Mouse Aorta Research Articles

Vasculo-Protective Effects of Standardized Black Chokeberry Extracts in Mice Aorta

Black chokeberry (Aronia melanocarpa <Michx.> Elliot) represents a rich source of dietary polyphenols and other bioactive phytochemicals with pleiotropic beneficial cardiovascular effects. The present study was aimed at evaluating the ex vivo effects of two black chokeberry extracts (BChEs), obtained from either dry (DryAr) or frozen (FrozAr) berries, on oxidative stress and vascular function in mice aortic rings after incubation with angiotensin 2 (Ang 2), lipopolysaccharide (LPS) and glucose (GLUC) in order to mimic renin–angiotensin system activation, inflammation and hyperglycemia. The identification of phenolic compounds was performed by means of liquid chromatography with a diode array detector coupled with mass spectrometry using the electrospray ionization interface. The BChE obtained from the FrozAr was rich in cyanidin glucoside, rutin and caffeic acid, while the one obtained from the dried berries was rich in rutin, caffeic acid and chlorogenic acid. Mice aortas were dissected and acutely incubated (12 h) with Ang2 (100 nM), LPS (1 µg/mL) or GLUC (400 mg/dL) in the presence vs. absence of the two BChEs (1, 10, 50, 75, 100, 500 µg/mL). Subsequently, the tissues were used for the assessment of (i) hydrogen peroxide (H2O2) and superoxide production (using two methods, spectrophotometry and immunofluorescence), (ii) H2O2 scavenger effect and (iii) vascular reactivity (using the organ bath/myograph system). After exposure to Ang2, LPS or GLUC, both types of extracts decreased the H2O2 and superoxide levels in a concentration-dependent manner starting from either 50 µg/mL or 100 µg/mL. Also, in the highest concentrations (100 µg/mL, 150 µg/mL and 500 µg/mL), both extracts elicited a significant scavenger effect on H2O2 (similar to catalase, the classic H2O2 scavenger). Moreover, at 100 µg/mL, both extracts were able to significantly improve vascular relaxation in all stimulated aortic rings. In conclusion, in mice aortas, black chokeberry extracts in acute application elicited a concentration-dependent vasculo-protective effect through the reduction of oxidative stress and the alleviation of endothelial dysfunction in ex vivo conditions that mimic cardio-metabolic diseases.

Itchy E3 Ubiquitin Ligase-Mediated Ubiquitination of Ferritin Light Chain Contributes to Endothelial Ferroptosis in Atherosclerosis

This research seeks to investigate the function and fundamental mechanisms of Itchy E3 ubiquitin ligase (ITCH), a HECT (homologous to E6AP carboxyl terminus)-type E3 ubiquitin ligase, in endothelial ferroptosis, particularly in the context of atherosclerosis, which has been underexplored. The levels of ITCH protein in the aortas of mice with atherosclerosis were analyzed. Constructs for ITCH RNA interference were generated and introduced into human aortic endothelial cells (HAECs). The findings indicated that ITCH protein expression was elevated in atherosclerotic mice and HAECs exposed to oxidized low-density lipoprotein (ox-LDL). ITCH downregulation significantly mitigated ox-LDL-induced endothelial injury and dysfunction. Reducing ITCH expression inhibited ox-LDL-induced endothelial ferroptosis. This study also revealed that ITCH mediates ox-LDL-induced ubiquitin-dependent degradation of ferritin light chain (FTL) in HAECs. The protective impact of ITCH knockdown against ox-LDL-induced ferroptosis and endothelial injury was reversed by FTL siRNA. Additionally, in vivo experiments showed that inhibiting ITCH reduced atherosclerosis progression and reversed ferroptosis in the aorta, with an associated increase in FTL protein expression in the aortas of mice. This study demonstrates that ITCH interacts with and regulates the stability of the FTL protein via the ubiquitin–proteasome system, contributing to ox-LDL-induced ferroptosis and endothelial cell dysfunction. Targeting components of the ITCH-FTL pathway holds potential as a therapeutic strategy against atherosclerosis.

ADAM10-mediated β-klotho degradation: a key to FGF21 resistance in senescent vascular endothelial cells.

Fibroblast growth factor 21 (FGF21) is a key regulator of metabolism and cardiovascular health. However, its upregulation in aging and age-related disorders suggests the presence of FGF21 resistance. This study aimed to elucidate the mechanisms underlying senescence-associated FGF21 resistance in human umbilical vein endothelial cells (HUVECs) and to explore potential therapeutic interventions. Transcriptomic analysis revealed a significant reduction in the number of FGF21-regulated genes in senescent HUVECs compared to young cells, indicating the onset of FGF21 resistance. In young HUVECs, FGF21 inhibited inflammatory cytokines and upregulated mitochondrial ribosomal protein genes. Conversely, senescent HUVECs showed a downregulation of β-klotho, an essential co-receptor for FGF21, at the cell membrane, concomitant with an upregulation of ADAM10, a protease involved in β-klotho degradation. These changes were also observed in aged mouse aortas. ADAM10 was shown to bind directly to β-klotho and promote its degradation, contributing to FGF21 resistance. Inhibition of ADAM10 or overexpression of β-klotho restored FGF21 responsiveness in senescent HUVECs. Moreover, pharmacologically high concentrations of FGF21 were effective in overcoming resistance and restoring its regulatory effects on senescent cells. These findings suggest that ADAM10-mediated degradation of β-klotho is a central mechanism in the development of FGF21 resistance, and that targeting the ADAM10/β-klotho axis could represent a novel therapeutic approach to restore FGF21 sensitivity. Pharmacological administration of FGF21 may hold promise in treating vascular aging and its associated cardiovascular pathologies.

