Caffeic Acid Prevents Vascular Oxidative Stress and Atherosclerosis against Atherosclerogenic Diet in Rats

Diet and lifestyle play a crucial role in the progress of some cardiovascular disorders (CVDs). Rising interest in natural products and their pharmacological investigations witnessed therapeutic potential against CVDs. Caffeic acid (CA) is an organic composite hydroxycinnamic acid derivative classified among phenolics. It is a secondary metabolite biosynthesized in all plant species in the form of ester conjugates. The reported pharmacological activities of CA are neuroprotective, cardioprotective, hypoglycemic, antioxidant, and immunomodulatory properties. This work is aimed to examine the outcome of CA in atherogenic diet- (Ath-) induced rat model on lipid profile changes and endothelium function. The method involves a study duration of 35 days utilizing (n = 6) male Wistar rats (180–200 g) that were fed either normal chow or Ath. Study groups are given (i) normal chow diet, (ii) Ath, (iii) Ath + CA (25 or 50 mg/kg, p.o.), (iv) normal chow diet + CA (50 mg/kg, p.o.), and (v) Ath + Atorvastatin (ATORVA) (5 mg/kg, p.o.). Blood samples were collected at the end of the study to measure serum lipid profile, alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, and tissue oxidative stress level. Hemodynamic parameters and aorta staining were performed. CA treatment ameliorated lipid profile and significantly reduced the oxidative stress level. Aorta staining examination revealed a marked reduction of the atherosclerotic lesions. These findings suggested that CA is an effective treatment approach for preventing atherosclerotic lesion progression attributed to protection against oxidative stress and various enzymatic activities in the Ath model.

MEF2A Is the Trigger of Resveratrol Exerting Protection on Vascular Endothelial Cell

Both resveratrol and myocyte enhancer factor 2A (MEF2A) may protect vascular endothelial cell (VEC) through activating the expression of SIRT1. However, the relationship between resveratrol and MEF2A is unclear. We aimed to investigate the deeper mechanism of resveratrol in protecting vascular endothelial cells and whether MEF2A plays a key role in the protective function of resveratrol. Human umbilical vein endothelial cell (HUVEC) was used for in vitro study, and small interfere RNA was used for silencing MEF2A. Silencing MEF2A in the vascular endothelium (VE) of ApoE−/− mice was performed by tail injection with adeno associated virus expressing si-mef2a-shRNA. The results showed that treatment of HUVEC with resveratrol significantly up-regulated MEF2A, and prevented H2O2-induced but not siRNA-induced down-regulation of MEF2A. Under various experimental conditions, the expression of SIRT1 changed with the level of MEF2A. Resveratrol could rescue from cell apoptosis, reduction of cell proliferation and viability induced by H2O2, but could not prevent against that caused by silencing MEF2A with siRNA. Silencing MEF2A in VE of apoE−/− mice decreased the expression of SIRT1, increased the plasma LDL-c, and abrogated the function of resveratrol on reducing triglyceride. Impaired integrity of VE and aggravated atherosclerotic lesion were observed in MEF2A silenced mice through immunofluorescence and oil red O staining, respectively. In conclusion, resveratrol enhances MEF2A expression, and the upregulation of MEF2A is required for the endothelial protective benefits of resveratrol in vitro via activating SIRT1. Our work has also explored the in vivo relevance of this signaling pathway in experimental models of atherosclerosis and lipid dysregulation, setting the stage for more comprehensive phenotyping in vivo and further defining the molecular mechanisms.

Histopathology-Based Deep-Learning Predicts Atherosclerotic Lesions in Intravascular Imaging

Background: Optical coherence tomography is a powerful modality to assess atherosclerotic lesions, but detecting lesions in high-resolution OCT is challenging and requires expert knowledge. Deep-learning algorithms can be used to automatically identify atherosclerotic lesions, facilitating identification of patients at risk. We trained a deep-learning algorithm (DeepAD) with co-registered, annotated histopathology to predict atherosclerotic lesions in optical coherence tomography (OCT).Methods: Two datasets were used for training DeepAD: (i) a histopathology data set from 7 autopsy cases with 62 OCT frames and co-registered histopathology for high quality manual annotation and (ii) a clinical data set from 51 patients with 222 OCT frames in which manual annotations were based on clinical expertise only. A U-net based deep convolutional neural network (CNN) ensemble was employed as an atherosclerotic lesion prediction algorithm. Results were analyzed using intersection over union (IOU) for segmentation.Results: DeepAD showed good performance regarding the prediction of atherosclerotic lesions, with a median IOU of 0.68 ± 0.18 for segmentation of atherosclerotic lesions. Detection of calcified lesions yielded an IOU = 0.34. When training the algorithm without histopathology-based annotations, a performance drop of >0.25 IOU was observed. The practical application of DeepAD was evaluated retrospectively in a clinical cohort (n = 11 cases), showing high sensitivity as well as specificity and similar performance when compared to manual expert analysis.Conclusion: Automated detection of atherosclerotic lesions in OCT is improved using a histopathology-based deep-learning algorithm, allowing accurate detection in the clinical setting. An automated decision-support tool based on DeepAD could help in risk prediction and guide interventional treatment decisions.

