Soluble CD40 Levels in Plasma Are Associated with Cardiovascular Disease and in Carotid Plaques with a Vulnerable Phenotype

Background and Purpose CD40 and CD40 ligand (CD40L) are costimulatory molecules of the tumor necrosis factor receptor superfamily and well known for their involvement in inflammatory diseases: atherosclerotic mouse models with disrupted CD40 signalling develop lesions of reduced size with a more stable plaque profile. This study investigated the potential of plasma and intraplaque levels of CD40 and CD40L as markers for cardiovascular disease (CVD) in humans and their association with plaque stability.Methods Soluble CD40 and CD40L (sCD40L) were measured in plasma in 1,437 subjects from The SUrrogate markers for Micro- and Macro-vascular hard endpoints for Innovative diabetes Tools (SUMMIT) cohort. Intra-plaque levels of sCD40 and sCD40L were measured in atherosclerotic plaque homogenates from 199 subjects of the Carotid Plaque Imaging Project (CPIP) cohort. Results Both plasma sCD40 and sCD40L levels were elevated in individuals with prevalent stroke, while sCD40 levels also were higher in individuals with a prior acute myocardial infarction. Plasma levels of sCD40 correlated with carotid intima-media thickness and total carotid plaque area and were associated with risk of cardiovascular events over a 3-year follow-up period. Intra-plaque levels of sCD40 and sCD40L were associated with plaque components characteristic for plaque vulnerability and extracellular matrix remodelling.Conclusions Higher plasma sCD40 and sCD40L levels are associated with prevalent CVD. Plasma sCD40 levels also correlate with the severity of carotid atherosclerosis and predict future cardiovascular events, while intra-plaque levels correlate with a vulnerable plaque phenotype. Our findings thus demonstrate that elevated levels of sCD40 and sCD40L are markers of CVD.

Two-faced Janus: the dual role of macrophages in atherosclerotic calcification

Calcification is an independent predictor of atherosclerosis-related cardiovascular events. Microcalcification is linked to inflamed, unstable lesions, in comparison to the fibrotic stable plaque phenotype generally associated with advanced calcification. This paradox relates to recognition that calcification presents in a wide spectrum of manifestations that differentially impact plaque’s fate. Macrophages, the main inflammatory cells in atherosclerotic plaque, have a multifaceted role in disease progression. They crucially control the mineralization process, from microcalcification to the osteoid metaplasia of bone-like tissue. It is a bilateral interaction that weighs heavily on the overall plaque fate but remains rather unexplored. This review highlights current knowledge about macrophage phenotypic changes in relation to and interaction with the calcifying environment. On the one hand, macrophage-led inflammation kickstarts microcalcification through a multitude of interlinked mechanisms, which in turn stimulates phenotypic changes in vascular cell types to drive microcalcification. Macrophages may also modulate the expression/activity of calcification inhibitors and inducers, or eliminate hydroxyapatite nucleation points. Contrarily, direct exposure of macrophages to an early calcifying milieu impacts macrophage phenotype, with repercussions for plaque progression and/or stability. Macrophages surrounding macrocalcification deposits show a more reparative phenotype, modulating extracellular matrix, and expressing osteoclast genes. This phenotypic shift favours gradual displacement of the pro-inflammatory hubs; the lipid necrotic core, by macrocalcification. Parallels to bone metabolism may explain many of these changes to macrophage phenotype, with advanced calcification able to show homeostatic osteoid metaplasia. As the targeted treatment of vascular calcification developing in atherosclerosis is thus far severely lacking, it is crucial to better understand its mechanisms of development.

