Development Of Atherosclerotic Lesions Research Articles

Keypoints in Cells and Atherogenesis

Endothelial dysfunction is the initial event in the atherogenic process, resulting from several events, such as expression of leukocyte binding sites, production of growth factors, chemotactic and vasoreactive molecules, ability to oxidize low-density lipoprotein (LDL) and respond to oxidized lipoproteins, ability to express procoagulant activity, and modulation of vascular permeability. Thus, the endothelium, when subjected to different conditions and factors, plays an active role in the development of atherosclerotic plaque. The expression of several adhesion molecules, such as intracellular adhesion molecule-1 (ICAM-1), E-selectin, vascular cell adhesion molecule-1 (VCAM-1), P-selectin in atherosclerotic plaques, mediating the interaction between endothelial cells and leukocytes, has been described. The modification of LDLs, in conjunction with other chemotactic factors produced by injured endothelial cells, recruit circulating monocytes into the subendothelial space, where they become macrophages. The proliferation of smooth muscle cells is an important event in the progression of arterial injury, occurring in 3 stages: smooth muscle cell replication still within the media layer; migration from the middle layer to the intima and proliferation within the intima. Apoptotic cells were evidenced in the atherosclerotic lesion, suggesting that apoptosis is part of the normal vascular healing process. The final stage of atherosclerotic lesion development is conversion of the fibrotic lesion to an advanced lesion, a lesion in which a thrombus forms as a result of plaque ulceration or intraplaque hemorrhage.

Antioxidant and Anti-Inflammatory Functions of High-Density Lipoprotein in Type 1 and Type 2 Diabetes.

(1) Background: high-density lipoproteins (HDLs) exhibit antioxidant and anti-inflammatory properties that play an important role in preventing the development of atherosclerotic lesions and possibly also diabetes. In turn, both type 1 diabetes (T1D) and type 2 diabetes (T2D) are susceptible to having deleterious effects on these HDL functions. The objectives of the present review are to expound upon the antioxidant and anti-inflammatory functions of HDLs in both diabetes in the setting of atherosclerotic cardiovascular diseases and discuss the contributions of these HDL functions to the onset of diabetes. (2) Methods: this narrative review is based on the literature available from the PubMed database. (3) Results: several antioxidant functions of HDLs, such as paraoxonase-1 activity, are compromised in T2D, thereby facilitating the pro-atherogenic effects of oxidized low-density lipoproteins. In addition, HDLs exhibit diminished ability to inhibit pro-inflammatory pathways in the vessels of individuals with T2D. Although the literature is less extensive, recent evidence suggests defective antiatherogenic properties of HDL particles in T1D. Lastly, substantial evidence indicates that HDLs play a role in the onset of diabetes by modulating glucose metabolism. (4) Conclusions and perspectives: impaired HDL antioxidant and anti-inflammatory functions present intriguing targets for mitigating cardiovascular risk in individuals with diabetes. Further investigations are needed to clarify the influence of glycaemic control and nephropathy on HDL functionality in patients with T1D. Furthermore, exploring the effects on HDL functionality of novel antidiabetic drugs used in the management of T2D may provide intriguing insights for future research.

Cholesterol trafficking, lysosomal function, and atherosclerosis.

Despite years of study and major research advances over the past 50 years, atherosclerotic diseases continue to rank as the leading global cause of death. Accumulation of cholesterol within the vascular wall remains the main problem and represents one of the early steps in the development of atherosclerotic lesions. There is a complex relationship between vesicular cholesterol transport and atherosclerosis, and abnormalities in cholesterol trafficking can contribute to the development and progression of the lesions. The dysregulation of vesicular cholesterol transport and lysosomal function fosters the buildup of cholesterol within various intracytoplasmic compartments, including lysosomes and lipid droplets. This, in turn, promotes the hallmark formation of foam cells, a defining feature of early atherosclerosis. Multiple cellular processes, encompassing endocytosis, exocytosis, intracellular trafficking, and autophagy, play crucial roles in influencing foam cell formation and atherosclerotic plaque stability. In this review, we highlight recent advances in the understanding of the intricate mechanisms of vesicular cholesterol transport and its relationship with atherosclerosis and discuss the importance of understanding these mechanisms in developing strategies to prevent or treat this prevalent cardiovascular disease.