Pleiotropic Effects of the Protease-Activated Receptor 1 (PAR1) Inhibitor, Vorapaxar, on Atherosclerosis and Vascular Inflammation

Background: Protease-activated receptor 1 (PAR1) and toll-like receptors (TLRs) are inflammatory mediators contributing to atherogenesis and atherothrombosis. Vorapaxar, which selectively antagonizes PAR1-signaling, is an approved, add-on antiplatelet therapy for secondary prevention. The non-hemostatic, platelet-independent, pleiotropic effects of vorapaxar have not yet been studied. Methods and Results: Cellular targets of PAR1 signaling in the vasculature were identified in three patient cohorts with atherosclerotic disease. Evaluation of plasma biomarkers (n = 190) and gene expression in endomyocardial biopsies (EMBs) (n = 12) revealed that PAR1 expression correlated with endothelial activation and vascular inflammation. PAR1 colocalized with TLR2/4 in human carotid plaques and was associated with TLR2/4 gene transcription in EMBs. In addition, vorapaxar reduced atherosclerotic lesion size in apolipoprotein E–knock out (ApoEko) mice. This reduction was associated with reduced expression of vascular adhesion molecules and TLR2/4 presence, both in isolated murine endothelial cells and the aorta. Thrombin-induced uptake of oxLDL was augmented by additional TLR2/4 stimulation and abrogated by vorapaxar. Plaque-infiltrating pro-inflammatory cells were reduced in vorapaxar-treated ApoEko mice. A shift toward M2 macrophages paralleled a decreased transcription of pro-inflammatory cytokines and chemokines. Conclusions: PAR1 inhibition with vorapaxar may be effective in reducing residual thrombo-inflammatory event risk in patients with atherosclerosis independent of its effect on platelets.

Chronic unilateral arm lymphedema correlates with increased intima-media thickness in the brachial artery

Summary: Drainage of the arterial wall via adventitial lymphatic vessels has been shown to play a pivotal role for vessel wall homeostasis. Also, retrograde cholesterol transport is ensured via this route, but no studies exist to demonstrate that lymphatic stasis would represent a mechanism to initiate atherosclerotic lesion formation in human arteries. To test this hypothesis, we embarked on a simple clinical experiment, assessing wall thickness in limb arteries with lymphedema after surgical intervention, with the contralateral limb serving as control. Using ultrasound imaging, the differential thickness was assessed separately for the three arterial wall layers. The potential of disease progression by lymphostasis was addressed by depiction of longitudinal results according to the time after lymph dissection.

DEVELOPMENT OF PERORAL HYPOLIPIDEMIC FORMULATION BASED ON SULFATED ARABINOGALACTAN IN THE FORM OF POTASSIUM SALT

An original heparinoid, sulfated arabinogalactan in the form of potassium salt, possessing anticoagulant and hypolipidemic activities, has been developed at the A.E. Favorsky Irkutsk Institute of Chemistry Siberian Branch of the Russian Academy of Sciences.The aim was to develop solid peroral dose forms (capsules and film-coated tablets) for the prevention and treatment of atherosclerotic lesion of blood vessels on the basis of potassium salt of sulfated arabinogalactan which would be suitable for further clinical trials of these forms.Materials and methods. The following materials were used in the work: sulfated arabinogalactan in the form of potassium salt, obtained at the A.E. Favorsky Irkutsk Institute of Chemistry Siberian Branch of the Russian Academy of Sciences; Ludipress®; AEROSIL® 200 Pharma; calcium stearate; Aquacoat ECD. The powder mixtures were briquetted followed by tableting and application of the finished film coating Aquacoat ECD, and encapsulation in hard gelatin capsules.Results. Composition and technological characteristics of capsules and film-coated tablets were determined using physico-chemical and technological properties of sulfated arabinogalactan in the form of potassium salt. Technological parameters and quality indicators were determined for the solid pharmaceutical dose forms in accordance with the requirements of the State Pharmacopoeia of the Russian Federation of the XIVth edition. Conclusion. The optimum compositions and technology for the preparation of capsules and film-coated tablets based on potassium salt of sulfated arabinogalactan for the prevention and treatment of atherosclerotic lesion of blood vessels, were developed. The data obtained were used for the regulatory documentation design.

An Inhibitor of Grp94 Inhibits OxLDL-Induced Autophagy and Apoptosis in VECs and Stabilized Atherosclerotic Plaques

