) is the major cause of death in people with type 1 diabetes mellitus (T1DM). Alterations in the HDL proteome associate with prevalent CVD in T1DM. We therefore determined which proteins carried by HDL are associated with incident CVD in T1DM patients. We conducted a case-cohort study of incident CVD in the prospective Coronary Artery Calcification in Type 1 Diabetes (CACTI) study. We randomly sampled 145 CACTI participants without prevalent CVD, 11 of whom subsequently developed a CVD event as a cohort, plus all additional T1DM participants in CACTI (N=36) who developed an incident CVD event (a total of 47 CVD cases). Using targeted MS/MS, we quantified 51 proteins in HDL isolated from baseline plasma samples. We used Cox proportional regression analysis and a case-cohort design to test associations of HDL proteins with incident CVD (myocardial infarction, coronary artery bypass grafting, angioplasty, or death from coronary heart disease). Only one HDL protein—SFTPB (pulmonary surfactant protein B)—was associated with incident CVD in T1DM in all of the models tested. In a fully adjusted model that controlled for smoking status, lipids and other risk factors, the hazard ratio was 2.17 per SD increase of SFTPB (95% confidence interval, 1.12-4.21, P=0.022). Plasma fractionation and targeted MS/MS quantification demonstrated that nearly 95% of plasma SFTPB is quantitatively bound to HDL. Our results show that elevated levels of SFTPB in HDL are strongly associated with incident CVD in CACTI subjects with T1DM independently of smoking status and a wide range of other confounding factors. Because HDL is the major carrier of SFTPB in plasma, our observations support the proposal that SFTPB carried by HDL is a novel marker of CVD risk in patients with T1DM.

Individuals with diabetes suffer from an elevated risk of coronary artery disease (CAD) events. Increased serum levels of apolipoprotein C3 (APOC3) predict incident CAD in individuals with type 1 diabetes (T1D). APOC3 is a small lipid-binding protein carried by lipoproteins, including VLDL, in circulation. Silencing of APOC3 in a mouse model of T1D-accelerated atherosclerosis has been demonstrated to prevent diabetes-accelerated atherosclerosis, strongly suggesting that APOC3 is a causal mediator. However, the exact mechanism whereby APOC3 promotes atherosclerosis in the setting of T1D is unclear. Mice with T1D demonstrated elevated levels of glucose, triglycerides, APOC3 (non-diabetic [ND] mice=442.6 μg/ml, diabetic [D] mice=755.9 μg/ml, pIl1b mRNA (APOC3=86.7-fold over control), and Nlrp3 mRNA (APOC3=13.8-fold over control), indicating activation of the NLRP3 inflammasome pathway. However, analysis of serum samples from subjects with T1D showed that virtually all APOC3 is bound to lipoproteins (primarily VLDL and HDL). To further confirm the effect of lipidated APOC3, we bound APOC3 to lipid-based small unilamellar vesicles to mimic the physiological state of APOC3 in plasma. The stimulatory effect of APOC3, measured by IL-1β release from mouse monocytes, was lost. Together, our findings suggest that although de-lipidated APOC3 has the ability to induce inflammasome activation in monocytes, virtually no APOC3 circulates in a lipid-free form in subjects with T1D.

Objective: Mice genetically deficient in endothelial nitric oxide synthase (Nos3 −/− ) have fasting hyperinsulinemia and hepatic insulin resistance, indicating the importance of Nos3 (nitric oxide synthase) in maintaining metabolic homeostasis. Although the current paradigm holds that these metabolic effects are derived specifically from the expression of Nos3 in the endothelium, it has been established that bone marrow–derived cells also express Nos3. The aim of this study was to investigate whether bone marrow–derived cell Nos3 is important in maintaining metabolic homeostasis. Approach and Results: To test the hypothesis that bone marrow–derived cell Nos3 contributes to metabolic homeostasis, we generated chimeric male mice deficient or competent for Nos3 expression in circulating blood cells. These mice were placed on a low-fat diet for 5 weeks, a time period which is known to induce hepatic insulin resistance in global Nos3-deficient mice but not in wild-type C57Bl/6 mice. Surprisingly, we found that the absence of Nos3 in the bone marrow–derived component is associated with hepatic insulin resistance and that restoration of Nos3 in the bone marrow–derived component in global Nos3-deficient mice is sufficient to restore hepatic insulin sensitivity. Furthermore, we found that overexpression of Nos3 in bone marrow–derived component in wild-type mice attenuates the development of hepatic insulin resistance during high-fat feeding. Finally, compared with wild-type macrophages, the loss of macrophage Nos3 is associated with increased inflammatory responses to lipopolysaccharides and reduced anti-inflammatory responses to IL-4, a macrophage phenotype associated with the development of hepatic and systemic insulin resistance. Conclusions: These results would suggest that the metabolic and hepatic consequences of high-fat feeding are mediated by loss of Nos3/nitric oxide actions in bone marrow–derived cells, not in endothelial cells.

