Adenosine kinase inhibition protects mice from abdominal aortic aneurysm via epigenetic modulation of VSMC inflammation.

AIM: Abdominal aortic aneurysm (AAA) is a common, serious vascular disease with no effective pharmacological treatment. The nucleoside adenosine plays an important role in modulating vascular homeostasis, which prompted us to determine whether adenosine kinase (ADK), an adenosine metabolizing enzyme, modulates AAA formation via control of intracellular adenosine level, and to investigate the underlying mechanisms. METHODS AND RESULTS: We used a combination of genetic and pharmacological approaches in murine models of AAA induced by calcium chloride (CaCl2) application or angiotensin II (Ang II) infusion to study the role of ADK in the development of AAA. In vitro functional assays were performed by knocking down ADK with adenovirus-short hairpin RNA in human vascular smooth muscle cells (VSMCs), and the molecular mechanisms underlying ADK function were investigated using RNA-sequencing, isotope tracing and chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP-qPCR). Heterozygous deficiency of Adk protected mice from CaCl2- and Ang II-induced AAA formation. Moreover, specific knockout of Adk in VSMCs prevented Ang II-induced AAA formation, as evidenced by reduced aortic extracellular elastin fragmentation, neovascularization and aortic inflammation. Mechanistically, ADK knockdown in VSMCs markedly suppressed the expression of inflammatory genes associated with AAA formation, and these effects were independent of adenosine receptors. Metabolic flux and ChIP-qPCR results showed that ADK knockdown in VSMCs decreased S-adenosylmethionine (SAM)-dependent transmethylation, thereby reducing H3K4me3 binding to the promoter regions of the genes that are associated with inflammation, angiogenesis and extracellular elastin fragmentation. Furthermore, the ADK inhibitor ABT702 protected mice from CaCl2-induced aortic inflammation, extracellular elastin fragmentation and AAA formation. CONCLUSION: Our findings reveal a novel role for ADK inhibition in attenuating AAA via epigenetic modulation of key inflammatory genes linked to AAA pathogenesis.

Single-Molecule Spatial Transcriptomics of Human Thoracic Aortic Aneurysms Uncovers Calcification-Related CARTPT-Expressing Smooth Muscle Cells.

BACKGROUND: Although single-cell RNA-sequencing is commonly applied to dissect the heterogeneity in human tissues, it involves the preparation of single-cell suspensions via cell dissociation, causing loss of spatial information. In this study, we employed high-resolution single-cell transcriptome imaging to reveal rare smooth muscle cell (SMC) types in human thoracic aortic aneurysm (TAA) tissue samples. METHODS: Single-molecule spatial distribution of transcripts from 140 genes was analyzed in fresh-frozen human TAA samples with region and sex-matched controls. In vitro studies and tissue staining were performed to examine human CART prepropeptide (CARTPT) regulation and function. RESULTS: We captured thousands of cells per sample including a spatially distinct CARTPT-expressing SMC subtype enriched in male TAA samples. Immunoassays confirmed human CART (cocaine- and amphetamine-regulated transcript) protein enrichment in male TAA tissue and truncated CARTPT secretion into cell culture medium. Oxidized low-density lipoprotein, a cardiovascular risk factor, induced CARTPT expression, whereas CARTPT overexpression in human aortic SMCs increased the expression of key osteochondrogenic transcription factors and reduced contractile gene expression. Recombinant human CART treatment of human SMCs further confirmed this phenotype. Alizarin red staining revealed calcium deposition in male TAA samples showing similar localization with human CART staining. CONCLUSIONS: Here, we demonstrate the feasibility of single-molecule imaging in uncovering rare SMC subtypes in the diseased human aorta, a difficult tissue to dissociate. We identified a spatially distinct CARTPT-expressing SMC subtype enriched in male human TAA samples. Our functional studies suggest that human CART promotes osteochondrogenic switch of aortic SMCs, potentially leading to medial calcification of the thoracic aorta.

Nanoassembly with self-regulated magnetic thermal therapy and controlled immuno-modulating agent release for improved immune response.

