Mouse Aorta Research Articles

Semi-quantitative elemental imaging of corrosion products from bioabsorbable Mg vascular implants in vivo

While metal materials historically have served as permanent implants and were designed to avoid degradation, next generation bioabsorbable metals for medical devices such as vascular stents are under development, which would elute metal ions and corrosion byproducts into tissues. The fate of these eluted products and their local distribution in vascular tissue largely under studied. In this study, we employ a high spatial resolution spectrometric imaging modality, laser ablation inductively coupled plasma time-of-flight mass spectrometry (LA-ICP-TOF-MS) to map the metal distribution, (herein refered to as laser ablation mapping, or LAM) from Mg alloys within the mouse vascular system and approximate their local concentrations. We used a novel rare earth element bearing Mg alloy (WE22) wire implanted within the abdominal aorta of transgenic hypercholesterolemic mice (APOE−/−) to simulate a bioabsorbable vascular prosthesis for up to 30 days. We describe qualitatively and semi-quantitatively implant-derived corrosion product presence throughout the tissue cross sections, and their approximate concentrations within the various vessel structures. Additionally, we report the spatial changes of corrosion products, which we postulate are mediated by phagocytic inflammatory cells such as macrophages (MΦ’s).

Licorice Extract Isoliquiritigenin Protects Endothelial Function in Type 2 Diabetic Mice.

Endothelial dysfunction occurs prior to atherosclerosis, which is an independent predictor of cardiovascular diseases (CVDs). Diabetes mellitus impairs endothelial function by triggering oxidative stress and inflammation in vascular tissues. Isoliquiritigenin (ISL), one of the major bioactive ingredients extracted from licorice, has been reported to inhibit inflammation and oxidative stress. However, the therapeutic effects of ISL on ameliorating type 2 diabetes (T2D)-associated endothelial dysfunction remain unknown. In our animal study, db/db male mice were utilized as a model for T2D-associated endothelial dysfunction, while their counterpart, heterozygote db/m+ male mice, served as the control. Mouse brain microvascular endothelial cells (mBMECs) were used for in vitro experiments. Interleukin-1β (IL-1β) was used to induce endothelial cell dysfunction. ISL significantly reversed the impairment of endothelium-dependent relaxations (EDRs) in db/db mouse aortas. ISL treatment decreased ROS (reactive oxygen species) levels in db/db mice aortic sections and IL-1β-treated endothelial cells. Encouragingly, ISL attenuated the overexpression of pro-inflammatory factors MCP-1, TNF-α, and IL-6 in db/db mouse aortas and IL-1β-impaired endothelial cells. The NOX2 (NADPH oxidase 2) overexpression was inhibited by ISL treatment. Notably, ISL treatment restored the expression levels of IL-10, SOD1, Nrf2, and HO-1 in db/db mouse aortas and IL-1β-impaired endothelial cells. This study illustrates, for the first time, that ISL attenuates endothelial dysfunction in T2D mice, offering new insights into the pharmacological effects of ISL. Our findings demonstrate the potential of ISL as a promising therapeutic agent for the treatment of vascular diseases, paving the way for the further exploration of novel vascular therapies.

Transcriptome meta-analysis of Kawasaki disease in humans and mice.

Kawasaki Disease (KD) affects young children less than five years old with severe blood vessel inflammation. Despite being treatable, the causes and mechanisms remain elusive. This study conducted a meta-analysis of RNA sequencing (RNA-seq) data from human and animal models to explore KD's transcriptomic profile and evaluate animal models. We retrieved bulk and single-cell RNA-seq data from Gene Expression Omnibus, with blood and coronary artery samples from KD patients, aorta samples from KD mouse models (Lactobacillus casei cell wall extract-injected mice), and their controls. Upon consistent quality control, we applied Fisher's exact test to assess differential gene expression, followed by an enrichment analysis of overlapping genes. These studies identified 400 differentially expressed genes in blood samples of KD patients compared to controls and 413 genes in coronary artery samples. The data from KD blood and KD coronary artery samples shared only 16 differentially expressed genes. Eighty-one genes overlapped between KD human coronary arteries and KD mouse aortas, and 67 of these 81 genes were regulated in parallel in both humans and mice: 30 genes were up-regulated, and 37 were down-regulated. These included previously identified KD-upregulated genes: CD74, SFRP4, ITGA4, and IKZF1. Gene enrichment analysis revealed significant alterations in the cardiomyopathy pathway. Single-cell RNAseq showed a few significant markers, with known KD markers like S100A9, S100A8, CD74, CD14, IFITM2, and IFITM3, being overexpressed in KD cohorts. Gene profiles obtained from KD human coronary artery are more compatible with data from aorta samples of KD mice than blood samples of KD humans, validating KD animal models for identifying therapeutic targets. Although blood samples can be utilized to discover novel biomarkers, more comprehensive single-cell sequencing is required to detail gene expression in different blood cell populations. This study identifies critical genes from human and mouse tissues to help develop new treatment strategies for KD.

