Intense impact of IL-1ß expressing inflammatory macrophages in acute aortic dissection.

There is no treatment for acute aortic dissection (AAD) targeting inflammatory cells. We aimed to identify the new therapeutic targets associated with inflammatory cells. We characterized the specific distribution of myeloid cells of both human type A AAD samples and a murine AAD model generated using angiotensin II (ANGII) and ß-aminopropionitrile (BAPN) by single-cell RNA sequencing (scRNA-seq). We also examined the effect of an anti-interleukin-1ß (IL-1ß) antibody in the murine AAD model. IL1B+ inflammatory macrophages and classical monocytes were increased in human AAD samples. Trajectory analysis demonstrated that IL1B+ inflammatory macrophages differentiated from S100A8/9/12+ classical monocytes uniquely observed in the aorta of AAD. We found increased infiltration of neutrophils and monocytes with the expression of inflammatory cytokines in the aorta and accumulation of inflammatory macrophages before the onset of macroscopic AAD in the murine AAD model. In blocking experiments using an anti-IL-1ß antibody, it improved survival of murine AAD model by preventing elastin degradation. We observed the accumulation of inflammatory macrophages expressing IL-1ß in both human AAD samples and in a murine AAD model. Anti-IL-1ß antibody could improve the mortality rate in mice, suggesting that it may be a treatment option for AAD.

Asiatic acid alleviates vascular remodeling in BAPN-induced aortic dissection through inhibiting NF-κB p65/CX3CL1 signaling.

Inflammation assumes a pivotal role in the aortic remodeling of aortic dissection (AD). Asiatic acid (AA), a triterpene compound, is recognized for its strong anti-inflammatory properties. Yet, its effects on ß-aminopropionitrile (BAPN)-triggered AD have not been clearly established. The objective is to determine whether AA attenuates adverse aortic remodeling in BAPN-induced AD and clarify potential molecular mechanisms. In vitro studies, RAW264.7 cells pretreated with AA were challenged with lipopolysaccharide (LPS), and then the vascular smooth muscle cells (VSMCs)-macrophage coculture system was established to explore intercellular interactions. To induce AD, male C57BL/6J mice at three weeks of age were administered BAPN at a dosage of 1 g/kg/d for four weeks. To decipher the mechanism underlying the effects of AA, RNA sequencing analysis was conducted, with subsequent validation of these pathways through cellular experiments. AA exhibited significant suppression of M1 macrophage polarization. In the cell coculture system, AA facilitated the transformation of VSMCs into a contractile phenotype. In the mouse model of AD, AA strikingly prevented the BAPN-induced increases in inflammation cell infiltration and extracellular matrix degradation. Mechanistically, RNA sequencing analysis revealed a substantial upregulation of CX3CL1 expression in BAPN group but downregulation in AA-treated group. Additionally, it was observed that the upregulation of CX3CL1 negated the beneficial impact of AA on the polarization of macrophages and the phenotypic transformation of VSMCs. Crucially, our findings revealed that AA is capable of downregulating CX3CL1 expression, accomplishing this by obstructing the nuclear translocation of NF-κB p65. The findings indicate that AA holds promise as a prospective treatment for adverse aortic remodeling by suppressing the activity of NF-κB p65/CX3CL1 signaling pathway.

Cannabidiol protects against acute aortic dissection by inhibiting macrophage infiltration and PMAIP1-induced vascular smooth muscle cell apoptosis.

Acute aortic dissection (AAD) progresses rapidly and is associated with high mortality; therefore, there remains an urgent need for pharmacological agents that can protect against AAD. Herein, we examined the therapeutic effects of cannabidiol (CBD) in AAD by establishing a suitable mouse model. In addition, we performed human AAD single-cell RNA sequencing and mouse AAD bulk RNA sequencing to elucidate the potential underlying mechanism of CBD. Pathological assays and in vitro studies were performed to verify the results of the bioinformatic analysis and explore the pharmacological function of CBD. In a ß-aminopropionitrile (BAPN)-induced AAD mouse model, CBD reduced AAD-associated morbidity and mortality, alleviated abnormal enlargement of the ascending aorta and aortic arch, and suppressed macrophage infiltration and vascular smooth muscle cell (VSMC) apoptosis. Bioinformatic analysis revealed that the pro-apoptotic gene PMAIP1 was highly expressed in human and mouse AAD samples, and CBD could inhibit Pmaip1 expression in AAD mice. Using human aortic VSMCs (HAVSMCs) co-cultured with M1 macrophages, we revealed that CBD alleviated HAVSMCs mitochondrial-dependent apoptosis by suppressing the BAPN-induced overexpression of PMAIP1 in M1 macrophages. PMAIP1 potentially mediates HAVSMCs apoptosis by regulating Bax and Bcl2 expression. Accordingly, CBD reduced AAD-associated morbidity and mortality and mitigated the progression of AAD in a mouse model. The CBD-induced effects were potentially mediated by suppressing macrophage infiltration and PMAIP1 (primarily expressed in macrophages)-induced VSMC apoptosis. Our findings offer novel insights into M1 macrophages and HAVSMCs interaction during AAD progression, highlighting the potential of CBD as a therapeutic candidate for AAD treatment.