Background: Oxidized low-density lipoprotein (oxLDL) induces vascular endothelial cell (VEC) injury and atherosclerosis through activating endoplasmic reticulum stress. Expression of glucose-regulated protein 94 (Grp94) is induced by endoplasmic reticulum stress and Grp94 is involved in cardiovascular diseases. This study aimed to determine the role of Grp94 in oxLDL-induced vascular endothelial cell injury and atherosclerosis.Methods and Results: An inhibitor of Grp94, HCP1, was used to investigate the role of Grp94 in oxLDL-induced VEC injury in human umbilical vein endothelial cells and atherosclerosis in apolipoprotein E−/− mice. Results showed that HCP1 inhibited autophagy and apoptosis induced by oxLDL in VECs. And we found that Grp94 might interact with adenosine monophosphate-activated protein kinase (AMPK) and activate its activity. HCP1 inhibited AMPK activity and overexpression of Grp94 blocked the effect of HCP1. Besides, HCP1 activated the activity of mechanistic target of rapamycin complex 1 (mTORC1), co-treatment with AMPK activator acadesine eliminated the effect of HCP1 on mTORC1 activity as well as autophagy. In apolipoprotein E−/− mice, HCP1 suppressed autophagy and apoptosis of atherosclerotic plaque endothelium. In addition, HCP1 increased the content of collagen, smooth muscle cells, and anti-inflammatory macrophages while reducing the activity of MMP-2/9 and pro-inflammatory macrophages in the atherosclerotic lesion.Conclusion: HCP1 inhibited oxLDL-induced VEC injury and promoted the stabilization of atherosclerotic plaque in apoE−/− mice. Grp94 might be a potential therapeutic target in the clinical treatment of atherosclerosis.

The cellular biology of atherosclerosis with atherosclerotic lesion classification and biomarkers

Background Atherosclerosis is a chronic lipid-driven inflammatory disease with infiltration of low-density lipoprotein and is considered as the pivotal step in plaque formation. The aim of the review is to get into the fine details of pathophysiologic mechanisms responsible for atherosclerosis with atherosclerotic lesion classification. It also provides a summary of current biomarkers other than the traditional risk factors so that new treatment modalities can emerge and reduce the morbidity and mortality associated with atherosclerosis. Main body In the classification of atherosclerosis made by American Heart Association (AHA), AHA Type I lesion is the earliest vascular change described microscopically. AHA Type II lesion is primarily composed of abundant macrophages. AHA Type III lesion is the earliest of progressive lesions, while AHA Type IV lesion consists of an acellular necrotic core. Various biomarkers are implicated in different stages of the pathophysiological mechanism of plaque formation and evolution. C Reactive Protein plays a direct role in promoting the inflammatory component of atherosclerosis. Fibrinogen was demonstrated to be elevated among patients with acute thrombosis. Higher leukocyte count is associated with a greater cardiovascular risk. Cytokines have been implicated in atheroma formation and complications. High rates of protease activated receptor expression are also induced by interleukin-6 secretion in atherosclerotic lesions and areas of vascular tissue injury. Cluster of differentiation 40 receptor and its ligand have been also detected in atherosclerotic plaques. Osteopontin, acidic phosphoprotein, and osteoprotegerin have emerged as novel markers of atherosclerotic plaque composition. There are also overproductions of matrix metalloproteinases in the rupture-prone regions and promote lipid-necrotic core formation in the atherosclerotic plaque. Myeloperoxidase has been proposed as a marker of plaque instability. Oxidized low-density lipoprotein receptor 1 provides a route of entry for oxidized low-density lipoprotein into the endothelium. A human atherosclerotic lesion also expresses lipoprotein-associated phospholipase A2. Short conclusion Atherosclerotic plaques are the battlefield between an unbalanced immune response and lipid accumulation in the intima of arteries. Most of the biomarkers associated with atherosclerosis are indicators of inflammatory response and will also be used for medical purposes.

Thymosin β4 preserves vascular smooth muscle phenotype in atherosclerosis via regulation of Low Density Lipoprotein Related Protein 1 (LRP1)

Objective: Atherosclerosis is a progressive, degenerative vascular disease and a leading cause of morbidity and mortality. In response to endothelial damage, platelet derived growth factor (PDGF)-BB induced phenotypic modulation of medial smooth muscle cells (VSMCs) promotes atherosclerotic lesion formation and destabilisation of the vessel wall. VSMC sensitivity to PDGF-BB is determined by endocytosis of Low density lipoprotein receptor related protein 1 (LRP1)-PDGFRβ complexes to balance receptor recycling with lysosomal degradation. Consequently, LRP1 is implicated in various arterial diseases. Having identified Tβ4 as a regulator of LRP1-mediated endocytosis to protect against aortic aneurysm, we sought to determine whether Tβ4 may additionally function to protect against atherosclerosis, by regulating LRP1-mediated growth factor signalling. Approach and Results: By single cell transcriptomic analysis, Tmsb4x, encoding Tβ4, strongly correlated with contractile gene expression and was significantly down-regulated in cells that adopted a modulated phenotype in atherosclerosis. We assessed susceptibility to atherosclerosis of global Tβ4 knockout mice using the ApoE-/- hypercholesterolaemia model. Inflammation, elastin integrity, VSMC phenotype and signalling were analysed in the aortic root and descending aorta. Tβ4KO; ApoE-/- mice develop larger atherosclerotic plaques than control mice, with medial layer degeneration characterised by accelerated VSMC phenotypic modulation. Defects in Tβ4KO; ApoE-/- mice phenocopied those in VSMC-specific LRP1 nulls and, moreover, were underpinned by hyperactivated LRP1-PDGFRβ signalling. Conclusions: We identify an atheroprotective role for endogenous Tβ4 in maintaining differentiated VSMC phenotype via LRP1-mediated PDGFRβ signalling.