PCSK9 expression in fibrous cap possesses a marker for rupture in advanced plaque.

BACKGROUND: To date, PCSK9 inhibitors are well known for eliminating cardiac and cerebral artery ischemia events by lowering the serum lipid level. However, the pathophysiological value of in-plaque PCSK9 expression is still unclear. METHODS: Advanced plaques removed by carotid endarterectomy were sectioned and stained to identify the PCSK9 expression pattern and its co-expression with rupture-relevant markers. To investigate the correlation of PCSK9 expression with regional blood shear flow, hemodynamic characteristics were analyzed using computational fluid dynamics, and representative parameters were compared between PCSK9 positive and negative staining plaques. To explore this phenomenon in vitro, human aortic vascular smooth muscle cells were used to overexpress and knock down PCSK9. The impacts of PCSK9 modulations on mechanical sensor activity were testified by western blot and immunofluorescence. Real-time polymerase chain reaction was used to evaluate the transcription levels of downstream rupture-prone effectors. RESULTS: PCSK9 distribution in plaque preferred cap and shoulder regions, residing predominantly in smooth muscle actin-positive cells. Cap PCSK9 expression correlated with fibrous cap thickness negatively and co-expressed with MMP-9, both pointing to the direction of plaque rupture. A hemodynamic profile indicated a rupture-prone feature of cap PCSK9 expression. In vitro, overexpression and knockdown of PCSK9 in human aortic vascular smooth muscle cells has positive modulation on mechanical sensor Yes-associated protein 1 (YAP) activity and transcription levels of its downstream rupture-prone effectors. Serial section staining verified in situ colocalization among PCSK9, YAP, and downstream effectors. CONCLUSIONS: Cap PCSK9 possesses a biomarker for rupture risk, and its modulation may lead to a novel biomechanical angle for plaque interventions.

Legumain knockout improved cognitive impairment via reducing neuroinflammation in right unilateral common carotid artery occlusion mice.

AIMS: Chronic cerebral hypoperfusion (CCH) is a state of chronic cerebral blood flow reduction, and it is the main cause of cognitive impairment and neurodegenerative diseases. The abnormal upregulation of legumain, a lysosomal cysteine protease, trigger synaptic plasticity impairment and neuroinflammation, which are involved in the underlying pathophysiology of CCH. At present, few studies have reported the role of legumain in cognitive impairment caused by CCH. In our study, we aimed to investigate the involvement of legumain knockout in cognitive function and neuroinflammation in a CCH mouse model. MAIN METHODS: In this study, right unilateral common carotid artery occlusion (rUCCAO) was used to simulate the pathological state of cerebral ischemic injury. Various behavioural tests were executed to assess cognitive performance. In vivo electrophysiological recordings were used to measure synaptic functions. Western blotting, Golgi staining, haematoxylin/eosin staining, and immunofluorescence assays were conducted to examine pathological changes and molecular mechanisms. KEY FINDINGS: The data showed that the level of legumain was significantly increased in the hippocampus of mice subjected to rUCCAO. Legumain knockout significantly improved cognitive function and synaptic plasticity induced by rUCCAO, suggesting that legumain knockout-regulation effectively protected against CCH-induced behavioural dysfunctions. Moreover, legumain knockout suppressed rUCCAO-induced microglial activation, reduced the abnormal expression of inflammatory cytokines and the inflammasome complex, and impeded the activation of P65 and pyroptosis. SIGNIFICANCE: These findings suggest that legumain is an effective regulator of CCH, and may be an ideal target for the development of cerebral ischemia treatments in the future.

H3K4 Methyltransferase Smyd3 Mediates Vascular Smooth Muscle Cell Proliferation, Migration, and Neointima Formation.

[Figure: see text].

Peptidylarginine Deiminase 4 as a Possible Biomarker of Plaque Instability in Carotid Artery Stenosis.

