Matrix metallopeptidase 9 contributes to the beginning of plaque and is a potential biomarker for the early identification of atherosclerosis in asymptomatic patients with diabetes.

Aims: To determine the roles of matrix metallopeptidase-9 (MMP9) on human coronary artery smooth muscle cells (HCASMCs) in vitro, early beginning of atherosclerosis in vivo in diabetic mice, and drug naïve patients with diabetes. Methods: Active human MMP9 (act-hMMP9) was added to HCASMCs and the expressions of MCP-1, ICAM-1, and VCAM-1 were measured. Act-hMMP9 (n=16) or placebo (n=15) was administered to diabetic KK.Cg-Ay/J (KK) mice. Carotid artery inflammation and atherosclerosis measurements were made at 2 and 10 weeks after treatment. An observational study of newly diagnosed drug naïve patients with type 2 diabetes mellitus (T2DM n=234) and healthy matched controls (n=41) was performed and patients had ultrasound of carotid arteries and some had coronary computed tomography angiogram for the assessment of atherosclerosis. Serum MMP9 was measured and its correlation with carotid artery or coronary artery plaques was determined. Results: In vitro, act-hMMP9 increased gene and protein expressions of MCP-1, ICAM-1, VCAM-1, and enhanced macrophage adhesion. Exogenous act-hMMP9 increased inflammation and initiated atherosclerosis in KK mice at 2 and 10 weeks: increased vessel wall thickness, lipid accumulation, and Galectin-3+ macrophage infiltration into the carotid arteries. In newly diagnosed T2DM patients, serum MMP9 correlated with carotid artery plaque size with a possible threshold cutoff point. In addition, serum MMP9 correlated with number of mixed plaques and grade of lumen stenosis in coronary arteries of patients with drug naïve T2DM. Conclusion: MMP9 may contribute to the initiation of atherosclerosis and may be a potential biomarker for the early identification of atherosclerosis in patients with diabetes. Clinical trial registration: https://clinicaltrials.gov, identifier NCT04424706.

Sorbs2 Deficiency and Vascular BK Channelopathy in Diabetes.

BACKGROUND: Vascular large conductance Ca2+-activated K+ (BK) channel, composed of the α-subunit (BK-α) and the ß1-subunit (BK-ß1), is a key determinant of coronary vasorelaxation and its function is impaired in diabetic vessels. However, our knowledge of diabetic BK channel dysregulation is incomplete. The Sorbs2 (Sorbin homology [SoHo] and Src homology 3 [SH3] domains-containing protein 2), is ubiquitously expressed in arteries, but its role in vascular pathophysiology is unknown. METHODS: The role of Sorbs2 in regulating vascular BK channel activity was determined using patch-clamp recordings, molecular biological techniques, and in silico analysis. RESULTS: Sorbs2 is not only a cytoskeletal protein but also an RNA-binding protein that binds to BK channel proteins and BK-α mRNA, regulating BK channel expression and function in coronary smooth muscle cells. Molecular biological studies reveal that the SH3 domain of Sorbs2 is necessary for Sorbs2 interaction with BK-α subunits, while both the SH3 and SoHo domains of Sorbs2 interact with BK-ß1 subunits. Deletion of the SH3 or SoHo domains abolishes the Sorbs2 effect on the BK-α/BK-ß1 channel current density. Additionally, Sorbs2 is a target gene of the Nrf2 (nuclear factor erythroid-2-related factor 2), which binds to the promoter of Sorbs2 and regulates Sorbs2 expression in coronary smooth muscle cells. In vivo studies demonstrate that Sorbs2 knockout mice at 4 months of age display a significant decrease in BK channel expression and function, accompanied by impaired BK channel Ca2+-sensitivity and BK channel-mediated vasodilation in coronary arteries, without altering their body weights and blood glucose levels. Importantly, Sorbs2 expression is significantly downregulated in the coronary arteries of db/db type 2 diabetic mice. CONCLUSIONS: Sorbs2, a downstream target of Nrf2, plays an important role in regulating BK channel expression and function in vascular smooth muscle cells. Vascular Sorbs2 is downregulated in diabetes. Genetic knockout of Sorbs2 manifests coronary BK channelopathy and vasculopathy observed in diabetic mice, independent of obesity and glucotoxicity.

EGR1 transcriptionally regulates SVEP1 to promote proliferation and migration in human coronary artery smooth muscle cells.