Inhibiting Lysyl Oxidases prevents pathologic cartilage calcification.

Lysyl oxidases (LOX(L)) are enzymes that catalyze the formation of cross-links in collagen and elastin fibers during physiologic calcification of bone. However, it remains unknown whether they may promote pathologic calcification of articular cartilage, an important hallmark of debilitating arthropathies. Here, we have studied the possible roles of LOX(L) in cartilage calcification, related and not related to their cross-linking activity. We first demonstrated that inhibition of LOX(L) by ß-aminoproprionitrile (BAPN) significantly reduced calcification in murine and human chondrocytes, and in joint of meniscectomized mice. These BAPN's effects on calcification were accounted for by different LOX(L) roles. Firstly, reduced LOX(L)-mediated extracellular matrix cross-links downregulated Anx5, Pit1 and Pit2 calcification genes. Secondly, BAPN reduced collagen fibrotic markers Col1 and Col3. Additionally, LOX(L) inhibition blocked chondrocytes hypertrophic differentiation (Runx2 and COL10), pro-inflammatory IL-6 release and reactive oxygen species (ROS) production, all triggers of chondrocyte calcification. Through unbiased transcriptomic analysis we confirmed a positive correlation between LOX(L) genes and genes for calcification, hypertrophy and extracellular matrix catabolism. This association was conserved throughout species (mouse, human) and tissues that can undergo pathologic calcification (kidney, arteries, skin). Overall, LOX(L) play a critical role in the process of chondrocyte calcification and may be therapeutic targets to treat cartilage calcification in arthropathies.

Selenium Deficiency Promotes Dilatation of the Aorta by Increasing Expression and Activity of Vascular Smooth Muscle Cell Derived Matrix Metalloproteinase-2.

OBJECTIVE: Selenium (Se) is a key part of the body's oxidation defence system. However, it is unclear whether Se affects the development of aortic aneurysm (AA). An animal experiment was conducted to clarify the role of Se in AA development. METHODS: C57BL/6N male mice were fed with a Se deficient (Se-D, < 0.05 mg/kg), Se adequate (Se-A, 0.2 mg/kg), or Se supplemented (Se-S, 1 mg/kg) diet for 8 weeks. Subsequently, an AA murine model (Se-D, n = 11; Se-A, n = 12; Se-S, n = 15) was established using angiotensin II (Ang II, 1 mg/kg/min) for four weeks plus ß-aminopropionitrile (BAPN, 1 mg/mL) for the first two weeks. Saline replaced Ang II, and BAPN was removed during the modelling process for sham mice (Se-A, n = 9). To determine whether Se deficiency promoted aortic dilation via matrix metalloproteinase-2 (MMP-2), the non-specific MMP inhibitor doxycycline (Dox, 100 mg/kg/day) was given to Se-D AA mice (n = 7) for two weeks. RESULTS: The maximum aortic diameter in Se-D AA model mice was significantly increased compared with Se-A AA model mice. MMP-2 expression and activity in the aortic media of Se-D AA model mice was significantly increased compared with Se-A AA model mice. A large number of vascular smooth muscle cells (VSMCs) were found aggregating in the media of the non-dilated aorta of Se-D AA model mice, which was completely inhibited by Dox. The percentage of VSMCs in aortic media of Se-D AA model mice was significantly higher than in Se-A AA model mice. The maximum aortic diameter and occurrence rate of AA in Se-D AA model mice with Dox were significantly reduced compared with Se-D AA model mice. CONCLUSION: Se deficiency promoted dilatation of the aorta in AA model mice by increasing expression and activity of VSMC derived MMP-2, causing abnormal aggregation and proliferation of VSMCs in aortic media.

