Immunoproteasomal Inhibition With ONX-0914 Attenuates Atherosclerosis and Reduces White Adipose Tissue Mass and Metabolic Syndrome in Mice.

BACKGROUND: Atherosclerosis is the major underlying pathology of cardiovascular disease and is driven by dyslipidemia and inflammation. Inhibition of the immunoproteasome, a proteasome variant that is predominantly expressed by immune cells and plays an important role in antigen presentation, has been shown to have immunosuppressive effects. METHODS: We assessed the effect of ONX-0914, an inhibitor of the immunoproteasomal catalytic subunits LMP7 (proteasome subunit ß5i/large multifunctional peptidase 7) and LMP2 (proteasome subunit ß1i/large multifunctional peptidase 2), on atherosclerosis and metabolism in LDLr-/- and APOE*3-Leiden.CETP mice. RESULTS: ONX-0914 treatment significantly reduced atherosclerosis, reduced dendritic cell and macrophage levels and their activation, as well as the levels of antigen-experienced T cells during early plaque formation, and Th1 cells in advanced atherosclerosis in young and aged mice in various immune compartments. Additionally, ONX-0914 treatment led to a strong reduction in white adipose tissue mass and adipocyte progenitors, which coincided with neutrophil and macrophage accumulation in white adipose tissue. ONX-0914 reduced intestinal triglyceride uptake and gastric emptying, likely contributing to the reduction in white adipose tissue mass, as ONX-0914 did not increase energy expenditure or reduce total food intake. Concomitant with the reduction in white adipose tissue mass upon ONX-0914 treatment, we observed improvements in markers of metabolic syndrome, including lowered plasma triglyceride levels, insulin levels, and fasting blood glucose. CONCLUSIONS: We propose that immunoproteasomal inhibition reduces 3 major causes underlying cardiovascular disease, dyslipidemia, metabolic syndrome, and inflammation and is a new target in drug development for atherosclerosis treatment.

Calpain inhibitor prevents atherosclerosis in apolipoprotein E knockout mice by regulating mRNA expression of genes related to cholesterol uptake and efflux.

PURPOSE: We previously reported that a calpain inhibitor (CAI) prevents the development of atherosclerosis in rats. This study aimed to investigate the effects of CAI (1 mg/kg) on atherosclerosis in apolipoprotein E knockout (ApoE KO) mice that were fed a high-fat diet (HFD) and explore the underlying mechanism by analyzing the expression of genes related to the uptake and efflux of cholesterol. METHODS: Atherosclerotic plaques were evaluated. The activity of calpain in the aorta and that of superoxide dismutase (SOD) in the serum were assessed. Lipid profiles in the serum and liver were examined. Serum oxidized low-density lipoprotein (oxLDL), malondialdehyde (MDA), tumor necrosis factor (TNF-α), and interleukin-6 (IL-6) levels were measured. The mRNA expressions of CD68, TNF-α, IL-6, CD36, scavenger receptor (SR-A), peroxisome proliferator-activated receptor gamma (PPAR-γ), liver-x-receptor alpha (LXR-α), and ATP-binding cassette transporter class A1 (ABCA1) in the aorta and peritoneal macrophages were also evaluated. RESULTS: CAI reduced calpain activity in the aorta. CAI also impeded atherosclerotic lesion formation and mRNA expression of CD68 in the aorta and peritoneal macrophages of ApoE KO mice compared with those of mice receiving HFD. However, CAI had no effect on body weight and lipid levels in both the serum and liver. CAI significantly decreased MDA, oxLDL, TNF-α, and IL-6 levels and increased SOD activity in the serum. Moreover, CAI significantly inhibited the mRNA expression of TNF-α and IL-6 genes in the aorta and peritoneal macrophages. In addition, CAI significantly downregulated the mRNA expression of scavenger receptors CD36 and SR-A and upregulated the expression of genes involved in the cholesterol efflux pathway, i.e., PPAR-γ, LXR-α, and ABCA1 in the aorta and peritoneal macrophages. CONCLUSIONS: CAI inhibited the development of atherosclerotic lesions in ApoE KO mice, and this effect might be related to the reduction of oxidative stress and inflammation and the improvement of cholesterol intake and efflux pathways.

Genetic Ablation of Transmembrane Prolyl 4-Hydroxylase Reduces Atherosclerotic Plaques in Mice.

[Figure: see text].

HSP25 Vaccination Attenuates Atherogenesis via Upregulation of LDLR Expression, Lowering of PCSK9 Levels and Curbing of Inflammation.