THE THERAPEUTIC POTENTIAL OF PRECONDITIONED MESENCHYMAL STEM CELLS IN ATHEROSCLEROSIS

Atherosclerosis (AS) is a multifactorial and inflammatory disease the cause of which is the formation of lipid and cholesterol plaques on the inner wall of blood vessels. Experimental and clinical studies have shown that the pathogenesis of atherosclerosis is, in particular, connected with inflammatory reactions. Recent studies reported that preconditioned mesenchymal stem cells (MSCs) possess an enhanced immunomodulatory potential. The purpose of the study was to investigate the therapeutic potential of preconditioned MSCs in the development of atherosclerosis. MSCs were isolated from the compact bone of mice by standard protocol. The characterization was conducted using flow cytometry, CFU assay and three-lineage differentiation. Cells were preconditioned with different combinations of cytokines and the optimal concentrations were determined by ELISA and by analysis of co-culturing with lymphocytes. The cytokine-preconditioned MSCs were administrated to mice with induced atherosclerosis. The lipids analysis, measuring of mouse spleens’ weights, ELISA and histological analysis of the aorta sections were done after 8-th weeks. We isolated MSCs and then confirmed the possession of the immunomodulatory properties. Preconditioning with the TNF-α cytokine effectively increased the immunomodulatory properties of MSCs compared to untreated MSCs in vitro. The results of in vivo study that the use of TNF-α-preconditioned MSCs reduced the development of atherosclerosis in mice compared to untreated MSCs: it increased the level of Treg cells in the spleen of mice; significantly reduced the levels of TNF-α and IFN-γ in blood serum; significantly reduced the spleen weights of mice; slightly decreased the total cholesterol levels and also increased HDL levels. Administration of TNF-α-preconditioned MSCs reduced the size of atherosclerotic plaques in the aorta of mice. The results of this study demonstrated the effect of cytokine-preconditioned MSCs in the inhibition of the development of atherosclerosis in a mouse model. However, for application as a therapy for suffered patients additional studies are needed. Besides, using of TNF-α preconditioned MSCs can be considered in other preclinical studies of the different diseases.

Asperuloside as a Novel NRF2 Activator to Ameliorate Endothelial Dysfunction in High Fat Diet-Induced Obese Mice.

Aims: Current treatments are inadequate in alleviating obesity-associated vascular diseases. The development of effective therapies to ameliorate endothelial dysfunction and attenuate oxidative stress is of utmost importance. Asperuloside (ASP), a bioactive compound extracted from Eucommia species, exhibits antiobesity properties. However, the effects of ASP on vasculopathy have not been investigated. Therefore, the effects of ASP on vascular dysfunction and related mechanisms were elucidated. Results: ASP significantly reversed the impaired endothelium-dependent relaxations (EDRs) in obese mice and interleukin (IL)-1β-treated aortas. ASP suppressed endothelial activation in obese mice aortas and IL-1β-treated endothelial cells. ASP attenuated oxidative stress, scavenged mitochondrial reactive oxygen species (ROS), and upregulated heme oxygenase-1 (HO-1) expression in endothelium, independent of its anti-inflammatory properties. HO-1 knockdown diminished the protective effects of ASP against impaired EDRs, ROS overproduction, and endothelial activation. Endothelial cell-specific nuclear factor erythroid 2-related factor 2 (Nrf2) knockdown eliminated the ASP-mediated vascular protective effects and endothelial HO-1 upregulation, emphasizing that ASP improves endothelial function by activating Nrf2/HO-1 signaling. ASP facilitated Nrf2 nuclear translocation and the direct binding of Nrf2 to antioxidant response element, thereby enhancing HO-1 transcription and scavenging ROS. The cellular thermal shift assay results provide the first experimental characterization of the direct binding of ASP to Nrf2. Conclusions: These findings demonstrate that ASP ameliorates obesity-associated endothelial dysfunction by activating Nrf2/HO-1 signaling and thereby maintaining redox hemostasis, suggesting its potential as a novel Nrf2-targeted therapeutic agent and dietary supplement for vasculopathy.

Olfr2-positive macrophages originate from monocytes proliferate in situ and present a pro-inflammatory foamy-like phenotype.