BACKGROUND AND PURPOSE: Neutrophil extracellular traps (NETs) exhibit pro-inflammatory and pro-thrombotic properties. However, they have only been reported as important regulators in atherosclerosis, especially in atherothrombosis. We investigated the presence of NETs and plaque instability in patients with carotid artery stenosis. MATERIAL AND METHODS: A total of 39 consecutive patients with carotid artery stenosis were evaluated. All patients underwent carotid artery stenting (CAS) with dual protection (simultaneous flow reversal + distal filter) and blood aspiration as a method of distal embolism prevention. Local arterial blood was aspirated at the stent site and peptidylarginine deiminase 4 (PAD4), which is essential for the formation of NETs, was measured. The relationships between PAD4 and the patient profile, blood examination and plaque data were investigated. RESULTS: The mean value of PAD4 in local arterial blood in CAS was 0.5 ng/ml. Bivariate analysis demonstrated that PAD4 was associated with the neutrophil to lymphocyte ratio (p = 0.007), high-density lipoprotein (p = 0.02), triglycerides to high-density lipoprotein ratio (p = 0.007), ulceration (p = 0.02) and plaque contrast enhancement on T1 black blood imaging (p = 0.03). In multiple linear regression analyses, PAD4 was correlated with the neutrophil to lymphocyte ratio (p = 0.01) and ulceration (p = 0.01, cut-off value: 0.49 odds ratio: 19.3). CONCLUSIONS: PAD4, representative of the presence of NETs, was high in carotid plaques with unstable features. The neutrophil to lymphocyte ratio in peripheral blood was suggested to be a biomarker of vulnerable plaques. Elucidating the role of NETs may aid in clarifying factors that promote the instability of carotid plaques.

Ruxolitinib attenuates intimal hyperplasia via inhibiting JAK2/STAT3 signaling pathway activation induced by PDGF-BB in vascular smooth muscle cells.

BACKGROUND: Cardiovascular diseases are associated with proliferation and phenotypic switch. Platelet-derived growth factor-BB (PDGF-BB) is a major initiating factor for proliferative vascular diseases, such as neointimal lesion formation, restenosis after angioplasty, and atherosclerosis. Ruxolitinib, a potent Janus kinase (JAK) 1 and 2 inhibitor, has been reported to significantly block the proliferation-related signaling pathway of JAK2/signal transducers and activators of transcription 3 (STAT3) and harbor a broad spectrum of anti-cancer activities, including proliferation inhibition, apoptosis induction, and anti-inflammation. However, the role of ruxolitinib in regulating PDGF-BB-induced VSMC proliferation remains to be elucidated. Thus, this study investigates the role of ruxolitinib in regulating PDGF-BB-induced VSMC proliferation and its underlying mechanisms. METHODS: In vivo, the medial thickness of the carotid artery was evaluated using a mouse carotid ligation model, ruxolitinib was administered orally to the mice every other day, and the mice were euthanized on day 28 to evaluate the therapeutic effects of ruxolitinib. Cell proliferation markers were measured using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blotting. In vitro, VSMCs were treated with ruxolitinib with or without PDGF-BB at an indicated time and concentration. Cell proliferation and apoptosis were measured using Cell Counting Kit-8 assay, MTS assays and flow cytometry. The JAK2/STAT3 signaling pathway involved in the effects of ruxolitinib on VSMCs was detected by western blotting with the specific pathway inhibitor AG490. RESULTS: In vivo, ruxolitinib significantly decreased the ratio-of-intima ratio (I/M ratio) by inhibiting the expression of PCNA and cyclinD1 (p <0.05). In vitro, ruxolitinib inhibited PDGF-BB-induced VSMC proliferation compared with the PDGF-BB treatment group (p <0.05). In addition, ruxolitinib inhibited the PDGF-BB-induced activation of the JAK2/STAT3 signaling pathway and decreased the expression of proliferation related-proteins cyclinD1 and PCNA in VSMCs (p <0.05). CONCLUSION: Our findings suggest that ruxolitinib inhibits VSMC proliferation in vivo and in vitro by suppressing the activation of the JAK2/STAT3 signaling pathway. Therefore, ruxolitinib has a therapeutic potential for proliferative vascular diseases.

Correlation between SHP-1 and carotid plaque vulnerability in humans.