Characterization of a novel model for atherosclerosis imaging: the apolipoprotein E-deficient rat

BackgroundThe apolipoprotein E-deficient (apoE−/−) mouse is a well-established model for studying atherosclerosis. However, its small size limits its use in longitudinal positron emission tomography (PET) imaging studies. Recently, the apoE−/− rat has emerged as an alternative. With this study, we investigate the feasibility of using apoE−/− rats as an in vivo model for longitudinal atherosclerotic PET/CT imaging.ResultsApoE−/− rats showed significantly higher [18F]FDG uptake than controls in the aortic arch (+ 18.5%, p < 0.001) and abdominal aorta (+ 31.0%, p < 0.001) at weeks 12, 26, and 51. ApoE−/− rats exhibited hypercholesterolemia, as evidenced by plasma cholesterol levels that were up to tenfold higher, and total hepatic cholesterol levels that were up to threefold higher than the control rats at the end of the study. Fast protein liquid chromatography cholesterol profiling indicated very high levels of pro-atherogenic apoB-containing very low-density lipoprotein and low-density lipoprotein fractions in the apoE−/− rats. Atherosclerotic lesions cover 19.9% of the surface of the aortic arch (p = 0.0013), and there was a significantly higher subendothelial accumulation of ED1-positive macrophages in the abdominal aorta of the apoE−/− rats compared to control rats (Ctrl) (p = 0.01). No differences in neutral sterols were observed but higher levels of bile acids were found in the apoE−/− rats.ConclusionThese data demonstrate early signs of hypercholesterolemia, high levels of bile acids, the development of atherosclerotic lesions, and macrophage accumulation in apoE−/− rats. Therefore, this model shows promise for atherosclerosis imaging studies.

Dapagliflozin decreases atherosclerotic plaque instability via regulating macrophage pyroptosis

Abstract Background Vulnerable plaques are characterized by infiltration of inflammatory cells, playing a key role in the progression of acute coronary events. It’s important to clarify the inflammatory mechanism of unstable plaque formation. Several clinical trials have demonstrated that dapagliflozin could reduce major adverse cardiac events in whether diabetic or non-diabetic patients. However, the underlying cardioprotective mechanism of dapagliflozin remains unclear. This study was aimed to investigate the role of dapagliflozin in regulating macrophage pyroptosis and vulnerable plaque formation. Methods 20 ApoE-/- mice (control) were fed with high fat diet while another 20 ApoE-/- mice were challenged with high fat diet plus dapagliflozin for 12 weeks. The extent and instability of atherosclerotic plaque was determined by oil-red staining, HE staining, immunofluorescence staining and electron microscopy. Changes in subsets of immune cells were evaluated by flow cytometry. Plasma cytokines were assessed by ELISA. Microarray analysis was applied to detect gene expressions while Western blot and real-time PCR was used to assess gene expression levels. Results Morphology studies revealed that dapagliflozin could inhibit plaque formation and reduce instability in ApoE-/- mice. FACS data showed that dapagliflozin could decrease CD11b+Ly6Chigh M1 macrophages differentiation and inhibit foam cells formation in ApoE-/- mice. Microarray analysis and in vitro studies exhibited that dapagliflozin could induce the down regulation of NLRP3, caspase-1, IL-1β, IL-18 and MMP-7/10/12/14 to retard macrophage pyroptosis and foam cell formation. Conclusions We have characterized a novel role for dapagliflozin in modulating atherosclerotic lesion development and progression. We envision that this study may provide several potential therapeutic targets for treatment of acute coronary syndromes.

Abstract 17384: Unraveling the Impact of VAMP-8 Deficiency on Atherogenesis: A Fresh Perspective on Platelet Involvement

Introduction and Objective: Recognized for their intricate role in atherogenesis, platelets interact with oxidized low-density lipoprotein (oxLDL), immune cells, and endothelial cells and release granule proteins and microparticles, contributing to the onset and advancement of atherosclerosis. Despite the challenges of antiplatelet treatment in primary prevention, exploring alternative platelet targets could provide key insights into their role in atherogenesis. This study pivots on the soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) proteins, notably vesicle associated membrane protein 8 (VAMP-8) - the primary vesicle membrane (v-SNARE) and vital facilitator of platelet secretion. Approach and Results: The profound impairment in α-granule, dense granule, and lysosome release due to VAMP-8 deficiency directed our investigation towards its potential role in atherogenesis. Using RNA-seq data from washed platelets of VAMP-8-/- mice versus wildtypes, we first analyzed the molecular signature of VAMP-8 deficiency. Gene ontology analysis revealed that the major differences in enriched genes between the two groups involved triglyceride biosynthetic process and platelet calcium homeostasis. We initiated atherosclerosis studies using ApoE -/- mice crossed with VAMP-8 -/- mice (n=11) alongside littermate controls (n=9), all fed a high cholesterol diet for 15 weeks. Remarkably, the deletion of VAMP-8 led to a substantial reduction in atherosclerotic lesion development when compared to the control group. Notably, this phenotype manifested irrespective of total cholesterol levels, as plasma cholesterol remained consistent across all groups. Conclusion: Our findings reveal that VAMP8 deficiency markedly mitigates atherogenesis, thereby proposing a novel perspective to explore the influence of platelet cargo secretion on the inception of atherosclerosis. We are currently conducting additional studies to uncover the underlying mechanisms responsible for these observations