A low-frequency variant of sushi, von Willebrand factor type A, EGF, and pentraxin domain-containing protein 1 (SVEP1) is associated with the risk of coronary artery disease, as determined by a genome-wide association study. SVEP1 induces vascular smooth muscle cell proliferation and an inflammatory phenotype to promote atherosclerosis. In the present study, qRT‒PCR demonstrated that the mRNA expression of SVEP1 was significantly increased in atherosclerotic plaques compared to normal tissues. Bioinformatics revealed that EGR1 was a transcription factor for SVEP1. The results of the luciferase reporter assay, siRNA interference or overexpression assay, mutational analysis and ChIP confirmed that EGR1 positively regulated the transcriptional activity of SVEP1 by directly binding to its promoter. EGR1 promoted human coronary artery smooth muscle cell (HCASMC) proliferation and migration via SVEP1 in response to oxidized low-density lipoprotein (ox-LDL) treatment. Moreover, the expression level of EGR1 was increased in atherosclerotic plaques and showed a strong linear correlation with the expression of SVEP1. Our findings indicated that EGR1 binding to the promoter region drive SVEP1 transcription to promote HCASMC proliferation and migration.

Impact of coronary artery calcium on mortality and cardiovascular events in metabolic syndrome and diabetes among younger adults.

AIMS: Whether coronary artery calcium (CAC) testing in younger individuals with metabolic syndrome (MetS) and diabetes mellitus (DM) helps predict cardiovascular disease (CVD) and death independent of traditional risk factors (RFs) remains less clear. METHODS AND RESULTS: We pooled data obtained from 5174 individuals aged 38-55 years from the CARDIA (Coronary Artery Risk Development in Young Adults; n = 3047, year 20) and MESA (Multi-Ethnic Study of Atherosclerosis; n = 2127, Visit 1) studies who completed computed tomography of CAC. The mean age (SD) of participants (44.7% men) was 47.3 (4.2) years. Multivariable Cox proportional hazards regression models were used to estimate hazard ratios (HRs) of CVD, coronary heart disease (CHD), and all-cause death. There were 1085 participants (21.0%) with prevalent CAC at baseline. A total of 461 (8.9%) had DM, 1025 (19.8%) had MetS without DM, and 3688 (71.3%) had neither condition. Over a median follow-up of 14.2 years, 256 (5.0%) participants died, and 304 (5.9%) CVD and 188 (3.6%) CHD events occurred. The CAC score was independently associated with incident CVD in those with DM (HR: 95% CI; 1.22: 1.08-1.38), MetS (1.18: 1.08-1.31), and neither condition (1.36: 1.26-1.46). The corresponding HRs for CAC ≥ 100 were 2.70 (1.25-5.83), 3.29 (1.87-5.79), and 6.30 (4.02-9.86), respectively. Similar associations for CHD and death were found. The impact of CAC ≥ 100 on CVD and CHD was lower in the presence of DM (P interaction < 0.05). The association of CAC with all outcomes in individuals with DM remained significant after adjusting with haemoglobin A1c levels. CONCLUSION: Coronary artery calcium score is independently associated with cardiovascular events and death over nearly 15 years after screening at ages 38-55 years, with a less pronounced impact on CVD and CHD events in the presence of DM.

In this pooled cohort, we aimed to analyse the relationship between coronary artery calcium (CAC) and incidence of cardiovascular disease (CVD), coronary heart disease (CHD), and all-cause mortality among younger individuals with diabetes mellitus (DM), metabolic syndrome (MetS), and neither condition. The CAC score was independently associated with incident CVD, CHD, and all-cause mortality in those with DM, MetS, and neither condition. The impact of CAC ≥ 100 on CVD and CHD events was lower in the presence of DM. The association of CAC with all outcomes in individuals with DM remained significant after adjusting with haemoglobin A1c levels.

Novel plasma biomarkers of coronary artery calcium incidence or progression: Insights from the prospective multi-ethnic Dallas Heart Study cohort.