Chronic inflammation contributes to cardiovascular disease. Increased levels of the inflammatory cytokine, TNF-α, are often present in conditions associated with cardiovascular disease risk, and TNF-α induces a number of pro-atherogenic effects in macrovascular endothelial cells, including expression of adhesion molecules and chemokines, and lipoprotein uptake and transcytosis to the subendothelial tissue. However, little is known about the roles of acyl-CoA synthetases (ACSLs), enzymes that esterify free fatty acids into their acyl-CoA derivatives, or about the effects of TNF-α on ACSLs in endothelial cells. Therefore, we investigated the effects of TNF-α on ACSLs and downstream lipids in cultured human coronary artery endothelial cells and human umbilical vein endothelial cells. We demonstrated that TNF-α induces ACSL1, ACSL3, and ACSL5, but not ACSL4, in both cell types. TNF-α also increased oleoyl-CoA levels, consistent with the increased ACSL3 expression. RNA-sequencing demonstrated that knockdown of ACSL3 had no marked effects on the TNF-α transcriptome. Instead, ACSL3 was required for TNF-α-induced lipid droplet formation in cells exposed to oleic acid. These results demonstrate that increased acyl-CoA synthesis as a result of ACSL3 induction is part of the TNF-α response in human macrovascular endothelial cells.

Objective: HDL (high-density lipoprotein) infusion reduces atherosclerosis in animal models and is being evaluated as a treatment in humans. Studies have shown either anti- or proinflammatory effects of HDL in macrophages, and there is no consensus on the underlying mechanisms. Here, we interrogate the effects of HDL on inflammatory gene expression in macrophages. Approach and Results: We cultured bone marrow–derived macrophages, treated them with reconstituted HDL or HDL isolated from APOA1 Tg ;Ldlr −/− mice, and challenged them with lipopolysaccharide. Transcriptional profiling showed that HDL exerts a broad anti-inflammatory effect on lipopolysaccharide-induced genes and proinflammatory effect in a subset of genes enriched for chemokines. Cholesterol removal by POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine) liposomes or β-methylcyclodextrin mimicked both pro- and anti-inflammatory effects of HDL, whereas cholesterol loading by POPC/cholesterol-liposomes or acetylated LDL (low-density lipoprotein) before HDL attenuated these effects, indicating that these responses are mediated by cholesterol efflux. While early anti-inflammatory effects reflect reduced TLR (Toll-like receptor) 4 levels, late anti-inflammatory effects are due to reduced IFN (interferon) receptor signaling. Proinflammatory effects occur late and represent a modified endoplasmic reticulum stress response, mediated by IRE1a (inositol-requiring enzyme 1a)/ASK1 (apoptosis signal-regulating kinase 1)/p38 MAPK (p38 mitogen-activated protein kinase) signaling, that occurs under conditions of extreme cholesterol depletion. To investigate the effects of HDL on inflammatory gene expression in myeloid cells in atherosclerotic lesions, we injected reconstituted HDL into Apoe −/− or Ldlr −/− mice fed a Western-type diet. Reconstituted HDL infusions produced anti-inflammatory effects in lesion macrophages without any evidence of proinflammatory effects. Conclusions: Reconstituted HDL infusions in hypercholesterolemic atherosclerotic mice produced anti-inflammatory effects in lesion macrophages suggesting a beneficial therapeutic effect of HDL in vivo.