Olfactory receptor 2 (Olfr2) has been identified in a minimum of 30% of vascular macrophages, and its depletion was shown to reduce atherosclerosis progression. Mononuclear phagocytes, including monocytes and macrophages within the vessel wall, are major players in atherosclerosis. Single-cell RNA sequencing studies revealed that atherosclerotic artery walls encompass several monocytes and vascular macrophages, defining at least nine distinct subsets potentially serving diverse functions in disease progression. This study investigates the functional phenotype and ontogeny of Olfr2-expressing vascular macrophages in atherosclerosis. Olfr2+ macrophages rapidly increase in Apoe-/- mice's aorta when fed a Western diet (WD). Mass cytometry showed that Olfr2+ cells are clustered within the CD64 high population and enriched for CD11c and Ccr2 markers. Olfr2+ macrophages express many pro-inflammatory cytokines, including Il1b, Il6, Il12, and Il23, and chemokines, including Ccl5, Cx3cl1, Cxcl9, and Ccl22. By extracting differentially expressed genes from bulk RNA sequencing (RNA-seq) of Olfr2+ vs. Olfr2- macrophages, we defined a signature that significantly mapped to single-cell data of plaque myeloid cells, including monocytes, subendothelial MacAir, and Trem2Gpnmb foamy macrophages. By adoptive transfer experiments, we identified that Olfr2 competent monocytes from CD45.1Apoe-/-Olfr2+/+ mice transferred into CD45.2Apoe-/-Olfr2-/- recipient mice fed WD for 12 weeks, accumulate in the atherosclerotic aorta wall already at 72 h, and differentiate in macrophages. Olfr2+ macrophages showed significantly increased BrdU incorporation compared to Olfr2- macrophages. Flow cytometry confirmed that at least 50% of aortic Olfr2+ macrophages are positive for BODIPY staining and have increased expression of both tumour necrosis factor and interleukin 6 compared to Olfr2- macrophages. Gene set enrichment analysis of the Olfr2+ macrophage signature revealed a similar enrichment pattern in human atherosclerotic plaques, particularly within foamy/TREM2hi-Mφ and monocytes. In summary, we conclude that Olfr2+ macrophages in the aorta originate from monocytes and can accumulate at the early stages of disease progression. These cells can undergo differentiation into MacAir and Trem2Gpnmb foamy macrophages, exhibiting proliferative and pro-inflammatory potentials. This dynamic behaviour positions them as key influencers in shaping the myeloid landscape within the atherosclerotic plaque.

Telomere stabilization by metformin mitigates the progression of atherosclerosis via the AMPK-dependent p-PGC-1α pathway.

Telomere dysfunction is a well-known molecular trigger of senescence and has been associated with various age-related diseases, including atherosclerosis. However, the mechanisms involved have not yet been elucidated, and the extent to which telomeres contribute to atherosclerosis is unknown. Therefore, we investigated the mechanism of metformin-induced telomere stabilization and the ability of metformin to inhibit vascular smooth muscle cell (VSMC) senescence caused by advanced atherosclerosis. The present study revealed that metformin inhibited the phenotypes of atherosclerosis and senescence in VSMCs. Metformin increased the phosphorylation of AMPK-dependent PGC-1α and thus increased telomerase activity and the protein level of TERT in OA-treated VSMCs. Mechanistically, the phosphorylation of AMPK and PGC-1α by metformin not only enhanced telomere function but also increased the protein level of TERT, whereas TERT knockdown accelerated the development of atherosclerosis and senescent phenotypes in OA-treated VSMCs regardless of metformin treatment. Furthermore, the in vivo results showed that metformin attenuated the formation of atherosclerotic plaque markers in the aortas of HFD-fed ApoE KO mice. Although metformin did not reduce plaque size, it inhibited the phosphorylation of the AMPK/PGC-1α/TERT signaling cascade, which is associated with the maintenance and progression of plaque formation, in HFD-fed ApoE KO mice. Accordingly, metformin inhibited atherosclerosis-associated phenotypes in vitro and in vivo. These observations show that the enhancement of telomere function by metformin is involved in specific signaling pathways during the progression of atherosclerosis. These findings suggest that telomere stabilization by metformin via the AMPK/p-PGC-1α pathway might provide a strategy for developing therapeutics against vascular diseases such as atherosclerosis.