BACKGROUND AND AIMS: Identifying the association of novel plasma biomarkers with coronary artery calcium (CAC) incidence or progression may provide insights into the pathophysiology of atherogenesis and plaque formation. METHODS: Participants of the Dallas Heart Study (DHS), a multi-ethnic cohort of ambulatory individuals at low-intermediate risk for future atherosclerotic cardiovascular disease (ASCVD), who had their blood tested for 31 biomarkers reflecting multiple pathophysiological pathways, underwent 2 serial non-contrast computed tomography assessments for CAC a median ∼7 years apart. The collected biomarkers were explored for association with CAC incidence or progression using univariate and multivariate analysis. RESULTS: A total of 1424 participants were included; mean age 43 years, 39 % male, and nearly half African-American. Over a 7-year interval between the two CAC measurements, 340 participants (23.9 %) had CAC incidence or progression, 105 (7.4 %) with incident CAC, and 309 (21.7 %) with CAC progression. Although several plasma biomarkers were associated with CAC incidence or progression in a univariate model, only soluble intercellular adhesion molecule-1 (sICAM-1), related to atherosclerosis by the inflammatory pathway, remained independently associated in a multivariate model adjusted for traditional risk factors. CONCLUSIONS: Further studies are needed to characterize the role of sICAM-1 in CAC evolvement to establish whether it has a pivotal mechanistic contribution or is rather an innocent bystander. Alternate measures of coronary atherosclerosis may be needed to elucidate contributors to atherosclerosis incidence or progression.

Asymmetric Dimethylarginine Enables Depolarizing Spikes and Vasospasm in Mesenteric and Coronary Resistance Arteries.

BACKGROUND: Increased vasoreactivity due to reduced endothelial NO bioavailability is an underlying feature of cardiovascular disease, including hypertension. In small resistance arteries, declining NO enhances vascular smooth muscle (VSM) reactivity partly by enabling rapid depolarizing Ca2+-based spikes that underlie vasospasm. The endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA) is metabolized by DDAH1 (dimethylarginine dimethylaminohydrolase 1) and elevated in cardiovascular disease. We hypothesized ADMA might enable VSM spikes and vasospasm by reducing NO bioavailability, which is opposed by DDAH1 activity and L-arginine. METHODS: Rat isolated small mesenteric arteries and myogenic rat-isolated intraseptal coronary arteries (RCA) were studied using myography, VSM intracellular recording, Ca2+ imaging, and DDAH1 immunolabeling. Exogenous ADMA was used to inhibit NO synthase and a selective DDAH1 inhibitor, NG-(2-methoxyethyl) arginine, to assess the functional impact of ADMA metabolism. RESULTS: ADMA enhanced rat-isolated small mesenteric arteries vasoreactivity to the α1-adrenoceptor agonist, phenylephrine by enabling T-type voltage-gated calcium channel-dependent depolarizing spikes. However, some endothelium-dependent NO-vasorelaxation remained, which was sensitive to DDAH1-inhibition with NG-(2-methoxyethyl) arginine. In myogenically active RCA, ADMA alone stimulated depolarizing Ca2+ spikes and marked vasoconstriction, while NO vasorelaxation was abolished. DDAH1 expression was greater in rat-isolated small mesenteric arteries endothelium compared with RCA, but low in VSM of both arteries. L-arginine prevented depolarizing spikes and protected NO-vasorelaxation in rat-isolated small mesenteric artery and RCA. CONCLUSIONS: ADMA increases VSM electrical excitability enhancing vasoreactivity. Endothelial DDAH1 reduces this effect, and low levels of DDAH1 in RCAs may render them susceptible to endothelial dysfunction contributing to vasospasm, changes opposed by L-arginine.

Expression of PCT, BNP and Inflammatory Factors in Children with Kawasaki Disease and Their Correlation with Coronary Artery Lesions.

Background: Kawasaki disease (KD), as one of the most common vascular diseases in children, will cause the risk of coronary artery lesions (CAL) without treatment. This study is to explore the expression of procalcitonin (PCT), brain natriuretic peptide (BNP), tumor necrosis factor-α (TNF-α), C-reactive protein (CRP) and interleukin-6 (IL-6) in children with KD and their correlation with CAL. Methods: 86 KD children in Baoding Hospital of Beijing Children's Hospital were selected as the study subjects from January 2020 to June 2021. According to whether CAL occurred, they were divided into the CAL group (n=30) and NCAL group (n=56). The clinical data of the two groups were collected from the medical record system. The levels of PCT and BNP were detected by chemiluminescence microparticle assay, the CRP level was detected by immunoturbidimetry, and the levels of TNF-α and IL-6 were detected by flow immunofluorescence method. The relationship of PCT, BNP, and inflammatory factors with CAL in KD children was explored by Pearson correlation analysis. Results: The comparative result of clinical data showed no overt difference in gender, disease types, age and blood routine indexes between the two groups, except for coronary artery diameter (P >.05). The levels of PCT, BNP, CRP, TNF-α and IL-6 in CAL group were (1.70±0.39) µg/L, (289.21±29.78) ng/L, (83.16±17.35) mg/L, (9.38±1.23) pg/mL and (59.97±0.97) ng/mL, respectively. The levels of PCT, BNP, CRP, TNF-α and IL-6 in NCAL group were (1.04±0.18) µg/L, (170.85±23.58) ng/L, (69.70±16.64) mg/L, (6.32±0.73) pg/mL and (44.16±11.97) ng/mL, respectively. The levels of each index in the CAL group were notably higher than in the NCAL group (P < .001). Pearson correlation analysis revealed that PCT, BNP, CRP, TNF-α and IL-6 were positively correlated with CAL in KD children (r=0.829, 0.865, 0.823, 0.894, 0.784, P < .001). Conclusion: The increase of PCT, BNP, and inflammatory factors has a certain warning effect on CAL in KD children. In clinical practice, health care professionals should strengthen the detection of PCT, BNP and inflammatory factors in KD children, carry out early monitoring of CAL in children with high expression of biomarkers, and formulate personalized preventive intervention based on the disease progress, so as to reduce the risk of cardiovascular disease. However, due to the limitations of research conditions and methods, the sample size of this study is small, which may affect the reliability and representativeness of the conclusion. In order to provide a new direction for the clinical prevention and treatment of the disease, future work will improve the research design, expand the sample size, and carry out more in-depth exploration on the prediction of CAL in KD children.