Loss-of-function mutations in the transcription factor CREB3L3 (CREBH) associate with severe hypertriglyceridemia in humans. CREBH is believed to lower plasma triglycerides by augmenting the activity of lipoprotein lipase (LPL). However, by using a mouse model of type 1 diabetes mellitus (T1DM), we found that greater liver expression of active CREBH normalized both elevated plasma triglycerides and cholesterol. Residual triglyceride-rich lipoprotein (TRL) remnants were enriched in apolipoprotein E (APOE) and impoverished in APOC3, an apolipoprotein composition indicative of increased hepatic clearance. The underlying mechanism was independent of LPL, as CREBH reduced both triglycerides and cholesterol in LPL-deficient mice. Instead, APOE was critical for CREBH's ability to lower circulating remnant lipoproteins because it failed to reduce TRL cholesterol in Apoe-/- mice. Importantly, individuals with CREB3L3 loss-of-function mutations exhibited increased levels of remnant lipoproteins that were deprived of APOE. Recent evidence suggests that impaired clearance of TRL remnants promotes cardiovascular disease in patients with T1DM. Consistently, we found that hepatic expression of CREBH prevented the progression of diabetes-accelerated atherosclerosis. Our results support the proposal that CREBH acts through an APOE-dependent pathway to increase hepatic clearance of remnant lipoproteins. They also implicate elevated levels of remnants in the pathogenesis of atherosclerosis in T1DM.

Summary Despite the decades-old knowledge that diabetes mellitus is a major risk factor for cardiovascular disease, the reasons for this association are only partially understood. While this association is true for both type 1 and type 2 diabetes, different pathophysiological processes may be responsible. Lipids and other risk factors are indeed important, whereas the role of glucose is less clear. This lack of clarity stems from clinical trials that do not unambiguously show that intensive glycemic control reduces cardiovascular events. Animal models have provided mechanisms that link diabetes to increased atherosclerosis, and evidence consistent with the importance of factors beyond hyperglycemia has emerged. We review clinical, pathological, and animal studies exploring the pathogenesis of atherosclerosis in humans living with diabetes and in mouse models of diabetes. An increased effort to identify risk factors beyond glucose is now needed to prevent the increased cardiovascular disease risk associated with diabetes.

Vein graft failure remains a common clinical challenge. We applied a systems approach in mouse experiments to discover therapeutic targets for vein graft failure.Global proteomics and high-dimensional clustering on multiple vein graft tissues were used to identify potential pathogenic mechanisms. The PPARs (peroxisome proliferator-activated receptors) pathway served as an example to substantiate our discovery platform. In vivo mouse experiments with macrophage-targeted PPARα small interfering RNA, or the novel, selective activator pemafibrate demonstrate the role of PPARα in the development and inflammation of vein graft lesions. In vitro experiments further included metabolomic profiling, quantitative polymerase chain reaction, flow cytometry, metabolic assays, and single-cell RNA sequencing on primary human and mouse macrophages.We identified changes in the vein graft proteome associated with immune responses, lipid metabolism regulated by the PPARs, fatty acid metabolism, matrix remodeling, and hematopoietic cell mobilization. PPARα agonism by pemafibrate retarded the development and inflammation of vein graft lesions in mice, whereas gene silencing worsened plaque formation. Pemafibrate also suppressed arteriovenous fistula lesion development. Metabolomics/lipidomics, functional metabolic assays, and single-cell analysis of cultured human macrophages revealed that PPARα modulates macrophage glycolysis, citrate metabolism, mitochondrial membrane sphingolipid metabolism, and heterogeneity.This study explored potential drivers of vein graft inflammation and identified PPARα as a novel potential pharmacological treatment for this unmet medical need.

