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Transketolase drives the development of aortic dissection by impairing mitochondrial bioenergetics
Abstract
Aim
Aortic dissection (AD) is a disease with rapid onset but with no effective therapeutic drugs yet. Previous studies have suggested that glucose metabolism plays a critical role in the progression of AD. Transketolase (TKT) is an essential bridge between glycolysis and the pentose phosphate pathway. However, its role in the development of AD has not yet been elucidated. In this study, we aimed to explore the role of TKT in AD.
Methods
We collected AD patients' aortic tissues and used high‐throughput proteome sequencing to analyze the main factors influencing AD development. We generated an AD model using BAPN in combination with angiotensin II (Ang II) and pharmacological inhibitors to reduce TKT expression. The effects of TKT and its downstream mediators on AD were elucidated using human aortic vascular smooth muscle cells (HAVSMCs).
Results
We found that glucose metabolism plays an important role in the development of AD and that TKT is upregulated in patients with AD. Western blot and immunohistochemistry confirmed that TKT expression was upregulated in mice with AD. Reduced TKT expression attenuated AD incidence and mortality, maintained the structural integrity of the aorta, aligned elastic fibers, and reduced collagen deposition. Mechanistically, TKT was positively associated with impaired mitochondrial bioenergetics by upregulating AKT/MDM2 expression, ultimately contributing to NDUFS1 downregulation.
Conclusion
Our results provide new insights into the role of TKT in mitochondrial bioenergetics and AD progression. These findings provide new intervention options for the treatment of AD.
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Transketolase (Tkt), an enzyme in pentose phosphate pathway, has been reported to regulate genome instability and cell survival in cancers. Yet, the role of Tkt after myocardial ischemic injury remains to be elucidated. Label-free proteomics revealed dramatic elevation of Tkt in murine hearts after myocardial infarction (MI). Lentivirus-mediated Tkt knockdown ameliorated cardiomyocyte apoptosis and preserved the systolic function after myocardial ischemic injury. In contrast, Tkt overexpression led to the opposite effects. Inducible conditional cardiomyocyte Tkt-knockout mice were generated, and cardiomyocyte-expressed Tkt was found to play an intrinsic role in the ischemic heart failure of these model mice. Furthermore, through luciferase assay and chromatin immunoprecipitation, Tkt was shown to be a direct target of transcription factor Krüppel-like factor 5 (Klf5). In cardiomyocytes under ischemic stress, Tkt redistributed into the nucleus. By binding with the full-length poly(ADP-ribose) polymerase 1 (Parp1), facilitating its cleavage, and activating apoptosis inducible factor (Aif) subsequently, nuclear Tkt demonstrated its non-metabolic functions. Overall, our study confirmed that elevated nuclear Tkt plays a noncanonical role in promoting cardiomyocyte apoptosis via the cleaved Parp1/Aif pathway, leading to the deterioration of cardiac dysfunction.
Transketolase (TKT) which is an important metabolic enzyme in the pentose phosphate pathway (PPP) participates in maintaining ribose 5-phosphate levels. TKT is necessary for maintaining cell growth. However, we found that in addition to this, TKT can also affect tumor progression through other ways. Our previous study indicate that TKT could promote the development of liver cancer by affecting bile acid metabolism. And in this study, we discovered that TKT expression was remarkably upregulated in colorectal cancer, abnormal high expression of TKT is associated with poor prognosis of colorectal cancer. Additionally, TKT promoted colorectal cancer cell growth and metastasis. Further study demonstrated that TKT interacted with GRP78 and promoted colorectal cancer cell glycolysis through increasing AKT phosphorylation, thereby enhancing colorectal cancer cell metastasis. Thus, TKT is expected to become an indicator for judging the prognosis of colorectal cancer, and provide a theoretical basis for drug development of new treatment targets for colorectal cancer.
Mitochondria are highly dynamic, maternally inherited cytoplasmic organelles, which fulfill cellular energy demand through the oxidative phosphorylation system. Besides, they play an active role in calcium and damage‐associated molecular patterns signaling, amino acid, and lipid metabolism, and apoptosis. Thus, the maintenance of mitochondrial integrity and homeostasis is extremely critical, which is achieved through continual fusion and fission. Mitochondrial fusion allows the transfer of gene products between mitochondria for optimal functioning, especially under metabolic and environmental stress. On the other hand, fission is crucial for mitochondrial division and quality control. The imbalance between these two processes is associated with various ailments such as cancer, neurodegenerative and cardiovascular diseases. This review discusses the molecular mechanisms that control mitochondrial fusion and fission and how the disruption of mitochondrial dynamics manifests into various disease conditions.