[Figure: see text].

ATP-based therapy prevents vascular calcification and extends longevity in a mouse model of Hutchinson-Gilford progeria syndrome.

Pyrophosphate deficiency may explain the excessive vascular calcification found in children with Hutchinson-Gilford progeria syndrome (HGPS) and in a mouse model of this disease. The present study found that hydrolysis products of ATP resulted in a <9% yield of pyrophosphate in wild-type blood and aortas, showing that eNTPD activity (ATP → phosphate) was greater than eNPP activity (ATP → pyrophosphate). Moreover, pyrophosphate synthesis from ATP was reduced and pyrophosphate hydrolysis (via TNAP; pyrophosphate → phosphate) was increased in both aortas and blood obtained from mice with HGPS. The reduced production of pyrophosphate, together with the reduction in plasma ATP, resulted in marked reduction of plasma pyrophosphate. The combination of TNAP inhibitor levamisole and eNTPD inhibitor ARL67156 increased the synthesis and reduced the degradation of pyrophosphate in aortas and blood ex vivo, suggesting that these combined inhibitors could represent a therapeutic approach for this devastating progeroid syndrome. Treatment with ATP prevented vascular calcification in HGPS mice but did not extend longevity. By contrast, combined treatment with ATP, levamisole, and ARL67156 prevented vascular calcification and extended longevity by 12% in HGPS mice. These findings suggest a therapeutic approach for children with HGPS.

A new activator of esterase D decreases blood cholesterol level through ESD/JAB1/ABCA1 pathway.

Excessively high cholesterol content in the blood leads to nonalcohol fatty liver disease (NAFLD) and arteriosclerosis. Although there are increasing publications and patent applications to lower blood cholesterol with small chemical molecules, limited effective drugs can be available in clinic. It is necessary to uncover new targets and drugs to alleviate high cholesterol. Esterase D (ESD) is abundant in liver and it remains unknown about its role in cholesterol metabolism. Here we reported that small chemical molecule fluorescigenic pyrazoline derivative 5 (FPD5), a new ESD activator, could effectively reverse high blood cholesterol level and prevent fatty liver and arteriosclerosis in apoE-/- mice fed the high-fat diet. We also observed that FPD5 could reduce oxidized low density lipoprotein (oxLDL)-induced formation of foam cells. To further investigate the mechanism of FPD5 action on blood cholesterol modulation, we found that ESD trigged by FPD5 was aggregated in lysosome and interacted with Jun activation domain binding protein 1 (JAB1). ESD served as a deacetylase to remove Thr89 acetylation of JAB1 and increased its activity; thus, promoting the ATP-binding cassette transporters A1 (ABCA1) to accelerate cholesterol efflux. Our findings demonstrate that FPD5 decreases blood cholesterol level to ameliorate NAFLD and arteriosclerosis through ESD/JAB1/ABCA1 pathway, and ESD functions as a novel nonclassical deacetylase that hydrolyzes serine/threonine acetyl group. Our findings not only highlight that FPD5 may be a pioneer drug for alleviating blood cholesterol but also indicate that ESD is a potential drug target that promotes cholesterol metabolism.

Dihydromyricetin ameliorates vascular calcification in chronic kidney disease by targeting AKT signaling.

Vascular calcification is highly prevalent in chronic kidney disease (CKD), and is characterized by transdifferentiation from contractile vascular smooth muscle cells (VSMCs) into an osteogenic phenotype. However, no effective and therapeutic option to prevent vascular calcification is yet available. Dihydromyricetin (DMY), a bioactive flavonoid isolated from Ampelopsis grossedentata, has been found to inhibit VSMCs proliferation and the injury-induced neointimal formation. However, whether DMY has an effect on osteogenic differentiation of VSMCs and vascular calcification is still unclear. In the present study, we sought to investigate the effect of DMY on vascular calcification in CKD and the underlying mechanism. DMY treatment significantly attenuated calcium/phosphate-induced calcification of rat and human VSMCs in a dose-dependent manner, as shown by Alizarin Red S staining and calcium content assay, associated with down-regulation of osteogenic markers including type I collagen (COL I), Runt-related transcription factor 2 (RUNX2), bone morphogenetic protein 2 (BMP2) and osteocalcin (OCN). These results were further confirmed in aortic rings ex vivo. Moreover, DMY ameliorated vascular calcification in rats with CKD. Additionally, we found that AKT signaling was activated during vascular calcification, whereas significantly inhibited by DMY administration. DMY treatment significantly reversed AKT activator-induced vascular calcification. Furthermore, inhibition of AKT signaling efficiently attenuated calcification, which was similar to that after treatment with DMY alone, and DMY had a better inhibitory effect on calcification as compared with AKT inhibitor. The present study demonstrated that DMY has a potent inhibitory role in vascular calcification partially by inhibiting AKT activation, suggesting that DMY may act as a promising therapeutic candidate for patients suffering from vascular calcification.