BACKGROUND: Advanced atherosclerotic plaques tend to indicate an increased risk of cerebral ischemic events. SH2 domain-containing protein tyrosine phosphatase 1 (SHP-1) is a class I classical nonreceptor protein tyrosine phosphatase associated with plaque stability, as shown by analysis of a Gene Expression Omnibus (GEO) dataset showing differences in mRNA levels. However, the correlation between SHP-1 and human carotid plaque stability at the protein level remains unclear. METHODS AND RESULTS: Thirty-nine carotid plaque tissue samples were acquired from 39 carotid artery stenosis patients after carotid endarterectomy. Hematoxylin and eosin, Masson trichrome, and CD68 staining was performed for pathological characterization, and immunohistochemical staining for SHP-1 was carried out. Within stable and unstable plaques, SHP-1 mainly accumulated in the necrotic area, plaque shoulder, and fibrous cap, similar to the distribution of CD68. A quantitative analysis of SHP-1 was carried out. The relative SHP-1-positive cell area was higher in the vulnerable group than in the stable group (P < .001). The number of symptomatic patients in the vulnerable group was no greater than that in the stable group (P = .098). Moreover, the integrated optical density (IOD)/area of SHP-1 was significantly higher in the vulnerable group than in the stable group (P < .001). Besides, SHP-1 colocalized with CD68 and vascular cell adhesion protein 1(VCAM-1). CONCLUSIONS: We demonstrate that SHP-1 expression increases during carotid atherosclerotic plaque progression. The protein expression of SHP-1 was related to an increase in plaque instability in not only symptomatic but also asymptomatic patients with carotid artery stenosis. SHP-1 may play a role in atherosclerosis progression by macrophage polarization-mediated efferocytosis. Furthermore, SHP-1 may become a promising biomarker for plaque vulnerability in the future.

Antioxidant enzymes expression in lymphocytes of patients undergoing carotid endarterectomy.

To remedy carotid artery stenosis and prevent stroke surgical intervention is commonly used, and the gold standard being carotid endarterectomy (CEA). During CEA cerebrovascular hemoglobin oxygen saturation decreases and when this decrease reaches critical levels it leads to cerebral hypoxia that causes neuronal damage. One of the proposed mechanism that affects changes during CEA and contribute to acute brain ischemia (ABI) is oxidative stress. The increased production of reactive oxygen species and reactive nitrogen species during ABI may cause an unregulated inflammatory response and further lead to structural and functional injury of neurons. Antioxidant activity are involved in the protection against neuronal damage after cerebral ischemia. We hypothesized that neuronal injury and poor outcomes in patients undergoing CEA may be results of oxidative stress that disturbed function of antioxidant enzymes and contributed to the DNA damage in lymphocytes.

Loss of CLOCK under high glucose upregulates ROCK1-mediated endothelial to mesenchymal transition and aggravates plaque vulnerability.

BACKGROUND AND AIMS: Carotid atherosclerotic plaque is one of the main sources of ischemic stroke, and endothelial-to-mesenchymal transition (EndMT) is a major feature of atherosclerosis. Rho-associated coiled-coil-containing protein kinase 1 (ROCK1) activation, stimulated by high glucose, plays an important role in EndMT, and circadian locomotor output cycles protein kaput (Clock) deficiency leads to hyperglycemia and enhanced atherosclerosis in ClockΔ19/Δ19apolipoprotein E (ApoE)-/- mice. These findings point to a mechanism whereby CLOCK exerts a protective effect against EndMT and atherosclerotic plaque accumulation. METHODS: Cultured human umbilical vein endothelial cells (HUVECs) were stimulated with 66 mM glucose for 120 h to induce EndMT. The expression of CLOCK and ROCK1 was assayed, as were their effects on EndMT. We also conducted molecular and morphometric examination of carotid artery plaques from patients with carotid artery stenosis to assess the clinical relevance of these findings. RESULTS: Upon EndMT, HUVECs exhibited decreased CLOCK expression and increased ROCK1 expression. Notably, CLOCK silencing increased high glucose-induced EndMT, migration ability, and ROCK1 activation, while overexpressing CLOCK attenuated these characteristics. Moreover, inhibition of ROCK1 largely blocked EndMT induced by high-glucose or transforming growth factor (TGF)-ß1 but failed to rescue the reduced CLOCK expression. The vulnerability of human carotid artery plaque was strongly correlated with loss of CLOCK expression, activation of TGF-ß/ROCK1 signaling, and the extent of EndMT. CONCLUSIONS: The data indicate that loss of protective endothelial CLOCK expression aggravates TGF-ß/ROCK1-modulated EndMT progression, which contributes to the vulnerability of human carotid plaque.