Endothelial cell Ass1 inhibits arteriosclerotic calcification in diabetes mellitus

Endothelial cell (EC) dysfunction is an important pathological feature of early calcification in diabetic plaques. Argininosuccinic synthase 1 (Ass1) is important in protecting EC activity. Therefore, this study aimed to explore the effect of endothelial Ass1 on calcification in diabetic plaques and its potential regulatory mechanism. In this study, serum Ass1 levels were measured in 84 patients, and the study showed that the serum Ass1 level in patients with diabetes was significantly decreased compared with the non-diabetic group, and the serum Ass1 level in patients with coronary artery calcification was significantly decreased compared with the non-coronary artery calcification group. The ApoE-/- mouse diabetic plaque calcification model and the mouse aortic endothelial cell (MAEC) calcification model were constructed, and the influence of endothelial cell Ass1 on diabetic plaque calcification was further investigated by adeno-associated virus and plasmid intervention. Molecular biology studies have shown that endothelial Ass1 overexpression can reduce plaque calcification and inhibit MAEC osteogenic differentiation in diabetic mice, and Ass1 has protective effects on EC and blood vessels in mice. 4D-label-free proteomic sequencing, bioinformatics analysis, and IP experiments were performed on ApoE-/- mouse aorta after adeno-associated virus intervention. It was found that the differential protein Ptk2b was closely related to vascular calcification (VC) and interacted with the target protein Ass1. The above studies indicate that endothelial Ass1 affects calcification formation in diabetic plaques, and the mechanism may be related to Ptk2b. Ass1 may be a new target for the treatment of diabetic VC.

Lateral shear interferometry for shape accuracy measurements of 3D-printed micro-optics

Abstract The technique of 3D-printed micro-optics has experienced significant advancements over the last few years [1, 2]. This progressive evolution has been marked by remarkable strides in precision and miniaturization, making it particularly attractive to applications in the field of medical endoscopy. One noteworthy application entails the fabrication of micro-lenses directly on optical fibers, thereby creating endoscopic devices with unparalleled capabilities. A significant attribute of these micro-optical systems lies in their capability for precise imaging and their adept navigation through exceptionally narrow biological structures. For instance, the application of the 3D-printed endoscopy technology has facilitated insertion into the aorta of mice, as well as severely diseased human carotid arteries [2, 3]. This unprecedented level of accessibility and adaptability holds immense promise for diagnostic and therapeutic interventions in the realm of cardiovascular medicine and beyond. In the future, it may also be conceivable for micro-lenses to serve not only as imaging tools, but also as integral components of 3D-printing processes aimed at repairing damaged human tissue with bio-materials. For micro-lens production and quality control, performance assessment is invariably necessary to assure diffraction limited imaging in the different optical applications. With confocal surface profiling, only the surface shape information of micro-optics can be obtained. However, not only the shape of 3D-printed optics is relevant for its quality, but also its refractive index distribution is crucial when determining its overall optical performance. Therefore, the measurement of the wavefront is decisive in evaluating lens performance. Lateral shear interferometry presents itself as a compact and precise method for wavefront measurements. The only essential component required in the set-up is a pair of glass plates with high surface quality. Consequently, the entire set-up can be notably streamlined, as there is no requirement to construct an interferometer reference arm. This advantage facilitates the development of a notably compact set-up, making lateral shear interferometry particularly advantageous for integration with microscope and 3D-printer, despite the requirement for more complex algorithms in analyzing lateral shear interferograms. In this paper, simulation of shear interferogram fringes corresponding to wavefronts exhibiting varying aberrations are discussed. Additionally, this research encompasses measurement and analysis of shear interferograms obtained from wave fronts transmitted through different lenses. Through meticulous examination of these experimental results, the characteristics and performance of the tested optical systems are elucidated, contributing to a deeper understanding of wavefront analysis methodologies and their applicability in optical characterization studies.

4-Octyl itaconate inhibits vascular calcification partially via modulation of HMOX-1 signaling