Cancer immunotherapy is an emerging cancer therapeutic method by activating the patient's immune system but suffers from low immunogenicity at tumor sites. Fever-like heat is known to modulate an immune-friendly tumor microenvironment. Here, temperature-responsive iron oxide nanoassemblies (IONAs) are developed by crosslinking iron oxide nanoparticles (IONPs) and loaded with JQ1 (JQ1/IONAs), an immuno-modulating agent known to down-regulate PD-L1. In the presence of an alternating magnetic field (AMF), the IONAs demonstrate a much more effective magnetic thermal effect than IONPs and are responsively disassembled to prevent overheating. Compared with IONPs + AMF (∼ 41 °C) and unresponsive nanoassemblies (uIONAs) + AMF (∼ 50 °C), the IONAs + AMF with a temperature heated around 45 °C show a much better immune response and anti-tumor effect. Further combining the mild thermal therapy with controlled release of JQ1, the JQ1/IONAs + AMF completely eradicate the primary tumors and trigger a strong immune effect to inhibit the distant tumor growth as well as prevent tumor recurrence and metastasis. Our JQ1/IONAs not only provide a magnetic thermal agent with effective heating and temperature self-regulation ability but also serve as a heat-triggered JQ1 carrier to spontaneously combine mild magnetic thermal therapy with immune checkpoint blockade therapy.

BAF60c prevents abdominal aortic aneurysm formation through epigenetic control of vascular smooth muscle cell homeostasis.

Abdominal aortic aneurysm (AAA) is a life-threatening vascular disease. BAF60c, a unique subunit of the SWItch/sucrose nonfermentable (SWI/SNF) chromatin remodeling complex, is critical for cardiac and skeletal myogenesis, yet little is known about its function in the vasculature and, specifically, in AAA pathogenesis. Here, we found that BAF60c was downregulated in human and mouse AAA tissues, with primary staining to vascular smooth muscle cells (VSMCs), confirmed by single-cell RNA-sequencing. In vivo studies revealed that VSMC-specific knockout of Baf60c significantly aggravated both angiotensin II- (Ang II-) and elastase-induced AAA formation in mice, with a significant increase in elastin degradation, inflammatory cell infiltration, VSMC phenotypic switch, and apoptosis. In vitro studies showed that BAF60c knockdown in VSMCs resulted in loss of contractile phenotype, increased VSMC inflammation, and apoptosis. Mechanistically, we demonstrated that BAF60c preserved VSMC contractile phenotype by strengthening serum response factor (SRF) association with its coactivator P300 and the SWI/SNF complex and suppressing VSMC inflammation by promoting a repressive chromatin state of NF-κB target genes as well as preventing VSMC apoptosis through transcriptional activation of KLF5-dependent B cell lymphoma 2 (BCL2) expression. Our identification of the essential role of BAF60c in preserving VSMC homeostasis expands its therapeutic potential in preventing and treating AAA.

Mouse Abdominal Aortic Aneurysm Model Induced by Perivascular Application of Elastase.

Abdominal aortic aneurysm (AAA), although primarily asymptomatic, is potentially life-threatening as the rupture of AAA usually has a devastating outcome. Currently, there are several distinct experimental models of AAA, each emphasizing a different aspect in the pathogenesis of AAA. The elastase-induced AAA model is the second most used rodent AAA model. This model involves direct infusion or application of porcine pancreatic elastase (PPE) to the infrarenal segment of the aorta. Due to technical challenges, most elastase-induced AAA model nowadays is performed with the external application rather than an intraluminal infusion of PPE. The infiltration of elastase will cause degradation of elastic lamellae in the medial layers, resulting in the loss of aortic wall integrity and subsequent dilation of the abdominal aorta. However, one disadvantage of the elastase-induced AAA model is the inevitable variation of how the surgery is performed. Specifically, the surgical technique of isolating the infrarenal segment of the aorta, the material used for aorta wrapping and PPE incubation, the enzymatic activity of PPE, and the time duration of PPE application can all be important determinants that affect the eventual AAA formation rate and aneurysm diameter. Notably, the difference in these factors from different studies on AAA can lead to reproducibility issues. This article describes a detailed surgical process of the elastase-induced AAA model through direct application of PPE to the adventitia of the infrarenal abdominal aorta in the mouse. Following this procedure, a stable AAA formation rate of around 80% in male and female mice is achievable. The consistency and reproducibility of AAA studies using an elastase-induced AAA model can be significantly enhanced by establishing a standard surgical procedure.

KLF11 Protects against Venous Thrombosis via Suppressing Tissue Factor Expression.