Loss of MLKL (Mixed Lineage Kinase Domain-Like Protein) Decreases Necrotic Core but Increases Macrophage Lipid Accumulation in Atherosclerosis.

OBJECTIVES: During the advancement of atherosclerosis, plaque cellularity is governed by the influx of monocyte-derived macrophages and their turnover via apoptotic and nonapoptotic forms of cell death. Previous reports have demonstrated that programmed necrosis, or necroptosis, of plaque macrophages contribute to necrotic core formation. Knockdown or inhibition of the necrosome components RIPK1 (receptor-interacting protein kinase 1) and RIPK3 (receptor-interacting protein kinase 3) slow atherogenesis, and activation of the terminal step of necroptosis, MLKL (mixed lineage kinase domain-like protein), has been demonstrated in advanced human atherosclerotic plaques. However, whether MLKL directly contributes to lesion development and necrotic core formation has not been investigated. Approaches and Results: MLKL expression was knocked down in atherogenic Apoe-knockout mice via the administration of antisense oligonucleotides. During atherogenesis, Mlkl knockdown decreased both programmed cell death and the necrotic core in the plaque. However, total lesion area remained unchanged. Furthermore, treatment with the MLKL antisense oligonucleotide unexpectedly reduced circulating cholesterol levels compared with control antisense oligonucleotide but increased the accumulation of lipids within the plaque and in vitro in macrophage foam cells. MLKL colocalized with the late endosome and multivesicular bodies in peritoneal macrophages incubated with atherogenic lipoproteins. Transfection with MLKL antisense oligonucleotide increased lipid localization with the multivesicular bodies, suggesting that upon Mlkl knockdown, lipid trafficking becomes defective leading to enhanced lipid accumulation in macrophages. CONCLUSIONS: These studies confirm the requirement for MLKL as the executioner of necroptosis, and as such a significant contributor to the necrotic core during atherogenesis. We also identified a previously unknown role for MLKL in regulating endosomal trafficking to facilitate lipid handling in macrophages during atherogenesis.

Inhibition of tissue-nonspecific alkaline phosphatase protects against medial arterial calcification and improves survival probability in the CKD-MBD mouse model.

Medial arterial calcification (MAC) is a major complication of chronic kidney disease (CKD) and an indicator of poor prognosis. Aortic overexpression of tissue-nonspecific alkaline phosphatase (TNAP) accelerates MAC formation. The present study aimed to assess whether a TNAP inhibitor, SBI-425, protects against MAC and improves survival probability in a CKD-mineral and bone disorder (MBD) mouse model. CKD-MBD mice were divided in three groups: vehicle, SBI-10, and SBI-30. They were fed a 0.2% adenine and 0.8% phosphorus diet from 14 to 20 weeks of age to induce CKD, followed by a high-phosphorus (0.2% adenine and 1.8% phosphorus) diet for another 6 weeks. At 14-20 weeks of age, mice in the SBI-10 and SBI-30 groups were given 10 and 30 mg/kg SBI-425 by gavage once a day, respectively, while vehicle-group mice were given distilled water as vehicle. Control mice were fed a standard chow (0.8% phosphorus) between the ages of 8 and 20 weeks. Computed tomography imaging, histology, and aortic tissue calcium content revealed that, compared to vehicle animals, SBI-425 nearly halted the formation of MAC. Mice in the control, SBI-10 and SBI-30 groups exhibited 100% survival, which was significantly better than vehicle-treated mice (57.1%). Aortic mRNA expression of Alpl, encoding TNAP, as well as plasma and aortic tissue TNAP activity, were suppressed by SBI-425 administration, whereas plasma pyrophosphate increased. We conclude that a TNAP inhibitor successfully protected the vasculature from MAC and improved survival rate in a mouse CKD-MBD model, without causing any adverse effects on normal skeletal formation and residual renal function. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Artemisinin Attenuated Atherosclerosis in High-Fat Diet-Fed ApoE-/- Mice by Promoting Macrophage Autophagy Through the AMPK/mTOR/ULK1 Pathway.