The activator protein-1 complex governs a vascular degenerative transcriptional programme in smooth muscle cells to trigger aortic dissection and rupture.

BACKGROUND AND AIMS: Stanford type A aortic dissection (AD) is a degenerative aortic remodelling disease marked by an exceedingly high mortality without effective pharmacologic therapies. Smooth muscle cells (SMCs) lining tunica media adopt a range of states, and their transformation from contractile to synthetic phenotypes fundamentally triggers AD. However, the underlying pathomechanisms governing this population shift and subsequent AD, particularly at distinct disease temporal stages, remain elusive. METHODS: Ascending aortas from nine patients undergoing ascending aorta replacement and five individuals undergoing heart transplantation were subjected to single-cell RNA sequencing. The pathogenic targets governing the phenotypic switch of SMCs were identified by trajectory inference, functional scoring, single-cell regulatory network inference and clustering, regulon, and interactome analyses and confirmed using human ascending aortas, primary SMCs, and a ß-aminopropionitrile monofumarate-induced AD model. RESULTS: The transcriptional profiles of 93 397 cells revealed a dynamic temporal-specific phenotypic transition and marked elevation of the activator protein-1 (AP-1) complex, actively enabling synthetic SMC expansion. Mechanistically, tumour necrosis factor signalling enhanced AP-1 transcriptional activity by dampening mitochondrial oxidative phosphorylation (OXPHOS). Targeting this axis with the OXPHOS enhancer coenzyme Q10 or AP-1-specific inhibitor T-5224 impedes phenotypic transition and aortic degeneration while improving survival by 42.88% (58.3%-83.3% for coenzyme Q10 treatment), 150.15% (33.3%-83.3% for 2-week T-5224), and 175.38% (33.3%-91.7% for 3-week T-5224) in the ß-aminopropionitrile monofumarate-induced AD model. CONCLUSIONS: This cross-sectional compendium of cellular atlas of human ascending aortas during AD progression provides previously unappreciated insights into a transcriptional programme permitting aortic degeneration, highlighting a translational proof of concept for an anti-remodelling intervention as an attractive strategy to manage temporal-specific AD by modulating the tumour necrosis factor-OXPHOS-AP-1 axis.

Mechanical Regulation of Retinal Vascular Inflammation and Degeneration in Diabetes.

Vascular inflammation is known to cause degeneration of retinal capillaries in early diabetic retinopathy (DR), a major microvascular complication of diabetes. Past studies investigating these diabetes-induced retinal vascular abnormalities have focused primarily on the role of molecular or biochemical cues. Here we show that retinal vascular inflammation and degeneration in diabetes are also mechanically regulated by the increase in retinal vascular stiffness caused by overexpression of the collagen-cross-linking enzyme lysyl oxidase (LOX). Treatment of diabetic mice with LOX inhibitor ß-aminopropionitrile (BAPN) prevented the increase in retinal capillary stiffness, vascular intracellular adhesion molecule-1 overexpression, and leukostasis. Consistent with these anti-inflammatory effects, BAPN treatment of diabetic mice blocked the upregulation of proapoptotic caspase-3 in retinal vessels, which concomitantly reduced retinal capillary degeneration, pericyte ghost formation, and the diabetes-induced loss of contrast sensitivity in these mice. Finally, our in vitro studies indicate that retinal capillary stiffening is sufficient to increase the adhesiveness and neutrophil elastase-induced death of retinal endothelial cells. By uncovering a link between LOX-dependent capillary stiffening and the development of retinal vascular and functional defects in diabetes, these findings offer a new insight into DR pathogenesis that has important translational potential.

Causal Role for Neutrophil Elastase in Thoracic Aortic Dissection in Mice.