Estradiol Protects White Matter of Male C57BL6J Mice against Experimental Chronic Cerebral Hypoperfusion.

BACKGROUND AND PURPOSE: Estradiol is a sex steroid hormone known to protect the brain against damage related to transient and global cerebral ischemia. In the present study, we leverage an experimental murine model of bilateral carotid artery stenosis (BCAS) to examine the putative effects of estradiol therapy on chronic cerebral hypoperfusion. We hypothesize that long-term estradiol therapy protects against white matter injury and declarative memory deficits associated with chronic cerebral hypoperfusion. METHODS: Adult male C57BL/6J mice underwent either surgical BCAS or sham procedures. Two days after surgery, the mice were given oral estradiol (Sham+E, BCAS+E) or placebo (Sham+P, BCAS+P) treatments daily for 31-34 days. All mice underwent Novel Object Recognition (NOR) testing 31-34 days after the start of oral treatments. Following sacrifice, blood was collected and brains fixed, sliced, and prepared for histological examination of white matter injury and extracellular signal-regulated kinase (ERK) expression. RESULTS: Animals receiving long-term oral estradiol therapy (BCAS-E2 and Sham-E2) had higher plasma estradiol levels than those receiving placebo treatment (BCAS-P and Sham-P). BCAS-E2 mice demonstrated less white matter injury (Klüver-Barrera staining) and performed better on the NOR task compared to BCAS-P mice. ERK expression in the brain was increased in the BCAS compared to sham cohorts. Among the BCAS mice, the BCAS-E2 cohort had a greater number of ERK + cells. CONCLUSION: This study demonstrates a potentially protective role for oral estradiol therapy in the setting of white matter injury and declarative memory deficits secondary to murine chronic cerebral hypoperfusion.

BICAO-induced ischaemia caused depressive-like behaviours and caspase-8/-9-dependent brain regional neural cell apoptosis in mice.

Introduction: Cerebral ischaemia-induced depression is among the most frequent neuropsychiatric consequences and adversely impact the prognosis and recovery of patients. Although several brain regions have been implied in the development of ischaemia-induced depression, the brain region-specific neural cell apoptosis pathways have not been clarified yet. Methods: In this study, bilateral internal carotid artery occlusion (BICAO) mouse model was established to induce cerebral ischaemia. Sucrose preference, tail suspension and forced swim tests were conducted on mice at 7, 21 and 30 days after BICAO treatment. In addition, brain regional ischaemic neuron loss was investigated by using immunofluorescent staining of neuronal nuclei (NeuN) and caspase-8/-9-dependent cell apoptosis was also examined by western blot analysis. Results: BICAO-induced cerebral ischaemia resulted in decreased sucrose preference and increased immobility times, which were representative depressive-like behaviours of mice until 30 days after BICAO treatment compared with Sham-operated mice. This outcome was associated with significant neuron loss by using immunofluorescent staining and increased cleavage levels of pro-caspase-3/-8/-9, but not pro-caspase-12, by western blot analysis in hypothalamus, midbrain, prefrontal cortex and hippocampus of mice. Conclusions: This study showed that BICAO-induced ischaemia caused depressive-like behaviours and caspase-8/-9-dependent neural cell apoptosis in several brain regions, including hypothalamus and midbrain of mice.

Inhibition of p110δ PI3K prevents inflammatory response and restenosis after artery injury.