Vascular calcification frequently occurs in patients with chronic conditions such as chronic kidney disease (CKD), diabetes, and hypertension and represents a significant cause of cardiovascular events. Thus, identifying effective therapeutic targets to inhibit the progression of vascular calcification is essential. 4-Octyl itaconate (4-OI), a derivative of itaconate, exhibits anti-inflammatory and antioxidant activity, both of which play an essential role in the progression of vascular calcification. However, the role and molecular mechanisms of 4-OI in vascular calcification have not yet been elucidated. In this study, we investigated the effects of exogenous 4-OI on vascular calcification using vascular smooth muscle cells (VSMCs), arterial rings, and mice. Alizarin red staining and western blot revealed that 4-OI inhibited calcification and osteogenic differentiation of human VSMCs. Similarly, 4-OI inhibited calcification of rat arterial rings and VitD3-overloaded mouse aortas. Mechanistically, RNA sequencing analysis revealed that 4-OI treatment is most likely to affect heme oxygenase 1 (HMOX-1) mRNA expression. The study demonstrated that 4-OI treatment increased HMOX-1 mRNA and protein levels, but suppressed inflammation and oxidative stress in VSMCs under osteogenic conditions. Moreover, 4-OI treatment. HMOX-1 knockdown by siRNA or treatment with the HMOX-1 inhibitor ZnPP9 significantly reversed the suppression effect on calcification of VSMCs and aortas of VitD3-overloaded mice by 4-OI. Furthermore, HMOX-1 knockdown by siRNA markedly abrogated the inhibitory effect of 4-OI on inflammation in VSMCs. These findings suggest that 4-OI alleviates vascular calcification and inhibits oxidative stress and inflammation through modulation of HMOX-1, indicating its potential as a therapeutic target for vascular calcification.

Generation of Transcript Length Variants and Reprogramming of mRNA Splicing During Atherosclerosis Progression in ApoE-Deficient Mice

Background. Variant 3′UTRs provide mRNAs with different binding sites for miRNAs or RNA-binding proteins (RBPs) allowing the establishment of new regulatory environments. Regulation of 3′UTR length impacts on the control of gene expression by regulating accessibility of miRNAs or RBPs to homologous sequences in mRNAs. Objective. Studying the dynamics of mRNA length variations in atherosclerosis (ATS) progression and reversion in ApoE-deficient mice exposed to a high-fat diet and treated with an αCD40-specific siRNA or with a sequence-scrambled siRNA as control. Methods. We gathered microarray mRNA expression data from the aortas of mice after 2 or 16 weeks of treatments, and used these data in a Bioinformatics analysis. Results. Here, we report the lengthening of the 5′UTR/3′UTRs and the shortening of the CDS in downregulated mRNAs during ATS progression. Furthermore, treatment with the αCD40-specific siRNA resulted in the partial reversion of the 3′UTR lengthening. Exon analysis showed that these length variations were actually due to changes in the number of exons embedded in mRNAs, and the further examination of transcripts co-expressed at weeks 2 and 16 in mice treated with the control siRNA revealed a process of mRNA isoform switching in which transcript variants differed in the patterns of alternative splicing or activated latent/cryptic splice sites. Conclusion. We document length variations in the 5′UTR/3′UTR and CDS of mRNAs downregulated during atherosclerosis progression and suggest a role for mRNA splicing reprogramming and transcript isoform switching in the generation of disease-related mRNA sequence diversity and variability.

Decoding the anti-hypertensive mechanism of α-mangostin based on network pharmacology, molecular docking and experimental validation.

Hypertension is a leading risk factor for disability and deaths worldwide. Evidence indicates that alpha-mangostin(α-MG) can reduce blood pressure and improve target organ damage. Nonetheless, its pharmacological targets and potential mechanisms of action remain inadequately elucidated. We used SwissTargetPrediction to identify α-MG's drug targets and DisGeNET, GeneCards, CTD, and GEO databases for hypertension-related targets, and then determined antihypertensive therapeutic targets of α-MG by intersecting these targets. GO functional enrichment analysis, KEGG pathway analysis, and disease association analysis were conducted using the DAVID database and R package "clusterprofile", visualized with Cytoscape software. The binding affinity of α-MG to identified targets was confirmed through molecular docking using Autodock Vina v.1.2.2 software. The impact of α-MG on target genes was validated using an Angiotensin II-induced hypertensive mouse model and RT-qPCR. A total of 51 potential antihypertensive therapeutic targets for α-MG were identified by intersecting 109 drug targets with 821 disease targets. Furthermore, 10 cellular component terms, 10 disease terms, and the top 20 enriched biological processes, molecular functions, and KEGG pathways related to α-MG's antihypertensive effects were documented. Molecular docking studies indicated a strong binding affinity of α-MG with the HSP90AA1 domain. In Ang II-induced hypertensive mice aorta, treatment with α-MG effectively reversed the aberrant mRNA expression of TNF, HSP90AA1, NFKB1, PPARG, SIRT1, PTGS2, and RELA. Our analyses showed that TNF, HSP90AA1, NFKB1, PPARG, SIRT1, PTGS2, and RELA might be α-MG's potential therapeutic targets for hypertension, laying groundwork for further investigation into its pharmacological mechanisms and clinical uses.

PIEZO1 attenuates Marfan syndrome aneurysm development through TGF-β signaling pathway inhibition via TGFBR2.