Krüppel-like factors (KLFs) play essential roles in multiple biological functions, including maintaining vascular homeostasis. KLF11, a causative gene for maturity-onset diabetes of the young type 7, inhibits endothelial activation and protects against stroke. However, the role of KLF11 in venous thrombosis remains to be explored. Utilizing stasis-induced murine deep vein thrombosis (DVT) model and cultured endothelial cells (ECs), we identified an increase of KLF11 expression under prothrombotic conditions both in vivo and in vitro. The expression change of thrombosis-related genes was determined by utilizing gain- and loss-of-function approaches to alter KLF11 expression in ECs. Among these genes, KLF11 significantly downregulated tumor necrosis factor-α (TNF-α)-induced tissue factor (TF) gene transcription. Using reporter gene assay, chromatin immunoprecipitation assay, and co-immunoprecipitation, we revealed that KLF11 could reduce TNF-α-induced binding of early growth response 1 (EGR1) to TF gene promoter in ECs. In addition, we demonstrated that conventional Klf11 knockout mice were more susceptible to developing stasis-induced DVT. These results suggest that under prothrombotic conditions, KLF11 downregulates TF gene transcription via inhibition of EGR1 in ECs. In conclusion, KLF11 protects against venous thrombosis, constituting a potential molecular target for treating thrombosis.

Renal Clearable Ultrasmall Single-Crystal Fe Nanoparticles for Highly Selective and Effective Ferroptosis Therapy and Immunotherapy.

Iron-based nanoparticles have attracted much attention because of their ability to induce ferroptosis via a catalyzing Fenton reaction and to further potentiate immunotherapy. However, current iron-based nanoparticles need to be used in cooperation with other treatments or be applied in a high dose for effective therapy because of their low reactive oxygen species production efficacy. Here, we synthesized ultrasmall single-crystal Fe nanoparticles (bcc-USINPs) that stayed stable in a normal physiological environment but were highly active in a tumor microenvironment because of the selective acidic etching of an Fe3O4 shell and the exposure of the Fe(0) core. The bcc-USINPs could efficiently induce tumor cell ferroptosis and immunogenetic cell death at a very low concentration. Intravenous injection of iRGD-bcc-USINPs at three doses of 1 mg/kg could effectively suppress the tumor growth, promote the maturation of dendritic cells, and trigger the adaptive T cell response. Combined with programmed death-ligand 1 (PD-L1) immune checkpoint blockade immunotherapy, the iRGD-bcc-USINP-mediated ferroptosis therapy greatly potentiated the immune response and developed strong immune memory. In addition, these USINPs were quickly renal excreted with no side effects in normal tissues. These iRGD-bcc-USINPs provide a simple, safe, effective, and selectively tumor-responsive Fe(0) delivery system for ferroptosis-based immunotherapy.

Dysregulated oxalate metabolism is a driver and therapeutic target in atherosclerosis.

Dysregulated glycine metabolism is emerging as a common denominator in cardiometabolic diseases, but its contribution to atherosclerosis remains unclear. In this study, we demonstrate impaired glycine-oxalate metabolism through alanine-glyoxylate aminotransferase (AGXT) in atherosclerosis. As found in patients with atherosclerosis, the glycine/oxalate ratio is decreased in atherosclerotic mice concomitant with suppression of AGXT. Agxt deletion in apolipoprotein E-deficient (Apoe-/-) mice decreases the glycine/oxalate ratio and increases atherosclerosis with induction of hepatic pro-atherogenic pathways, predominantly cytokine/chemokine signaling and dysregulated redox homeostasis. Consistently, circulating and aortic C-C motif chemokine ligand 5 (CCL5) and superoxide in lesional macrophages are increased. Similar findings are observed following dietary oxalate overload in Apoe-/- mice. In macrophages, oxalate induces mitochondrial dysfunction and superoxide accumulation, leading to increased CCL5. Conversely, AGXT overexpression in Apoe-/- mice increases the glycine/oxalate ratio and decreases aortic superoxide, CCL5, and atherosclerosis. Our findings uncover dysregulated oxalate metabolism via suppressed AGXT as a driver and therapeutic target in atherosclerosis.

Catechol-metal coordination-mediated nanocomposite hydrogels for on-demand drug delivery and efficacious combination therapy.