Astragaloside Attenuates Atherosclerosis Coupled Inflammation Through miR-101/Mitogen-Activated Protein Kinase Phosphatase-1 (MKP-1)/p38 Signaling in Mice

This study aimed at elucidating the effect of astragaloside on atherosclerosis coupled inflammation and potential mechanism in mice. C57BL/6J mice were maintained in high-fat diet (HFD) for 12 weeks to induce atherosclerosis, with or without treatment with astragaloside (50 mg/kg). In turn, serum biochemical parameters in mice were also evaluated. Multiple tissue stain assay, including HE, were employed to assess the pathological alterations in arteries, and blood inflammation mediators were examined using ELISA. Expressions of microRNA101 (miR-101), p-p38 and mitogen-activated protein kinase phosphatase-1 (MKP-1) in the arteries were evaluated by qPCR and Western blot. Finally, AML-193 cells were transfected by miR-101 mimics and inhibitors. Expression of miR-101, MKP-1 and downstream inflammation cytokines were then analyzed. High-fat diet (HFD) mice with astragaloside treatment exhibited reduced atherosclerotic plaques size evaluated by oil red o, improved hepatocyte steatosis, and increased collagen fibers in atherosclerotic plaques for more stable plaque. Further, astragaloside treatment suppressed miR-101 transcription and enhanced MKP-1 expression, thus restraining the secretion of inflammation factors in vitro. Moreover, the inhibited impact of astragaloside in inflammatory factors production was ineffective in the presence of miR-101 mimics in AML-193 cells stimulated by LPS. Astragaloside exerted an anti-inflammatory role through miR-101/MKP-1/p38 signaling, for reducing atherosclerotic plaques and alleviate inflammation damage in mice and AML-193 cell.

SPECT/CT imaging of inflammation and calcification in human carotid atherosclerosis to identify the plaque at risk of rupture

Background Calcification and inflammation are atherosclerotic plaque compositional biomarkers that have both been linked to stroke risk. The aim of this study was to evaluate their co-existing prevalence in human carotid plaques with respect to plaque phenotype to determine the value of hybrid imaging for the detection of these biomarkers. Methods Human carotid plaque segments, obtained from endarterectomy, were incubated in [111In]In-DOTA-butylamino-NorBIRT ([111In]In-Danbirt), targeting Leukocyte Function-associated Antigen-1 (LFA-1) on leukocytes. By performing SPECT/CT, both inflammation from DANBIRT uptake and calcification from CT imaging were assessed. Plaque phenotype was classified using histology. Results On a total plaque level, comparable levels of calcification volume existed with different degrees of inflammation and vice versa. On a segment level, an inverse relationship between calcification volume and inflammation was evident in highly calcified segments, which classify as fibrocalcific, stable plaque segments. In contrast, segments with little or no calcification presented with a moderate to high degree of inflammation, often coinciding with the more dangerous fibrous cap atheroma phenotype. Conclusion Calcification imaging alone can only accurately identify highly calcified, stable, fibrocalcific plaques. To identify high-risk plaques, with little or no calcification, hybrid imaging of calcification and inflammation could provide diagnostic benefit.

Regularities of plaque stabilization in various scenarios of neointimal calcification and vascularization

Aim. To study the relationships between phenotypes of extracranial arteries' plaques (stable/unstable), their calcification and its causes, in particular, vascularization.Material and methods. The study included 88 patients: patients (n=44) with ischemic stroke and those (n=44) with chronic brain ischemia. In all subjects, the parameters of systemic mineral homeostasis were assessed (total and ionized calcium, phosphate, total protein, albumin, and calcification propensity). Atherosclerotic plaques have been obtained during carotid endarterectomy, fixed in formalin, postfixed in 1% osmium tetroxide, stained in 2% osmium tetroxide, dehydrated in ascending ethanol series and acetone, stained with 2% alcoholic uranyl acetate and embedded into epoxy resin with its further polymerization. Epoxy resin blocks were grinded, polished, counterstained with Reynolds' lead citrate and sputter coated with carbon. Sample visualization was performed employing backscattered scanning electron microscopy. Number and area of calcium deposits and neointimal vessels were quantified using ImageJ. Statistical analysis was carried out using Mann-Whitney U-test and Spearman's rank correlation coefficient.Results. It was found that area of neointimal calcification, but not number of calcium deposits, was associated with the stable plaque phenotype. The stabilizing effect of calcification was manifested in retarding stenosis associated with plaque rupture and stroke. Calcification extent directly correlated with total and local plaque vascularization, which have been associated with unstable and stable plaque phenotype, respectively. In addition, plaque calcification negatively correlated with total protein and albumin, thereby reflecting the impaired systemic mineral homeostasis.Conclusion. Atherosclerotic plaque calcification and active local vascularization reduce stenosis extent and stabilize plaque. In contrast, total plaque calcification contributes to the atherosclerosis progression and promotes major acute cardiovascular events.

