Inhibition of PFKFB3 Hampers the Progression of Atherosclerosis and Promotes Plaque Stability.

AIMS: 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB)3-mediated glycolysis is pivotal in driving macrophage- and endothelial cell activation and thereby inflammation. Once activated, these cells play a crucial role in the progression of atherosclerosis. Here, we analyzed the expression of PFKFB3 in human atherosclerotic lesions and investigated the therapeutic potential of pharmacological inhibition of PFKFB3 in experimental atherosclerosis by using the glycolytic inhibitor PFK158. METHODS AND RESULTS: PFKFB3 expression was higher in vulnerable human atheromatous carotid plaques when compared to stable fibrous plaques and predominantly expressed in plaque macrophages and endothelial cells. Analysis of advanced plaques of human coronary arteries revealed a positive correlation of PFKFB3 expression with necrotic core area. To further investigate the role of PFKFB3 in atherosclerotic disease progression, we treated 6-8 weeks old male Ldlr -/- mice. These mice were fed a high cholesterol diet for 13 weeks, of which they were treated for 5 weeks with the glycolytic inhibitor PFK158 to block PFKFB3 activity. The incidence of fibrous cap atheroma (advanced plaques) was reduced in PFK158-treated mice. Plaque phenotype altered markedly as both necrotic core area and intraplaque apoptosis decreased. This coincided with thickening of the fibrous cap and increased plaque stability after PFK158 treatment. Concomitantly, we observed a decrease in glycolysis in peripheral blood mononuclear cells compared to the untreated group, which alludes that changes in the intracellular metabolism of monocyte and macrophages is advantageous for plaque stabilization. CONCLUSION: High PFKFB3 expression is associated with vulnerable atheromatous human carotid and coronary plaques. In mice, high PFKFB3 expression is also associated with a vulnerable plaque phenotype, whereas inhibition of PFKFB3 activity leads to plaque stabilization. This data implies that inhibition of inducible glycolysis may reduce inflammation, which has the ability to subsequently attenuate atherogenesis.

Elevated Lp(a) (Lipoprotein[a]) Levels Increase Risk of 30-Day Major Adverse Cardiovascular Events in Patients Following Carotid Endarterectomy.

BACKGROUND AND PURPOSE: General population studies have shown that elevated Lp(a) (lipoprotein[a]) levels are an emerging risk factor for cardiovascular disease and subsequent cardiovascular events. The role of Lp(a) for the risk of secondary MACE in patients undergoing carotid endarterectomy (CEA) is unknown. Our objective is to assess the association of elevated Lp(a) levels with the risk of secondary MACE in patients undergoing CEA. METHODS: Lp(a) concentrations were determined in preoperative blood samples of 944 consecutive patients with CEA included in the Athero-Express Biobank Study. During 3-year follow-up, major adverse cardiovascular events (MACE), consisting of myocardial infarction, stroke, and cardiovascular death, were documented. RESULTS: After 3 years follow-up, Kaplan-Meier cumulative event rates for MACE were 15.4% in patients with high Lp(a) levels (>137 nmol/L; >80th cohort percentile) and 10.2% in patients with low Lp(a) levels (≤137 nmol/L; ≤80th cohort percentile; log-rank test: P=0.047). Cox regression analyses adjusted for conventional cardiovascular risk factors revealed a significant association between high Lp(a) levels and 3-year MACE with an adjusted hazard ratio of 1.69 (95% CI, 1.07-2.66). One-third of MACE occurred within 30 days after CEA, with an adjusted hazard ratio for the 30-day risk of MACE of 2.05 (95% CI, 1.01-4.17). Kaplan-Meier curves from time point 30 days to 3 years onward revealed no significant association between high Lp(a) levels and MACE. Lp(a) levels were not associated with histological carotid plaque characteristics. CONCLUSIONS: High Lp(a) levels (>137 nmol/L; >80th cohort percentile) are associated with an increased risk of 30-day MACE after CEA. This identifies elevated Lp(a) levels as a new potential risk factor for secondary cardiovascular events in patients after carotid surgery. Future studies are required to investigate whether Lp(a) levels might be useful in guiding treatment algorithms for carotid intervention.

Surmounting the endothelial barrier for delivery of drugs and imaging tracers.