Marfan syndrome (MFS) is a hereditary disorder primarily caused by mutations in the FBN1 gene. Its critical cardiovascular manifestation is thoracic aortic aneurysm (TAA), which poses life-threatening risks. Owing to the lack of effective pharmacological therapies, surgical intervention continues to be the current definitive treatment. In this study, the role of Piezo-type mechanosensitive ion channel component 1 (Piezo1) in MFS was investigated and the activation of PIEZO1 was identified as a potential treatment for MFS. PIEZO1 expression was detected in MFS mice (Fbn1C1041G/+) and patients. Piezo1 conditional knockout mice in vascular smooth muscle cells of MFS mice (MFS × CKO) was generated, and bioinformatics analysis and experiments in vitro and in vivo were performed to investigate the role of Piezo1 in MFS. PIEZO1 expression decreased in the aortas of MFS mice; MFS × CKO mice showed aggravated TAA, inflammation, extracellular matrix remodelling, and TGF-β pathway activation compared to MFS mice. Mechanistically, PIEZO1 knockout exacerbated the activation of the TGF-β signalling pathway by inhibiting the endocytosis and autophagy of TGF-β receptor 2 mediated by Rab GTPase 3C. Additionally, the pharmacological activation PIEZO1 through Yoda1 prevented TGF-β signalling pathway activation and reversed TAA in MFS mice. Piezo1 deficiency aggravates MFS aneurysms by promoting TGF-β signalling pathway activation via TGF-β receptor 2 endocytosis and a decrease in autophagy. These data suggest that PIEZO1 may be a potential therapeutic target for MFS treatment.

The Role of Sex in the Effects of Smoking and Nicotine in Cardiovascular Function, Atherosclerosis, and Inflammation.

Cigarette smoke (CS) invokes an inflammatory response associated with vascular dysfunction and atherosclerosis. The role of sex and nicotine in CS effects on cardiovascular function and atherosclerosis is unexplored. Male and female C57Bl/6 WT (wild type) and ApoE-/- mice were exposed to CS and nicotine with access to chow and water ad libitum for 16 weeks to fill this gap. Heart rate and endothelial function were measured in the aorta of WT mice, while the lipid profile, cytokines, chemokines, and plaque area and composition were assessed in ApoE-/- mice. CS increased heart rate similarly in both sexes and induced a more substantial impairment in endothelial function in males and more plaque in females than nicotine. Necrotic core areas were similar for both treatments in both sexes, while females had higher collagen deposition across treatments. Both treatments elevated senescence-associated GLB1/-galactosidase (SA-GLB1) and interleukin 17A (IL17A) similarly in both sexes. CS upregulated cholesterol in both sexes, triglycerides, VLDL, HDL, and C-X-C motif chemokine ligand-5 (CXCL5/LIX) only in males, and LDL and IL1A only in females. Additionally, nicotine metabolism showed sex-specific responses to nicotine but not smoking. Findings suggest sex influences cardiovascular function and atherosclerosis following exposure to nicotine and CS. The purpose of this study was to fill the existing literature gap through assessment of the differential sex effects of CS and nicotine on vascular function and atherosclerosis to identify sex-specific risk factors. We show sex-specific differences in endothelial function, plaque, inflammation, and extracellular matrix (ECM) regulators with exposure to CS and nicotine, which underscore the importance of assessing sex in tobacco and nicotine exposure studies. This study also shows the negative effect of oral nicotine administration as many oral dissolvable nicotine products, like pouches and gum, are becoming increasingly popular among adolescents and young adults.

Abstract 4120623: Sphingosine Kinase 1 Is Integral For Elastin Deficiency-induced Arterial Hypermuscularization

Introduction: Defective elastic lamellae and smooth muscle cell (SMC) accumulation are characteristics of diverse obstructive arterial diseases (e.g., atherosclerosis, pulmonary hypertension, and supravalvular aortic stenosis [SVAS]) as well as physiological closure of the ductus arteriosus (DA). Mechanistic links between defective elastin and SMC proliferation are not well elucidated. Methods: Immunostaining for proliferation marker Ki67 was performed on wild-type (WT) and Eln (-/-) mouse aortas at embryonic day (E) 13.5 and E15.5 because elastin (ELN) is expressed in the mouse aorta from E14. Bulk RNA-seq was conducted on mouse aortic SMCs isolated from WT or Eln (-/-) embryos at E15.5. As sphingosine kinase 1 (SPHK1) was found as a highly promising candidate, its expression was evaluated in human SVAS patient aortas and in mouse Eln (-/-) aortas and WT DA. S1P receptor 1 (S1PR1) activity was assessed in aortic SMCs from S1pr1 (knock-in/knock-in) , H2B-GFP mice. Genetic deletion of Sphk1 in SMCs was performed on the elastin mutant background. Pharmacological SPHK1 inhibition was evaluated on both elastin mutants and WT embryos. Regulatory mechanisms of SPHK1 were assessed by mRNA stability assay and TRANSFAC database analysis. Results: SMC hyperproliferation was first observed in Eln (-/-) aorta at E15.5, prior to morphological differences. Bulk RNA-seq revealed that Sphk1 is the most upregulated transcript in Eln (-/-) aortic SMCs at E15.5. Reduced ELN increases SPHK1 levels in SMCs of human patient aortas and mouse aorta and DA. S1PR1 expression and activity are increased by elastin insufficiency. SMC Sphk1 deletion attenuates SMC proliferation and muscularization in the elastin-defective aorta, leading to extended viability of Eln (-/-) mice. Similarly, pharmacological SPHK1 inhibition ameliorates elastin aortopathy but leads to patent DA in WT mice. mRNA stability assay indicated Sphk1 is upregulated by enhanced transcription. TRANSFAC and bulk RNA-seq data suggested that transcription factor early growth response 1 (Egr1) induces Sphk1 transcription. Indeed, EGR1 was upregulated in elastin mutant aortas and DA. Conclusions: Elastin deficiency upregulates SPHK1 transcription, leading to SMC proliferation and hypermuscularization in elastin aortopathy as well as during physiological DA closure. Inhibiting SPHK1 is a promising therapeutic strategy for elastin aortopathy and for select congenital heart diseases in which patent DA maintains circulation.