Artemisinin is an endoperoxide sesquiterpene lactone from Artemisia annua L with multiple beneficial effects, including anti-inflammation, antioxidant, and vascular protection. Recent studies have found that inflammation along with autophagy deficiency in macrophages is the possible reason for foam cell accumulation in the intima, which leads to atherosclerotic plaque formation. The primary aims of this study were to explore the inhibiting effect of artemisinin on atherosclerosis in high-fat diet-fed ApoE mice and investigate the probable mechanism. Artemisinin (50 and 100 mg/kg, intragastric administration) treatment effectively inhibited foamy macrophage transformation and decreased atherosclerotic plaque formation in atherosclerotic mice. Moreover, artemisinin promoted AMP-activated protein kinase (AMPK) activation, inhibited mammalian target of rapamycin (mTOR) and uncoordinated-51-like kinase 1 (ULK1) phosphorylation, and increased LC-3II accumulation and P62 degradation, thereby enhancing macrophage autophagy. Besides, the inhibiting effect of artemisinin on mTOR and ULK1 phosphorylation could be abrogated by AMPK knockdown, suggesting AMPK was the essential target of artemisinin on promoting macrophage autophagy. Our study indicated that artemisinin alleviated atherosclerotic lesions by accelerating macrophage autophagy through the AMPK/mTOR/ULK1 pathway.

Inhibition of JAK2 Suppresses Myelopoiesis and Atherosclerosis in Apoe-/- Mice.

OBJECTIVE: Increased myelopoiesis has been linked to risk of atherosclerotic cardiovascular disease (ACD). Excessive myelopoiesis can be driven by dyslipidemia and cholesterol accumulation in hematopoietic stem and progenitor cells (HSPC) and may involve increased signaling via Janus kinase 2 (JAK2). Constitutively activating JAK2 mutants drive biased myelopoiesis and promote development of myeloproliferative neoplasms (MPN) or clonal hematopoiesis, conditions associated with increased risk of ACD. JAK2 inhibitors have been developed as a therapy for MPNs. The potential for JAK2 inhibitors to protect against atherosclerosis has not been tested. We therefore assessed the impact of JAK2 inhibition on atherogenesis. METHODS: A selective JAK2 inhibitor TG101348 (fedratinib) or vehicle was given to high-fat high-cholesterol Western diet (WD)-fed wild-type (WT) or Apoe-/- mice. Hematopoietic cell profiles, cell proliferation, and atherosclerosis in WT or Apoe-/- mice were assessed. RESULTS: TG101348 selectively reversed neutrophilia, monocytosis, HSPC, and granulocyte-macrophage progenitor (GMP) expansion in Apoe-/- mice with decreased cellular phosphorylated STAT5 and ERK1/2 and reduced cell cycling and BrdU incorporation in HSPCs, indicating inhibition of JAK/STAT signaling and cell proliferation. Ten-week WD feeding allowed the development of marked aortic atherosclerosis in Apoe-/- mice which was substantially reduced by TG101348. CONCLUSIONS: Selective JAK2 inhibition reduces atherogenesis by suppressing excessive myelopoiesis in hypercholesterolemic Apoe-/- mice. These findings suggest selective JAK2 inhibition as a potential therapeutic approach to decrease ACD risk in patients with increased myelopoiesis and leukocytosis.

Inhibition of JAK2/STAT3/SOCS3 signaling attenuates atherosclerosis in rabbit.

BACKGROUND: Previous studies have indicated that the JAK/STAT signaling pathway is involved in modulating arterial adventitia inflammation response. In this study, we designed experiments to further investigate the effect of JAK2/STAT3/SOCS3 signaling in rabbit atherosclerosis process. METHODS: Atherosclerosis was induced in the abdominal arteries of rabbits by balloon injury of the aorta supplemented by the atherogenic diet. Simultaneously, in the process of atherosclerosis, animals underwent either ruxolitinib treatment or not for 12 weeks. At the end of the experimental period, all rabbits were sacrificed. The plaque areas in abdominal artery, the lipid burden of plaque and the calcium burden of plaque were detected by H&E staining, Oil Red O staining and Alizarin Red staining, respectively. In addition, rabbit plasma lipids and inflammatory cytokines were measured by biochemical test kits or ELISA kits. Finally, the expression and phosphorylation levels of JAK2/STAT3/SOCS3 pathway-related proteins were detected by RT-qPCR, western blot and immunohistochemistry assays. RESULTS: H&E staining and CT scan analysis showed that rabbit atherosclerosis model was constructed successfully. Ruxolitinib, an inhibitor of the Janus kinase 2 (JAK2), substantially reduced the area of atherosclerotic plaques in rabbits treated with high fat diet and balloon injury of the aorta. Moreover, ruxolitinib significantly decreased IL-6, IL-1ß, IFN-γ and TNF-α, but increased IL-10 and IL-17 levels in plasma of atherosclerotic rabbits. Additionally, ruxolitinib reduced plasma TC, TG and LDL-C contents and AIP value, while enhanced HDL-C level in atherosclerotic rabbits. Furthermore, we found that JAK2 and STAT3 phosphorylation were up-regulated in rabbits with atherosclerosis when compared with those of the control group, followed by the expression of SOCS3 was also increased due to the activation of JAK2 and STAT3. Interestingly, ruxolitinib could inactivate JAK2 and STAT3 pathway and decrease SOCS3 expression. CONCLUSION: Taken together, the inhibition of JAK2/STAT3/SOCS3 signaling pathway may be a novel method for the clinical treatment of artery atherosclerosis.