BACKGROUND: Thoracic aortic dissection (TAD) is a life-threatening aortic disease without effective medical treatment. Increasing evidence has suggested a role for NE (neutrophil elastase) in vascular diseases. In this study, we aimed at investigating a causal role for NE in TAD and exploring the molecular mechanisms involved. METHODS: ß-aminopropionitrile monofumarate was administrated in mice to induce TAD. NE deficiency mice, pharmacological inhibitor GW311616A, and adeno-associated virus-2-mediated in vivo gene transfer were applied to explore a causal role for NE and associated target gene in TAD formation. Multiple functional assays and biochemical analyses were conducted to unravel the underlying cellular and molecular mechanisms of NE in TAD. RESULTS: NE aortic gene expression and plasma activity was significantly increased during ß-aminopropionitrile monofumarate-induced TAD and in patients with acute TAD. NE deficiency prevents ß-aminopropionitrile monofumarate-induced TAD onset/development, and GW311616A administration ameliorated TAD formation/progression. Decreased levels of neutrophil extracellular traps, inflammatory cells, and MMP (matrix metalloproteinase)-2/9 were observed in NE-deficient mice. TBL1x (F-box-like/WD repeat-containing protein TBL1x) has been identified as a novel substrate and functional downstream target of NE in TAD. Loss-of-function studies revealed that NE mediated inflammatory cell transendothelial migration by modulating TBL1x-LTA4H (leukotriene A4 hydrolase) signaling and that NE regulated smooth muscle cell phenotype modulation under TAD pathological condition by regulating TBL1x-MECP2 (methyl CpG-binding protein 2) signal axis. Further mechanistic studies showed that TBL1x inhibition decreased the binding of TBL1x and HDAC3 (histone deacetylase 3) to MECP2 and LTA4H gene promoters, respectively. Finally, adeno-associated virus-2-mediated Tbl1x gene knockdown in aortic smooth muscle cells confirmed a regulatory role for TBL1x in NE-mediated TAD formation. CONCLUSIONS: We unravel a critical role of NE and its target TBL1x in regulating inflammatory cell migration and smooth muscle cell phenotype modulation in the context of TAD. Our findings suggest that the NE-TBL1x signal axis represents a valuable therapeutic for treating high-risk TAD patients.

Urate-Lowering Therapy Inhibits Thoracic Aortic Aneurysm and Dissection Formation in Mice.

BACKGROUND: Thoracic aortic aneurysm and dissection (TAAD) is a highly lethal vascular disease without effective drug therapy. Whether elevated serum concentrations of uric acid are involved in TAAD development remains unclear. METHODS: Serum uric acid levels were detected in different TAAD mouse models and patients. The urate-lowering drug allopurinol was administered in the drinking water of TAAD mice. Adenine diet-induced mice were established to investigate the role of hyperuricemia in TAAD formation and RNA-sequencing of thoracic aortas from these mice was performed. RESULTS: We found serum uric acid levels were elevated in various mouse TAAD models, including mice fed a ß-aminopropionitrile diet, Marfan mice with fibrillin-1 haploinsufficiency (Fbn1C1041G/+), and ApoE-/- mice infused with Ang II (angiotensin II), as well as in patients with TAAD. Administration of urate-lowering drug allopurinol in the drinking water significantly alleviated TAAD formation in ß-aminopropionitrile-treated mice, Fbn1C1041G/+ mice, and Ang II-infused ApoE-/- mice. Moreover, an adenine diet was used to induce hyperuricemia in mice. Intriguingly, a 4-week adenine diet feeding directly induced TAAD formation characterized by increased maximal thoracic aortic diameters and severe elastin degradation, which were ameliorated by allopurinol. Unbiased RNA-sequencing in mouse thoracic aortas suggested that FcγR (Fc gamma receptor) was upregulated upon adenine diet, but reciprocally repressed by allopurinol. Mechanistically, hyperuricemia activated FcγR-mediated ERK1/2 (extracellular signal-regulated kinase 1/2) phosphorylation to induce macrophage inflammation and TAAD development, which was abrogated by allopurinol or FcγR deficiency. CONCLUSIONS: This study uncovered an important and previously unrecognized role of hyperuricemia in mediating the pathogenesis of TAAD, and uric acid-lowering drug may represent a promising therapeutic approach for TAAD.

Combination Therapy of Lox Inhibitor and Stimuli-Responsive Drug for Mechanochemically Synergistic Breast Cancer Treatment.