Abstract 4131674: ADP-Ribosylation In a Mouse Model of Atherosclerosis: a Potential Novel Link Between Dyslipidemia and Inflammation in Cardiovascular Disease

Background: Inflammation and lipid accumulation are major features of atherosclerosis, a leading cause of death and morbidity worldwide. Our previous study recognized ADP-ribosylation, a post-translational modification, as a novel regulator of macrophage activation. We also have established mass spectrometry-based ADP-ribosylation proteomics. Using this technology, we evaluated the completely uncharacterized role of ADP-ribosylation in atherogenesis. We hypothesized that ADP-ribosylated proteins circulate from liver, accumulate in aorta and promote atherogenesis. Methods and Results: We harvested the aorta, liver, and plasma of LDL receptor-deficient ( Ldlr -/- ) mice that were on a regular chow or high-fat diet for 3 or 6 months (n=40/condition). To increase ADP-ribosyl peptide signals in the aorta, we applied our novel recently optimized ion mobility mass spectrometry strategy to generate ADP-ribosylation proteomics data. We analyzed 160 mice aortas and identified 3 APOA1 and 3 APOE ADP-ribosylated peptides in both the aorta and the liver (Fig. A). In addition, these peptides were differentially abundant in the aorta of HFD-fed mice, compared to controls (i.e, APOA1 ARPALEDLR peptide relative abundance (Fig. B)). Using the same mouse plasma, we then validated the presence of ADP-ribosylated APOA1 and ADP-ribosylated APOE in HDL and chylomicron/VLDL/LDL fractions (Western blot), respectively. This finding indicates that classical apolipoproteins circulate as ADP-ribosylated forms, representing a completely novel class of modified apolipoproteins. Immunohistochemistry confirmed the enrichment of aortic lesions in macrophages and ADP-ribosylation signal (5-fold increase, p=0.0006). Conclusions: This work provides the first in vivo evidence that ADP-ribosylation occurs in atherosclerotic lesions, which may originate from the liver via circulating blood (Fig. C). Future studies will examine whether ADP-ribosylation of apolipoproteins, specifically APOA1, alters anti-atherogenic functions of HDL.

Senolysis potentiates endothelial progenitor cell adhesion to and integration into the brain vasculature

BackgroundOne of the most severe consequences of ageing is cognitive decline, which is associated with dysfunction of the brain microvasculature. Thus, repairing the brain vasculature could result in healthier brain function.MethodsTo better understand the potential beneficial effect of endothelial progenitor cells (EPCs) in vascular repair, we studied the adhesion and integration of EPCs using the early embryonic mouse aorta–gonad–mesonephros – MAgEC 10.5 endothelial cell line. The EPC interaction with brain microvasculature was monitored ex vivo and in vivo using epifluorescence, laser confocal and two-photon microscopy in healthy young and old animals. The effects of senolysis, EPC activation and ischaemia (two-vessel occlusion model) were analysed in BALB/c and FVB/Ant: TgCAG-yfp_sb #27 mice.ResultsMAgEC 10.5 cells rapidly adhered to brain microvasculature and some differentiated into mature endothelial cells (ECs). MAgEC 10.5-derived endothelial cells integrated into microvessels, established tight junctions and co-formed vessel lumens with pre-existing ECs within five days. Adhesion and integration were much weaker in aged mice, but were increased by depleting senescent cells using abt-263 or dasatinib plus quercetin. Furthermore, MAgEC 10.5 cell adhesion to and integration into brain vessels were increased by ischaemia and by pre-activating EPCs with TNFα.ConclusionsCombining progenitor cell therapy with senolytic therapy and the prior activation of EPCs are promising for improving EPC adhesion to and integration into the cerebral vasculature and could help rejuvenate the ageing brain.

OxLDL/LOX-1 mediated sex, age, stiffness, and endothelial dependent alterations in mouse thoracic aortic vascular reactivity.