Empagliflozin prevents oxidative stress in human coronary artery endothelial cells via the NHE/PKC/NOX axis.

BACKGROUND: Empagliflozin (EMPA) ameliorates reactive oxygen species (ROS) generation in human endothelial cells (ECs) exposed to 10 % stretch, but the underlying mechanisms are still unclear. Pathological stretch is supposed to stimulate protein kinase C (PKC) by increasing intracellular calcium (Ca2+), therefore activating nicotinamide adenine dinucleotide phosphate oxidase (NOX) and promoting ROS production in human ECs. We hypothesized that EMPA inhibits stretch-induced NOX activation and ROS generation through preventing PKC activation. METHODS: Human coronary artery endothelial cells (HCAECs) were pre-incubated for 2 h before exposure to cyclic stretch (5 % or 10 %) with either vehicle, EMPA or the PKC inhibitor LY-333531 or PKC siRNA. PKC activity, NOX activity and ROS production were detected after 24 h. Furthermore, the Ca2+ chelator BAPTA-AM, NCX inhibitor ORM-10962 or NCX siRNA, sodium/potassium pump inhibitor ouabain and sodium hydrogen exchanger (NHE) inhibitor cariporide were applied to explore the involvement of the NHE/Na+/NCX/Ca2+ in the ROS inhibitory capacity of EMPA. RESULTS: Compared to 5 % stretch, 10 % significantly increased PKC activity, which was reduced by EMPA and PKC inhibitor LY-333531. EMPA and LY-333531 showed a similar inhibitory capacity on NOX activity and ROS generation induced by 10 % stretch, which was not augmented by combined treatment with both drugs. PKC-ß knockdown inhibits the NOX activation induced by Ca2+ and 10 % stretch. BAPTA, pharmacologic or genetic NCX inhibition and cariporide reduced Ca2+ in static HCAECs and prevented the activation of PKC and NOX in 10%-stretched cells. Ouabain increased ROS generation in cells exposed to 5 % stretch. CONCLUSION: EMPA reduced NOX activity via attenuation of the NHE/Na+/NCX/Ca2+/PKC axis, leading to less ROS generation in HCAECs exposed to 10 % stretch.

Transient receptor potential channel TRPM4 favors oxidized low-density lipoprotein-induced coronary endothelial cell dysfunction via a mechanism involving ferroptosis.

Accelerating the repair of damaged endothelium can effectively inhibit the progression of atherosclerosis (AS). Transient receptor potential channel TRPM4 is a non-selective cation channel activated by internal Ca2+, which is expressed in endothelial cells. This study aimed to reveal the potential role of TRPM4 in AS along with the mechanism. Human coronary artery endothelial cells (HCAECs) induced by ox-LDL was regarded as an in vitro model. The impacts of TRPM4 knockdown on cellular inflammation response, oxidative stress, normal endothelial function and lipid peroxidation were evaluated. Given that ferroptosis promotes AS progression, the effects of TRPM4 on intracellular iron ions and ferroptosis-related proteins was determined. Afterwards, HCAECs were treated with ferroptosis inducer erastin, and the influence of ferroptosis in the cellular model was revealed. TRPM4 was elevated in response to ox-LDL treatment in HCAECs. TRPM4 knockdown reduced the inflammation response, oxidative stress and lipid peroxidation caused by ox-LDL, and maintained the normal function of HCAECs. Erastin treatment destroyed the impacts of TRPM4 knockdown that are beneficial for cells to resist ox-LDL, showing the enhancement of the above adverse factors. Together, this study found that TRPM4 knockdown reduced ox-LDL-induced inflammation, oxidative stress, and dysfunction in HCAECs, possibly via a mechanism involving Fe2+ and ferroptosis-related proteins.