Hydrogels have drawn considerable attention in the field of drug delivery, yet their poor mechanical strength and uncontrollable drug release behavior have hindered further applications in clinical practice. Taking utility of metal-ligand coordination for structurally reinforcing the hydrogel network, we report design and synthesis of magnetic nanocomposite hydrogels (HA-DOPA·MNPs) that are crosslinked by DOPA-Fe(III) coordination existing between dopamine-conjugated hyaluronan (HA-DOPA) and iron oxide magnetic nanoparticles (MNPs). The MNPs in the nanocomposite hydrogel not only serve as structural crosslinkers, but also facilitate magnetic hyperthermia and on-demand release of doxorubicin (DOX) in HA-DOPA·MNPs/DOX hydrogels, for release rate of DOX accelerates when external alternating magnetic field (AMF) is ON, and it restores to a slow pace when AMF is OFF. Importantly, HA-DOPA·MNPs/DOX hydrogel shows a longer retention time than HA-DOPA/DOX gel or DOX solution in vivo. Further experiments confirm the efficacious anticancer potency of HA-DOPA·MNPs/DOX in vitro and in vivo, that is mediated by a combination therapy consisting of chemotherapy (DOX) and hyperthermia (MNPs). In contrast, single-modality treatment (DOX or hyperthermia only) fails to show an equivalent efficacy at the same dose. STATEMENT OF SIGNIFICANCE: This study reports the design of a class of magnetic nanocomposite hydrogel (HA-DOPA·MNPs) that was structurally reinforced by DOPA-Fe (III) coordination between HA-DOPA and iron oxide MNPs. On one hand, MNPs served as crosslinking centers for structurally reinforcing the nanocomposite hydrogel; on the other hand, MNPs facilitated temperature rise under an external MNPs, which prompted on-demand drug release as well as a combination therapy. Comparing to single modality treatment (chemotherapy or hyperthermia alone), the HA-DOPA·MNPs/DOX formulation with AMF demonstrated better efficacy against proliferation of tumor cells (A375) both in vitro and in vivo. We believe that design of HA-DOPA·MNPs/DOX hydrogel in this report provides a general approach to fabricate structurally-reinforced nanocomposite hydrogels for on-demand drug delivery and efficacious combination therapy.

Single-cell RNA sequencing reveals the cellular heterogeneity of aneurysmal infrarenal abdominal aorta.

AIMS: The artery contains numerous cell types which contribute to multiple vascular diseases. However, the heterogeneity and cellular responses of these vascular cells during abdominal aortic aneurysm (AAA) progression have not been well characterized. METHODS AND RESULTS: Single-cell RNA sequencing was performed on the infrarenal abdominal aortas (IAAs) from C57BL/6J mice at Days 7 and 14 post-sham or peri-adventitial elastase-induced AAA. Unbiased clustering analysis of the transcriptional profiles from >4500 aortic cells identified 17 clusters representing nine-cell lineages, encompassing vascular smooth muscle cells (VSMCs), fibroblasts, endothelial cells, immune cells (macrophages, T cells, B cells, and dendritic cells), and two types of rare cells, including neural cells and erythrocyte cells. Seurat clustering analysis identified four smooth muscle cell (SMC) subpopulations and five monocyte/macrophage subpopulations, with distinct transcriptional profiles. During AAA progression, three major SMC subpopulations were proportionally decreased, whereas the small subpopulation was increased, accompanied with down-regulation of SMC contractile markers and up-regulation of pro-inflammatory genes. Another AAA-associated cellular response is immune cell expansion, particularly monocytes/macrophages. Elastase exposure induced significant expansion and activation of aortic resident macrophages, blood-derived monocytes and inflammatory macrophages. We also identified increased blood-derived reparative macrophages expressing anti-inflammatory cytokines suggesting that resolution of inflammation and vascular repair also persist during AAA progression. CONCLUSION: Our data identify AAA disease-relevant transcriptional signatures of vascular cells in the IAA. Furthermore, we characterize the heterogeneity and cellular responses of VSMCs and monocytes/macrophages during AAA progression, which provide insights into their function and the regulation of AAA onset and progression.

Porous yolk-shell Fe/Fe3O4 nanoparticles with controlled exposure of highly active Fe(0) for cancer therapy.

The iron-based Fenton-type reaction has drawn tremendous attention in cancer therapy. Compared with oxidized iron, Fe(0) possesses high catalytic activity but unstable for biomedical application. Here, we report a new strategy to stabilize Fe(0) via a porous yolk shell nanostructure of Fe/Fe3O4 (PYSNPs) in normal physiological condition, and to control the release of Fe(0) in tumor microenvironment for enhanced cancer therapy. These PYSNPs display superior tumor inhibition with the IC50 down to 20 µg/mL (over 1 mg/mL for iron oxide nanoparticles as control) for HepG2 cell. A single intravenous injection of as low as 1 mg/kg dosage is effective to suppress tumor growth in vivo. Moreover, the disintegration of PYSNPs in the acidic tumor microenvironment could cause significant change in MRI signal for contrast-enhanced diagnosis. Of note, the resulting Fe3O4 fragments are renal clearable with minimized side effect. In all, this work represented a nanoplatform to stabilize and selectively deliver Fe(0) for highly effective cancer therapy.