Chemotherapy based on small molecule drugs, hormones, cycline kinase inhibitors, and monoclonal antibodies has been widely used for breast cancer treatment in the clinic but with limited efficacy, due to the poor specificity and tumor microenvironment (TME)-caused diffusion barrier. Although monotherapies targeting biochemical cues or physical cues in the TME have been developed, none of them can cope with the complex TME, while mechanochemical combination therapy remains largely to be explored. Herein, a combination therapy strategy based on an extracellular matrix (ECM) modulator and TME-responsive drug for the first attempt of mechanochemically synergistic treatment of breast cancer is developed. Specifically, based on overexpressed NAD(P)H quinone oxidoreductase 1 (NQO1) in breast cancer, a TME-responsive drug (NQO1-SN38) is designed and it is combined with the inhibitor (i.e., ß-Aminopropionitrile, BAPN) for Lysyl oxidases (Lox) that contributes to the tumor stiffness, for mechanochemical therapy. It is demonstrated that NQO1 can trigger the degradation of NQO1-SN38 and release SN38, showing nearly twice tumor inhibition efficiency compared with SN38 treatment in vitro. Lox inhibition with BAPN significantly reduces collagen deposition and enhances drug penetration in tumor heterospheroids in vitro. It is further demonstrated that the mechanochemical therapy showed outstanding therapeutic efficacy in vivo, providing a promising approach for breast cancer therapy.

Involvement of lysyl oxidase in the pathogenesis of arterial stiffness in chronic kidney disease.

Patients with chronic kidney disease (CKD) are at increased risk for adverse cardiovascular events. CKD is associated with increases in arterial stiffness, whereas improvements in arterial stiffness correlate with better survival. However, arterial stiffness is increased early in CKD, suggesting that there might be additional factors, unique to kidney disease, that increase arterial stiffness. Lysyl oxidase (LOX) is a key mediator of collagen cross linking and matrix remodeling. LOX is predominantly expressed in the cardiovascular system, and its upregulation has been associated with increased tissue stiffening and extracellular matrix remodeling. Thus, this study was designed to evaluate the role of increased LOX activity in inducing aortic stiffness in CKD and whether ß-aminopropionitrile (BAPN), a LOX inhibitor, could prevent aortic stiffness by reducing collagen cross linking. Eight-week-old male C57BL/6 mice were subjected to 5/6 nephrectomy (Nx) or sham surgery. Two weeks after surgery, mice were randomized to BAPN (300 mg/kg/day in water) or vehicle treatment for 4 wk. Aortic stiffness was assessed by pulse wave velocity (PWV) using Doppler ultrasound. Aortic levels of LOX were assessed by ELISA, and cross-linked total collagen levels were analyzed by mass spectrometry and Sircol assay. Nx mice showed increased PWV and aortic wall remodeling compared with control mice. Collagen cross linking was increased in parallel with the increases in total collagen in the aorta of Nx mice. In contrast, Nx mice that received BAPN treatment showed decreased cross-linked collagens and PWV compared with that received vehicle treatment. Our results indicated that LOX might be an early and key mediator of aortic stiffness in CKD.NEW & NOTEWORTHY Arterial stiffness in CKD is associated with adverse cardiovascular outcomes. However, the mechanisms underlying increased aortic stiffness in CKD are unclear. Herein, we demonstrated that 1) increased aortic stiffness in CKD is independent of hypertension and calcification and 2) LOX-mediated changes in extracellular matrix are at least in part responsible for increased aortic stiffness in CKD. Prevention of excess LOX may have therapeutic potential in alleviating increased aortic stiffness and improving cardiovascular disease in CKD.

A Polymeric Extracellular Matrix Nanoremodeler for Activatable Cancer Photo-Immunotherapy.

Cancer immunotherapy has shown tremendous potential to train the intrinsic immune system against malignancy in the clinic. However, the extracellular matrix (ECM) in tumor microenvironment is a formidable barrier that not only restricts the penetration of therapeutic drugs but also prevents the infiltration of antitumor immune cells. We herein report a semiconducting polymer-based ECM nanoremodeler (SPNcb) to combine photodynamic antitumor activity with cancer-specific inhibition of collagen-crosslinking enzymes (lysyl oxidase (LOX) family) for activatable cancer photo-immunotherapy. SPNcb is self-assembled from an amphiphilic semiconducting polymer conjugated with a LOX inhibitor (ß-aminopropionitrile, BAPN) via a cancer biomarker (cathepsin B, CatB)-cleavable segment. BAPN can be exclusively activated to inhibit LOX activity in the presence of the tumor-overexpressed CatB, thus blocking collagen crosslinking and decreasing ECM stiffness. Such an ECM nanoremodeler synergizes immunogenic phototherapy and checkpoint blockade immunotherapy to improve the tumor infiltration of cytotoxic T cells, inhibiting tumor growth and metastasis.

Knock-Out of the Five Lysyl-Oxidase Family Genes Enables Identification of Lysyl-Oxidase Pro-Enzyme Regulated Genes.