Elevated plasma levels of oxidized low-density lipoprotein (oxLDL) are a risk factor and key component that accelerates and worsens cardiovascular disease fueling inflammation, plaque buildup and vascular damage. OxLDL can elicit its detrimental action via lectin-like oxLDL receptor 1 (LOX-1). In this study, we determined whether oxLDL, via LOX-1, alters aortic vascular reactivity and determined if sex and age differences exist. Thoracic aortic endothelium-intact or -denuded ring segments were isolated from 7 to 12months old intact C57BL/6J female and male mice and pre-incubated with oxLDL ex vivo (50ug/dL; 2h). Using wire myography, cumulative concentration-response curves to phenylephrine (PE) were generated to determine contractile responses. From these curves, the EC50 was determined and used to contract rings to assess acetylcholine (ACh) dependent relaxation. Calculated aortic stiffness and remodeling were also assessed. BI-0115 (10 M; selective LOX-1 inhibitor) was used to determine LOX-1 dependence. We observed differential sex, age, endothelial cell, and LOX-1 dependent alterations to the efficacy of PE-induced contractile responses and ACh-mediated vasorelaxation in thoracic aortic rings following oxLDL exposure. Additionally, we observed a distinct sex and age effect on thoracic aortic stiffness following exposure to oxLDL. There was also a sex effect on calculated vessel diameter, as well as an age effect on oxLDL-mediated aortic remodeling that was LOX-1 dependent. Thus, LOX-1 inhibition and the resulting attenuation of oxLDL/endothelial-mediated alterations in aortic function suggests that there are differential sex differences in the role of oxLDL/LOX-1 in the thoracic aorta of middle-aged male and female mice. NEW and NOTEWORTHY. We investigated the effects of oxLDL via the LOX-1 receptor on murine thoracic aortic vasoreactivity, stiffness, and remodeling across age and sex. Acute exposure to oxLDL led to altered vasoreactivity, endothelial dysfunction, and changes in aortic stiffness and remodeling. These effects were in-part age, sex, endothelial, and LOX-1 dependent. This study reveals potential complex interactions in oxLDL/LOX-1-mediated vascular responses that could serve as potential therapeutic intervention for vascular diseases such as atherosclerosis and stroke.

Targeting GPR39 in structure-based drug discovery reduces Ang II-induced hypertension.

The endothelium-dependent vascular injury, a primary pathological feature of angiotensin II (Ang II)-induced hypertension. This study aimed to explore the role and underlying mechanisms of G protein-coupled receptor 39 (GPR39) in the pathogenesis of Ang II-induced hypertension. For in vivo studies, GPR39 knockout (KO) mice (C57BL/6 J, male) were generated and administered Ang II for 4 weeks. GPR39 expression was upregulated in the aorta of hypertensive patients and mice. The ablation of GPR39 mitigated vascular fibrosis, augmented endothelium-dependent vasodilation, and inhibited endothelial inflammation, oxidative stress, and apoptosis in mice. Additionally, GPR39 KO decreased NOD-like receptor protein 3 (Nlrp3) gene expression in Ang II-stimulated endothelial cells. Notably, Nlrp3 activation counteracted the therapeutic benefits of GPR39 KO. We identified the potential ligand of GPR39 using structure-based high throughput virtual screening (HTVS) and validated its antihypertensive function in vitro and in vivo. The small molecule ligand Z1780628919 of GPR39 can also reduce Ang II-induced hypertension and improve vascular function. GPR39 KO and the small molecule ligand Z1780628919 potentially downregulates Nlrp3, thereby mitigating vascular fibrosis, endothelial inflammation, oxidative stress, and apoptosis. This effect contributes to the alleviation of Ang II-induced hypertension and the rectification of vascular dysfunctions. These findings suggest new avenues for therapeutic intervention.

Itga9 protects against thoracic aortic aneurysm or dissection

Abstract Background The thoracic aortic aneurysm or dissection (TAAD) is a fatal cardiovascular disease with unclear pathogenic mechanisms. Integrins are important in coordinating the transmembrane connections between the extracellular matrix (ECM) and cytoskeleton, contributing to pathological processes of various diseases. Integrin α9 (Itga9), one of the integrin family subunits, has emerged as critical for vascular homeostasis. However, the contribution of Itga9 to TAAD remains elusive. Purpose The present study aimed to determine the role of Itga9 in the development of TAAD and its underlying mechanisms. Methods Western blotting and immunofluorescence staining were used to evaluate ITGA9 expression in the human Standford-A AAD tissues and rodent model of TAAD. Beta-aminopropionitrile monofumarate (BAPN) was used to induce TAAD mouse model. Vascular smooth muscle cell (VSMC)-specific Itga9 knockout mice or adeno-associated virus (AAV)-treated Itga9 overexpressed mice were generated to detect the role of Itga9 in the progression of TAAD. RNA-sequencing analysis was performed to discern possible transcriptome changes in the aorta tissues of VSMC-specific Itga9 knockout mice after BAPN treatment. Primary mouse aortic VSMC culture, flow cytometry analysis, EdU incorporation assay, transmission electron microscopy, and autophagy flux experiments were performed to evaluate the function of Itga9 on VSMCs and uncover its downstream target. Results ITGA9 expression levels were reduced in the aorta tissues of Standford-A AAD patients and BAPN-induced TAAD mice. VSMC-specific deletion of Itga9 exacerbated the incidence of BAPN-induced TAAD in mice. Itga9 deficiency downregulated Valosin-containing protein (Vcp) expression in mice aorta and caused reduced autophagy biomarkers of LC3 after BAPN treatment. In the primary aortic VSMCs, Itga9 deficiency resulted in repressed autophagy, accelerated apoptosis, and reduced proliferation ability. Mechanistically, Itga9 recruited the transcription factor Pre-B-cell leukemia transcription factor 1 (Pbx1) to activate the Vcp and promote VSMC autophagy. Consistently, overexpression of Itga9 in vitro and in vivo rescued the autophagic change and protected against the TAAD progression by upregulating the expression of Pbx1 and Vcp. Conclusions Our findings indicate a critical role of Itga9 in the regulation of VSMC autophagy by controlling Pbx1-Vcp. The Itga9-Pbx1-Vcp axis may represent a novel therapeutic target against TAAD.