Abstract 17634: CCN2 Deficiency in Vascular Smooth Muscle Cells Drives the Progression of Atherosclerosis

Introduction: Atherogenesis involves a complex interaction between immune cells and lipids, processes greatly influenced by the vascular smooth muscle cells (VSMCs) phenotype. Cellular communication network factor 2 (CCN2) is a matricellular protein with an established role in maintaining tissue and organ homeostasis. Various studies have demonstrated the importance of CCN2 in regulating cardiovascular diseases, however its role in atherosclerosis remains to be investigated. Hypothesis: SMC-restricted CCN2 deficiency exacerbates atherosclerosis development in murine models through the modulation of SMC phenotype switching. Aim: Determine the role of VSMC-derived CCN2 in regulating atherosclerosis progression. Methods: In this study, CCN2 was deleted in VSMCs using Cre-lox technology. To induce atherosclerosis, mice were rendered LDLR deficient via AAV8-PCSK9 injection, followed by administration of a high-fat diet for 20 weeks. Aortic atherosclerotic lesion development was then assessed along with single-cell RNA sequencing to map the molecular effects of CCN2 deficiency on the VSMCs phenotype. Gain and loss of function studies in VSMCs were performed to assess the impact of CCN2 alteration on VSMC phenotype switching. Results: SMC-specific CCN2 knockout (SMC-CCN2-KO) mice demonstrated exquisite susceptibility to atherosclerosis formation as evidenced by a significant increase of aortic lipid-rich plaques in aortic roots, arch, thoracic and abdominal aorta. Concomitant with this, a profound vascular inflammation was seen in SMC-CCN2-KO mice. Importantly, these phenotypic changes were associated with a dramatic shift in VSMC phenotype towards a proliferating, lipid-accumulating and macrophage-like cell phenotype. Mechanistic studies point to the requisite role of the KLF4 pathway in VSMC transdifferentiation in SMC-CCN2-KO mice, findings corroborated by cell culture experiments in which KLF4-dependent phenotypic changes were observed in CCN2-null human primary aortic VSMCs. Conclusions: Our findings show that CCN2 deficiency in VSMCs promotes atherosclerosis development through affecting the VSMC phenotypic transition into macrophage-like cells by activation of the KLF4 signaling pathway.

Abstract 17310: Epsins Promote Atherosclerosis Through Vascular Smooth Muscle Phenotypic Modulation

Hypothesis: VSMC play crucial roles in atherosclerosis via phenotypic switching. The trans-differentiation of VSMC into other cell types might contribute to atherosclerotic lesion development, progression, and the subsequent diseases such as myocardial infarction or stroke. Epsins, a family of endocytic adaptors, are crucial for atherosclerosis development and progression; yet, the role of epsins in VSMC phenotypic modulation is unknown. Methods and Results: To decipher the role of VSMC epsins in regulating atheroma development and progression, we created WT and SMC-specific inducible epsin1/2 double knockout (SMC-iDKO) mice on ApoE -/- background fed a western diet. Using single-cell RNA-seq analysis, we found VSMC and EC population significantly changed in SMC-iDKO/ApoE -/- mice compared to ApoE -/- mice. Using immunofluorescent and FACS analysis, we observed that both VSMC and EC marker genes expressions were up-regulated in DKO-VSMCs indicating that DKO-SMCs were maintaining their SMC phenotype and may further trans-differentiating into ECs via a process called MEndoT, mesenchymal to endothelial transition. Given the critical role of KLF4 in regulating VSMCs phenotype during atheroma progression, we observed diminished KLF4 expression in DKO-SMCs compared to WT treated with oxLDL. Furthermore, we found that epsins interacted with KLF4 with Epsin-UIM domain and their interaction improved KLF4 stability and promoted KLF4 to transfer into nuclei. In this study, we found that epsins were highly expressed and positively correlated with the lesion severity in VSMCs in atherosclerotic. Using immunofluorescent and Oil Red O stainings, we observed reduced lipid accumulation, lesion size and macrophage infiltration but elevated VSMCs in the cap of lesions in SMC-iDKO/ApoE -/- mice compared with ApoE -/- mice. Conclusions: In conclusions, we demonstrated an unexpected role of epsins in regulating phenotypic switching by repressing SM-contractile and EC marker genes expression through an epigenetic regulatory mechanism. Our data suggest that epsins may be a therapeutic target for treating occlusive vascular diseases, and uncover regulatory pathways for therapeutic targeting of SMC transitions in atherosclerotic cardiovascular disease.