Scutellarin exerts protective effects against atherosclerosis in rats by regulating the Hippo-FOXO3A and PI3K/AKT signaling pathways.

Atherosclerosis (AS), a progressive disorder, is one of the tough challenges in the clinic. Scutellarin, an extract from Herba Erigerontis, is found to have oxygen-free radicals scavenging effects and antioxidant effects. In this study, we aimed to investigate the anti-AS effects of scutellarin is related to controlling the Hippo-FOXO3A and PI3K/AKT signal pathway. To establish an AS model, the rats in the scutellarin and model groups were intraperitoneally injected with vitamin D 3 and then fed a high-fat diet for 12 weeks. In addition, in vitro angiotensin II-induced apoptosis of human aortic endothelial cells (HAECs) were used to establish models. Scutellarin significantly reduced blood lipid levels and increased antioxidase levels in both models. Additionally, scutellarin inhibited reactive oxygen species generation and apoptosis in HAECs. The impaired vascular barrier function was restored by using scutellarin in AS rats and in HAECs cells characterized by inhibiting mammalian sterile-20-like kinases 1 (Mst1) phosphorylation, Yes-associated protein (YAP) phosphorylation, forkhead box O3A (FOXO3A) phosphorylation at serine 207, nuclear translocation of FOXO3A, and upregulating protein expression of AKT and FOXO3A phosphorylation at serine 253. Scutellarin significantly reduced Bcl-2 interacting mediator of cell death (Bim), caspase-3, APO-1, CD95 (Fas), and Bax: Bcl-2-associated X (Bax) levels and activated Bcl-2: B-cell lymphoma-2 (Bcl-2). Scutellarin also significantly inhibited the expression of Mst1, YAP, FOXO3A at the messenger RNA level. When Mst1 was overexpressed or phosphoinositide 3-kinases suppressed, the effects of scutellarin were significantly blocked. In conclusion, the results of the present study suggest that scutellarin exerts protective effects against AS by inhibiting endothelial cell injury and apoptosis by regulating the Hippo-FOXO3A and PI3K/AKT signal pathways.

PINK1/Parkin-mediated mitophagy promotes apelin-13-induced vascular smooth muscle cell proliferation by AMPKα and exacerbates atherosclerotic lesions.

Aberrant proliferation of vascular smooth muscle cells (VSMC) is a critical contributor to the pathogenesis of atherosclerosis (AS). Our previous studies have demonstrated that apelin-13/APJ confers a proliferative response in VSMC, however, its underlying mechanism remains elusive. In this study, we aimed to investigate the role of mitophagy in apelin-13-induced VSMC proliferation and atherosclerotic lesions in apolipoprotein E knockout (ApoE-/-) mice. Apelin-13 enhances human aortic VSMC proliferation and proliferative regulator proliferating cell nuclear antigen expression in dose and time-dependent manner, while is abolished by APJ antagonist F13A. We observe the engulfment of damage mitochondria by autophagosomes (mitophagy) of human aortic VSMC in apelin-13 stimulation. Mechanistically, apelin-13 increases p-AMPKα and promotes mitophagic activity such as the LC3I to LC3II ratio, the increase of Beclin-1 level and the decrease of p62 level. Importantly, the expressions of PINK1, Parkin, VDAC1, and Tom20 are induced by apelin-13. Conversely, blockade of APJ by F13A abolishes these stimulatory effects. Human aortic VSMC transfected with AMPKα, PINK1, or Parkin and subjected to apelin-13 impairs mitophagy and prevents proliferation. Additional, apelin-13 not only increases the expression of Drp1 but also reduces the expressions of Mfn1, Mfn2, and OPA1. Remarkably, the mitochondrial division inhibitor-1(Mdivi-1), the pharmacological inhibition of Drp1, attenuates human aortic VSMC proliferation. Treatment of ApoE-/- mice with apelin-13 accelerates atherosclerotic lesions, increases p-AMPKα and mitophagy in aortic wall in vivo. Finally, PINK1-/- mutant mice with apelin-13 attenuates atherosclerotic lesions along with defective in mitophagy. PINK1/Parkin-mediated mitophagy promotes apelin-13-evoked human aortic VSMC proliferation by activating p-AMPKα and exacerbates the progression of atherosclerotic lesions.