Inflammatory cells play key roles in restenosis upon vascular surgical procedures such as bypass grafts, angioplasty and stent deployment but the molecular mechanisms by which these cells affect restenosis remain unclear. The p110δ isoform of phosphoinositide 3-kinase (PI3K) is mainly expressed in white blood cells. Here, we have investigated whether p110δ PI3K is involved in the pathogenesis of restenosis in a mouse model of carotid injury, which mimics the damage following arterial grafts. We used mice in which p110δ kinase activity has been disabled by a knockin (KI) point mutation in its ATP-binding site (p110δD910A/D910A PI3K mice). Wild-type (WT) and p110δD910A/D910A mice were subjected to longitudinal carotid injury. At 14 and 30 days after carotid injury, mice with inactive p110δ showed strongly decreased infiltration of inflammatory cells (including T lymphocytes and macrophages) and vascular smooth muscle cells (VSMCs), compared with WT mice. Likewise, PI-3065, a p110δ-selective PI3K inhibitor, almost completely prevented restenosis after artery injury. Our data showed that p110δ PI3K plays a main role in promoting neointimal thickening and inflammatory processes during vascular stenosis, with its inhibition providing significant reduction in restenosis following carotid injury. p110δ-selective inhibitors, recently approved for the treatment of human B-cell malignancies, therefore, present a new therapeutic opportunity to prevent the restenosis upon artery injury.

LIPOPROTEIN ASSOCIATED PHOSPHOLIPASE A2 AS A MARKER OF VULNERABLE ATHEROSCLEROTIC PLAQUE IN PATIENTS WITH INTERNAL CAROTID ARTERY STENOSIS.

The aim of this study was to compare the concentration of inflammatory vascular markers and morphological structure of atherosclerotic plaque in symptomatic and asymptomatic patients with the stenosis of internal carotid artery (ICA). The research was carried out in 70 patients with hemodynamically significant stenosis of ICA out of which 40 (57%) were asymptomatic patients and 30 (43%) were symptomatic patients, of which 20 patients (66%) have had a stroke, or transient ischemic attack (TIA), 10 patients (33%). All the patients were indicated to carotid endarterectomy as a surgical prevention of stroke. All the patients were taken their blood for biochemical testing (T-Chol, LDL, HDL, TG, Fibrinogen, CRP and specific markers IL-4 and Lp-PLA2) early morning prior to surgery. The highest concentrations of T-Chol, LDL, HDL, CRP and Fibrinogen were measured in symptomatic patients, however, these did not feature a significant difference compared with the group of asymptomatic patients (P>0.05). Significant difference was found in IL-4 (P<0.001) and in Lp-PLA2 (P<0.001). When evaluating concentration of tracked parameters in patients with soft atherosclerotic plaque and patients with calcified atherosclerotic plaque, significant differences were found in these markers: TG (P<0.05), CRP (P<0.01), IL-4 (P<0.001) and Lp-PLA2 (P<0.001). The paper deals with higher concentrations of Lp-PLA2 in patients with a soft atherosclerotic plaque. Higher concentration of Lp-PLA2 and systemic inflammatory markers (CRP, IL-4) could be used along with ultrasonography to detect mainly asymptomatic patients who are in urgent need of surgical or endovascular treatment as a prevention of stroke.

iNOS-Derived Nitric Oxide Induces Integrin-Linked Kinase Endocytic Lysosome-Mediated Degradation in the Vascular Endothelium.