Increased endothelial cell uptake of erythrocyte-derived extracellular vesicles carrying arginase-1 induces endothelial dysfunction in type 2 diabetes

Abstract Background Recently, we have demonstrated that red blood cells (RBCs) from individuals with type 2 diabetes (T2D-RBCs) induce endothelial dysfunction. However, the mechanism by which RBCs communicate with the vessel is unknown. Extracellular vesicles (EVs) are actively secreted by practically all cell types, including RBCs, and represent a novel mechanism of intercellular communication. However, the involvement of EVs from RBC in the development of endothelial dysfunction remains to be elucidated. Purpose This study was designed to test the hypothesis that EVs are key players in the communication and the transfer of signalling between RBCs and the vascular endothelium to induce endothelial dysfunction in T2D. Methods RBCs from T2D patients and age-matched healthy controls (H-RBCs) were incubated for 18h with Krebs-Henseleit buffer (20% haematocrit) for EV release. RBC-derived EVs in the conditioned medium were isolated using a membrane affinity column. The EVs were co-incubated with mouse aortae to evaluate endothelium-dependent relaxation and with endothelial cells for expression analysis. The uptake of the EVs by endothelial cells and their content of arginase-1 were determined. The functional involvement of arginase was investigated using pharmacological interventions and expression analyses. All animal experiments were performed according to the principles of laboratory animal care (NIH Publication no. 85-23 revised 1985) and human procedures according to the declaration of Helsinki with approval by the Swedish Ethical Review Authority. Results The uptake of EVs derived from T2D-RBCs by endothelial cells was 2-fold greater than that of EVs from H-RBCs (Fig. 1A-B). Inhibiting the uptake of EVs derived from T2D-RBCs by the addition of heparin during the co-incubation rescued the endothelial function (Fig. 1C). Arginase-1 was detected in RBC-derived EVs (Fig. 2A). Arginase-1 mRNA and protein levels were increased in endothelial cells following co-incubation with EVs derived from T2D-RBCs (Fig. 2B-D). Additionally, the increase in arginase-1 protein induced by EVs derived from T2D-RBCs in endothelial cells was observed also following mRNA silencing for arginase-1 (Fig. 2E-F). Finally, mouse aortae co-incubated with EVs derived from T2D-RBCs in the presence or absence of the arginase inhibitor 2(S)-amino-6-boronohexanoic acid significantly attenuated the impairment in endothelial function induced by EVs derived from T2D-RBCs (Fig. 2G). Conclusion Increased uptake of RBC-derived EVs by the endothelial cells is an important feature of the endothelial dysfunction induced by these EVs in T2D. In addition, these EVs carry arginase-1 protein to induce endothelial dysfunction. The mechanism underlying the increased uptake of EVs in target cells is of importance to identify in future studies, as it could lead to new treatment strategies.

Oral low-dose rapamycin treatment prevents aortic aneurysms in marfan mice

Abstract Background/Introduction Marfan syndrome (MFS) is a hereditary connective tissue disorder caused by fibrillin-1 (FBN1) gene mutations. Life-threatening complications are cardiovascular, e.g. thoracic aortic aneurysms and dissections. The pathophysiological cause is the increased release of transforming growth factor β-1 (TGF-β1) from the FBN1-deficient extracellular matrix (ECM) and structural disintegrity. Overactivation of the mechanistic target of rapamycin (mTOR) pathway has been demonstrated in the murine mgR/mgR model of MFS. Short-term administration of rapamycin significantly reduced aortic aneurysm formation and increased the life span in these mice. Purpose To assess the effect of an early continuous oral low-dose rapamycin treatment on the aortic aneurysm formation in the FBN1 hypomorphic mgR/mgR mouse model. Methods Male and female 3-4 week-old mgR/mgR mice were orally administered vehicle or rapamycin through diet (8mg/kg/KG) and sacrificed after 11 weeks. Rapamycin concentration was measured by liquid chromatography-mass spectrometry in whole blood. Aortic diameter was studied using echocardiography. Histopathological and immunofluorescence analyses were performed using aortic tissue sections and techniques. Results Blood rapamycin concentration following oral administration remained below detection level. Rapamycin normalized the aortic wall thickness and the diameter in female mgR/mgR mice to levels of the littermate control mice but not in male mgR/mgR mice. The circumferentially distributed elastin fibres remained significantly intact in sections of the ascending aorta of female mice. Echocardiography revealed that rapamycin-treated female mice stayed below a cut-off value for aortic aneurysms of 2 mm inner aortic diameter eight weeks after starting therapy. In contrast, vehicle-treated female animals already exceeded this value after five weeks. In female but not in male mice, the abundance of the mTOR downstream target phosphorylated ribosomal protein S6 was effectively suppressed by low-dose rapamycin up to levels detected in control animals. At the same time, extracellular matrix proteins like the proteoglycan aggrecan (ACAN) and the related chondroitin sulfate were significantly decreased in female mice. The abundance of ADAMTS5 transglutaminase-2 and xylosyltransferase-1, key enzymes involved in the turnover of proteoglycans and biosynthesis of glycosaminoglycan chains, was improved by the rapamycin treatment in female mice. No decrease in mTOR activity could be detected in male mgR/mgR animals. Here, the ACAN and chondroitin sulphate levels also remained at the level of the vehicle-treated animals. Conclusions Chronic low-dose rapamycin treatment reduced aneurysm formation only in female mgR/mgR mice, since the dose was too low for male mgR/mgR mice, by affecting the composition and organization of key ECM components essential for maintaining aortic homeostasis and mechanical properties.