Relationship of Neutrophil-to-Lymphocyte Ratio with Carotid Plaque Vulnerability and Occurrence of Vulnerable Carotid Plaque in Patients with Acute Ischemic Stroke

Background. Carotid plaque is an undefined risk factor in ischemic stroke and is driven by inflammation. Mounting evidence suggests that neutrophil-to-lymphocyte ratio (NLR) is crucial not only for cerebrovascular events but also in atherosclerosis progression. Here, we aimed to explore the association between the admission NLR and carotid plaque vulnerability as well as the occurrence of vulnerable carotid plaque detected by carotid ultrasonography in patients with acute ischemic stroke (AIS) among Chinese. Methods. We conducted a retrospective study composed of 588 patients with AIS and 309 healthy controls free of carotid plaque in the Department of Neurology in The Second Hospital of Lanzhou University from March 2014 to February 2015. All patients were classified as nonplaque, stable plaque, and vulnerable plaque groups on the basis of carotid ultrasonography results. The baseline information was collected and compared among the four different groups. The correlation between variables and carotid plaque vulnerability was tested by Spearman linear correlation analysis. To identify the independent predictors for vulnerable carotid plaque, univariate and multivariate logistic regression analysis was performed. Results. The comparisons of age, sex proportion, history of hypertension, diabetes, and smoking, the levels of HDL-C, Lp(a), BMI, SBP, DBP, Fib, CRP, leukocyte, and NLR among the four groups showed a statistically significant difference ( P < 0.05 ); in particular, the NLR was significantly higher in the vulnerable plaque group as compared to the control ( P = 0.043 ), nonplaque ( P = 0.022 ), and stable plaque groups ( P = 0.015 ). The Spearman correlation analysis presented a positive correlation between carotid plaque vulnerability and age ( r = 0.302 ; P < 0.001 ), SBP ( r = 0.163 ; P < 0.001 ), and NLR ( r = 0.087 ; P = 0.034 ), while the lymphocyte was negatively related to the carotid plaque vulnerability ( r = − 0.089 ; P = 0.030 ). The multivariate logistic regression analysis adjusted for confounding factors revealed that age (odds ratio [OR], 1.042; 95% confidence interval [CI], 1.025-1.060; P < 0.001 ), male gender (OR, 2.005; 95% CI, 1.394-2.884; P < 0.001 ), diabetes (OR, 1.481; 95% CI, 1.021-2.149; P = 0.039 ), SBP (OR, 1.012; 95% CI, 1.003-1.021; P = 0.010 ), and NLR (OR, 1.098; 95% CI, 1.018-1.184; P = 0.015 ) are independent predictors of vulnerable carotid plaque in patients with AIS. Conclusion. The admission NLR is a novel and meaningful biomarker that can be used in predicting carotid plaque vulnerability and the presence of vulnerable carotid plaque assessed by carotid ultrasonography in patients with AIS among Chinese.

Evaluation of capillary density in psoriasis: An intrapatient study and literature review