Abstract P449: A Murine Model of Mid-Thoracic Aortic Coarctation

Vascular pressure can directly affect cardiovascular structure and function. Understanding the mechanisms, however, is complicated because conditions or interventions that chronically raise blood pressure (e.g. excess angiotensin II) can also have direct effects on the vasculature that occur independent of changes in blood pressure. One approach to mitigating this problem is to obstruct flow in a vascular segment sufficiently to reduce blood pressure in the segment downstream of the obstruction, while pressure upstream either stays the same or increases. As a result, all factors other than pressure are expected to be the same in upstream and downstream vascular segments. We have found evidence that differences in intravascular pressure can also affect closely adjacent tissues, specifically perivascular adipose tissue (PVAT). To test this idea in vivo , we developed a method to produce chronic, partial occlusion (or coarctation) of the mid-thoracic aorta in mice. This exposes upstream aortic PVAT depots to significantly higher aortic pressure than downstream aortic PVAT depots. The key to the method is producing a stable occlusion with a diameter small enough to produce a pressure gradient but not so small that the downstream flow is inadequate to support tissue oxygenation. To this end, a thoracotomy was performed to expose the thoracic aorta in adult mice anesthetized with isoflurane and artificially ventilated. A coarctation of the mid-thoracic aorta was produced by tying a ligature around the aorta and a blunted needle on the aorta, then removing the needle. In other mice, a silicone rubber or nitrile O-ring of varying dimensions was placed around the aorta. Mice were allowed to recover for varying periods of time (weeks), then the degree of coarctation was evaluated using both imaging methods (ultrasound) and direct measurement of the pressure gradient (catheters placed in a carotid and a femoral artery) under isoflurane anesthesia. In a group of 9 male mice with coarctation and 4 male mice with sham coarctation, we found an average mean arterial pressure gradient of 11.7 ± 5.0 mmHg (mean ± SEM) in coarct mice and 1.6 ± 2.0 mmHg in sham mice. Systolic pressure gradients averaged 14.0 ± 5.9 mmHg in coarct mice and 1.0 ± 3.2 mmHg in sham mice. These results suggest it is possible to produce a stable pressure gradient across the thoracic aorta of mice as a tool to explore the impact of arterial pressure on vascular and PVAT structure and function.

Abstract 70: Cellular senescence as a driver of preeclampsia

Background: Preeclampsia (PE) is a severe hypertensive disorder of pregnancy of unknown pathophysiology. We previously have reported the presence of senescence in kidney and fat tissue of PE women, as well as accelerated epigenetic aging, which prompted us to postulate that cellular senescence may drive development of PE. Methods: We prospectively collected sera from pregnant women throughout pregnancy, and using multiplex Luminex analysis, measured 50 markers of the senescence-associated secretory phenotype (SASP). Comparisons were made between women who developed PE (n=10) vs. normotensive pregnancies (n=10). We then injected human PE serum into the IL-10KO mouse to induce PE and assess whether PE is associated with development of senescence. We measured plasma p16, a circulating indicator of cellular senescence, before and after inducing PE. We also compared plasma p16 and expressions of senescence-associated genes in the hearts and aortas of regular pregnant (PG, no PE serum injection) and PE mice (n=4). Results: There was a significant increase in several SASP markers in sera of women who developed PE, beginning in the first trimester, through delivery. Notably, eotaxin, MCP1, GROb, MIF, MMP2, TNFRII, IGFBP2 were significantly increased in PE women compared to their normotensive peers throughout all trimesters. Mice injected with PE serum exhibited all features of PE, as reported earlier. When measuring plasma p16 before and after PE serum injection, animals had higher values upon inducing PE. Plasma samples of PE mice demonstrated higher p16 concentrations compared to the PG group. The aortas of PE mice exhibited increased expressions of senescence associated genes p16, il6, and mcp1, whereas in the hearts, p16, Il1a and il8 were upregulated. Conclusion: The SASP is present very early in human pregnancy, even before PE has developed. Injection of SASP-enriched PE serum into IL-10KO mice induces cellular senescence in different tissues resulting in a PE phenotype. These results suggest that senescence may be an underlying mechanism of PE.

Abstract P183: Peripheral Blood Mononuclear Cells from Patients with Systemic Lupus Erythematosus Induce Inflammation and Salt Sensitivity of Blood Pressure in Humanized Mice