The five lysyl-oxidase genes share similar enzymatic activities and contribute to tumor progression. We have knocked out the five lysyl-oxidase genes in MDA-MB-231 breast cancer cells using CRISPR/Cas9 in order to identify genes that are regulated by LOX but not by other lysyl-oxidases and in order to study such genes in more mechanistic detail in the future. Re-expression of the full-length cDNA encoding LOX identified four genes whose expression was downregulated in the knock-out cells and rescued following LOX re-expression but not re-expression of other lysyl-oxidases. These were the AGR2, STOX2, DNAJB11 and DNAJC3 genes. AGR2 and STOX2 were previously identified as promoters of tumor progression. In addition, we identified several genes that were not downregulated in the knock-out cells but were strongly upregulated following LOX or LOXL3 re-expression. Some of these, such as the DERL3 gene, also promote tumor progression. There was very little proteolytic processing of the re-expressed LOX pro-enzyme in the MDA-MB-231 cells, while in the HEK293 cells, the LOX pro-enzyme was efficiently cleaved. We introduced point mutations into the known BMP-1 and ADAMTS2/14 cleavage sites of LOX. The BMP-1 mutant was secreted but not cleaved, while the LOX double mutant dmutLOX was not cleaved or secreted. However, even in the presence of the irreversible LOX inhibitor ß-aminoproprionitrile (BAPN), these point-mutated LOX variants induced the expression of these genes, suggesting that the LOX pro-enzyme has hitherto unrecognized biological functions.

A facile turn-on chemiluminescence probe for sensitive imaging on aminopeptidase N activity.

Aminopeptidase N, as a target for drug discovery, shows marked relationships with many diseases, especially liver injury and cancer. Here, we explored a chemiluminescence (CL) probe for sensing APN by tethering the APN-specific substrate group to the ortho-acrylated phenoxy-dioxetane scaffold. In this way, two CL probes (APN-CL and BAPN-CL) were designed with noncapped leucine and butoxy-carbonyl capped leucine as the protecting group to preserve the chemiexcitation energy. The uncovered leucine was demonstrated to be essential for detection of APN activity by comparing the CL intensity of two CL probes. Probe APN-CL was turned on upon APN cleavage, resulting in a high chemiluminescent emission, whereas the chemiexcitation energy of probe BAPN-CL was still restrained even with the high-level APN. The result was further elucidated by molecular docking simulations. Probe APN-CL exhibited a fast response and high sensitivity with a detection limit of 0.068 U/L, and an excellent specificity for the discrimination of APN from biological ions, small molecules, and other proteases commonly found in living system. By virtue of good stability and cell viability, probe APN-CL imaged abnormal levels of APN in tumour cells and tumour-bearing mice. Moreover, this probe APN-CL could be easily used to evaluate APN inhibitors and APN levels in plasma samples from 20 patients. Overall, as a facile and cost-effective probe, APN-CL will be a promising alternative in the early diagnosis of pathologies and for cost-effective screening of inhibitors.

Intramural injection of pluronic gel loaded with drugs to alleviate arterial injury.

BACKGROUND: Balloon angioplasty, stent implantation, and application of an arterial clamp during surgery can induce artery injury such as elastin breaks and endothelium injury, but there is little research focused on the injury induced by these therapeutic manipulations. We established a simple and reproducible small animal aortic injury model and examined intramural injection as a potential therapeutic method to alleviate injury. MATERIALS AND METHODS: The abdominal aorta of male Sprague Dawley (SD) rats or C57BL/6 J mice was clamped sequentially throughout its length. Transforming growth factor ß1 (TGFß1), SB431542, lipopolysaccharide (LPS), Necrostatin-1 (Nec-1), rapamycin, or MHY1485 contained in Pluronic gel was injected intramurally at day 0 or day 7. Animals were fed with chow containing 0.25% beta-aminopropionitrile (BAPN) to evaluate the influence of BAPN. All samples were harvested and examined by immunohistochemistry and immunofluorescence. RESULTS: The clamped rat aorta showed luminal dilation, elastin fiber breaks, neointimal hyperplasia, and dissection (days 0-90). Intramural injection of TGFß1, rapamycin and Nec-1 showed a protective effect on the injured aorta, whereas SB431542, MHY1485 and LPS showed more severe wall damage. The aortic lumen in rats fed with BAPN was significantly larger than in control rats (day 7). Mouse aorta showed similar injury with neointimal hyperplasia and elastin fiber breaks. CONCLUSIONS: The rodent arterial injury model is reproducible and may mimic early changes of arterial injury. The model accommodates intramural injection of different drugs that may show mechanisms of arterial injury. Although this is a preliminary animal model, the intramural injection method may have potential clinical application in the future.