ADCY9 (Adenylate Cyclase Type 9) Inactivation Protects From Atherosclerosis Only in the Absence of CETP (Cholesteryl Ester Transfer Protein).

BACKGROUND: Pharmacogenomic studies have shown that ADCY9 genotype determines the effects of the CETP (cholesteryl ester transfer protein) inhibitor dalcetrapib on cardiovascular events and atherosclerosis imaging. The underlying mechanisms responsible for the interactions between ADCY9 and CETP activity have not yet been determined. METHODS: Adcy9-inactivated ( Adcy9Gt/Gt) and wild-type (WT) mice, that were or not transgenic for the CETP gene (CETPtg Adcy9Gt/Gt and CETPtg Adcy9WT), were submitted to an atherogenic protocol (injection of an AAV8 [adeno-associated virus serotype 8] expressing a PCSK9 [proprotein convertase subtilisin/kexin type 9] gain-of-function variant and 0.75% cholesterol diet for 16 weeks). Atherosclerosis, vasorelaxation, telemetry, and adipose tissue magnetic resonance imaging were evaluated. RESULTS: Adcy9Gt/Gt mice had a 65% reduction in aortic atherosclerosis compared to WT ( P<0.01). CD68 (cluster of differentiation 68)-positive macrophage accumulation and proliferation in plaques were reduced in Adcy9Gt/Gt mice compared to WT animals ( P<0.05 for both). Femoral artery endothelial-dependent vasorelaxation was improved in Adcy9Gt/Gt mice (versus WT, P<0.01). Selective pharmacological blockade showed that the nitric oxide, cyclooxygenase, and endothelial-dependent hyperpolarization pathways were all responsible for the improvement of vasodilatation in Adcy9Gt/Gt ( P<0.01 for all). Aortic endothelium from Adcy9Gt/Gt mice allowed significantly less adhesion of splenocytes compared to WT ( P<0.05). Adcy9Gt/Gt mice gained more weight than WT with the atherogenic diet; this was associated with an increase in whole body adipose tissue volume ( P<0.01 for both). Feed efficiency was increased in Adcy9Gt/Gt compared to WT mice ( P<0.01), which was accompanied by prolonged cardiac RR interval ( P<0.05) and improved nocturnal heart rate variability ( P=0.0572). Adcy9 inactivation-induced effects on atherosclerosis, endothelial function, weight gain, adipose tissue volume, and feed efficiency were lost in CETPtg Adcy9Gt/Gt mice ( P>0.05 versus CETPtg Adcy9WT). CONCLUSIONS: Adcy9 inactivation protects against atherosclerosis, but only in the absence of CETP activity. This atheroprotection may be explained by decreased macrophage accumulation and proliferation in the arterial wall, and improved endothelial function and autonomic tone.

Lysyl oxidase-like 2 depletion is protective in age-associated vascular stiffening.