The endothelium is crucial for maintaining vascular homeostasis and functions as a barrier between blood components and tissue. In atherosclerosis, this barrier function is impaired, which is characterized by secretion of chemoattractants and cytokines, upregulation of adhesion molecules and increased vascular permeability. This facilitates enhanced leukocyte migration through the vessel wall. Fortunately, we can utilize these features to our advantage by using nanomedicine to deliver drugs and imaging tracers into the interstitial space. This provides us with targeted, local delivery of therapeutic agents, which enhances the specificity and efficacy of these agents and thus, could be used to inhibit disease progression. Additionally, delivery of imaging tracers in the interstitial space will give us insight into the vulnerability of atherosclerotic plaques by targeting resident macrophages and activated endothelial cells, providing pivotal information that is currently lacking in the clinic. In this review, we discuss how the endothelial barrier is affected during atherosclerosis and how to surmount this barrier for successful delivery of nanomedicine carrying drugs and imaging tracers to both the endothelium and macrophages.

Short-term regulation of hematopoiesis by lipoprotein(a) results in the production of pro-inflammatory monocytes.

BACKGROUND: Lipoproteins are important regulators of hematopoietic stem and progenitor cell (HSPC) biology, predominantly affecting myelopoiesis. Since myeloid cells, including monocytes and macrophages, promote the inflammatory response that propagates atherosclerosis, it is of interest whether the atherogenic low-density lipoprotein (LDL)-like particle lipoprotein(a) [Lp(a)] contributes to atherogenesis via stimulating myelopoiesis. METHODS & RESULTS: To assess the effects of Lp(a)-priming on long-term HSPC behavior we transplanted BM of Lp(a) transgenic mice, that had been exposed to elevated levels of Lp(a), into lethally-irradiated C57Bl6 mice and hematopoietic reconstitution was analyzed. No differences in HSPC populations or circulating myeloid cells were detected ten weeks after transplantation. Likewise, in vitro stimulation of C57Bl6 BM cells for 24 h with Lp(a) did not affect colony formation, total cell numbers or myeloid populations 7 days later. To assess the effects of elevated levels of Lp(a) on myelopoiesis, C57Bl6 bone marrow (BM) cells were stimulated with lp(a) for 24 h, and a marked increase in granulocyte-monocyte progenitors, pro-inflammatory Ly6high monocytes and macrophages was observed. Seven days of continuous exposure to Lp(a) increased colony formation and enhanced the formation of pro-inflammatory monocytes and macrophages. Antibody-mediated neutralization of oxidized phospholipids abolished the Lp(a)-induced effects on myelopoiesis. CONCLUSION: Lp(a) enhances the production of inflammatory monocytes at the bone marrow level but does not induce cell-intrinsic long-term priming of HSPCs. Given the short-term and direct nature of this effect, we postulate that Lp(a)-lowering treatment has the capacity to rapidly revert this multi-level inflammatory response.

Potent lipoprotein(a) lowering following apolipoprotein(a) antisense treatment reduces the pro-inflammatory activation of circulating monocytes in patients with elevated lipoprotein(a).

AIMS: Elevated lipoprotein(a) [Lp(a)] is strongly associated with an increased cardiovascular disease (CVD) risk. We previously reported that pro-inflammatory activation of circulating monocytes is a potential mechanism by which Lp(a) mediates CVD. Since potent Lp(a)-lowering therapies are emerging, it is of interest whether patients with elevated Lp(a) experience beneficial anti-inflammatory effects following large reductions in Lp(a). METHODS AND RESULTS: Using transcriptome analysis, we show that circulating monocytes of healthy individuals with elevated Lp(a), as well as CVD patients with increased Lp(a) levels, both have a pro-inflammatory gene expression profile. The effect of Lp(a)-lowering on gene expression and function of monocytes was addressed in two local sub-studies, including 14 CVD patients with elevated Lp(a) who received apolipoprotein(a) [apo(a)] antisense (AKCEA-APO(a)-LRx) (NCT03070782), as well as 18 patients with elevated Lp(a) who received proprotein convertase subtilisin/kexin type 9 antibody (PCSK9ab) treatment (NCT02729025). AKCEA-APO(a)-LRx lowered Lp(a) by 47% and reduced the pro-inflammatory gene expression in monocytes of CVD patients with elevated Lp(a), which coincided with a functional reduction in transendothelial migration capacity of monocytes ex vivo (-17%, P < 0.001). In contrast, PCSK9ab treatment lowered Lp(a) by 16% and did not alter transcriptome nor functional properties of monocytes, despite an additional reduction of 65% in low-density lipoprotein cholesterol (LDL-C). CONCLUSION: Potent Lp(a)-lowering following AKCEA-APO(a)-LRx, but not modest Lp(a)-lowering combined with LDL-C reduction following PCSK9ab treatment, reduced the pro-inflammatory state of circulating monocytes in patients with elevated Lp(a). These ex vivo data support a beneficial effect of large Lp(a) reductions in patients with elevated Lp(a).