OBJECTIVE: ILK (integrin-linked kinase) plays a key role in controlling vasomotor tone and is decreased in atherosclerosis. The objective of this study is to test whether nitric oxide (NO) regulates ILK in vascular remodeling. APPROACH AND RESULTS: We found a striking correlation between increased levels of inducible nitric oxide and decreased ILK levels in human atherosclerosis and in a mouse model of vascular remodeling (carotid artery ligation) comparing with iNOS (inducible NO synthase) knockout mice. iNOS induction produced the same result in mouse aortic endothelial cells, and these effects were mimicked by an NO donor in a time-dependent manner. We found that NO decreased ILK protein stability by promoting the dissociation of the complex ILK/Hsp90 (heat shock protein 90)/eNOS (endothelial NO synthase), leading to eNOS uncoupling. NO also destabilized ILK signaling platform and lead to decreased levels of paxillin and α-parvin. ILK phosphorylation of its downstream target GSK3-ß (glycogen synthase kinase 3 beta) was decreased by NO. Mechanistically, NO increased ILK ubiquitination mediated by the E3 ubiquitin ligase CHIP (C terminus of HSC70-interacting protein), but ILK ubiquitination was not followed by proteasome degradation. Alternatively, NO drove ILK to degradation through the endocytic-lysosomal pathway. ILK colocalized with the lysosome marker LAMP-1 (lysosomal-associated membrane protein 1) in endothelial cells, and inhibition of lysosome activity with chloroquine reversed the effect of NO. Likewise, ILK colocalized with the early endosome marker EEA1 (early endosome antigen 1). ILK endocytosis proceeded via dynamin because a specific inhibitor of dynamin (Dyngo 4a) was able to reverse ILK endocytosis and its lysosome degradation. CONCLUSIONS: Endocytosis regulates ILK signaling in vascular remodeling where there is an overload of inducible NO, and thus its inhibition may represent a novel target to fight atherosclerotic disease.

Carotid disease at age 73 and cognitive change from age 70 to 76 years: A longitudinal cohort study.

Cognitive decline and carotid artery atheroma are common at older ages. In community-dwelling subjects, we assessed cognition at ages 70, 73 and 76 and carotid Doppler ultrasound at age 73, to determine whether carotid stenosis was related to cognitive decline. We used latent growth curve models to examine associations between four carotid measures (internal carotid artery stenosis, velocity, pulsatility and resistivity indices) and four cognitive ability domains (memory, visuospatial function, crystallised intelligence, processing speed) adjusted for cognitive ability at age 11, current age, gender and vascular risk factors. Amongst 866 participants, carotid stenosis (median 12.96%) was not associated with cognitive abilities at age 70 or cognitive decline from age 70 to 76. Increased ICA pulsatility and resistivity indices were associated with slower processing speed (both P < 0.001) and worse visuospatial function ( P = 0.036, 0.031, respectively) at age 70, and declining crystallised intelligence from ages 70 to 76 ( P = 0.008, 0.006, respectively). The findings suggest that vascular stiffening, rather than carotid luminal narrowing, adversely influences cognitive ageing and provides a potential target for ameliorating age-related cognitive decline.

Variants in COX-2, PTGIS, and TBXAS1 Are Associated with Carotid Artery or Intracranial Arterial Stenosis and Neurologic Deterioration in Ischemic Stroke Patients.

BACKGROUND: Eicosanoids may play a role in ischemic stroke (IS). However, the association of variants in eicosanoid genes with symptomatic carotid artery or intracranial arterial stenosis and neurologic deterioration (ND) is not fully understood. The aim of the present study was to investigate the association of 11 variants in eicosanoid genes with symptomatic carotid artery or intracranial arterial stenosis and ND. METHODS: Eleven variants in eicosanoid genes were examined using mass spectrometry method in 297 IS patients. The symptomatic carotid artery or intracranial arterial stenosis was assessed by computed tomographic angiography. Platelet aggregation and platelet-leukocyte aggregates were measured. The primary outcome was ND within 10 days of admission. ND was defined as an increase of 2 or more points in National Institutes of Health Stroke Scale score. RESULTS: Among 297 IS patients, 182 (61.3%) cases had symptomatic carotid artery or intracranial arterial stenosis, and 88 (29.6%) patients experienced ND within 10 days after admission. Symptomatic carotid artery or intracranial arterial stenosis was significantly associated with higher ND (P < .001). Rs20417CC, rs41708TT, and rs5629CC were independent risk factors for symptomatic carotid artery or intracranial arterial stenosis and ND, and associated with higher platelet aggregation and platelet-leukocyte aggregates. CONCLUSIONS: Symptomatic carotid artery or intracranial arterial stenosis was associated with higher ND. Rs20417CC, rs41708TT, and rs5629CC were not only independent risk factors for symptomatic carotid artery or intracranial arterial stenosis, but also independent risk predictors for ND.