Control of coronary vascular cell fate in development and regeneration.

The coronary vasculature consists of a complex hierarchal network of arteries, veins, and capillaries which collectively function to perfuse the myocardium. However, the pathways controlling the temporally and spatially restricted mechanisms underlying the formation of this vascular network remain poorly understood. In recent years, the increasing use and refinement of transgenic mouse models has played an instrumental role in offering new insights into the cellular origins of the coronary vasculature, as well as identifying a continuum of transitioning cell states preceding the full maturation of the coronary vasculature. Coupled with the emergence of single cell RNA sequencing platforms, these technologies have begun to uncover the key regulatory factors mediating the convergence of distinct cellular origins to ensure the formation of a collectively functional, yet phenotypically diverse, vascular network. Furthermore, improved understanding of the key regulatory factors governing coronary vessel formation in the embryo may provide crucial clues into future therapeutic strategies to reactivate these developmentally functional mechanisms to drive the revascularisation of the ischaemic adult heart.

EGFR activation differentially affects the inflammatory profiles of female human aortic and coronary artery endothelial cells.

Endothelial cells (EC) are key players in vascular function, homeostasis and inflammation. EC show substantial heterogeneity due to inter-individual variability (e.g. sex-differences) and intra-individual differences as they originate from different organs or vessels. This variability may lead to different responsiveness to external stimuli. Here we compared the responsiveness of female human primary EC from the aorta (HAoEC) and coronary arteries (HCAEC) to Epidermal Growth Factor Receptor (EGFR) activation. EGFR is an important signal integration hub for vascular active substances with physiological and pathophysiological relevance. Our transcriptomic analysis suggested that EGFR activation differentially affects the inflammatory profiles of HAoEC and HCAEC, particularly by inducing a HCAEC-driven leukocyte attraction but a downregulation of adhesion molecule and chemoattractant expression in HAoEC. Experimental assessments of selected inflammation markers were performed to validate these predictions and the results confirmed a dual role of EGFR in these cells: its activation initiated an anti-inflammatory response in HAoEC but a pro-inflammatory one in HCAEC. Our study highlights that, although they are both arterial EC, female HAoEC and HCAEC are distinguishable with regard to the role of EGFR and its involvement in inflammation regulation, what may be relevant for vascular maintenance but also the pathogenesis of endothelial dysfunction.

Diabetic state of human coronary artery endothelial cells results in altered effects of bone mesenchymal stem cell-derived extracellular vesicles.

Human bone mesenchymal stem cell-derived extracellular vesicles (HBMSC-EV) have been used successfully in animal models of myocardial ischemia, yet have dampened effects in metabolic syndrome through unknown mechanisms. This study demonstrates the basal differences between non-diabetic human coronary artery endothelial cells (HCAEC) and diabetic HCAEC (DM-HCAEC), and how these cells respond to the treatment of HBMSC-EV. HCAEC and DM-HCAEC were treated with HBMSC-EV for 6 h. Proteomics, western blot analysis, and tube formation assays were performed. Key metabolic, growth, and stress/starvation cellular responses were significantly altered in DM-HCAEC in comparison to that of HCAEC at baseline. Proteomics demonstrated increased phosphorus metabolic process and immune pathways and decreased RNA processing and biosynthetic pathways in DM-HCAEC. Similar to previous in vivo findings, HCAEC responded to the HBMSC-EV with regenerative and anti-inflammatory effects through the upregulation of multiple RNA pathways and downregulation of immune cell activation pathways. In contrast, DM-HCAEC had a significantly diminished response to HBMSC-EV, likely due to the baseline abnormalities in DM-HCAEC. To achieve the full benefits of HBMSC-EV and for a successful transition of this potential therapeutic agent to clinical studies, the abnormalities found in DM-HCAEC will need to be further studied.

NFIL3 aggravates human coronary artery endothelial cell injury by promoting ITGAM transcription in Kawasaki disease.

