Blood flow patterns regulate PCSK9 secretion via MyD88-mediated pro-inflammatory cytokines.

AIMS: Blood flow patterns play an important role in the localization of atherosclerosis in the sense that low-flow state is pro-atherogenic, and helical flow is protective against atherosclerosis. Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates cholesterol metabolism via low-density lipoprotein receptor (LDLr) degradation and is highly expressed in the atherosclerotic tissues. This study was designed to investigate the role of different blood flow patterns in the regulation of PCSK9 expression. METHODS AND RESULTS: We designed an experimental model guider to generate stable helical flow. Our data showed that compared with normal flow, low-flow state induces whereas helical flow inhibits PCSK9 expression in the rabbit thoracic aorta in an inflammatory state. Our data also identified that TLR4-MyD88-NF-κB signalling plays an important role in PCSK9 expression. On the other hand, TRIF pathway had almost no effect. Further studies showed that the signals downstream of NF-κB, such as pro-inflammatory cytokines (IL-1ß, IL-18, MCP-1, IL-6, TNF-α, IL-12, IFNγ, and GM-CSF) directly influence PCSK9 expression. Interestingly, high fat diet further enhanced PCSK9 expression in an inflammatory milieu. CONCLUSIONS: These observations suggest a link between abnormal flow patterns and PCSK9 expression in inflammatory states, which may qualify helical flow and pro-inflammatory cytokines as potential targets to treat PCSK9-related cardiovascular diseases.

USP20 (Ubiquitin-Specific Protease 20) Inhibits TNF (Tumor Necrosis Factor)-Triggered Smooth Muscle Cell Inflammation and Attenuates Atherosclerosis.

Objective- Signaling that activates NFκB (nuclear factor κB) in smooth muscle cells (SMCs) is integral to atherosclerosis and involves reversible ubiquitination that activates proteins downstream of proatherogenic receptors. Deubiquitination of these proteins is mediated by USP20 (ubiquitin-specific protease 20), among other deubiquitinases. We sought to determine whether USP20 activity in SMCs decreases atherosclerosis. Approach and Results- To address this question, we used male Ldlr-/- mice without (control) or with SMC-specific expression of murine USP20 (SMC-USP20-transgenic) or its dominant-negative (DN; C154S/H643Q) mutant (SMC-DN-USP20-transgenic). Before the appearance of intimal macrophages, NFκB activation in aortic medial SMCs was greater in SMC-DN-USP20-transgenic than in control mice. After 16 weeks on a Western diet, SMC-DN-USP20-transgenic mice had 46% greater brachiocephalic artery atheroma area than control mice. Congruently, aortic atherosclerosis assessed en face was 21% greater than control in SMC-DN-USP20-transgenic mice and 13% less than control in SMC-USP20-transgenic mice. In response to TNF (tumor necrosis factor), SMCs from SMC-DN-USP20-transgenic mice showed ≈3-fold greater NFκB activation than control SMCs. Silencing USP20 in SMCs with siRNA (small interfering RNA) augmented NFκB activation by ≈50% in response to either TNF or IL-1ß (interleukin-1ß). Coimmunoprecipitation experiments revealed that USP20 associates with several components of the TNFR1 (TNF receptor-1) signaling pathway, including RIPK1 (receptor-interacting protein kinase 1), a critical checkpoint in TNF-induced NFκB activation and inflammation. TNF evoked ≈2-fold more RIPK1 ubiquitination in SMC-DN-USP20-transgenic than in control SMCs, and RIPK1 was deubiquitinated by purified USP20 in vitro. Conclusions- USP20 attenuates TNF- and IL-1ß-evoked atherogenic signaling in SMCs, by deubiquitinating RIPK1, among other signaling intermediates.

Relationship between inflammation and progression of an abdominal aortic aneurysm in a rabbit model based on 18F-FDG PET/CT imaging.