The cyclic GMP–AMP synthase (cGAS)–stimulator of interferon genes (STING) signaling exert essential regulatory function in microbial-and onco-immunology through the induction of cytokines, primarily type I interferons. Recently, the aberrant and deranged signaling of the cGAS–STING axis is closely implicated in multiple sterile inflammatory diseases, including heart failure, myocardial infarction, cardiac hypertrophy, nonalcoholic fatty liver diseases, aortic aneurysm and dissection, obesity, etc. This is because of the massive loads of damage-associated molecular patterns (mitochondrial DNA, DNA in extracellular vesicles) liberated from recurrent injury to metabolic cellular organelles and tissues. Interestingly, the cGAS–STING pathway crosstalk with essential intracellular homeostasis processes like apoptosis, autophagy and also regulate cellular metabolism. Targeting derailed STING signaling has become necessary for chronic inflammatory diseases. Meanwhile, excessive type I interferons signaling impact on cardiovascular and metabolic health remain entirely elusive. In this review, we summarize the intimate connection between the cGAS–STING pathway and cardiovascular and metabolic disorders. We also discuss some potential small molecule inhibitors for the pathway. Importantly, this review will provide insight to stimulate interest in and support future research into understanding this signaling axis in cardiovascular and metabolic tissues and diseases.
Mitochondrial dysfunction has been suggested to be the key factor in the development and progression of cardiac hypertrophy. The onset of mitochondrial dysfunction and the mechanisms underlying the development of cardiac hypertrophy (CH) are incompletely understood. The present study is based on the use of multiple bioinformatics analyses for the organization and analysis of scRNA-seq and microarray datasets from a transverse aortic constriction (TAC) model to examine the potential role of mitochondrial dysfunction in the pathophysiology of CH. The results showed that NADH:ubiquinone oxidoreductase core subunit S1- (Ndufs1-) dependent mitochondrial dysfunction plays a key role in pressure overload-induced CH. Furthermore, in vivo animal studies using a TAC mouse model of CH showed that Ndufs1 expression was significantly downregulated in hypertrophic heart tissue compared to that in normal controls. In an in vitro model of angiotensin II- (Ang II-) induced cardiomyocyte hypertrophy, Ang II treatment significantly downregulated the expression of Ndufs1 in cardiomyocytes. In vitro mechanistic studies showed that Ndufs1 knockdown induced CH; decreased the mitochondrial DNA content, mitochondrial membrane potential (MMP), and mitochondrial mass; and increased the production of mitochondrial reactive oxygen species (ROS) in cardiomyocytes. On the other hand, Ang II treatment upregulated the expression levels of atrial natriuretic peptide, brain natriuretic peptide, and myosin heavy chain beta; decreased the mitochondrial DNA content, MMP, and mitochondrial mass; and increased mitochondrial ROS production in cardiomyocytes. The Ang II-mediated effects were significantly attenuated by overexpression of Ndufs1 in rat cardiomyocytes. In conclusion, our results demonstrate downregulation of Ndufs1 in hypertrophic heart tissue, and the results of mechanistic studies suggest that Ndufs1 deficiency may cause mitochondrial dysfunction in cardiomyocytes, which may be associated with the development and progression of CH.
1. Introduction
Cardiac hypertrophy (CH) is a pathophysiological response characterized by increased thickness of the ventricular wall, greater myocardial cell volume, and enhanced myocardial contractility during the early stage of overload pressure [1]. Primarily, CH is the compensatory response for preservation of cardiac function; however, persistent CH is often associated with disturbed energy metabolism, deteriorated cardiac function, and interstitial fibrosis, which will eventually progress into heart failure [2, 3]. Heart failure caused by CH has been shown to be an independent risk factor for various cardiovascular diseases [4, 5]. To date, the pathophysiology underlying the progression of myocardial hypertrophy remains elusive. Thus, determination of potential molecular mechanisms is necessary for identification of novel and effective therapies to attenuate myocardial hypertrophy.
The contraction and relaxation of cardiomyocytes require a sufficient energy supply to meet the workload demand, and mitochondria are important primary organelles for energy production in cardiomyocytes [6–8]. Mitochondrial dysfunction was shown to be closely associated with the development of heart failure [9–11]. Under pathological conditions of CH, the activities of ATP synthase and mitochondrial oxidative phosphorylation complex are attenuated, which results in reduced production of ATP [12, 13]. Moreover, attenuated mitochondrial dynamics, reduced mitochondrial volume, and abnormal mitochondrial morphology were detected in cardiomyocytes in CH [14, 15]. Mitochondrial dysfunction was shown to increase the production of reactive oxygen species (ROS) via impaired electron transport chains, which can lead to increased oxidative stress and decreased energy production in cardiomyocytes [9, 16, 17]. Thus, restoration of impaired mitochondrial functions will provide novel strategies to attenuate the progression of CH. NADH:ubiquinone oxidoreductase core subunit S1 (Ndufs1) is one of the core subunits of mitochondrial complex I that regulates mitochondrial oxidative phosphorylation and ROS production [18–20]. However, the detailed role of Ndufs1 in the pathophysiology of CH is largely unknown.