OBJECTIVE: High expression of nuclear factor interleukin-3 (NFIL3) and integrin Alpha M (ITGAM) was found in serum samples from Kawasaki disease (KD) patients through bioinformatics analysis. Hence, this study aimed to explore the biological functions of NFIL3 and ITGAM in KD serum-stimulated human coronary artery endothelial cells (HCAECs). METHODS: The differentially-expressed genes in KD were analyzed through bioinformatics analysis. Serum samples were obtained from 18 KD patients and 18 healthy volunteers, followed by detection of NFIL3 and ITGAM levels in KD serum. After HCAECs were transfected with sh-NFIL3, sh-ITGAM, or sh-NFIL3 + oe-ITGAM and underwent 24-h KD serum stimulation, cell viability and apoptosis and the levels of inflammation-related factors were measured. The binding between NFIL3 and ITGAM was validated by dual-luciferase and chromatin immunoprecipitation (ChIP) assays. RESULTS: NFIL3 and ITGAM were up-regulated in serum from KD patients and KD serum-stimulated HCAECs. Down-regulation of NFIL3 or ITGAM inhibited KD serum-induced cell apoptosis and inflammatory response of HCAECs and promoted cell viability. Mechanistically, NFIL3 promoted ITGAM transcription level. Up-regulation of ITGAM reversed the improvement of NFIL3 down-regulation on KD serum-induced HCAEC injury. CONCLUSION: NFIL3 aggravated KD serum-induced HCAEC injury by promoting ITGAM transcription, which provided new insights into the treatment of KD.

Effect of Flavonoids on MCP-1 Expression in Human Coronary Artery Endothelial Cells and Impact on MCP-1-Dependent Migration of Human Monocytes.

The monocyte chemoattractant protein-1 (MCP-1), also known as chemokine (CC motif) ligand 2 (CCL2), is involved in the formation, progression, and destabilization of atheromatous plaques. Flavonoids, found in fruits and vegetables, have been associated with various health-promoting properties, including antioxidant, anti-inflammatory, and cardioprotective effects. In the present study, the flavonoids quercetin, kaempferol, and luteolin, but not cannflavin A, were shown to substantially inhibit interleukin (IL)-1ß-induced MCP-1 mRNA and protein expression in human coronary artery endothelial cells (HCAEC). At the functional level, conditioned medium (CM) from IL-1ß-stimulated HCAEC caused an increase in the migration of THP-1 monocytes compared with CM from unstimulated HCAEC. However, this induction was suppressed when IL-1ß-treated HCAEC were coincubated with quercetin, kaempferol, or luteolin. The functional importance of MCP-1 in IL-1ß-induced monocyte migration was supported by experiments showing that neutralization of MCP-1 in the CM of IL-1ß-treated HCAEC led to a significant inhibition of migration. In addition, a concentration-dependent induction of monocyte migration in the presence of recombinant MCP-1 was demonstrated. Collectively, the flavonoids quercetin, kaempferol, and luteolin were found to exert potential antiatherogenic effects in HCAEC, challenging further studies with these compounds.

Non-Psychoactive Phytocannabinoids Inhibit Inflammation-Related Changes of Human Coronary Artery Smooth Muscle and Endothelial Cells.

Atherosclerosis is associated with vascular smooth muscle cell proliferation, chronic vascular inflammation, and leukocyte adhesion. In view of the cardioprotective effects of cannabinoids described in recent years, the present study investigated the impact of the non-psychoactive phytocannabinoids cannabidiol (CBD) and tetrahydrocannabivarin (THCV) on proliferation and migration of human coronary artery smooth muscle cells (HCASMC) and on inflammatory markers in human coronary artery endothelial cells (HCAEC). In HCASMC, CBD and THCV at nontoxic concentrations exhibited inhibitory effects on platelet-derived growth factor-triggered proliferation (CBD) and migration (CBD, THCV). When interleukin (IL)-1ß- and lipopolysaccharide (LPS)-stimulated HCAEC were examined, both cannabinoids showed a concentration-dependent decrease in the expression of vascular cell adhesion molecule-1 (VCAM-1), which was mediated independently of classical cannabinoid receptors and was not accompanied by a comparable inhibition of intercellular adhesion molecule-1. Further inhibitor experiments demonstrated that reactive oxygen species, p38 mitogen-activated protein kinase activation, histone deacetylase, and nuclear factor κB (NF-κB) underlie IL-1ß- and LPS-induced expression of VCAM-1. In this context, CBD and THCV were shown to inhibit phosphorylation of NF-κB regulators in LPS- but not IL-1ß-stimulated HCAEC. Stimulation of HCAEC with IL-1ß and LPS was associated with increased adhesion of monocytes, which, however, could not be significantly abolished by CBD and THCV. In summary, the results highlight the potential of the non-psychoactive cannabinoids CBD and THCV to regulate inflammation-related changes in HCASMC and HCAEC. Considering their effect on both cell types studied, further preclinical studies could address the use of CBD and THCV in drug-eluting stents for coronary interventions.