Salt sensitivity of blood pressure (SSBP) is an independent risk factor for cardiovascular disease (CVD), even in normotensive people. Systemic lupus erythematosus (SLE) is also a risk factor for CVD and hypertension. SLE and CVD are on the rise, and it is not known if this is attributable to the increased dietary salt in the modern diet and SSBP. Isolevuglandins (IsoLGs) adduct self-proteins forming neoantigens in antigen-presenting cells (APC), activate T cells, and produce cytokines that promote sodium retention, kidney damage, SLE, and SSBP. We hypothesized the adoptive transfer of peripheral blood mononuclear cells (PBMCs) from patients with SLE into immunodeficient mice (NSG/MHCI/MHCII-/-) would increase inflammation and SSBP after high salt feeding. In vitro studies were performed using PBMCs from patients with SLE and treated with and without high salt, and myeloid cell activation and cytokine expression were measured with flow cytometry. We humanized mice with PBMCs from patients with SLE (n=7) and controls without autoimmune disease (n=7) in animal studies. The mice were fed a 4% high-salt diet for two weeks. Blood pressure was measured with radiotelemetry, immune phenotyping was performed with flow cytometry, and vascular reactivity was assessed in mesenteric arteries using wire myography. A high salt diet significantly increased systolic blood pressure in lupus mice compared to the controls (133.0 vs.116.4 mmHg, p=0.001). APC infiltration (macrophages, monocytes, dendritic cells), proinflammatory markers, and oxidative stress (Isolevuglandins, Nox2) were significantly higher in the kidneys of lupus mice. In lupus mice, classical monocyte and proinflammatory markers were significantly elevated in the spleen lymphocytes, and proinflammatory markers were increased in the kidney, spleen, and aorta in lupus mice. These mice also exhibited mesenteric artery vascular dysfunction (p<0.05). Lupus mice tended to excrete more urinary albumin than the controls (25.89 vs. 17.24 µg/ml p=0.142), suggesting kidney injury. High salt treatment in vitro increased intermediate and nonclassical monocytes with increased CD86, IsoLG, and TNF-alpha expression compared to normal salt-treated cells. In conclusion, our findings suggest that immune cells from patients with SLE, in response to high salt, increase SSBP and the proinflammatory milieu in the kidney, aorta, and spleen; impair vascular function; and worsen albuminuria in immunodeficient mice.

Aminolysis-mediated single-step surface functionalization of poly (butyl cyanoacrylate) microbubbles for ultrasound molecular imaging

Molecular ultrasound imaging with actively targeted microbubbles (MB) proved promising in preclinical studies but its clinical translation is limited. To achieve this, it is essential that the actively targeted MB can be produced with high batch-to-batch reproducibility with a controllable and defined number of binding ligands on the surface. In this regard, poly (n-butyl cyanoacrylate) (PBCA)-based polymeric MB have been used for US molecular imaging, however, ligand coupling was mostly done via hydrolysis and carbodiimide chemistry, which is a multi-step procedure with poor reproducibility and low MB yield. Herein, we developed a single-step coupling procedure resulting in high MB yields with minimal batch-to-batch variation. Actively targeted PBCA-MB were generated using an aminolysis protocol, wherein amine-containing cRGD was added to the MB using lithium methoxide as a catalyst. We confirmed the successful conjugation of cRGD on the MB surface, while preserving their structure and acoustic signal. Compared to the conventional hydrolysis protocol, aminolysis resulted in higher MB yields and better reproducibility of coupling efficiency. Optical imaging revealed that under flow conditions, cRGD- and rhodamine-labelled MB, generated by aminolysis, specifically bind to tumor necrosis factor-alpha (TNF-α) activated endothelial cells in vitro. Furthermore, US molecular imaging demonstrated a markedly higher binding of the cRGD-MB than of control MB in TNF-α activated mouse aortas and 4T1 tumors in mice. Thus, using the aminolysis based conjugation approach, important refinements on the production of cRGD-MB could be achieved that will facilitate the production of clinical-scale formulations with excellent binding and ultrasound imaging performance.Graphical

Abstract P309: Vasodilating Effects of Macrolides: ex vivo and in vivo study

Background: Anecdotal reports showed a decrease in blood pressure (BP) values in subjects assuming macrolide antibiotics when co-administered with calcium channel blockers. Recently, in the MAPA study, in patients with primary hypertension given a single dose of macrolide a decrease in BP was observed. However, whether macrolides induce per se vasorelaxation remains unknown. Aim: To investigate if macrolides induce vasorelaxation and to identify the underlying mechanisms. Methods: The effects of 3 macrolides (roxithromycin, azithromycin, clarithromycin) were investigated in a wire myograph in aortas isolated from C57BL/6J mice, following standard protocols. Relaxation/contraction was evaluated in phenylephrine pre-contracted aorta segments exposed to different macrolide concentrations [10 -10 M to 10 -5 M]. Longer-term effects of azithromycin (50 mg/Kg/d intraperitoneally for 14 days) were investigated in vivo in a mouse model of Ang II-induced hypertension. BP was measured with the tail-cuff method; functional and structural/mechanical properties of the aorta and mesenteric arteries were evaluated with wire or pressure myograph, as appropriate. Results: After exposure to macrolides, phenylephrine pre-contracted mice aorta segments showed relaxation (maximum effect: -15-20% vs DMSO). The vasodilating effect was abolished by the removal of the endothelium, and was blunted by L-NAME. In the vivo studies treatment with azithromycin lowered BP values and prevented Ang II-induced endothelial dysfunction. Conclusions: Macrolides exert a vasodilatory action that involves endothelium-dependent NO release. In a model of Ang II-mediated hypertension, azithromycin exerted a BP-lowering effect.