Endothelial TFEB (Transcription Factor EB) Improves Glucose Tolerance via Upregulation of IRS (Insulin Receptor Substrate) 1 and IRS2.

OBJECTIVE: Vascular endothelial cells (ECs) play a critical role in maintaining vascular homeostasis. Aberrant EC metabolism leads to vascular dysfunction and metabolic diseases. TFEB (transcription factor EB), a master regulator of lysosome biogenesis and autophagy, has protective effects on vascular inflammation and atherosclerosis. However, the role of endothelial TFEB in metabolism remains to be explored. In this study, we sought to investigate the role of endothelial TFEB in glucose metabolism and underlying molecular mechanisms. Approach and Results: To determine whether endothelial TFEB is critical for glucose metabolism in vivo, we utilized EC-selective TFEB knockout and EC-selective TFEB transgenic mice fed a high-fat diet. EC-selective TFEB knockout mice exhibited significantly impaired glucose tolerance compared with control mice. Consistently, EC-selective TFEB transgenic mice showed improved glucose tolerance. In primary human ECs, small interfering RNA-mediated TFEB knockdown blunts Akt (AKT serine/threonine kinase) signaling. Adenovirus-mediated overexpression of TFEB consistently activates Akt and significantly increases glucose uptake in ECs. Mechanistically, TFEB upregulates IRS1 and IRS2 (insulin receptor substrate 1 and 2). TFEB increases IRS2 transcription measured by reporter gene and chromatin immunoprecipitation assays. Furthermore, we found that TFEB increases IRS1 protein via downregulation of microRNAs (miR-335, miR-495, and miR-548o). In vivo, Akt signaling in the skeletal muscle and adipose tissue was significantly impaired in EC-selective TFEB knockout mice and consistently improved in EC-selective TFEB transgenic mice on high-fat diet. CONCLUSIONS: Our data revealed a critical role of TFEB in endothelial metabolism and suggest that TFEB constitutes a potential molecular target for the treatment of vascular and metabolic diseases.

Glycine-based treatment ameliorates NAFLD by modulating fatty acid oxidation, glutathione synthesis, and the gut microbiome.

Nonalcoholic fatty liver disease (NAFLD) including nonalcoholic steatohepatitis (NASH) has reached epidemic proportions with no pharmacological therapy approved. Lower circulating glycine is consistently reported in patients with NAFLD, but the causes for reduced glycine, its role as a causative factor, and its therapeutic potential remain unclear. We performed transcriptomics in livers from humans and mice with NAFLD and found suppression of glycine biosynthetic genes, primarily alanine-glyoxylate aminotransferase 1 (AGXT1). Genetic (Agxt1 -/- mice) and dietary approaches to limit glycine availability resulted in exacerbated diet-induced hyperlipidemia and steatohepatitis, with suppressed mitochondrial/peroxisomal fatty acid ß-oxidation (FAO) and enhanced inflammation as the underlying pathways. We explored glycine-based compounds with dual lipid/glucose-lowering properties as potential therapies for NAFLD and identified a tripeptide (Gly-Gly-L-Leu, DT-109) that improved body composition and lowered circulating glucose, lipids, transaminases, proinflammatory cytokines, and steatohepatitis in mice with established NASH induced by a high-fat, cholesterol, and fructose diet. We applied metagenomics, transcriptomics, and metabolomics to explore the underlying mechanisms. The bacterial genus Clostridium sensu stricto was markedly increased in mice with NASH and decreased after DT-109 treatment. DT-109 induced hepatic FAO pathways, lowered lipotoxicity, and stimulated de novo glutathione synthesis. In turn, inflammatory infiltration and hepatic fibrosis were attenuated via suppression of NF-κB target genes and TGFß/SMAD signaling. Unlike its effects on the gut microbiome, DT-109 stimulated FAO and glutathione synthesis independent of NASH. In conclusion, impaired glycine metabolism may play a causative role in NAFLD. Glycine-based treatment attenuates experimental NAFLD by stimulating hepatic FAO and glutathione synthesis, thus warranting clinical evaluation.