Single-cell transcriptomics reveals subtype-specific molecular profiles in Nrf2-deficient macrophages from murine atherosclerotic aortas.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcriptional regulator of antioxidant and anti-inflammatory response in all cell types. It also activates the transcription of genes important for macrophage function. Nrf2 activity declines with age and has been closely linked to atherosclerosis, but its specific role in this vascular pathology is not clear. Atherosclerotic plaques contain several macrophage subsets with distinct, yet not completely understood, functions in the lesion development. The aim of this study was to analyze the transcriptome of diverse Nrf2-deficient macrophage subpopulations from murine atherosclerotic aortas. Mice with transcriptionally inactive Nrf2 in Cdh5-expressing cells (Nrf2 Cdh5tKO) were used in the experiments. These mice lack transcriptional Nrf2 activity in endothelial cells, but also in a proportion of leukocytes. We confirmed that the bone marrow-derived and tissue-resident macrophages isolated from Nrf2 Cdh5tKO mice exhibit a significant decline in Nrf2 activity. Atherosclerosis was induced in Nrf2 Cdh5tKO and appropriate control mice via adeno-associated viral vector (AAV)-mediated overexpression of murine proprotein convertase subtilisin/kexin type 9 (Pcsk9) in the liver and high-fat diet feeding. After 21 weeks, live aortic cells were sorted on FACS and single-cell RNA sequencing (scRNA-seq) was performed. Unsupervised clustering singled out 13 distinct aortic cell types. Among macrophages, 9 subclusters were identified. Differential gene expression analysis revealed cell subtype-specific expression patterns. A subset of inflammatory macrophages from atherosclerotic Nrf2 Cdh5tKO mice demonstrated downregulation of DNA replication genes (e.g. Mcm7, Lig1, Pola1) concomitant with upregulation of DNA damage sensor Atr gene. Atherosclerotic Nrf2 Cdh5tKO Lyve1+ resident macrophages showed strong upregulation of IFN-stimulated genes, as well as changes in the expression of death pathways-associated genes (Slc40a1, Bcl2a1). Furthermore, we observed subtype-specific expression of core ferroptosis genes (e.g. Cp, Hells, Slc40a1) in inflammatory versus tissue resident macrophages. This observation suggested a link between ferroptosis and inflammatory microenvironment appearing at a very early stage of atherogenesis. Our findings indicate that Nrf2 deficiency in aortic macrophages leads to subtype-specific transcriptomic changes associated with inflammation, iron homeostasis, cell injury or death pathways. This may help understanding the role of aging-associated decline of Nrf2 activity and the function of specific macrophage subtypes in atherosclerotic lesion development.

Nogo-B mediates endothelial oxidative stress and inflammation to promote coronary atherosclerosis in pressure-overloaded mouse hearts