OBJECTIVE: To explore the relationship between abdominal aortic aneurysm development and inflammation in the rabbit through the establishment of a rabbit infrarenal abdominal aortic aneurysm model and the use of 18F-FDG PET/CT imaging. METHODS: Twenty male New Zealand rabbits were administered an elastase intracavity perfusion to induce an infrarenal abdominal aortic aneurysm model. Prior to surgery, the rabbits underwent abdominal aorta ultrasonic testing and blood collection from the ear veins. Of the original 20 rabbits, 10 rabbits were euthanized two weeks after the operation following ultrasonic testing, PET/CT scanning and blood collection, and their arterial tissue samples were prepared for pathological and immunohistochemical staining. The remaining 10 rabbits were euthanized four weeks after the operation following ultrasonic testing, PET/CT scanning and blood collection, and the arterial tissue samples were prepared for pathological and immunohistochemical staining. RESULTS: Compared with the preoperative measurement, the maximum growth rate of the aneurysm diameter is 89.21 ± 0.02% (the absolute increase in diameter is 2.040 ± 0.376 mm) two weeks after the operation. Compared with the two-week postoperative value, the maximum growth rate of the aneurysm diameter is 15.8 ± 0.01% (the absolute increase in diameter is 0.684 ± 0.115 mm) four weeks after the operation. Compared with the preoperative values, the blood MMP-2 and MMP-9 levels significantly increase two weeks after surgery, P < 0.05. Compared with the two-week postoperative values, the blood MMP-2 and MMP-9 levels significantly decrease after four weeks post-surgery, P < 0.05. At two weeks after the operation, the SUVmax and the TBR of the 18F-FDG PET/CT of the AAA wall are 0.90 ± 0.03 and 1.19 ± 0.09, respectively. At four weeks after the operation, the SUVmax and the TBR of the 18F-FDG PET/CT of the AAA wall are 0.35 ± 0.05 and 1.15 ± 0.12, respectively. Compared with two weeks after the operation, the SUVmax significantly decreases at four weeks after the operation, P < 0.05. Compared with two weeks after the operation, there is no significant difference in the TBR at four weeks after the operation, P > 0.05. Immunohistochemical staining shows that the CD68-positive cell rate at four weeks after the operation significantly decreases ( P < 0.05) compared with the CD68-positive cell rate at two weeks after the operation. CONCLUSION: In the early stages of abdominal aortic aneurysm development, the inflammatory response of the arterial wall is significant, the local metabolic activity is strengthened, the SUVmax value of 18F-FDG is high, and the abdominal aortic aneurysm diameter experiences rapid growth. In the later stages of abdominal aortic aneurysm development, the diameter continues to increase; however, there are decreases in the wall inflammatory response, the local metabolic activity, and the SUVmax value of 18F-FDG. Thus, inflammation plays an important role in the early development of abdominal aortic aneurysm.

Differential effects of NOX4 and NOX1 on immune cell-mediated inflammation in the aortic sinus of diabetic ApoE-/- mice.

Oxidative stress and inflammation are central mediators of atherosclerosis particularly in the context of diabetes. The potential interactions between the major producers of vascular reactive oxygen species (ROS), NADPH oxidase (NOX) enzymes and immune-inflammatory processes remain to be fully elucidated. In the present study we investigated the roles of the NADPH oxidase subunit isoforms, NOX4 and NOX1, in immune cell activation and recruitment to the aortic sinus atherosclerotic plaque in diabetic ApoE(-/-) mice. Plaque area analysis showed that NOX4- and NOX1-derived ROS contribute to atherosclerosis in the aortic sinus following 10 weeks of diabetes. Immunohistochemical staining of the plaques revealed that NOX4-derived ROS regulate T-cell recruitment. In addition, NOX4-deficient mice showed a reduction in activated CD4(+) T-cells in the draining lymph nodes of the aortic sinus coupled with reduced pro-inflammatory gene expression in the aortic sinus. Conversely, NOX1-derived ROS appeared to play a more important role in macrophage accumulation. These findings demonstrate distinct roles for NOX4 and NOX1 in immune-inflammatory responses that drive atherosclerosis in the aortic sinus of diabetic mice.

Trimethylamine N-Oxide Promotes Vascular Inflammation Through Signaling of Mitogen-Activated Protein Kinase and Nuclear Factor-κB.

BACKGROUND: The choline-derived metabolite trimethylamine N-oxide (TMAO) has been demonstrated to contribute to atherosclerosis and is associated with coronary artery disease risk. METHODS AND RESULTS: We explored the impact of TMAO on endothelial and smooth muscle cell function in vivo, focusing on disease-relevant outcomes for atherogenesis. Initially, we observed that aortas of LDLR(-/-) mice fed a choline diet showed elevated inflammatory gene expression compared with controls. Acute TMAO injection at physiological levels was sufficient to induce the same inflammatory markers and activate the well-known mitogen-activated protein kinase, extracellular signal-related kinase, and nuclear factor-κB signaling cascade. These observations were recapitulated in primary human aortic endothelial cells and vascular smooth muscle cells. We also found that TMAO promotes recruitment of activated leukocytes to endothelial cells. Through pharmacological inhibition, we further showed that activation of nuclear factor-κB signaling was necessary for TMAO to induce inflammatory gene expression in both of these relevant cell types as well as endothelial cell adhesion of leukocytes. CONCLUSIONS: Our results suggest a likely contributory mechanism for TMAO-dependent enhancement in atherosclerosis and cardiovascular risks.