Vascular stiffening and its sequelae are major causes of morbidity and mortality in the elderly. The increasingly accepted concept of "smooth muscle cell (SMC) stiffness syndrome" along with matrix deposition has emerged in vascular biology to account for the mechanical phenotype of arterial aging, but the molecular targets remain elusive. In this study, using an unbiased proteomic analysis, we identified lysyl oxidase-like 2 (LOXL2) as a critical SMC mediator for age-associated vascular stiffening. We tested the hypothesis that loss of LOXL2 function is protective in aging-associated vascular stiffening. We determined that exogenous and endogenous nitric oxide markedly decreased LOXL2 abundance and activity in the extracellular matrix of isolated SMCs and LOXL2 endothelial cells suppress LOXL2 abundance in the aorta. In a longitudinal study, LOXL2+/- mice were protected from age-associated increase in pulse-wave velocity, an index of vascular stiffening, as occurred in littermate wild-type mice. Using isolated aortic segments, we found that LOXL2 mediates vascular stiffening in aging by promoting SMC stiffness, augmented SMC contractility, and vascular matrix deposition. Together, these studies establish LOXL2 as a nodal point for a new therapeutic approach to treat age-associated vascular stiffening. NEW & NOTEWORTHY Increased central vascular stiffness augments risk of major adverse cardiovascular events. Despite significant advances in understanding the genetic and molecular underpinnings of vascular stiffening, targeted therapy has remained elusive. Here, we show that lysyl oxidase-like 2 (LOXL2) drives vascular stiffening during aging by promoting matrix remodeling and vascular smooth muscle cell stiffening. Reduced LOXL2 expression protects mice from age-associated vascular stiffening and delays the onset of isolated systolic hypertension, a major consequence of stiffening.

Ablation of Myeloid ADK (Adenosine Kinase) Epigenetically Suppresses Atherosclerosis in ApoE-/- (Apolipoprotein E Deficient) Mice.

Objective- Monocyte-derived foam cells are one of the key players in the formation of atherosclerotic plaques. Adenosine receptors and extracellular adenosine have been demonstrated to modulate foam cell formation. ADK (adenosine kinase) is a major enzyme regulating intracellular adenosine levels, but its functional role in myeloid cells remains poorly understood. To enhance intracellular adenosine levels in myeloid cells, ADK was selectively deleted in novel transgenic mice using Cre-LoxP technology, and foam cell formation and the development of atherosclerotic lesions were determined. Approach and Results- ADK was upregulated in macrophages on ox-LDL (oxidized low-density lipoprotein) treatment in vitro and was highly expressed in foam cells in atherosclerotic plaques. Atherosclerotic mice deficient in ADK in myeloid cells were generated by breeding floxed ADK (ADKF/F) mice with LysM-Cre (myeloid-specific Cre recombinase expressing) mice and ApoE-/- (apolipoprotein E deficient) mice. Mice absent ADK in myeloid cells exhibited much smaller atherosclerotic plaques compared with controls. In vitro assays showed that ADK deletion or inhibition resulted in increased intracellular adenosine and reduced DNA methylation of the ABCG1 (ATP-binding cassette transporter G1) gene. Loss of methylation was associated with ABCG1 upregulation, enhanced cholesterol efflux, and eventually decreased foam cell formation. Conclusions- Augmentation of intracellular adenosine levels through ADK knockout in myeloid cells protects ApoE-/- mice against atherosclerosis by reducing foam cell formation via the epigenetic regulation of cholesterol trafficking. ADK inhibition is a promising approach for the treatment of atherosclerotic diseases.

Myeloid HMG-CoA (3-Hydroxy-3-Methylglutaryl-Coenzyme A) Reductase Determines Atherosclerosis by Modulating Migration of Macrophages.

Objective- Inhibition of HMGCR (3-hydroxy-3-methylglutaryl-coenzyme A reductase) is atheroprotective primarily by decreasing plasma LDL (low-density lipoprotein)-cholesterol. However, it is unknown whether inhibition of HMGCR in myeloid cells contributes to this atheroprotection. We sought to determine the role of myeloid HMGCR in the development of atherosclerosis. Approach and Results- We generated mice with genetically reduced Hmgcr in myeloid cells ( Hmgcr m-/m-) using LysM (Cre) and compared various functions of their macrophages to those of Hmgcr fl/fl control mice. We further compared the extent of atherosclerosis in Hmgcr m-/ m- and Hmgcr fl/fl mice in the absence of Ldlr (LDL receptor). Hmgcr m-/ m- macrophages and granulocytes had significantly lower Hmgcr mRNA expression and cholesterol biosynthesis than Hmgcr fl/fl cells. In vitro, Hmgcr m-/ m- monocytes/macrophages had reduced ability to migrate, proliferate, and survive compared with Hmgcr fl/fl monocytes/macrophages. However, there was no difference in ability to adhere, phagocytose, store lipids, or polarize to M1 macrophages between the 2 types of macrophages. The amounts of plasma membrane-associated small GTPase proteins, such as RhoA (RAS homolog family member A), were increased in Hmgcr m-/ m- macrophages. In the setting of Ldlr deficiency, Hmgcr m-/ m- mice developed significantly smaller atherosclerotic lesions than Hmgcr fl/fl mice. However, there were no differences between the 2 types of mice either in plasma lipoprotein profiles or in the numbers of proliferating or apoptotic cells in the lesions in vivo. The in vivo migration of Hmgcr m-/ m- macrophages to the lesions was reduced compared with Hmgcr fl/fl macrophages. Conclusions- Genetic reduction of HMGCR in myeloid cells may exert atheroprotective effects primarily by decreasing the migratory activity of monocytes/macrophages to the lesions.