AimsEndothelial dysfunction plays a pivotal role in atherosclerosis, but the detailed mechanism remains incomplete understood. Nogo-B is an endoplasmic reticulum (ER)-localized protein mediating ER-mitochondrial morphology. We previously showed endothelial Nogo-B as a key regulator of endothelial function in the setting of hypertension. Here, we aim to further assess the role of Nogo-B in coronary atherosclerosis in ApoE−/− mice with pressure overload. Methods and resultsWe generated double knockout (DKO) mouse models of systemically or endothelium-specifically excising Nogo-A/B gene on an ApoE−/− background. After 7 weeks of transverse aortic constriction (TAC) surgery, compared to ApoE−/− mice DKO mice were resistant to the development of coronary atherosclerotic lesions and plaque rapture. Sustained elevation of Nogo-B and adhesion molecules (VCAM-1/ICAM-1), early markers of atherosclerosis, was identified in heart tissues and endothelial cells (ECs) isolated from TAC ApoE−/− mice, changes that were significantly repressed by Nogo-B deficiency. In cultured human umbilical vein endothelial cells (HUVECs) exposure to inflammatory cytokines (TNF-α, IL-1β), Nogo-B was upregulated and activated reactive oxide species (ROS)-p38-p65 signaling axis. Mitofusin 2 (Mfn2) is a key protein tethering ER to mitochondria in ECs, and we showed that Nogo-B expression positively correlated with Mfn2 protein level. And Nogo-B deletion in ECs or in ApoE−/− mice reduced Mfn2 protein content and increased ER-mitochondria distance, reduced ER-mitochondrial Ca2+ transport and mitochondrial ROS generation, and prevented VCAM-1/ICAM-1 upregulation and EC dysfunction, eventually restrained atherosclerotic lesions development. ConclusionOur study revealed that Nogo-B is a critical modulator in promoting endothelial dysfunction and consequent pathogenesis of coronary atherosclerosis in pressure overloaded hearts of ApoE−/− mice. Nogo-B may hold the promise to be a common therapeutic target in the setting of hypertension.

IL-1 Beta-A Biomarker for Ischemic Stroke Prognosis and Atherosclerotic Lesions of the Internal Carotid Artery.

Background and Objectives: Stroke is a leading cause of mortality and morbidity worldwide. Treatment of this pathology is still under development and its risk factors remain to be determined. Therefore, we aim to determine the role of interleukin-1 beta in atherosclerotic lesions of the internal carotid artery as a risk factor for stroke and the role of this biomarker in stroke prognosis. Materials and Methods: This study enrolled 56 patients diagnosed with ischemic stroke in the anterior vascular territory (AVT) and posterior vascular territory (PVT). All the patients had venous blood collected at admission and 7 days after the onset of the cerebral ischemia in order to determine the plasma concentration of interleukin-1 beta. At the same time, an extracranial carotid ultrasound was performed. Results: The interleukin-1 beta collected at admission was positively correlated with the NIHSS at admission (Pearson index 0.424), and both measurements were correlated with carotid stenosis (Spearmen correlation index of 0.529 and 0.653, respectively). Conclusions: Interleukin-1 beta could be a reliable biomarker for stroke prognosis and the development of atherosclerotic lesions of the internal carotid.

GIP_HUMAN [22-51] Peptide Encoded by the Glucose-Dependent Insulinotropic Polypeptide (GIP) Gene Suppresses Insulin Expression and Secretion in INS-1E Cells and Rat Pancreatic Islets.

GIP_HUMAN [22-51] is a recently discovered peptide that shares the same precursor molecule with glucose-dependent insulinotropic polypeptide (GIP). In vivo, chronic infusion of GIP_HUMAN [22-51] in ApoE-/- mice enhanced the development of aortic atherosclerotic lesions and upregulated inflammatory and proatherogenic proteins. In the present study, we evaluate the effects of GIP_HUMAN [22-51] on insulin mRNA expression and secretion in insulin-producing INS-1E cells and isolated rat pancreatic islets. Furthermore, we characterize the influence of GIP_HUMAN [22-51] on cell proliferation and death and on Nf-kB nuclear translocation. Rat insulin-producing INS-1E cells and pancreatic islets, isolated from male Wistar rats, were used in this study. Gene expression was evaluated using real-time PCR. Cell proliferation was studied using a BrdU incorporation assay. Cell death was quantified by evaluating histone-complexed DNA fragments. Insulin secretion was determined using an ELISA test. Nf-kB nuclear translocation was detected using immunofluorescence. GIP_HUMAN [22-51] suppressed insulin (Ins1 and Ins2) in INS-1E cells and pancreatic islets. Moreover, GIP_HUMAN [22-51] promoted the translocation of NF-κB from cytoplasm to the nucleus. In the presence of a pharmacological inhibitor of NF-κB, GIP_HUMAN [22-51] was unable to suppress Ins2 mRNA expression. Moreover, GIP_HUMAN [22-51] downregulated insulin secretion at low (2.8 mmol/L) but not high (16.7 mmol/L) glucose concentration. By contrast, GIP_HUMAN [22-51] failed to affect cell proliferation and apoptosis. We conclude that GIP_HUMAN [22-51] suppresses insulin expression and secretion in pancreatic β cells without affecting β cell proliferation or apoptosis. Notably, the effects of GIP_HUMAN [22-51] on insulin secretion are glucose-dependent.