We have shown that elevated HDL cholesterol (HDL-C) levels (≥80 mg/dL) associate with increased CAD risk in women with T1D. Furthermore, HDL-C levels are not necessarily cardioprotective in nondiabetic individuals, suggesting that the cholesterol content of HDL does not accurately assess CAD risk. We thus determined whether two other HDL metrics, HDL particle concentration (HDL-P) and cholesterol efflux capacity (CEC), predicted incident CAD in a large cohort of childhood onset T1D (n=550). At baseline, mean age was 27.8 yrs, T1D duration was 19.6 yrs and 49% were women. HDL-P was assessed by calibrated ion mobility analysis and CEC was quantified with validated assays in cAMP stimulated J774 cells and in ABCA1-expressing BHK cells. CAD was defined as CAD death, myocardial infarction and/or revascularization. During a median follow-up of 26 yrs, we observed 203 incident CAD cases (36.9%). At baseline, incident cases were older, more likely to have smoked, have hypertension, longer duration of T1D, higher non-HDL-C and markers of kidney disease and inflammation. Cases were also more likely to have lower HDL-P concentrations but similar CEC to non-cases. Cox models with backward elimination were constructed for each HDL metric, allowing for potential confounding variables, including HDL-C. In separate multivariable models, higher concentrations of total HDL-P (T-HDL-P, HR=0.88, 95% CI: 0.83-0.92) and 3 major HDL subpopulations - small (S-HDL-P, HR=0.78, 0.67-0.92), medium (M-HDL-P, HR=0.79, 0.71-0.88) and large-sized (L-HDL-P, HR=0.74, 0.61-0.89) particles - associated with a lower CAD incidence whereas HDL-C did not. Higher total CEC in J774 cells also predicted lower CAD risk (HR=0.74, 0.55-0.996) after similar adjustments, but became non-significant after further adjusting for HDL-P. ABCA1-specific CEC did not predict CAD. Our results suggest that increased HDL-P levels are cardioprotective in T1D, in contrast to HDL-C and CEC which did not independently predict CAD. Disclosure T. Costacou: None. T. Vaisar: Consultant; Self; AstraZeneca. K. Bornfeldt: None. T. J. Orchard: None. J. Heinecke: None. Funding National Institutes of Health (HL130153, DK34818)

The increased cardiovascular disease (CVD) risk associated with type 2 diabetes (T2D) is likely due in part to diabetic dyslipidemia—a condition characterized by elevated plasma levels of triglyceride‐rich lipoproteins (TRLs), smaller denser LDL particles, and decreased HDL cholesterol (1,2). Lifestyle modifications and statins are first-line interventions for CVD risk reduction in individuals with T2D, but such individuals remain at considerable risk for cardiovascular events (3). This residual CVD risk appears to be linked to abnormal metabolism of TRLs (3)—chylomicrons, VLDL, and their respective remnant lipoprotein particles (RLPs), many of which are present as intermediate-density lipoproteins (4). Thus, there is a need for developing effective and safe alternative approaches/medications to prevent and reduce diabetic dyslipidemia. In this issue of Diabetes , Roger et al. (5) report a potential new therapeutic strategy to simultaneously reduce LDL and VLDL levels by using a hybrid compound that demonstrates dual inhibition of cannabinoid-1 receptor (CB1R) and inducible nitric oxide synthase (iNOS) in a mouse model. The elevated TRLs in T2D are generally attributed to hepatic overproduction of apolipoprotein B (APOB)-containing VLDL, increased intestinal secretion of APOB48 (a truncated form of APOB100 in chylomicrons and their remnants), and dysregulated clearance of TRLs (1,2,6,7), as shown by Fig. 1. Insulin resistance and the resulting elevated fatty acids are believed to be responsible for the abnormal TRL metabolism in T2D (1,2,7). For example, inhibition of the transcription factor FoxO1 by insulin-induced Akt activation represses expression of microsomal triglyceride transfer …

OBJECTIVE A subset of people with long-standing type 1 diabetes (T1D) appears to be protected from microvascular and macrovascular complications. Previous studies have focused on improved abilities to respond to glucose and its downstream effects as protective mechanisms. It is unclear whether lipoproteins play a role in the vascular health of these people. We therefore determined whether HDL particle concentration, size, function, and/or protein composition associate with protection from vascular complications. RESEARCH DESIGN AND METHODS We studied two independent cross-sectional cohorts with T1D: the T1D Exchange Living Biobank (n = 47) and the Joslin Medalist Study (n = 100). Some of the subjects had vascular complications, whereas others never exhibited vascular complications, despite an average duration of diabetes in the cohorts of 45 years. We assessed HDL particle size and concentration by calibrated ion mobility analysis, the HDL proteome by targeted mass spectrometry, and HDL function ex vivo by quantifying cholesterol efflux capacity and inhibition of monocyte adhesion to endothelial cells. RESULTS In both cohorts, people without vascular complications exhibited significantly higher concentrations of medium-sized HDL particles (M-HDL) independently of total and HDL cholesterol levels. While no consistent differences in HDL functions were observed ex vivo, people without vascular complications had higher levels of HDL-associated paraoxonase 1 (PON1), an enzyme that inhibits atherosclerosis in animal models. CONCLUSIONS Elevated concentrations of M-HDL particles and elevated levels of HDL-associated PON1 may contribute to long-term protection from the vascular complications of diabetes by pathways that are independent of total cholesterol and HDL cholesterol.