A phase 2 randomized, double-blind, placebo-controlled study of the effect of VIA-2291, a 5-lipoxygenase inhibitor, on vascular inflammation in patients after an acute coronary syndrome.

OBJECTIVE: Arachidonate 5-lipoxygenase (5-LO) is a key enzyme in the synthesis of leukotrienes. VIA-2291 is a potent 5-LO inhibitor, which has been shown to reduce hsCRP and noncalcified coronary plaque volume following an acute coronary syndrome (ACS). We aim to evaluate the effect of VIA-2291 on vascular inflammation compared to placebo using FDG-PET. METHODS: A Phase II, randomized, double-blind, parallel-group study was conducted in 52 patients with recent ACS assigned 1:1 to either 100 mg VIA-2291 or placebo for 24 weeks. The primary outcome was the effect of VIA-2291 relative to placebo on arterial inflammation detected by (18)fluorodeoxyglucose positron emission tomography (FDG-PET) within the index vessel after 24 weeks of daily treatment, compared to baseline. RESULTS: VIA-2291 was relatively well tolerated and was associated with a significant inhibition of the potent chemo-attractant LTB4, with a mean inhibition of activity of 92.8% (p<0.0001) at 6 weeks in the VIA-2291 group, without further significant change in inhibition at 24 weeks. However, for VIA-2291 was not associated with significant difference in inflammation (target-to-background ratio) compared to placebo at 24 weeks or 6 weeks of treatment. Further, VIA-2291 was not associated with a significant reduction in hsCRP from baseline after either 6 or 24 weeks of treatment. CONCLUSIONS: VIA-2291 is well-tolerated and effectively reduces leukotriene production. However, inhibition of 5-LO with VIA-2291 is not associated with significant reductions in vascular inflammation (by FDG-PET) or in blood inflammatory markers. Accordingly, this study does not provide evidence to support a significant anti-inflammatory effect of VIA-2291 in patients with recent ACS.

Suppression of abdominal aortic aneurysm formation by inhibition of prolyl hydroxylase domain protein through attenuation of inflammation and extracellular matrix disruption.

In the present study we sought to determine the effect of CoCl2, an inhibitor of PHD (prolyl hydroxylase domain protein), on the development of AAA (abdominal aortic aneurysm). AAA was induced in C57BL/6 mice by periaortic application of CaCl2 (AAA group). NaCl (0.9%)-treated mice were used as a sham control (SHAM group). Mice were treated with 0.05% CoCl2 in the drinking water (AAA/CoCl2 group). At 1 and 6 weeks after the operation, aortic tissue was excised for further examination. After 6 weeks of CaCl2 treatment, aortic diameter and macrophage infiltration into the aortic adventitia were increased in the AAA group compared with the SHAM group. Treatment with CoCl2 reduced the aneurysmal size and macrophage infiltration compared with the AAA group. Aortic expression of inflammatory cytokines and MCP-1 (monocyte chemoattractant protein-1) and the activities of MMP-9 (matrix metalloproteinase-9) and MMP-2 were enhanced in the AAA group and attenuated in the AAA/CoCl2 group. Expression of cytokines and the activities of MMPs were already increased after 1 week of CaCl2 treatment, but were suppressed by CoCl2 treatment in association with reduced NF-κB (nuclear factor κB) phosphorylation. Treatment with CoCl2 in mice prevented the development of CaCl2-induced AAA in association with reduced inflammation and ECM (extracellular matrix) disruption. The results of the present study suggest that PHD plays a critical role in the development of AAA and that there is a therapeutic potential for PHD inhibitors in the prevention of AAA development.

LRP1 protects the vasculature by regulating levels of connective tissue growth factor and HtrA1.