The Complementary Effects of Dabigatran Etexilate and Exercise Training on the Development and Stability of the Atherosclerotic Lesions in Diabetic ApoE Knockout Mice.

Aim: To determine the complementary effects of dabigatran etexilate (DE), exercise training (ET), and combination (DE + ET) on the development and stability of the atherosclerotic lesions in diabetic apoE knockout (apoE-/-) mice. Methods: In 48 male apoE-/- diabetic mice, streptozotocin (STZ) was induced for 5 consecutive days. Mice received a high-fat diet (HFD) for 8 weeks and then were randomized into four groups (1. Control/CG, 2. DEG: HFD with DE, 3. ETG: ET on treadmill, 4. DE + ETG: combination DE and ET treatment). At the end of the eighth week, all mice were euthanatized and morphometry of the aortic lesions at the level of aortic valve was obtained. Collagen, elastin, MCP-1, TNF-a, matrix metalloproteinases (MMP-2,-3,-9), and TIMP-1 concentrations within plaques at the aortic valve were determined. Results: All active groups had significantly smaller aorta stenosis (DEG:7.9 ± 2.2%, ETG:17.3 ± 5.3%, DE + ETG:7.1 ± 2.7%) compared to CG (23.3 ± 5.5% p < 0.05), reduced the relative intra-plaque content of MCP-1, macrophages, MMP-3, and MMP-9, and considerably increased collagen, elastin, and TIMP-1 (p < 0.05). Group 4 showed the most pronounced results (p < 0.05). Both DEG and DE + ETG significantly reduced MMP-2 and TNF-a concentrations compared to ETG and CG (p < 0.010). Conclusion: DE and ET treatment of diabetic apoE-/- mice resulted in complementary amelioration of atherosclerotic lesions development and stability, mediated by the anti-inflammatory modulation of both DE and ET.

Metyrapone Treatment Protects Low-Density Lipoprotein Receptor Knockout Mice against Hypercorticosteronemia Development without Changing Atherosclerosis Susceptibility.

The steroid 11beta-hydroxylase inhibitor metyrapone is able to effectively reverse the hypercortisolemia detected in human Cushing's Syndrome patients. In this current preclinical study, we investigated whether metyrapone monotherapy can also reverse the hypercortisolemia-associated increase in atherosclerotic cardiovascular disease risk. In this instance, female low-density lipoprotein receptor knockout mice fed a cholic acid-containing high cholesterol/high fat diet to induce the development of hypercorticosteronemia and atherosclerotic lesions were treated twice daily with 100 mg/kg metyrapone for 4 weeks. Metyrapone effectively protected against hypercorticosteronemia development with endpoint plasma corticosterone levels remaining 43% lower than in controls (p < 0.01). Gene expression analysis in livers and adrenals validated that glucocorticoid receptor signaling was also reduced. Importantly, metyrapone treatment did not impact plasma cholesterol levels or alter atherosclerotic plaque areas or lesional collagen contents. However, metyrapone induced significant systemic lymphocytopenia as evident from marked decreases in splenic white pulp contents and thymus weights (-48% and -41%, respectively; p < 0.001). In conclusion, we have shown that treatment with metyrapone diminishes hypercorticosteronemia without affecting atherosclerosis susceptibility in cholic acid-containing high cholesterol/high fat diet-fed low-density lipoprotein receptor knockout mice. These preclinical findings highlight that restoring plasma glucocorticoid levels to normal is not necessarily sufficient to overcome the cardiovascular co-morbidities associated with human Cushing's disease.

Ethanolic Extract of Moringa oleifera Leaves Reduces The Expression of ICAM-1 in Aortic Tissues of Metabolic Syndrome Induced Rattus norvegicus