Increased levels of legumain in plasma and plaques from patients with carotid atherosclerosis.

BACKGROUND AND AIMS: The cysteine protease legumain has been shown to be up-regulated in unstable atherosclerotic plaques. This study aims to further elucidate legumain in atherosclerosis, by examining legumain in plasma and carotid plaques from patients with carotid stenosis. Furthermore, legumain secretion from monocyte-derived macrophages treated with atherogenic lipids during macrophage polarization was studied. METHODS: Plasma levels of legumain from patients with carotid stenosis (n = 254), healthy controls (n = 91), and secreted from monocyte-derived macrophages were assessed by enzyme-linked-immunosorbent assay. Quantitative PCR and immunoblotting of legumain were performed on isolated plaques and legumain localization was visualized by immunohistochemistry and fluorescence microscopy. Monocyte-derived macrophages polarized to M1 or M2 macrophages were treated with VLDL, oxLDL or cholesterol crystals (CC) and the level of legumain analysed. RESULTS: Patients with carotid stenosis had significantly higher levels of plasma legumain compared with healthy controls (median 2.0 versus 1.5 ng/ml, respectively; p = 0.003), although there was no correlation between the level of legumain and the degree of stenosis, and legumain was not an independent factor to identify patients with carotid plaques. Moreover, patients with symptoms the last 2 months had higher expressions of mature legumain, cystatin C and E/M, and the macrophage markers CD80 (M1) and CD163 (M2). Legumain co-localized with both M1 and M2 macrophages within plaques, whereas legumain mRNA expression was significantly higher (p < 0.0001) in plaques compared to non-atherosclerotic arteries (controls). Furthermore, in vitro studies showed significantly increased secretion of legumain from pro-inflammatory M1 compared to pro-resolving M2 macrophages (p = 0.014), and particularly in M1 treated with CC. In plaques, legumain was localized to structures resembling foam cells. CONCLUSIONS: Legumain is increased in both plasma and plaques of patients with carotid stenosis and might be a new and early biomarker of atherosclerosis.

The relationship between serum paraoxonase levels and carotid atherosclerotic plaque formation in Alzheimer's patients.

Low paraoxonase 1 (PON1) activity and carotid atherosclerosis have been suggested to be important risk factors for dementia. However, the studies to date could not fully clarify the relationship between PON1, carotid atherosclerosis and dementia. The present study aimed to measure carotid atherosclerosis and PON1 activity in Alzheimer's Disease and to evaluate the relationship between them. The study included 25 Alzheimer's patients and 25 control subjects, for a total of 50 individuals. The study measured the serum PON1 activity and other biochemical parameters and carotid atherosclerotic plaque values of the participants. The mean paraoxonase activity (31.06±2.31U/L) was significantly lower in the Alzheimer's group compared to the control group (59.05±7.05U/L) (P<0.001). Nonetheless, the carotid plaque values were significantly higher in the patient group (3.02±0.52mm) compared to the control group (1.84±0.45mm) (P<0.001). Furthermore, there was a negative correlation (81.0%) between PON1 activity and carotid plaque in the overall study group (P<0.05). Also serum homocystein level was higher in the patient group (22.15±7.05) compared to the control group (13.30±3.32). In conclusion, our findings show inverse association between PON1 activity and carotid atherosclerosis in Alzheimer patients: the lower the PON1 activity the more progressed the atherosclerotic process in AD.

The ubiquitin-activating enzyme E1 as a novel therapeutic target for the treatment of restenosis.