Antibiotic-induced gut microbiota disruption promotes vascular calcification by reducing short-chain fatty acid acetate

BackgroundVascular calcification is a common vascular lesion associated with high morbidity and mortality from cardiovascular events. Antibiotics can disrupt the gut microbiota (GM) and have been shown to exacerbate or attenuate several human diseases. However, whether antibiotic-induced GM disruption affects vascular calcification remains unclear.MethodsAntibiotic cocktail (ABX) treatment was utilized to test the potential effects of antibiotics on vascular calcification. The effects of antibiotics on GM and serum short-chain fatty acids (SCFAs) in vascular calcification mice were analyzed using 16 S rRNA gene sequencing and targeted metabolomics, respectively. Further, the effects of acetate, propionate and butyrate on vascular calcification were evaluated. Finally, the potential mechanism by which acetate inhibits osteogenic transformation of VSMCs was explored by proteomics.ResultsABX and vancomycin exacerbated vascular calcification. 16 S rRNA gene sequencing and targeted metabolomics analyses showed that ABX and vancomycin treatments resulted in decreased abundance of Bacteroidetes in the fecal microbiota of the mice and decreased serum levels of SCFAs. In addition, supplementation with acetate was found to reduce calcium salt deposition in the aorta of mice and inhibit osteogenic transformation in VSMCs. Finally, using proteomics, we found that the inhibition of osteogenic transformation of VSMCs by acetate may be related to glutathione metabolism and ubiquitin-mediated proteolysis. After adding the glutathione inhibitor Buthionine sulfoximine (BSO) and the ubiquitination inhibitor MG132, we found that the inhibitory effect of acetate on VSMC osteogenic differentiation was weakened by the intervention of BSO, but MG132 had no effect.ConclusionABX exacerbates vascular calcification, possibly by depleting the abundance of Bacteroidetes and SCFAs in the intestine. Supplementation with acetate has the potential to alleviate vascular calcification, which may be an important target for future treatment of vascular calcification.

Gasdermin D deficiency attenuates development of ascending aortic dissections in a novel mouse model.

Thoracic aortic dissection (TAD) is a silent killer. Approximately two-thirds of the cases occur in the ascending aorta (i.e. type A dissection) and majority of them are unrelated to genetic mutations. However, animal models of spontaneous type A dissection are not widely available. In the present study, a novel mouse TAD model was created. Further, the role of gasdermin D (GSDMD) in TAD development was evaluated. TADs were created by treating ascending aorta of adult mice (C57BL/6J) with active elastase (40.0 U/ml) and β-aminopropionitrile (Act E+BAPN). The temporal progress of the TAD pathology was rigorously characterized by histological evaluation and scanning electron microscopy, while potential mechanisms explored with bulk RNA sequencing of specimens collected at multiple timepoints. With this novel TAD model, further experiments were performed with Gsdmd -/- mice to evaluate its impact on TAD formation. The ascending aorta challenged with Act E+BAPN developed pathology characterized by an early onset of intimomedial tears (complete penetration) and intramural hematoma, followed by progressive medial loss and aortic dilation. Ingenuity Pathway Analysis and functional annotation of differentially expressed genes suggested that a unique inflammatory micro-environment, rather than general inflammation, promoted the onset of TADs by specifically recruiting neutrophils to the aortic wall, while the pathology at the advanced stage was driven by T-cell mediated immune injury. Gsdmd -/- attenuated medial loss, adventitial fibrosis, and dilation of TADs. This protective effect was associated with a reduced number of TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) positive cells and T-cells in TADs. A novel mouse TAD model was created in the ascending aorta. It produces a unique microenvironment to activate different immune cell subsets, promoting onset and subsequent remodeling of TADs. Consistently, Gsdmd -/- attenuates TAD development, with modulation of cell death and T-cell response likely acting as the underlying mechanism.

Berberine Inhibits Ferroptosis and Stabilizes Atherosclerotic Plaque through NRF2/SLC7A11/GPX4 Pathway.