BAF60a Deficiency in Vascular Smooth Muscle Cells Prevents Abdominal Aortic Aneurysm by Reducing Inflammation and Extracellular Matrix Degradation.

OBJECTIVE: Currently, there are no approved drugs for abdominal aortic aneurysm (AAA) treatment, likely due to limited understanding of the primary molecular mechanisms underlying AAA development and progression. BAF60a-a unique subunit of the SWI/SNF (switch/sucrose nonfermentable) chromatin remodeling complex-is a novel regulator of metabolic homeostasis, yet little is known about its function in the vasculature and pathogenesis of AAA. In this study, we sought to investigate the role and underlying mechanisms of vascular smooth muscle cell (VSMC)-specific BAF60a in AAA formation. Approach and Results: BAF60a is upregulated in human and experimental murine AAA lesions. In vivo studies revealed that VSMC-specific knockout of BAF60a protected mice from both Ang II (angiotensin II)-induced and elastase-induced AAA formation with significant suppression of vascular inflammation, monocyte infiltration, and elastin fragmentation. Through RNA sequencing and pathway analysis, we found that the expression of inflammatory response genes in cultured human aortic smooth muscle cells was significantly downregulated by small interfering RNA-mediated BAF60a knockdown while upregulated upon adenovirus-mediated BAF60a overexpression. BAF60a regulates VSMC inflammation by recruiting BRG1 (Brahma-related gene-1)-a catalytic subunit of the SWI/SNF complex-to the promoter region of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) target genes. Furthermore, loss of BAF60a in VSMCs prevented the upregulation of the proteolytic enzyme cysteine protease CTSS (cathepsin S), thus ameliorating ECM (extracellular matrix) degradation within the vascular wall in AAA. CONCLUSIONS: Our study demonstrated that BAF60a is required to recruit the SWI/SNF complex to facilitate the epigenetic regulation of VSMC inflammation, which may serve as a potential therapeutic target in preventing and treating AAA.

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.

Rapamycin prevents thoracic aortic aneurysm and dissection in mice.

OBJECTIVE: The purpose of this study was to investigate whether rapamycin inhibits the development of thoracic aortic aneurysm and dissection (TAAD) in mice. METHODS: Three-week-old C57BL/6J male mice were fed a normal diet and randomized into a control group (n = 6), ß-aminopropionitrile fumarate (BAPN) group (Gp A; n = 15), BAPN plus rapamycin (5 mg) group (Gp B; n = 8), and BAPN plus rapamycin (10 mg) group (Gp C; n = 8). Gp A, Gp B, and Gp C were administered BAPN (1 g/kg/d) for 4 weeks. One week after BAPN administration, Gp B and Gp C were treated with rapamycin (5 mg/kg/d or 10 mg/kg/d) through gavage for 21 days. Thoracic aortas were harvested for Western blot and immunofluorescence staining at day 14 and for morphologic and histologic analyses at day 28. RESULTS: BAPN treatment induced TAAD formation in mice. The incidence of TAAD in control, Gp A, Gp B, and Gp C mice was 0%, 80%, 25%, and 37.5%, respectively. Smaller thoracic aortic diameters (ascending aorta and arch) were observed in Gp B and Gp C mice than in Gp A mice (Gp B vs Gp A: ascending aorta, ex vivo, 1.07 ± 0.21 mm vs 1.80 ± 0.67 mm [P < .05]; aortic arch, ex vivo, 1.51 ± 0.40 mm vs 2.70 ± 1.06 mm [P < .05]; Gp C vs Gp A: ascending aortas, ex vivo, 1.10 ± 0.33 mm vs 1.80 ± 0.67 mm [P < .05]; aortic arch, ex vivo, 1.55 ± 0.56 mm vs 2.70 ± 1.06 mm [P < .05]). TAAD mice exhibited elastin fragmentation, abundant inflammatory cell infiltration, and significantly increased matrix metalloproteinase production in the aorta, and rapamycin treatment alleviated these changes. The protein levels of p-S6K and p-S6 in TAAD aortic tissues increased significantly, whereas they were suppressed by rapamycin. CONCLUSIONS: Rapamycin suppressed TAAD formation, probably by inhibition of mechanistic target of rapamycin signaling and reduction of inflammatory cell infiltration and matrix metalloproteinase 9 production. Targeting of the mechanistic target of rapamycin signaling pathway using rapamycin may be a favorable modulation for the clinical treatment of TAAD.