Dexamethasone reduces the formation of thoracic aortic aneurysm and dissection in a murine model.

Thoracic aortic aneurysm and dissection (TAAD) is a life-threatening vascular disease with no effective pharmaceutical therapies currently available. Inflammation plays a key role in the progression of aneurysms. Dexamethasone (DEX), a synthetic glucocorticoid, has showed alleviating effects on cells in vitro from TAAD patients. Here we performed a study aiming at investigating the protective role of DEX in a ß-aminopropionitrile monofumarate (BAPN)-induced TAAD mouse model. DEX (dose: 0.04 mg/kg/day) treatment significantly reduced the aortic diameter and inhibited TAAD formation. DEX reduced infiltration of macrophages and neutrophils, apoptosis of vascular smooth muscle cells (VSMCs), expression of metalloproteinase 2/9, and extracellular matrix degradation in BAPN-treated TAAD mice. Furthermore, DEX therapy downregulated the expression of p-p65 in macrophages and VSMCs, which suggested that DEX might ameliorate BAPN-induced TAAD by suppressing NF-κB signaling. Therefore, DEX therapy attenuates the progression of BAPN-induced TAAD murine model and could be used as an effective adjuvant therapy for treating TAAD.

A validated mouse model capable of recapitulating the protective effects of female sex hormones on ascending aortic aneurysms and dissections (AADs).

Fewer females develop AADs (ascending aortic aneurysms and dissections) and the reasons for this protection remain poorly understood. The present study seeks to develop a mouse model that may be utilized to address this sexual dimorphism. Adult normolipidemic mice were challenged with BAPN (ß-aminopropionitrile), AngII (angiotensin II), or BAPN + AngII. An initial protocol optimization found that 0.2% BAPN in drinking water plus AngII-infusion at 1,000 ng kg-1  min-1 produced favorable rates of AAD rupture (~50%) and dilation (~40%) in 28 days. Using these dosages, further experiments revealed that BAPN is toxic to naïve mature aortas and it acted synergistically with AngII to promote aortic tears and dissections. BAPN + AngII provoked early infiltration of myeloid cells and subsequent recruitment of lymphoid cells to the aortic wall. AADs established with BAPN + AngII, but not AngII alone, continued to expand after the cessation of AngII-infusion. This indefinite growth precipitated a 61% increase in the AAD diameter in 56 days. More importantly, with the optimized protocol, significant differences in AAD dilation (p = .012) and medial degeneration (p = .036) were detected between male and female mice. Treatment of ovariectomized mice with estradiol protected AAD formation (p = .014). In summary, this study developed a powerful mouse AAD model that can be used to study the sexual dimorphism in AAD formation.

Blocking Interleukin-1 Beta Reduces the Evolution of Thoracic Aortic Dissection in a Rodent Model.

OBJECTIVE: Thoracic aortic dissection (TAD) is associated with matrix changes, biochemical changes, and inflammatory markers like interleukin-1 beta (IL-1ß). However, the exact mechanism remains unknown. This study aimed to investigate the role of IL-1ß, matrix metalloproteinase (MMP)-2, MMP-9, smooth muscle cell apoptosis, and elastic fibre fracture in the development of TAD in a rat model. METHODS: The TAD rat model was induced by ß-aminopropionitrile (BAPN). TAD was investigated in 112 male Sprague-Dawley rats, which were equally divided into four groups of 28 rats (Control, BAPN, BAPN + IL-1ß, and BAPN + IL-1ß antibody). Systolic blood pressure, survival, and the development of TAD were measured after six weeks. Expression of IL-1ß, MMP-2, and MMP-9 was measured by Western blot. Apoptosis, aortic elastin concentration, and biomechanical characteristics were measured by the TdT mediated dUTP nick end labelling assay, Victoria blue staining, and in vitro testing. RESULTS: During six weeks, the mortality was 0% (0/28) in the control group, 53.6% (15/28) in the BAPN group (p < .001 compared with the control group), 75.0% (21/28) in the BAPN + IL-1ß group (p = .007 compared with the BAPN group), and 35.7% (10/28) in the BAPN + IL-1ß antibody group (p = .023 compared with BAPN group and p < .001 compared with the BAPN + IL-1ß group). IL-1ß treatment deteriorates BAPN induced mortality and aneurysm expansion, which were attenuated by anti-IL-1ß treatment. In BAPN + IL-1ß group, stress and strain parameters were decreased by 13.5%-53.5% and elastin content was decreased by 14%, and IL-1ß, MMP-2, and MMP-9 were expressed higher by 117%, 108%, and 75% when compared with the rats in the BAPN group. Contrarily, in the BAPN + IL-1ß antibody group, the above changes could be completely (strain, elastin content, and expression of MMP-2) or partly (elasticity modulus, stress, and expression of MMP-9) blocked by anti-IL-1ß treatment. CONCLUSION: IL-1ß plays a critical role in TAD formation by altering the expression of MMP-2 and MMP-9, degrading the aortic wall matrix, causing elastic fibre rupture, and changing the stress or strain of the aortic wall. Anti-IL-1ß reduces the later effects and could be one of the molecular targets for prognosis and drug treatment of TAD in the future.