Inflammasome Activation Aggravates Cutaneous Xanthomatosis and Atherosclerosis in ACAT1 (Acyl-CoA Cholesterol Acyltransferase 1) Deficiency in Bone Marrow.

Objective- ACAT1 (Acyl-CoA cholesterol acyltransferase 1) esterifies cellular free cholesterol, thereby converting macrophages to cholesteryl ester-laden foam cells in atherosclerotic lesions and cutaneous xanthoma. Paradoxically, however, loss of ACAT1 in bone marrow causes the aggravation of atherosclerosis and the development of severe cutaneous xanthoma in hyperlipidemic mice. Recently, it has been reported that cholesterol crystals activate NLRP3 (NACHT, LRR [leucine-rich repeats], and PYD [pyrin domain] domain-containing protein 3) inflammasomes, thereby contributing to the development of atherosclerosis. The present study aimed to clarify the role of NLRP3 inflammasomes in the worsening of atherosclerosis and cutaneous xanthoma induced by ACAT1 deficiency. Approach and Results- Ldlr-null mice were transplanted with bone marrow from WT (wild type) mice and mice lacking ACAT1, NLRP3, or both. After the 4 types of mice were fed high-cholesterol diets, we compared their atherosclerosis and skin lesions. The mice transplanted with Acat1-null bone marrow developed severe cutaneous xanthoma, which was filled with numerous macrophages and cholesterol clefts and had markedly increased expression of inflammatory cytokines, and increased atherosclerosis. Loss of NLRP3 completely reversed the cutaneous xanthoma, whereas it improved the atherosclerosis only partially. Acat1-null peritoneal macrophages showed enhanced expression of CHOP (C/EBP [CCAAT/enhancer binding protein] homologous protein) and TNF-α (tumor necrosis factor-α) but no evidence of inflammasome activation, after treatment with acetylated LDL (low-density lipoprotein). Conclusions- Elimination of ACAT1 in bone marrow-derived cells aggravates cutaneous xanthoma and atherosclerosis. The development of cutaneous xanthoma is induced mainly via the NLRP3 inflammasome activation.

Lipoprotein Lipase Deficiency Impairs Bone Marrow Myelopoiesis and Reduces Circulating Monocyte Levels.

OBJECTIVE: Tissue macrophages induce and perpetuate proinflammatory responses, thereby promoting metabolic and cardiovascular disease. Lipoprotein lipase (LpL), the rate-limiting enzyme in blood triglyceride catabolism, is expressed by macrophages in atherosclerotic plaques. We questioned whether LpL, which is also expressed in the bone marrow (BM), affects circulating white blood cells and BM proliferation and modulates macrophage retention within the artery. APPROACH AND RESULTS: We characterized blood and tissue leukocytes and inflammatory molecules in transgenic LpL knockout mice rescued from lethal hypertriglyceridemia within 18 hours of life by muscle-specific LpL expression (MCKL0 mice). LpL-deficient mice had ≈40% reduction in blood white blood cell, neutrophils, and total and inflammatory monocytes (Ly6C/Ghi). LpL deficiency also significantly decreased expression of BM macrophage-associated markers (F4/80 and TNF-α [tumor necrosis factor α]), master transcription factors (PU.1 and C/EBPα), and colony-stimulating factors (CSFs) and their receptors, which are required for monocyte and monocyte precursor proliferation and differentiation. As a result, differentiation of macrophages from BM-derived monocyte progenitors and monocytes was decreased in MCKL0 mice. Furthermore, although LpL deficiency was associated with reduced BM uptake and accumulation of triglyceride-rich particles and macrophage CSF-macrophage CSF receptor binding, triglyceride lipolysis products (eg, linoleic acid) stimulated expression of macrophage CSF and macrophage CSF receptor in BM-derived macrophage precursor cells. Arterial macrophage numbers decreased after heparin-mediated LpL cell dissociation and by genetic knockout of arterial LpL. Reconstitution of LpL-expressing BM replenished aortic macrophage density. CONCLUSIONS: LpL regulates peripheral leukocyte levels and affects BM monocyte progenitor differentiation and aortic macrophage accumulation.