To investigate potential mechanisms of anti-atherosclerosis by berberine (BBR) using ApoE-/- mice. Eight 8-week-old C57BL/6J mice were used as a blank control group (normal), and 56 8-week-old AopE-/- mice were fed a high-fat diet for 12 weeks, according to a completely random method, and were divided into the model group, BBR low-dose group (50 mg/kg, BBRL), BBR medium-dose group (100 mg/kg, BBRM), BBR high-dose group (150 mg/kg, BBRH), BBR+nuclear factor erythroid 2-related factor 2 (NRF2) inhibitor group (100 mg/kg BBR+30 mg/kg ML385, BBRM+ML385), NRF2 inhibitor group (30 mg/kg, ML385), and positive control group (2.5 mg/kg, atorvastatin), 8 in each group. After 4 weeks of intragastric administration, samples were collected and serum, aorta, heart and liver tissues were isolated. Biochemical kits were used to detect serum lipid content and the expression levels of malondialdehyde (MDA) and superoxide dismutase (SOD) in all experimental groups. The pathological changes of atherosclerosis (AS) were observed by aorta gross Oil Red O, aortic sinus hematoxylin-eosin (HE) and Masson staining. Liver lipopathy was observed in mice by HE staining. The morphology of mitochondria in aorta cells was observed under transmission electron microscope. Flow cytometry was used to detect reactive oxygen species (ROS) expression in aorta of mice in each group. The content of ferrous ion Fe2+ in serum of mice was detected by biochemical kit. The mRNA and protein relative expression levels of NRF2, glutathione peroxidase 4 (GPX4) and recombinant solute carrier family 7 member 11 (SLC7A11) were detected by quantitative real time polymerase chain reaction (RT-qPCR) and Western blot, respectively. BBRM and BBRH groups delayed the progression of AS and reduced the plaque area (P<0.01). The characteristic morphological changes of ferroptosis were rarely observed in BBR-treated AS mice, and the content of Fe2+ in BBR group was significantly lower than that in the model group (P<0.01). BBR decreased ROS and MDA levels in mouse aorta, increased SOD activity (P<0.01), significantly up-regulated NRF2/SLC7A11/GPX4 protein and mRNA expression levels (P<0.01), and inhibited lipid peroxidation. Compared with the model group, the body weight, blood lipid level and aortic plaque area of ML385 group increased (P<0.01); the morphology of mitochondria showed significant ferroptosis characteristics; the serum Fe2+, MDA and ROS levels increased (P<0.05 or P<0.01), and the activity of SOD decreased (P<0.01). Compared with BBRM group, the iron inhibition effect of BBRM+ML385 group was significantly weakened, and the plaque area significantly increased (P<0.01). Through NRF2/SLC7A11/GPX4 pathway, BBR can resist oxidative stress, inhibit ferroptosis, reduce plaque area, stabilize plaque, and exert anti-AS effects.

Empagliflozin improves aortic injury in obese mice by regulating fatty acid metabolism.

Empagliflozin has been shown in clinical studies to lower the risk of adverse cardiovascular events. Using proteomics, the current study aims to determine whether empagliflozin reduces aortic alterations in obese mice and to investigate its molecular mechanism of action. We constructed obese mice and then treated them with empagliflozin. Changes in the weight of the mice were recorded. Blood glucose and lipid levels were measured in each group of mice, and changes in pulse wave velocity and aortic structure were recorded. In addition, changes in aortic protein expression were detected by proteomics and analyzed bioinformatically. Proteomics results showed that 507 differentially expressed proteins (DEPs) were identified in the comparison of normal and obese mice, while 90 DEPs were identified in the comparison of obese and empagliflozin-treated mice. Examination of these three groups revealed that DEPs were largely associated with the digestion of unsaturated fats. Among them, empagliflozin significantly reduced the expression of fatty acid synthase (FASN), acyl-CoA desaturase 3 (SCD3), ACSL1. and ACSL5 in the aorta of obesity-induced mice, and there was a close relationship between the four. Empagliflozin reduced the protein expression of FASN, SCD3, ACSL1, and ACSL5 in the aorta of obese mice and improved aortic fatty acid metabolism and reduced vascular stiffness for vasoprotective effects.

Assessing Atherosclerosis by Super-Resolution Imaging of HClO in Foam Cells Using a Ratiometric Fluorescent Probe.

Atherosclerosis (AS) is the leading cause of cardiovascular disease. Foam cells, with elevated lipid droplets (LDs) and HClO levels, are the main components of the atherosclerotic plaques that are characteristic of AS. Super-resolution imaging can be used to visualize the distribution of LDs in foam cells at the nanometer level, facilitating the identification of LDs and HClO. In the present study, we report the development of a ratiometric fluorescent probe, SFL-HClO, for super-resolution imaging of LDs and HClO. Super-resolution imaging with this probe revealed the precise structure of LDs at the suborganelle level. Moreover, the fluorescence behavior of SFL-HClO on the surface of LDs verified its excellent performance in detecting HClO in the foam cells. SFL-HClO can sequentially and specifically respond to LDs and HClO via "turn-on" and ratiometric signal output, respectively, thus contributing to precise imaging of foam cells. Importantly, we demonstrate that SFL-HClO can be used to report on upregulated HClO in atherosclerotic plaques in the aorta of AS mice, providing a suitable fluorescent tool for early atherosclerotic disease assessment.