OBJECTIVE: Low-density lipoprotein receptor-related protein 1 (LRP1) is a large endocytic and signaling receptor that is abundant in vascular smooth muscle cells. Mice in which the lrp1 gene is deleted in smooth muscle cells (smLRP1(-/-)) on a low-density lipoprotein receptor-deficient background display excessive platelet derived growth factor-signaling, smooth muscle cell proliferation, aneurysm formation, and increased susceptibility to atherosclerosis. The objectives of the current study were to examine the potential of LRP1 to modulate vascular physiology under nonatherogenic conditions. APPROACH AND RESULTS: We found smLRP1(-/-) mice to have extensive in vivo aortic dilatation accompanied by disorganized and degraded elastic lamina along with medial thickening of the arterial vessels resulting from excess matrix deposition. Surprisingly, this was not attributable to excessive platelet derived growth factor-signaling. Rather, quantitative differential proteomic analysis revealed that smLRP1(-/-) vessels contain a 4-fold increase in protein levels of high-temperature requirement factor A1 (HtrA1), which is a secreted serine protease that is known to degrade matrix components and to impair elastogenesis, resulting in fragmentation of elastic fibers. Importantly, our study discovered that HtrA1 is a novel LRP1 ligand. Proteomics analysis also identified excessive accumulation of connective tissue growth factor, an LRP1 ligand and a key mediator of fibrosis. CONCLUSIONS: Our findings suggest a critical role for LRP1 in maintaining the integrity of vessels by regulating protease activity as well as matrix deposition by modulating HtrA1 and connective tissue growth factor protein levels. This study highlights 2 new molecules, connective tissue growth factor and HtrA1, which contribute to detrimental changes in the vasculature and, therefore, represent new target molecules for potential therapeutic intervention to maintain vessel wall homeostasis.

AKT2 confers protection against aortic aneurysms and dissections.

RATIONALE: Aortic aneurysm and dissection (AAD) are major diseases of the adult aorta caused by progressive medial degeneration of the aortic wall. Although the overproduction of destructive factors promotes tissue damage and disease progression, the role of protective pathways is unknown. OBJECTIVE: In this study, we examined the role of AKT2 in protecting the aorta from developing AAD. METHODS AND RESULTS: AKT2 and phospho-AKT levels were significantly downregulated in human thoracic AAD tissues, especially within the degenerative medial layer. Akt2-deficient mice showed abnormal elastic fibers and reduced medial thickness in the aortic wall. When challenged with angiotensin II, these mice developed aortic aneurysm, dissection, and rupture with features similar to those in humans, in both thoracic and abdominal segments. Aortas from Akt2-deficient mice displayed profound tissue destruction, apoptotic cell death, and inflammatory cell infiltration that were not observed in aortas from wild-type mice. In addition, angiotensin II-infused Akt2-deficient mice showed significantly elevated expression of matrix metalloproteinase-9 (MMP-9) and reduced expression of tissue inhibitor of metalloproteinase-1 (TIMP-1). In cultured human aortic vascular smooth muscle cells, AKT2 inhibited the expression of MMP-9 and stimulated the expression of TIMP-1 by preventing the binding of transcription factor forkhead box protein O1 to the MMP-9 and TIMP-1 promoters. CONCLUSIONS: Impaired AKT2 signaling may contribute to increased susceptibility to the development of AAD. Our findings provide evidence of a mechanism that underlies the protective effects of AKT2 on the aortic wall and that may serve as a therapeutic target in the prevention of AAD.

Effects of p38 mitogen-activated protein kinase inhibition on vascular and systemic inflammation in patients with atherosclerosis.