Atherogenic Lipoprotein(a) Increases Vascular Glycolysis, Thereby Facilitating Inflammation and Leukocyte Extravasation.

RATIONALE: Patients with elevated levels of lipoprotein(a) [Lp(a)] are hallmarked by increased metabolic activity in the arterial wall on positron emission tomography/computed tomography, indicative of a proinflammatory state. OBJECTIVE: We hypothesized that Lp(a) induces endothelial cell inflammation by rewiring endothelial metabolism. METHODS AND RESULTS: We evaluated the impact of Lp(a) on the endothelium and describe that Lp(a), through its oxidized phospholipid content, activates arterial endothelial cells, facilitating increased transendothelial migration of monocytes. Transcriptome analysis of Lp(a)-stimulated human arterial endothelial cells revealed upregulation of inflammatory pathways comprising monocyte adhesion and migration, coinciding with increased 6-phophofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB)-3-mediated glycolysis. ICAM (intercellular adhesion molecule)-1 and PFKFB3 were also found to be upregulated in carotid plaques of patients with elevated levels of Lp(a). Inhibition of PFKFB3 abolished the inflammatory signature with concomitant attenuation of transendothelial migration. CONCLUSIONS: Collectively, our findings show that Lp(a) activates the endothelium by enhancing PFKFB3-mediated glycolysis, leading to a proadhesive state, which can be reversed by inhibition of glycolysis. These findings pave the way for therapeutic agents targeting metabolism aimed at reducing inflammation in patients with cardiovascular disease.

Lipoprotein(a) as Orchestrator of Calcific Aortic Valve Stenosis.

Aortic valve stenosis (AVS) is the most prevalent valvular heart disease in the Western World with exponentially increased incidence with age. If left untreated, the yearly mortality rates increase up to 25%. Currently, no effective pharmacological interventions have been established to treat or prevent AVS. The only treatment modality so far is surgical or transcatheter aortic valve replacement (AVR). Lipoprotein(a) [Lp(a)] has been implicated as a pivotal player in the pathophysiology of calcification of the valves. Patients with elevated levels of Lp(a) have a higher risk of hospitalization or mortality due to the presence of AVS. Multiple studies indicated Lp(a) as a likely causal and independent risk factor for AVS. This review discusses the most important findings and mechanisms related to Lp(a) and AVS in detail. During the progression of AVS, Lp(a) enters the aortic valve tissue at damaged sites of the valves. Subsequently, autotaxin converts lysophosphatidylcholine in lysophosphatidic acid (LysoPA) which in turn acts as a ligand for the LysoPA receptor. This triggers a nuclear factor-κB cascade leading to increased transcripts of interleukin 6, bone morphogenetic protein 2, and runt-related transcription factor 2. This progresses to the actual calcification of the valves through production of alkaline phosphatase and calcium depositions. Furthermore, this review briefly mentions potentially interesting therapies that may play a role in the treatment or prevention of AVS in the near future.

Oral vancomycin treatment does not alter markers of postprandial inflammation in lean and obese subjects.