Cathelicidin (LL-37) causes expression of inflammatory factors in coronary artery endothelial cells of Kawasaki disease by activating TLR4-NF-κB-NLRP3 signaling.

BACKGROUND: Kawasaki disease (KD) is a type of vasculitis with an unidentified etiology. Cathelicidin (LL-37) may be involved in the development of the KD process; therefore, further research to investigate the molecular mechanism of LL-37 involvement in KD is warranted. METHODS: Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, NLRP3, and LL-37 in the sera of healthy subjects, children with KD, and children with pneumonia. Subsequently, human recombinant LL-37 or/and toll-like receptors 4 (TLR4)-specific inhibitor TAK-242 stimulated human coronary artery endothelial cells (HCAECs), CCK-8 was used to detect cell proliferation, flow cytometry to detect apoptosis, transmission electron microscopy to observe cytoskeletal changes, Transwell to measure cell migration ability, ELISA to detect inflammatory factor levels, Western blot analysis to analyze protein levels of toll-like receptors 4 (TLR4) and NF-κB p-65, and quantitative real-time polymerase chain reaction (qRT-PCR) to determine LL-37, NLRP3 mRNA levels. RESULTS: In this study, we found that the level of LL-37 was highly expressed in the serum of children with KD, and after LL-37 stimulation, apoptosis was significantly increased in HCAECs, and the expression levels of TLR4, NLRP3 and inflammatory factors in cells were significantly enhanced. Intervention with the TLR4-specific inhibitor TAK-242 significantly alleviated the LL-37 effects on cellular inflammation, TLR4, NLRP3 promotion effect. CONCLUSIONS: Our data suggest that LL-37 induces an inflammatory response in KD coronary endothelial cells via TLR4-NF-κB-NLRP3, providing a potential target for the treatment of KD.

Association of apolipoproteins C-I and C-II truncations with coronary heart disease and progression of coronary artery calcium: Multi-Ethnic Study of Atherosclerosis.

BACKGROUND AND AIMS: Higher truncated-to-native proteoform ratios of apolipoproteins (apo) C-I (C-I'/C-I) and C-II (C-II'/C-II) are associated with less atherogenic lipid profiles. We examined prospective relationships of C-I'/C-II and C-II'/C-II with coronary heart disease (CHD) and coronary artery calcium (CAC). METHODS: ApoC-I and apoC-II proteoforms were measured by mass spectrometry immunoassay in 5790 MESA baseline plasma samples. CHD events (myocardial infarction, resuscitated cardiac arrest, fatal CHD, n = 434) were evaluated for up to 17 years. CAC was measured 1-4 times over 10 years for incident CAC (if baseline CAC = 0), and changes (follow-up adjusted for baseline) in CAC score and density (if baseline CAC>0). RESULTS: C-II'/C-II was inversely associated with CHD (n = 434 events) after adjusting for non-lipid cardiovascular risk factors (Hazard ratio: 0.89 [95% CI: 0.81-0.98] per SD), however, the association was attenuated after further adjustment for HDL levels (0.93 [0.83-1.03]). There was no association between C-I'/C-I and CHD (0.98 [0.88-1.08]). C-II'/C-II was positively associated with changes in CAC score (3.4% [95%CI: 0.6, 6.3]) and density (6.3% [0.3, 4.2]), while C-I'/C-I was inversely associated with incident CAC (Risk ratio: 0.89 [95% CI: 0.81, 0.98]) in fully adjusted models that included plasma lipids. Total apoC-I and apoC-II concentrations were not associated with CHD, incident CAC or change in CAC score. CONCLUSIONS: Increased apoC-II truncation was associated with reduced CHD, possibly explained by differences in lipid metabolism. Increased apoC-I and apoC-II truncations were also associated with less CAC progression and/or development of denser coronary plaques.

Advanced glycation end products impair coronary artery BK channels via AMPK/Akt/FBXO32 signaling pathway.