OBJECTIVES: This study sought to determine the effects of a p38 mitogen-activated protein kinase inhibitor, losmapimod, on vascular inflammation, by (18)F-fluorodeoxyglucose (FDG) positron emission tomography/computed tomography imaging. BACKGROUND: The p38 mitogen-activated protein kinase cascade plays an important role in the initiation and progression of inflammatory diseases, including atherosclerosis. METHODS: Patients with atherosclerosis on stable statin therapy (n = 99) were randomized to receive losmapimod 7.5 mg once daily (lower dose [LD]), twice daily (higher dose [HD]) or placebo for 84 days. Vascular inflammation was assessed by FDG positron emission tomography/computed tomography imaging of the carotid arteries and aorta; analyses focused on the index vessel (the artery with the highest average maximum tissue-to-background ratio [TBR] at baseline). Serum inflammatory biomarkers and FDG uptake in visceral and subcutaneous fat were also measured. RESULTS: The primary endpoint, change from baseline in average TBR across all segments in the index vessel, was not significantly different between HD and placebo (ΔTBR: -0.04 [95% confidence interval [CI]: -0.14 to +0.06], p = 0.452) or LD and placebo (ΔTBR: -0.02 [95% CI: -0.11 to +0.06], p = 0.579). However, there was a statistically significant reduction in average TBR in active segments (TBR ≥1.6) (HD vs. placebo: ΔTBR: -0.10 [95% CI: -0.19 to -0.02], p = 0.0125; LD vs. placebo: ΔTBR: -0.10 [95% CI: -0.18 to -0.02], p = 0.0194). The probability of a segment being active was also significantly reduced for HD when compared with placebo (OR: 0.57 [95% CI: 0.41 to 0.81], p = 0.002). Within the HD group, reductions were observed in placebo-corrected inflammatory biomarkers including high-sensitivity C-reactive protein (% reduction: -28% [95% CI: -46 to -5], p = 0.023) as well as FDG uptake in visceral fat (ΔSUV: -0.05 [95% CI: -0.09 to -0.01], p = 0.018), but not subcutaneous fat. CONCLUSIONS: Despite nonsignificant changes for the primary endpoint of average vessel TBR, HD losmapimod reduced vascular inflammation in the most inflamed regions, concurrent with a reduction in inflammatory biomarkers and FDG uptake in visceral fat. These results suggest a systemic anti-inflammatory effect. (A Study to Evaluate the Effects of 3 Months Dosing With GW856553, as Assessed FDG-PET/CT Imaging; NCT00633022).

[The biochemical and clinical aspects of the participation of granulocytes and their proteinases in a vascular wall lesion]. / Biokhimicheskie i klinicheskie aspekty uchastiia granulotsitov i ikh proteinaz v porazhenii stenki sosudov.

Some components of plasma and granulocyte proteolytic system, granulocyte activation degree were evaluated in 15 patients with nonspecific aortic arteritis (NAA) and 12 hereditary hypercholesterolemia (HHC) patients. Catepsin G activity in blood plasma of NAA and HHC patients was higher than in donors 8-9 and 6-fold, respectively (p < 0.001). Granulocyte collagenase activity, spontaneous NBT test values in them were higher than in donors 6 and 4-fold (p < 0.02 and p < 0.01, respectively), 2-fold (p < 0.005 and p < 0.001, respectively). The data obtained confirm the suggestion on the presence of an inflammatory component not only in pathogenesis of NAA, but also in HHC causation.

[Immunohistochemical and histoenzymological characteristics of the arterial intimal cells in nonspecific aorto-arteritis]. / Immunogistokhimicheskaia i gistoénzimologicheskaia kharakteristika kletok intimy arterii pri nespetsificheskom aorto-arteriite.

Enzymatic activity of cells, antigenic cellular markers and extracellular matrix of the hyperplastic intima of the aorta and carotid arteries was investigated in non-specific aorto-arteritis by immunomorphological and histochemical techniques. The cells of subendothelial layer of thickened arterial intima contained smooth muscle cell myosin, gave positive reactions to myosin ATP-ase and revealed high activity of thiamine pyrophosphatase. Fibronectin and type IV and V collagen were located in close proximity to these cells. The data obtained make it possible to consider these cells as modified smooth muscle cells. Type III collagen was the prevalent type of extracellular matrix of the thickened intima. A great number of blood vessels of the capillary and precapillary types have been found to penetrate into the intima from the adventitia. A possible role of pericytes surrounding newly formed capillaries as the precursors of subendothelial cell population in the hyperplastic intima is discussed.

[Potential clinical use of an acid-stable proteinase inhibitor from human urine]. / Vozmozhnosti klinicheskogo primeneniia kislotostabil'nogo ingibitora proteinaz iz mochi cheloveka.

The method for the preparation of a specific proteinase adsorbent (acid-resistant human urinary proteinase inhibitor--UPI--immobilized on sepharose) has been developed. Proteinase adsorption using this adsorbent has proved highly effective in the treatment of acute pancreatitis with plasmasorption. High adsorption capacity of UPI-sepharose from plasma samples of patients with acute nonspecific aortoarteritis has been demonstrated in vitro. Determination of UPI activity in the human urine is an informative test for early tissue damage, as compared to plasma middle molecules assay. The former test may be used as a diagnostic technique in patients with parenchymal kidney injury and increased arterial pressure.