Intake of a high-fat meal induces a systemic inflammatory response in the postprandial which is augmented in obese subjects. However, the underlying mechanisms of this response have not been fully elucidated. We aimed to assess the effect of gut microbiota modulation on postprandial inflammatory response in lean and obese subjects. Ten lean and ten obese subjects with metabolic syndrome received oral vancomycin 500 mg four times per day for 7 days. Oral high-fat meal tests (50 g fat/m2 body surface area) were performed before and after vancomycin intervention. Gut microbiota composition, leukocyte counts, plasma lipopolysaccharides (LPS), LPS-binding protein (LBP), IL-6 and MCP-1 concentrations and monocyte CCR2 and cytokine expression were determined before and after the high-fat meal. Oral vancomycin treatment resulted in profound changes in gut microbiota composition and significantly decreased bacterial diversity in both groups (phylogenetic diversity pre- versus post-intervention: lean, 56.9 ± 7.8 vs. 21.4 ± 6.6, P < 0.001; obese, 53.9 ± 7.8 vs. 21.0 ± 5.9, P < 0.001). After intervention, fasting plasma LPS significantly increased (lean, median [IQR] 0.81 [0.63-1.45] EU/mL vs. 2.23 [1.33-3.83] EU/mL, P = 0.017; obese, median [IQR] 0.76 [0.45-1.03] EU/mL vs. 1.44 [1.11-4.24], P = 0.014). However, postprandial increases in leukocytes and plasma LPS were unaffected by vancomycin in both groups. Moreover, we found no changes in plasma LBP, IL-6 and MCP-1 or in monocyte CCR2 expression. Despite major vancomycin-induced disruption of the gut microbiota and increased fasting plasma LPS, the postprandial inflammatory phenotype in lean and obese subjects was unaffected in this study.

N-Glycosylation Defects in Humans Lower Low-Density Lipoprotein Cholesterol Through Increased Low-Density Lipoprotein Receptor Expression.

BACKGROUND: The importance of protein glycosylation in regulating lipid metabolism is becoming increasingly apparent. We set out to further investigate this by studying patients with type I congenital disorders of glycosylation (CDGs) with defective N-glycosylation. METHODS: We studied 29 patients with the 2 most prevalent types of type I CDG, ALG6 (asparagine-linked glycosylation protein 6)-deficiency CDG and PMM2 (phosphomannomutase 2)-deficiency CDG, and 23 first- and second-degree relatives with a heterozygous mutation and measured plasma cholesterol levels. Low-density lipoprotein (LDL) metabolism was studied in 3 cell models-gene silencing in HepG2 cells, patient fibroblasts, and patient hepatocyte-like cells derived from induced pluripotent stem cells-by measuring apolipoprotein B production and secretion, LDL receptor expression and membrane abundance, and LDL particle uptake. Furthermore, SREBP2 (sterol regulatory element-binding protein 2) protein expression and activation and endoplasmic reticulum stress markers were studied. RESULTS: We report hypobetalipoproteinemia (LDL cholesterol [LDL-C] and apolipoprotein B below the fifth percentile) in a large cohort of patients with type I CDG (mean age, 9 years), together with reduced LDL-C and apolipoprotein B in clinically unaffected heterozygous relatives (mean age, 46 years), compared with 2 separate sets of age- and sex-matched control subjects. ALG6 and PMM2 deficiency led to markedly increased LDL uptake as a result of increased cell surface LDL receptor abundance. Mechanistically, this outcome was driven by increased SREBP2 protein expression accompanied by amplified target gene expression, resulting in higher LDL receptor protein levels. Endoplasmic reticulum stress was not found to be a major mediator. CONCLUSIONS: Our study establishes N-glycosylation as an important regulator of LDL metabolism. Given that LDL-C was also reduced in a group of clinically unaffected heterozygotes, we propose that increasing LDL receptor-mediated cholesterol clearance by targeting N-glycosylation in the LDL pathway may represent a novel therapeutic strategy to reduce LDL-C and cardiovascular disease.

The Role of (Modified) Lipoproteins in Vascular Function: A Duet Between Monocytes and the Endothelium.

Over the last century, many studies have demonstrated that low-density lipoprotein (LDL) is a key risk factor of cardiovascular diseases (CVD) related to atherosclerosis. Thus, for these CVD patients, LDL lowering agents are commonly used in the clinic to reduce the risk for CVD. LDL, upon modification, will develop distinct inflammatory and proatherogenic potential, leading to impaired endothelial integrity, influx of immune cells and subsequent increased foam cell formation. LDL can also directly affect peripheral monocyte composition, rendering them in a more favorable position to migrate and accumulate in the subendothelial space. It has become apparent that other lipoprotein particles, such as triglyceride- rich lipoproteins or remnants (TRL) and lipoprotein(a) [Lp(a)] may also impact on atherogenic pathways. Evidence is accumulating that Lp(a) can promote peripheral monocyte activation, eventually leading to increased transmigration through the endothelium. Similarly, remnant cholesterol has been identified to play a key role in endothelial dysfunction and monocyte behavior. In this review, we will discuss recent developments in understanding the role of different lipoproteins in the context of inflammation at both the level of the monocyte and the endothelium.