Introduction: Metabolic syndrome poses a risk for the development of atherosclerotic lesions, which are associated with various cardiovascular diseases. Moringa leaves are known for their antioxidant and anti-inflammatory properties. ICAM-1 serves as an early marker of atherosclerosis. This study aims to investigate how the dosage of ethanolic extract from Moringa leaves affects the expression of ICAM-1 in the aortic tissue of Wistar rats with metabolic syndrome. Methods: This study employed a laboratory experimental design with a posttest only control group. The research involved 30 Wistar rats, divided into five groups: the normal control group (G1), the positive control group (G2), and three experimental groups (G3, G4, and G5). The experimental groups (G3, G4, and G5) were induced with metabolic syndrome and administered ethanolic extract of Moringa leaves at doses of 150 mg/kg, 250 mg/kg, and 350 mg/kg, respectively. The expression of aortic ICAM-1 was evaluated using the Intensity Distribution Score (IDS) and analyzed using one-way ANOVA and Post-hoc Tukey HSD test. Results: Increasing the dosage of ethanolic leaf extract of Moringa oleifera reduces the IDS value of ICAM-1 in the aortic tissue of Wistar rats with a significance of p&lt;0.05.. Conclusion: The higher dosage of ethanolic leaf extract from Moringa oleifera Lamk has the potential to decrease ICAM-1 expression in the aortic tissue of Wistar rats with metabolic syndrome. The author hopes that Moringa leaf extract can be utilized as a supplement to prevent the formation of atherosclerotic plaques and cardiovascular diseases resulting from atherosclerotic plaques, such as thromboembolism and stroke.

Inhibition of GSDMD activation by Z-LLSD-FMK or Z-YVAD-FMK reduces vascular inflammation and atherosclerotic lesion development in ApoE-/- mice.

Pyroptosis is a form of pro-inflammatory cell death that can be mediated by gasdermin D (GSDMD) activation induced by inflammatory caspases such as caspase-1. Emerging evidence suggests that targeting GSDMD activation or pyroptosis may facilitate the reduction of vascular inflammation and atherosclerotic lesion development. The current study investigated the therapeutic effects of inhibition of GSDMD activation by the novel GSDMD inhibitor N-Benzyloxycarbonyl-Leu-Leu-Ser-Asp(OMe)-fluoromethylketone (Z-LLSD-FMK), the specific caspase-1 inhibitor N-Benzyloxycarbonyl-Tyr-Val-Ala-Asp(OMe)-fluoromethylketone (Z-YVAD-FMK), and a combination of both on atherosclerosis in ApoE-/- mice fed a western diet at 5weeks of age, and further determined the efficacy of these polypeptide inhibitors in bone marrow-derived macrophages (BMDMs). In vivo studies there was plaque formation, GSDMD activation, and caspase-1 activation in aortas, which increased gradually from 6 to 18weeks of age, and increased markedly at 14 and 18weeks of age. ApoE-/- mice were administered Z-LLSD-FMK (200µg/day), Z-YVAD-FMK (200µg/day), a combination of both, or vehicle control intraperitoneally from 14 to 18weeks of age. Treatment significantly reduced lesion formation, macrophage infiltration in lesions, protein levels of vascular cell adhesion molecule-1 and monocyte chemoattractant protein-1, and pyroptosis-related proteins such as activated caspase-1, activated GSDMD, cleaved interleukin(IL)-1β, and high mobility group box 1 in aortas. No overt differences in plasma lipid contents were detected. In vitro treatment with these polypeptide inhibitors dramatically decreased the percentage of propidium iodide-positive BMDMs, the release of lactate dehydrogenase and IL-1β, and protein levels of pyroptosis-related proteins both in supernatants and cell lysates elevated by lipopolysaccharide + nigericin. Notably however, there were no significant differences in the above-mentioned results between the Z-LLSD-FMK group and the Z-YVAD-FMK group, and the combination of both did not yield enhanced effects. These findings indicate that suppression of GSDMD activation by Z-LLSD-FMK or Z-YVAD-FMK reduces vascular inflammation and lesion development in ApoE-/- mice.

Modification of low-density lipoproteins by low molecular weight carbonyl products of free-radical oxidation of lipids and carbohydrates plays a key role in atherosclerotic lesion of the vascular wall and in endothelial dysfunction

The review presents evidence of the participation of low-density lipoproteins (LDL) modified by low molecular weight dicarbonyl compounds formed during freeradical oxidation of lipids (malondialdehyde) and carbohydrates in the development of endothelial dysfunction and atherosclerotic vascular lesions. The authors believe that it is they, and not oxidized (hydroperoxide-containing) LDL, that are the main factors of pathogenesis. The role of dicarbonyl-modified LDL in LOX-1 dependent induction of processes leading to the development of endothelial dysfunction is discussed. The results of studies proving that damage to the glycocalyx (a layer of macromolecules that prevent the development of endothelial dysfunction) covering the luminal surface of the endothelium is caused by hyperproduction of reactive oxygen species. Ways of pharmacological correction of free-radical oxidation processes are discussed, due to which inhibition of atherogenesis and diabetogenesis can be achieved.