Postnatal deficiency of ADAMTS1 ameliorates thoracic aortic aneurysm and dissection in mice.

NEW FINDINGS: What is the central question of this study? Thoracic aortic aneurysm and dissection (TAAD) is characterized by extracellular matrix remodelling and an inflammatory response. Evidence suggests that ADAMTS1 is closely associated with TAAD development, but whether it contributes to the pathophysiology of TAAD remains unknown. What is the main finding and its importance? We generated inducible postnatal ADAMTS1 knockout mice and found that ADAMTS1 deficiency attenuated ß-aminopropionitrile-dependent TAAD formation and rupture. Furthermore, ADAMTS1 deficiency suppressed neutrophil and macrophage infiltration by inhibiting inflammatory cytokine levels and macrophage migration during the early stage of ß-aminopropionitrile-induced TAAD. ADAMTS1 could be a new therapeutic target for TAAD. ABSTRACT: Thoracic aortic aneurysm and dissection (TAAD), as a life-threatening cardiovascular disease, is characterized by extracellular matrix remodelling and an inflammatory response. A disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1) is an inflammation-related protein that is able to degrade extracellular matrix proteins in arteries. Herein, we investigated whether ADAMTS1 contributes to the pathophysiology of TAAD in mice. Using the mouse model of ß-aminopropionitrile (BAPN)-induced TAAD, we found that ADAMTS1 expression was upregulated beginning in the early stage of TAAD development and localized predominantly in the aortic adventitia. ADAMTS1-floxed mice and whole-body tamoxifen-inducible ADAMTS1 knockout mice (ADAMTS1flox/flox Ubc-CreERT2+ , ADAMTS1 KO) were generated to investigate the direct causal role of ADAMTS1 in TAAD development. The incidence and rupture rates of BAPN-induced TAAD in ADAMTS1 KO mice were significantly lower than those in ADAMTS1flox/flox mice (45.5 versus 81.8% and 18.2 versus 42.4%, respectively). Aortas from BAPN-treated ADAMTS1flox/flox mice displayed profound destruction of the elastic lamellae, abundant neutrophil and macrophage accumulation in the adventitia, obviously increased neutrophil proportions in peripheral blood and significantly increased expression of inflammatory factors in the early stage of TAAD induction, all of which were markedly suppressed in ADAMTS1 KO mice. Furthermore, ADAMTS1-deficient macrophages exhibited abrogated migration capacity both in vivo and in vitro. In conclusion, ADAMTS1 plays a crucial role in postnatal TAAD formation and rupture by regulating inflammatory responses, suggesting that ADAMTS1 might be a new therapeutic target for TAAD.

Novel Adipokine, FAM19A5, Inhibits Neointima Formation After Injury Through Sphingosine-1-Phosphate Receptor 2.

BACKGROUND: Obesity plays crucial roles in the development of cardiovascular diseases. However, the mechanisms that link obesity and cardiovascular diseases remain elusive. Compelling evidence indicates that adipokines play an important role in obesity-related cardiovascular diseases. Here, we found a new adipokine-named family with sequence similarity 19, member A5 (FAM19A5), a protein with unknown function that was predicted to be distantly related to the CC-chemokine family. We aimed to test whether adipose-derived FAM19A5 regulates vascular pathology on injury. METHODS: DNA cloning, protein expression, purification, and N-terminal sequencing were applied to characterize FAM19A5. Adenovirus infection and siRNA transfection were performed to regulate FAM19A5 expression. Balloon and wire injury were performed in vivo on the rat carotid arteries and mouse femoral arteries, respectively. Bioinformatics analysis, radioactive ligand-receptor binding assays, receptor internalization, and calcium mobilization assays were used to identify the functional receptor for FAM19A5. RESULTS: We first characterized FAM19A5 as a secreted protein, and the first 43 N-terminal amino acids were the signal peptides. Both FAM19A5 mRNA and protein were abundantly expressed in the adipose tissue but were downregulated in obese mice. Overexpression of FAM19A5 markedly inhibited vascular smooth muscle cell proliferation and migration and neointima formation in the carotid arteries of balloon-injured rats. Accordingly, FAM19A5 silencing in adipocytes significantly promoted vascular smooth muscle cell activation. Adipose-specific FAM19A5 transgenic mice showed greater attenuation of neointima formation compared with wild-type littermates fed with or without Western-style diet. We further revealed that sphingosine-1-phosphate receptor 2 was the functional receptor for FAM19A5, with a dissociation constant (Kd) of 0.634 nmol/L. Inhibition of sphingosine-1-phosphate receptor 2 or its downstream G12/13-RhoA signaling circumvented the suppressive effects of FAM19A5 on vascular smooth muscle cell proliferation and migration. CONCLUSIONS: We revealed that a novel adipokine, FAM19A5, was capable of inhibiting postinjury neointima formation via sphingosine-1-phosphate receptor 2-G12/13-RhoA signaling. Downregulation of FAM19A5 during obesity may trigger cardiometabolic diseases.