Metabolism: The road to inflammation and atherosclerosis.

PURPOSE OF REVIEW: Evidence accumulates suggesting that cellular metabolic alterations fuel and dictate the inflammatory state of cells. In this review, we provide an overview of the observed metabolic reprogramming in endothelial cells and innate immune cells upon interaction with modified lipoproteins, thereby contributing to the progression of atherosclerosis. RECENT FINDINGS: Inflammatory endothelial cells at sites exposed to disturbed flow patterns show increased glycolytic activity. Atherogenic factors further enhance these metabolic changes by upregulating the mitochondrial energy production and thereby facilitating increased energy expenditure. Metabolic alterations are pivotal for monocyte and macrophage function as well. Exposure to atherogenic particles such as oxidized phospholipids lead to a regulatory metabolic pro-inflammatory phenotype, mediated via Toll-like receptor (TLR) 2 and the transcription factor erythroid 2-related factor (Nrf) 2. Translational studies highlighted the importance of metabolic alterations, as atherosclerotic plaques in the carotid arteries showed an increased glycolytic signature. SUMMARY: Alterations in cellular metabolism play an important role in controlling and steering the inflammatory state of both endothelial cells and immune cells. Targeting glycolysis may therefore provide an interesting route to attenuate the progression of atherosclerosis.

Nile Red Quantifier: a novel and quantitative tool to study lipid accumulation in patient-derived circulating monocytes using confocal microscopy.

The inflammatory profile of circulating monocytes is an important biomarker for atherosclerotic plaque vulnerability. Recent research revealed that peripheral lipid uptake by monocytes alters their phenotype toward an inflammatory state and this coincides with an increased lipid droplet (LD) content. Determination of lipid content of circulating monocytes is, however, not very well established. Based on Nile Red (NR) neutral LD imaging, using confocal microscopy and computational analysis, we developed NR Quantifier (NRQ), a novel quantification method to assess LD content in monocytes. Circulating monocytes were isolated from blood and used for the NR staining procedure. In monocytes stained with NR, we clearly distinguished, based on 3D imaging, phospholipids and exclusively intracellular neutral lipids. Next, we developed and validated NRQ, a semi-automated quantification program that detects alterations in lipid accumulation. NRQ was able to detect LD alterations after ex vivo exposure of isolated monocytes to freshly isolated LDL in a time- and dose-dependent fashion. Finally, we validated NRQ in patients with familial hypercholesterolemia and obese subjects in pre- and postprandial state. In conclusion, NRQ is a suitable tool to detect even small differences in neutral LD content in circulating monocytes using NR staining.

The maturation of a 'neural-hematopoietic' inflammatory axis in cardiovascular disease.

PURPOSE OF REVIEW: Atherogenesis is the result of a complex interplay between lipids and innate immune cells, which are descendants of upstream progenitors residing in hematopoietic organs. In this review, we will discuss recent advances in the connection between hematopoiesis and atherogenesis. RECENT FINDINGS: The relevance of a neural-hematopoietic axis was recently supported by the demonstration of a correlation between metabolic activity in the amygdala and the bone marrow. During follow-up, both amygdalar and bone marrow activities also predicted cardiovascular risk in patients, lending further support to a connection between neural stress and cardiovascular events mediated via increased hematopoietic activity.In parallel, functional changes in hematopoietic stem cells may also convey cardiovascular risk. In experimental models, knock-out of the ten-eleven translocation 2 (TET2) gene leading to monocyte-macrophage hyperresponsiveness, was associated with accelerated atherogenesis in murine experiments. In humans, whole-exome sequencing reporting on the 'clonal hematopoiesis of indeterminate potential' gene substantiated a two-fold elevated risk for developing coronary heart disease compared with noncarriers. SUMMARY: Recent studies support the relevance of a 'neural-hematopoietic' inflammatory axis and clonal hematopoiesis as drivers of atherogenesis in humans. These data warrant further studies addressing the role of novel 'hematopoietic' targets for the treatment of patients with increased cardiovascular risk.