Background: Advanced glycation end products (AGEs) impair vascular physiology in Diabetes mellitus (DM). However, the underlying mechanisms remain unclear. Vascular large conductance calcium-activated potassium (BK) channels play important roles in coronary arterial function.Purpose: Our study aimed to investigate the regulatory role of AGEs in BK channels.Research Design: Using gavage of vehicle (V, normal saline) or aminoguanidine (A) for 8 weeks, normal and diabetic rats were divided into four groups: C+V group, DM+V group, C+A group, and DM+A group.Study Sample: Coronary arteries from different groups of rats and human coronary smooth muscle cells were used in this study.Data Collection and Analysis: Data were presented as mean ± SEM (standard error of mean). Student's t-test was used to compare data between two groups. One-way ANOVA with post-hoc LSD analysis was used to compare data between multiple groups.Results: Compared to the C+V group, vascular contraction induced by iberiotoxin (IBTX), a BK channel inhibitor, was impaired, and BK channel densities decreased in the DM+V group. However, aminoguanidine administration reduced the impairment. Protein expression of BK-ß1, phosphorylation of adenosine 5'-monophosphate-activated protein kinase (AMPK), and protein kinase B (PKB or Akt) were down-regulated, while F-box protein 32 (FBXO32) expression increased in the DM+V group and in high glucose (HG) cultured human coronary smooth muscle cells. Treatment with aminoguanidine in vitro and in vivo could reverse the above protein expression. The effect of aminoguanidine on the improvement of BK channel function by inhibiting the generation of AGEs was reversed by adding MK2206 (Akt inhibitor) or Compound C (AMPK inhibitor) in HG conditions in vitro.Conclusions: AGEs aggravate BK channel dysfunction via the AMPK/Akt/FBXO32 signaling pathway.

Higher exercise capacity, but not omega-3 fatty acid consumption, predicts lower coronary artery calcium scores in women and men with coronary artery disease.

BACKGROUND AND AIMS: Higher coronary artery calcium (CAC) scores are associated with increased cardiovascular (CVD) events and mortality. Exercise capacity is predictive of CVD events. Our aim was to examine the relationship between exercise capacity and CAC in women and men. METHODS: CAC was measured in 203 men and 38 women with clinical coronary artery disease using multidetector coronary tomography. They were randomized to 3.36 g eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) daily or none for 30 months. Maximal exercise treadmill testing was performed at baseline with calculation of metabolic equivalents of task (METs) achieved as a measure of exercise capacity. RESULTS: Despite similar ages at baseline (64.0 ± 6.7 vs 62.7 ± 7.8 years, respectively, p = 0.225), women had lower CAC scores compared to men: 106.7 Agatston units [AU] vs 535.3, respectively, p < 0.001, and at every age (p < 0.001). Female CAC scores did not equal those of men until women were 20 years older. Higher levels of METs were associated with lower CAC scores in both women and men. After multivariate adjustment, METs was the most important predictor of CAC score in women at baseline and 30 months (p = 0.001 and 0.029, respectively) whereas only age predicted in men (p = 0.019 and 0.004, respectively). Annual CAC progression was significantly greater in men compared to women (94.8 AU/year vs 38.0, respectively, p = 0.014). No difference was observed in CAC progression in the EPA + DHA group compared to control in either men or women. CONCLUSIONS: The association of higher METs with lower CAC scores in both women and men supports recommending exercise to maximize cardiorespiratory fitness as this may minimize CAC scores and thus, potentially decrease risk for CVD events. This may be especially important for women since METs independently predicted baseline and 30 month CAC in women.

A Noonan-like pediatric patient with a de novo CBL pathogenic variant and an RNF213 polymorphism p.R4810K presenting with cardiopulmonary arrest due to left main coronary artery ostial atresia.

Left main coronary artery ostial atresia (LMCAOA) is an extremely rare condition. Here, we report the case of a 14-year-old boy with Noonan syndrome-like disorder in whom LMCAOA was detected following cardiopulmonary arrest. The patient had been diagnosed with Noonan syndrome-like disorder with a pathogenic splice site variant of CBL c.1228-2 A > G. He suddenly collapsed when he was running. After administering two electric shocks using an automated external defibrillator, the patient's heartbeat resumed. Cardiac catheterization confirmed the diagnosis of LMCAOA. Left main coronary artery angioplasty was performed. The patient was discharged without neurological sequelae. Brain magnetic resonance imaging revealed asymptomatic Moyamoya disease. In addition, RNF213 c.14429 G > A p.R4810K was identified. There are no reports on congenital coronary malformations of compound variations of RNF213 and CBL. In contrast, the RNF213 p.R4810K polymorphism has been established as a risk factor for angina pectoris and myocardial infarction in adults, and several congenital coronary malformations due to genetic abnormalities within the RAS/MAPK signaling pathway have been reported. This report aims to highlight the risk of sudden death in patients with RASopathy and RNF213 p.R4810K polymorphism and emphasize the significance of actively searching for coronary artery morphological abnormalities in these patients.