In vitro study on hepatitis B virus infecting human choriocarcinoma JEG3 cells and its mechanism.

AIM: To build a hepatitis B virus (HBV)-infected human trophoblast cell model in vitro and determine the mechanism of intrauterine HBV infection. METHODS: Serum from hepatitis B-infected patients containing HBV DNA >10(9) was drawn, subsequently inoculated into human trophoblast cells in vitro (JEG3) and passage-cultured. The supernatants and intracellular HBV viral load of inoculated cells were tested by real-time PCR, and HBV DNA was determined by Southern blot. RESULTS: From inoculation of the 1st passage JEG3 cells, the supernatant viral load of the 1st passage was seen increasing over time, which peaked at 120 h, whereas the HBV viral load was seen decreasing gradually in subsequent passages, and tested negative after the 6th passage. In addition, infected cells of HBV DNA were tested by Southern blot, and showed continual expression in the subsequent cell passages 1-5 while passage 6 was negative. HBsAg was tested as positive from different passages 1-5, and its concentration was also seen decreasing with each subsequent passage cultured until the 6th passage when it tested negative. CONCLUSION: HBV could infect human trophoblast cells (JEG3) in vitro, and it showed continual expression in subsequent cell passages 1-5.

[Tumor necrosis factor-alpha, caspase-3 expression and hepatocyte apoptosis in fulminanting hepatic failure].

OBJECTIVE: To study the role of tumor necrosis factor-alpha (TNFalpha) and caspase-3 expression on hepatocyte apoptosis in experimental model of fulminant hepatic failure (FHF). METHODS: Mouse experimental model of FHF was induced by lipopolysaccharide (LPS) and D-galactosamine (D-GalN). Serum TNFalpha level and TNFalpha mRNA expression in liver were tested by ELISA and reverse transcriptase PCR (RT-PCR) method, respectively. The expression of caspase-3 in liver tissue was determined by in situ hybridization. Hepatocyte apoptosis was examined by DNA agarose gel electrophoresis and TUNEL method. TNFalpha, caspase-3 and hepatocyte apoptosis were observed in different stages after drug administration. In addition, we observed the changes of above variables after pretreatment with anti-TNFalpha IgG1. RESULTS: TNFalpha mRNA expression in liver increased significantly (0.91 +/- 0.75, that of normal control was 0.32 +/- 0.10) 2 to 4 hours after administration of LPS and D-GalN. The level of serum TNFalpha increased [(320.50 +/- 86.57) ng/L; that of normal control was (16.66 +/- 7.01) ng/L] and caspase-3 expressed to a slight extent, too. Typical manifestation of hepatocyte apoptosis appeared 8h after drug administration. 8 hours after drug administration the level of serum ALT and total bilirubin (TBil) remarkably increased [(560.66 +/- 60.20) U/L and (163.66 +/- 34.51) micro mol/L, respectively, that of normal control was (23.56 +/- 8.03) U/L and (14.90 +/- 4.80) micro mol/L, respectively] and the expression of caspase-3 reached the peak. 12 hours after drug administration, hepatocyte apoptosis and necrosis coexisted, and the level of serum ALT and TBil reached a peak [(668.30 +/- 78.69) U/L and (203.17 +/- 19.92) micro mol/L, respectively] whereas the expression of caspase-3 already decreased. Hepatocyte apoptosis and liver injury and the expression of caspase-3 could be blocked by antagonizing TNFalpha. CONCLUSIONS: TNFalpha played an important role in hepatocyte apoptosis and liver injury in fulminant hepatic failure. The hepatocyte apoptosis induced by TNFalpha is correlated with the activation of caspase-3. Hepatocyte apoptosis occurs earlier than necrosis.