PCSK9 monoclonal antibodies reverse the pro-inflammatory profile of monocytes in familial hypercholesterolaemia.

AIMS: Migration of monocytes into the arterial wall contributes to arterial inflammation and atherosclerosis progression. Since elevated low-density lipoprotein cholesterol (LDL-C) levels have been associated with activation of plasma monocytes, intensive LDL-C lowering may reverse these pro-inflammatory changes. Using proprotein convertase subtilisin/kexin type 9 (PCSK9) monoclonal antibodies (mAbs) which selectively reduce LDL-C, we studied the impact of LDL-C lowering on monocyte phenotype and function in patients with familial hypercholesterolaemia (FH) not using statins due to statin-associated muscle symptoms. METHODS AND RESULTS: We assessed monocyte phenotype and function using flow cytometry and a trans-endothelial migration assay in FH patients (n = 22: LDL 6.8 ± 1.9 mmol/L) and healthy controls (n = 18, LDL 2.9 ± 0.8 mmol/L). Monocyte chemokine receptor (CCR) 2 expression was approximaterly three-fold higher in FH patients compared with controls. C-C chemokine receptor type 2 (CCR2) expression correlated significantly with plasma LDL-C levels (r = 0.709) and was positively associated with intracellular lipid accumulation. Monocytes from FH patients also displayed enhanced migratory capacity ex vivo. After 24 weeks of PCSK9 mAb treatment (n = 17), plasma LDL-C was reduced by 49%, which coincided with reduced intracellular lipid accumulation and reduced CCR2 expression. Functional relevance was substantiated by the reversal of enhanced migratory capacity of monocytes following PCSK9 mAb therapy. CONCLUSIONS: Monocytes of FH patients have a pro-inflammatory phenotype, which is dampened by LDL-C lowering by PCSK9 mAb therapy. LDL-C lowering was paralleled by reduced intracellular lipid accumulation, suggesting that LDL-C lowering itself is associated with anti-inflammatory effects on circulating monocytes.

Remnant Cholesterol Elicits Arterial Wall Inflammation and a Multilevel Cellular Immune Response in Humans.

OBJECTIVE: Mendelian randomization studies revealed a causal role for remnant cholesterol in cardiovascular disease. Remnant particles accumulate in the arterial wall, potentially propagating local and systemic inflammation. We evaluated the impact of remnant cholesterol on arterial wall inflammation, circulating monocytes, and bone marrow in patients with familial dysbetalipoproteinemia (FD). APPROACH AND RESULTS: Arterial wall inflammation and bone marrow activity were measured using 18F-FDG PET/CT. Monocyte phenotype was assessed with flow cytometry. The correlation between remnant levels and hematopoietic activity was validated in the CGPS (Copenhagen General Population Study). We found a 1.2-fold increase of 18F-FDG uptake in the arterial wall in patients with FD (n=17, age 60±8 years, remnant cholesterol: 3.26 [2.07-5.71]) compared with controls (n=17, age 61±8 years, remnant cholesterol 0.29 [0.27-0.40]; P<0.001). Monocytes from patients with FD showed increased lipid accumulation (lipid-positive monocytes: Patients with FD 92% [86-95], controls 76% [66-81], P=0.001, with an increase in lipid droplets per monocyte), and a higher expression of surface integrins (CD11b, CD11c, and CD18). Patients with FD also exhibited monocytosis and leukocytosis, accompanied by a 1.2-fold increase of 18F-FDG uptake in bone marrow. In addition, we found a strong correlation between remnant levels and leukocyte counts in the CGPS (n=103 953, P for trend 5×10-276). In vitro experiments substantiated that remnant cholesterol accumulates in human hematopoietic stem and progenitor cells coinciding with myeloid skewing. CONCLUSIONS: Patients with FD have increased arterial wall and cellular inflammation. These findings imply an important inflammatory component to the atherogenicity of remnant cholesterol, contributing to the increased cardiovascular disease risk in patients with FD.