Cyclodextrin Prevents Abdominal Aortic Aneurysm via Activation of Vascular Smooth Muscle Cell Transcription Factor EB.

BACKGROUND: Abdominal aortic aneurysm (AAA) is a severe aortic disease with a high mortality rate in the event of rupture. Pharmacological therapy is needed to inhibit AAA expansion and prevent aneurysm rupture. Transcription factor EB (TFEB), a master regulator of autophagy and lysosome biogenesis, is critical to maintain cell homeostasis. In this study, we aim to investigate the role of vascular smooth muscle cell (VSMC) TFEB in the development of AAA and establish TFEB as a novel target to treat AAA. METHODS: The expression of TFEB was measured in human and mouse aortic aneurysm samples. We used loss/gain-of-function approaches to understand the role of TFEB in VSMC survival and explored the underlying mechanisms through transcriptome and functional studies. Using VSMC-selective Tfeb knockout mice and different mouse AAA models, we determined the role of VSMC TFEB and a TFEB activator in AAA in vivo. RESULTS: We found that TFEB is downregulated in both human and mouse aortic aneurysm lesions. TFEB potently inhibits apoptosis in VSMCs, and transcriptome analysis revealed that TFEB regulates apoptotic signaling pathways, especially apoptosis inhibitor B-cell lymphoma 2. B-cell lymphoma 2 is significantly upregulated by TFEB and is required for TFEB to inhibit VSMC apoptosis. We consistently observed that TFEB deficiency increases VSMC apoptosis and promotes AAA formation in different mouse AAA models. Furthermore, we demonstrated that 2-hydroxypropyl-ß-cyclodextrin, a clinical agent used to enhance the solubility of drugs, activates TFEB and inhibits AAA formation and progression in mice. Last, we found that 2-hydroxypropyl-ß-cyclodextrin inhibits AAA in a VSMC TFEB-dependent manner in mouse models. CONCLUSIONS: Our study demonstrated that TFEB protects against VSMC apoptosis and AAA. TFEB activation by 2-hydroxypropyl-ß-cyclodextrin may be a promising therapeutic strategy for the prevention and treatment of AAA.

Therapeutic Effect of Rapamycin on Aortic Dissection in Mice.

Aortic dissection (AD) is a serious clinical condition that is unpredictable and frequently results in fatal outcome. Although rapamycin, an inhibitor of mechanistic target of rapamycin (mTOR), has been reported to be effective in preventing aortopathies in mouse models, its mode of action has yet to be clarified. A mouse AD model that was created by the simultaneous administration of ß-aminopropionitrile (BAPN) and angiotensin II (AngII) for 14 days. Rapamycin treatment was started either at day 1 or at day 7 of BAPN+AngII challenge, and continued throughout the observational period. Rapamycin was effective both in preventing AD development and in suppressing AD progression. On the other hand, gefitinib, an inhibitor of growth factor signaling, did not show such a beneficial effect, even though both rapamycin and gefitinib suppressed cell cycle activation in AD. Rapamycin suppressed cell cycle-related genes and induced muscle development-related genes in an AD-related gene expression network without a major impact on inflammation-related genes. Rapamycin augmented the activation of Akt1, Akt2, and Stat3, and maintained the contractile phenotype of aortic smooth muscle cells. These findings indicate that rapamycin was effective both in preventing the development and in suppressing the progression of AD, indicating the importance of the mTOR pathway in AD pathogenesis.