AIMS: The ubiquitin-activating enzyme E1 (UBA1, E1), the apex of the ubiquitin proteasome pathway, plays a critical role in protein degradation and in pathological processes. Whether UBA1 participates the development of vascular restenosis remains unknown. This study aims to determine the role of UBA1 in the development of balloon injury induced neointimal formation. METHODS AND RESULTS: Immunostaining and western blots were used to examine the expression of the ubiquitinated protein in the injured carotid after angioplasty. Higher levels of ubiquitinated protein were observed in the neointima. Local delivery of potent chemical UBA1 inhibitor PYR-41 (100 µM) and UBA1 shRNA lentivirus both resulted in a substantial decrease in intimal hyperplasia at 2 weeks and 4 weeks after balloon injury. UBA1 inhibition also reduced Ki-67 positive cell percentage and inflammatory response in the carotid artery wall. We further determined that in vitro UBA1 inhibition was able to ameliorate TNF-α-induced nuclear factor-kappa B (NF-κB) activation by reducing IκB degradation in vascular smooth muscle cells (VSMCs). UBA1 inhibition also led to the accumulation of short-lived proteins such as p53, p21 and c-jun, which may account for the UBA1 inhibition-induced cell cycle delay. Thus, VSMCs proliferation was blocked. CONCLUSIONS: UBA1 inhibition effectively suppresses neointimal thickening through its anti-proliferative and anti-inflammatory effects. Our results provide further evidence that the ubiquitin-proteasome system is a potential new target for the prevention of vascular restenosis.

The Early Effect of Carotid Artery Stenting on Antioxidant Capacity and Oxidative Stress in Patients with Carotid Artery Stenosis.

The treatment of carotid artery stenosis is associated with the risk of complications, which may include stroke after carotid artery stenting (CAS) and myocardial infarction after carotid endarterectomy (CEA). The imbalance between prooxidative mechanisms and antioxidant capacity creates a milieu of factors, which may increase the risk of complications after endovascular procedures. We have examined 43 consecutive patients with carotid artery stenosis. Sera were analyzed for the activity of paraoxonase (PON) and arylesterase (ARE), sulfhydryl groups (SG), malondialdehyde (MDA), and conjugated dienes (CD) concentrations by means of spectrophotometric methods before and next day after CAS. We have found lowered PON (P = 0.0032), increase in ARE activity (P = 0.0058), and decrease in sulfhydryl groups concentration (P = 0.0267). No effect on absolute MDA and CD concentrations was observed. The degree of carotid artery stenosis correlated negatively with PON/ARE ratio after CAS (rS = -0.507, P = 0.0268). To conclude, CAS influences both enzymatic (differently, PON and ARE activity) and nonenzymatic antioxidant defense. Females are more susceptible to lipid peroxidation after CAS. PON/ARE ratio after CAS correlated with the degree of carotid artery stenosis. The changes (deltas) in ARE activity, SG, and MDA concentrations correlated with the severity of neurological deficit and disability.

Increased Number of Mast Cells in Atherosclerotic Lesions Correlates with the Presence of Myeloid but not Plasmacytoid Dendritic Cells as well as Pro-inflammatory T Cells.

BACKGROUND: Atherosclerosis is an inflammatory disease of the vessel wall promoted by different immune cells and inflammatory mediators. METHODS: In this study, 26 human plaques and 12 control vessels without atherosclerosis were immunohistochemically stained to analyze the emergence of mast cells dependent on plaque morphology and to correlate mast cell occurrence with the emergence of myeloid as well as plasmacytoid dendritic cells. Also, mast cell emergence was correlated with the number of pro-inflammatory T cells. For this, plaques were classified as stable or unstable according to established histological criteria. RESULTS: As expected, atherosclerotic lesions showed significantly higher numbers of tryptase+, chymase+, and cathepsin G+ mast cells compared to control vessels, particularly in lesions with unstable morphology. As a novel finding, we detected significant correlations between mast cells and myeloid dendritic cells (fascin, CD83, r > 0.3, p < 0.01), but not plasmacytoid dendritic cells (CD123, CD304). Also, we observed significant correlations of mast cells and different subgroups of pro-inflammatory T cells (CD3, CD8, CD161, CD25; r > 0.35, p < 0.05). CONCLUSIONS: Overall, the higher number of mast cells in plaques, particularly with unstable morphology, suggests that mast cells might be involved in the progression of atherosclerosis. The correlation of mast cells with other immune cells that are pivotal in atherogenesis, e.g., myeloid dendritic cells and pro-inflammatory T cells, also suggests an interplay leading to plaque destabilization. Therefore, modulating local mast cell function and invasion into the plaque might be a therapeutic tool for plaque stabilization.