Discovery of an embryonically derived bipotent population of endothelial-macrophage progenitor cells in postnatal aorta

Converging evidence indicates that extra-embryonic yolk sac is the source of both macrophages and endothelial cells in adult mouse tissues. Prevailing views are that these embryonically derived cells are maintained after birth by proliferative self-renewal in their differentiated states. Here we identify clonogenic endothelial-macrophage (EndoMac) progenitor cells in the adventitia of embryonic and postnatal mouse aorta, that are independent of Flt3-mediated bone marrow hematopoiesis and derive from an early embryonic CX3CR1+ and CSF1R+ source. These bipotent progenitors are proliferative and vasculogenic, contributing to adventitial neovascularization and formation of perfused blood vessels after transfer into ischemic tissue. We establish a regulatory role for angiotensin II, which enhances their clonogenic and differentiation properties and rapidly stimulates their proliferative expansion in vivo. Our findings demonstrate that embryonically derived EndoMac progenitors participate in local vasculogenic responses in the aortic wall by contributing to the expansion of endothelial cells and macrophages postnatally.

Effects of a high-phosphate diet on vascular calcification and abdominal aortic aneurysm in mice.

Vascular aging is an important risk factor for cardiovascular diseases, including abdominal aortic aneurysm (AAA) and pathological aortic dilatation, playing a critical role in the morbidity and mortality of older adults. Vascular calcification, a phenotype of vascular aging, is frequently associated with AAA. However, this association remains unclear owing to the lack of animal models. This study investigated the effects of a high-phosphate diet (HPD), a prominent trigger of vascular calcification in AAA. Eight-week-old male mice were fed either a normal diet (ND; Ca 1.18%/P 1.07% = 1.10) or an HPD (Ca 1.23%/P 1.65% = 0.75) for 4 weeks. Subsequently, AAA was induced using CaCl2 application and angiotensin II (AngII) infusion for 4 weeks. The HPD resulted in more pronounced AAA formation than did the ND. Importantly, vascular calcification was observed only in the aorta of the HPD mice. Enhanced Runt-related transcription factor 2 expression and apoptosis (downregulation of growth arrest-specific gene 6/pAkt survival pathway), two major mechanisms of vascular calcification, were also observed. Furthermore, increased IL-6 and F4/80 expression was observed in the aorta of HPD mice. In RAW264.7 cells, inorganic phosphate enhanced IL-6 and IL-1β expression under AngII priming. Ferric citrate, a phosphate binder, significantly inhibited HPD-induced AAA formation. These findings suggest that HPD induces vascular calcification and AAA formation, possibly through inflammation. This murine model suggests that vascular calcification induced by phosphate burden may be a therapeutic target for vascular diseases, including AAA. Geriatr Gerontol Int 2024; ••: ••-••.

Age-Disturbed Vascular Extracellular Matrix Links to Abdominal Aortic Aneurysms.

Abdominal aortic aneurysm (AAA) is a common but life-threatening vascular condition in men at an advanced age. However, the underlying mechanisms of age-increased incidence and mortality of AAA remain elusive. Here, we performed RNA sequencing (RNA-seq) of mouse aortas from males (young: 3-month, n = 4 vs old: 23-month, n = 4) and integrated with the data sets of human aortas (young: 20-39, n = 47 vs old: 60-79 years, n = 92) from GTEx project and the data set (GSE183464) for AAA to search for age-shifted aortic aneurysm genes, their relevant biological processes, and signaling pathways. Angiotensin II-induced AAA in mice was used to verify the critical findings. We found 1001 genes transcriptionally changed with ages in both mouse and human. Most age-increased genes were enriched intracellularly and the relevant biological processes included mitochondrial function and translational controls, whereas the age-decreased genes were largely localized in extracellular regions and cell periphery and the involved biological processes were associated with extracellular matrix (ECM). Fifty-one were known genes for AAA and found dominantly in extracellular region. The common age-shifted vascular genes and known aortic aneurysm genes had shared functional influences on ECM organization, apoptosis, and angiogenesis. Aorta with angiotensin II-induced AAA exhibited similar phenotypic changes in ECM to that in old mice. Together, we present a conserved transcriptional signature for aortic aging and provide evidence that mitochondrial dysfunction and the imbalanced ribosomal homeostasis act likely as driven-forces for aortic aging and age-disturbed ECM is the substrate for developing AAA.