Endothelial Gata6 deletion reduces monocyte recruitment and proinflammatory macrophage formation and attenuates atherosclerosis through Cmpk2-Nlrp3 pathways

Endothelial dysfunction results in chronic vascular inflammation, which is critical for the development of atherosclerotic diseases. Transcription factor Gata6 has been reported to regulate vascular endothelial cell activation and inflammation in vitro. Here, we aimed to explore the roles and mechanisms of endothelial Gata6 in atherogenesis.Endothelial cell (EC) specific Gata6 deletion was generated in the ApoeKO hyperlipidemic atherosclerosis mouse model. Atherosclerotic lesion formation, endothelial inflammatory signaling, and endothelial-macrophage interaction were examined in vivo and in vitro by using cellular and molecular biological approaches. EC-GATA6 deletion mice exhibited a significant decrease in monocyte infiltration and atherosclerotic lesion compared to littermate control mice. Cytosine monophosphate kinase 2 (Cmpk2) was identified as a direct target gene of GATA6 and EC-GATA6 deletion decreased monocyte adherence, migration and pro-inflammatory macrophage foam cell formation through regulation of the CMPK2-Nlrp3 pathway. Endothelial target delivery of Cmpk2-shRNA by intercellular adhesion molecule 2 (Icam-2) promoter-driven AAV9 carrying the shRNA reversed the Gata6 upregulation mediated elevated Cmpk2 expression and further Nlrp3 activation and thus attenuated atherosclerosis. In addition, C–C motif chemokine ligand 5 (Ccl5) was also identified as a direct target gene of Gata6 to regulate monocyte adherence and migration influencing atherogenesis.This study provides direct in vivo evidence of EC-GATA6 involvement in the regulation of Cmpk2-Nlrp3, as well as Ccl5, on monocyte adherence and migration in atherosclerosis development and advances our understanding of the in vivo mechanisms of atherosclerotic lesion development, and meanwhile provides opportunities for future therapeutic interventions.

A cyclic azapeptide ligand of the scavenger receptor CD36/SR-B2 reduces the atherosclerotic lesion progression and enhances plaque stability in apolipoprotein E-deficient mice.

Atherosclerosis is a chronic inflammatory disease of the arterial walls that develops at predisposed sites. As a major risk factor for adverse cardiovascular pathology, atherosclerosis can progress to myocardial infarction and stroke, due to the rupture of unstable atherosclerotic lesions. Macrophage uptake of modified lipoproteins and metabolic dysfunction contributes significantly to the initiation and development of atherosclerotic lesions. The cluster of differentiation 36 receptor [CD36 (SR-B2)] plays a key role in atherosclerotic lesion progression and acts as an efferocytic molecule in the resolution of advanced plaque. In previous studies, linear azapeptide CD36 ligands were shown to exhibit anti-atherosclerotic properties. In the present study, a novel potent and selective macrocyclic azapeptide CD36 ligand, MPE-298, has proven effective in protecting against atherosclerosis progression. Features of greater plaque stability were observed after 8 weeks of daily injections with the cyclic azapeptide in apolipoprotein E-deficient mice fed a high-fat high-cholesterol diet.

Lipid Effects of Experimental Oophorectomy and Cardiovascular Risk Association

In view of the controversy between hormonal replacement in postmenopausal women and decreased cardiovascular morbidity and mortality, and the effects of estrogens associated with progesterones, or progesterone alone, we developed a protocol to evaluate the repercussions of hormone therapy with progesterone in oophorectomized rabbits submitted to atherogenic diet, on the development of atherosclerotic lesions induced in animals. Forty New Zealand rabbits were oophorectomized and divided into three groups. After 90 days, when the animals were sacrificed and the lesions that developed in the aorta and coronary arteries were evaluated. The results showed an increase in serum cholesterol in the four groups of animals. In groups II, III and IV, there was an increase of approximately fifteen times the baseline values, with no significant differences between these groups observed in 5 time courses. Regarding triglyceride levels, we found that group III showed a significant increase in values in Time 4, but this difference was not verified in Time 5. When quantitatively analyzing the area occupied by the lesion, we found that there were no statistically significant differences between the groups submitted to the atherogenic diet, observing that the coronary arteries presented a lower number of compromised arteries