Background Dilated and tortuous vessels within elongated dermal papillae represent a histopathological clue of psoriasis. However, the number of dilated capillaries (capillary density) in psoriasis remains undefined as the results from the available studies differ significantly. Objectives To evaluate the capillary density in psoriasis using dermoscopy and horizontal histopathological sections (HHS), two techniques that share the horizontal view of the skin, and to compare the results with the existing data. Methods Twenty adult patients with stable plaque psoriasis were enrolled and, in each patient, a target area of the examined plaque, previously engraved by gently rotating a 5-mm biopsy punch device, underwent dermoscopy and biopsy for HHS. In all examined fields, capillary density was evaluated in a centered 4-mm diameter area, counting the number of red dots at dermoscopy and of dermal papillae at HHS. Results A total of 20 target lesions located on the trunk, arms and tights were evaluated. The mean capillary density resulting from dermoscopy was 43.02±6.60/mm 2 whereas that from HHS was 50.30±9.05/mm 2. These data showed a statistically significant difference (p = 0.006), with a strong correlation at Pearson’s test (r = 0.88). Conclusions Our results when compared with those from the existing literature showed some differences. The peculiarity of our work is represented by the precise measurement and correlation of the capillary density using two different methods, as the preliminary skin engraving allowed a perfect match between the area undergoing dermoscopy and that of skin sampling for HHS. Compared to dermoscopy in which deep-located vessels might have gone undetected, HHS seems to reflect more precisely and reliably the real capillary density showing an average of 50 capillaries/mm 2 that in a common 5x5 cm psoriatic patch corresponds to an average of 125.000 capillaries. These results highlight the extraordinary potential of psoriatic skin to develop such a complex and intricate vascular network.

Deficiency of endothelial CD40 induces a stable plaque phenotype and limits inflammatory cell recruitment to atherosclerotic lesions in mice

Objectives: The co-stimulatory CD40L-CD40 dyad exerts a critical role in atherosclerosis by modulating leukocyte accumulation into developing atherosclerotic plaques. The requirement for cell-type specific expression of both molecules, however, remains elusive. Here, we evaluate the contribution of CD40 expressed on endothelial cells (ECs) in a mouse model of atherosclerosis. Approach & Results: Atherosclerotic plaques of Apolipoprotein E deficient (Apoe-/-) mice and humans displayed increased expression of CD40 on ECs compared to controls. To interrogate the role of CD40 on ECs in atherosclerosis, we induced EC-specific (BmxCreERT2-driven) deficiency of CD40 in Apoe-/- mice. After feeding a chow diet for 25 weeks, EC-specific deletion of CD40 (iEC-CD40) ameliorated plaque lipid deposition and lesional macrophage accumulation but increased intimal smooth muscle cell and collagen content, while atherosclerotic lesion size did not change. Leukocyte adhesion to the vessel wall was impaired in iEC-CD40-deficient mice as demonstrated by intravital microscopy. In accord, expression of vascular adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1 in the vascular endothelium declined after deletion of CD40. In vitro, antibody-mediated inhibition of human endothelial CD40 significantly abated monocyte adhesion on ECs. Conclusions: Endothelial deficiency of CD40 in mice promotes structural features associated with a stable plaque phenotype in humans and decreases leukocyte adhesion. These results suggest that endothelial-expressed CD40 contributes to inflammatory cell migration and consecutive plaque formation in atherogenesis.

Effects of Nicorandil on Inflammation, Apoptosis and Atherosclerotic Plaque Progression

Nicorandil, a balanced vasodilator, is used in the second-line therapy of angina pectoris. In this study, we aimed to illuminate the effects of nicorandil on inflammation, apoptosis, and atherosclerotic plaque progression. Twenty-five LDL-R -/- mice were fed a high-fat diet for 14 weeks. After 6 weeks mice were randomly allocated to treatment with nicorandil (10 mg/kg/day) or tap water. Nicorandil treatment led to a more stable plaque phenotype, displaying an increased thickness of the fibrous cap (p = 0.014), a significant reduction in cholesterol clefts (p = 0.045), and enhanced smooth muscle cell content (p = 0.009). In endothelial cells nicorandil did not reduce the induction of adhesion molecules or proinflammatory cytokines. In H2O2 challenged endothelial cells, pretreatment with nicorandil significantly reduced the percentage of late apoptotic/necrotic cells (p = 0.016) and the ratio of apoptotic to living cells (p = 0.036). Atherosclerotic lesions of animals treated with nicorandil exhibited a significantly decreased content of cleaved caspase-3 (p = 0.034), lower numbers of apoptotic nuclei (p = 0.040), and reduced 8-oxogunanine staining (p = 0.039), demonstrating a stabilizing effect of nicorandil in established atherosclerotic lesions. We suggest that nicorandil has a positive effect on atherosclerotic plaque stabilization by reducing apoptosis.