The effect of CD38 inhibition in age-associated vascular dysfunction

Abstract Introduction Aging is characterized by age-related vascular dysfunction, mainly caused by endothelial dysfunction and arterial stiffness. Levels of nicotinamide adenine dinucleotide (NAD) decrease with aging and accordingly, NAD is thought to be involved in the progression of age-related vascular dysfunction. Recent studies reported the involvement of CD38 in NAD regulation; indeed, its inhibition significantly prolongs the lifespan of progeroid and aged mice. However, whether CD38 is involved in the progression of age-related vascular dysfunction remains to be investigated. Methods CD38 expression was investigated in aortic homogenates of young (12-16 weeks) and aged (18-24 months) C57BL/6 wild-type mice, as well as in vitro in young (passage 5-7) and senescent (passage 14-15) primary human aortic endothelial cells (HAECs). Ex vivo tension myograph experiments were performed on thoracic aortas isolated from aged C57BL/6 mice (22-24 months old) pre-incubated for 1 hour with CD38-specific inhibitor, 78c, or DMSO as control. The effect of CD38 inhibition was also observed in vitro in senescent HAECs. Results A significant upregulation of CD38 expression was detected in the aortas of aged mice compared to the young ones as well as in senescent HAECs compared to young cells. Ex vivo tension myograph experiments showed a significant increase in endothelium-dependent relaxation responses to acetylcholine upon CD38 inhibition. The improvement of endothelial function was prevented by the addition of endothelial nitric oxide synthase (eNOS) inhibitor (L-NAME), but not by the COXs inhibitor, indomethacin, indicating that the observed effect achieved by CD38 inhibition is eNOS dependent. The involvement of the eNOS pathway was further supported by the in vitro finding in HAECs of increased eNOS activation upon CD38 inhibition. Conclusion The herein-reported data suggests an involvement of CD38 in the development of age-related endothelial dysfunction. Further studies are ongoing to investigate the underlying molecular mechanisms as well as the effect of long-term CD38 inhibitor supplementation on the progression of age-related vascular dysfunction.

DNMT3A CHIP-driver mutations ignite the bone marrow

Abstract Background Clonal hematopoiesis of indeterminate potential (CHIP) is an acquired, genetic cardiovascular (CV) risk factor affecting about 10% of the general population at the age of 70 years. Most CHIP-driver mutations affect the epigenetic regulator gene DNMT3A. Experimental studies suggest a causal link between CHIP and inflammation-driven atherosclerosis, but the underlying mechanisms of how few mutant clones provoke adverse clinical outcomes in patients remain obscure. Methods Single monocytes and bone marrow cells from DNMT3A-mutation carriers undergoing coronary angiography and open-heart surgery underwent RNA and genomic DNA sequencing in parallel on a single cell level. This approach enabled us to compare mutant and non-mutant cells directly within carriers, and to non-carrier cells. Atherosclerotic chimeric mice reconstituted with a mixture of Dnmt3a-deficient (CD45.2) and -competent (CD45.1) bone marrow served as an experimental model to compare mutant to non-mutant macrophages in atherosclerotic lesions. Results Surprisingly, RNA/gDNA parallel sequencing revealed that gene expression profiles of mutant monocytes resembled those of non-mutant monocytes in carriers of DNMT3A-CHIP mutations (n=4). Collectively, however, monocytes of DNMT3A mutation carriers were more inflammatory than those of matched non-carrier patients. Mutant hematopoietic stem and progenitor cells, analyzed by RNA/gDNA parallel sequencing in two carriers, expressed more activation and inflammatory markers compared to non-mutant cells within the same bone marrow, and intraindividual differences declined as cells differentiated into monocytes. Macrophages were isolated from atherosclerotic aortas of mixed bone marrow chimeric mice based on CD45.1 (non-mutant) or CD45.2 (mutant or non-mutant controls) expression and subjected to single cell RNA sequencing. Inflammation markers were higher expressed in non-mutant macrophages within carrier mice than in non-mutant macrophages of non-carrier chimeras, and showed relatively small differences compared to Dnmt3a-deficient macrophages. Conclusions Our experimental findings in humans and mice support a pathomechanistic model in which few DNMT3A-CHIP mutation-carrying hematopoietic stem cells stimulate non-mutant cells within the bone marrow, thereby augmenting inflammation downstream in all circulating monocytes and their progeny in atherosclerotic lesions.