In the present study, we initially demonstrated the deregulation of Ndufs1 in heart tissue of mice with CH by analyzing the GSE95140 scRNA-seq dataset. The expression of Ndusf1 was confirmed in heart tissue in a mouse model of CH. Furthermore, in vitro studies determined the molecular mechanisms of Ndusf1-mediated CH. The present study may provide novel insight into the role of Ndusf1 in the pathophysiology of CH.
2. Materials and Methods
2.1. Analysis of scRNA-seq and Microarray Datasets
RNA sequencing data for single cardiomyocytes were downloaded from the GSE95140 dataset of the GEO database [21]. This dataset is based on the GPL17021 platform and contains 396 single-cardiomyocyte transcriptomes of mice after transverse aortic constriction (TAC) or sham operation assayed on day 3 (D3), week 1 (W1), week 2 (W2), week 4 (W4), and week 8 (W8). The expression in each cell was detected by using the “DropletUtils” package. Gene expression in the cells was calculated using the “QC-Metrics” function in the “scater” package [22]. and were used for subsequent filtering. After filtering, the expression matrix of each sample was normalized by using the “NormalizeData” function of the “Seurat” package (version 3.0) [23]. The genes with the most pronounced differences between the cells were selected using the “FindVariableFeatures” function of the “Seurat” package. The “ScaleData” function was used to convert the expression data to linear scale. Then, principal component analysis (PCA) was performed using the “RunPCA” function of the “Seurat” package. Principal components (PCs) with were selected. “RunUMAP” of the “Seurat” package was employed to perform UMAP dimensionality reduction analysis. The “FindAllMarkers” function of the “Seurat” package was used to define the criteria for identification of differentially expressed genes (DEGs) as follows: cell population expression , , and .
The differentially expressed genes were validated using the GSE24454 microarray dataset. In this dataset, mice were sacrificed 4 weeks after aortic banding (AB) or sham procedure (sAB) and subsequent debanding, including banding and subsequent debanding (DB3) or sham procedure and subsequent debanding (sDB3); the data were obtained at various time points up to day 3 [24]. Thus, the CEL raw data and corresponding annotation platform file were downloaded and preprocessed by background adjustment, normalization, probe summarization, and log2 transformation of the expression values using the “Affy” package in R.
2.2. Gene Ontology (GO) Term Enrichment Analysis
GO enrichment analysis was performed using the “clusterProfiler” package in R [25]. Notably, the major GO terms of DE genes in biological processes, molecular functions, cellular components, and pathways were evaluated. The Benjamini-Hochberg method was used to adjust the original values. The GO terms corresponding to the DE genes were enriched with the threshold of correction value < 0.05. Additionally, the enrichment analyses of the biological processes of the hub genes were carried out with the ToppGene tool (https://toppgene.cchmc.org/), which is a web-based analytic tool used for functional enrichment analysis of the gene lists [26]. Additionally, the cellular compartment-specific protein-protein interaction network was constructed by the ComPPI database (https://comppi.linkgroup.hu/) [27].
2.3. Gene Set Enrichment Analysis (GSEA)
GSEA was used to assess the Kyoto Encyclopedia of Genes and Genomes (KEGG) maps involved in TCA-induced CH development based on time series analysis [28]. Initially, the Kolmogorov-Smirnov method was used to determine the enrichment score (ES); then, the statistical significance of ES was assessed using the empirical phenotype replacement test procedure. The enrichment score (NES) was derived by normalization of ES for each gene set. The false discovery rate (FDR) of each NES was determined.
2.4. Gene Set Variation Analysis (GSVA)
The GSVA package of R was used to analyze the activation of the gene sets by unsupervised and nonparametric scoring calculations [29]. The hub pathway-related scores were calculated by the GSVA method in each cell based on the transcription expression matrix after assigning various groups in the TAC model. Significant differences in GSVA scores between various groups were assessed by one-way ANOVA.
2.5. Animals and Surgical Intervention
All animal experiments were approved by the Animal Ethics Committee of Sun Yat-sen University (SYSU-IACUC-2020-000469). Sixteen male C57BL/6 mice (8 weeks old) were purchased from Sun Yat-sen University, and the mice were randomly divided into two groups, including the sham () and TAC groups (). Before operation, the animals were anaesthetized by intraperitoneal injection with . After the animals reached general anesthesia, a small incision was made in the second intercostal space at the left upper sternal border to open the chest cavity, and the animals were subjected to respiratory ventilation. After exposure of the aortic arch, TAC was performed by tying a 7-0 nylon suture ligature against a 27-gauge needle between the left common carotid artery and the brachiocephalic artery. Then, the needle was quickly retracted to complete the partial constriction procedure. Sham-operated mice were subjected to the same surgical procedures without transverse aortic constriction. The chest was closed with 5-0 nonabsorbable sutures. Postoperatively, the animals were subcutaneously injected with 1.0 mg/kg buprenorphine to relieve postoperative pain every 12 h for 3 consecutive days. The mice were closely monitored every day for body weight and any signs of labored breathing or postoperative pain.
2.6. Echocardiography
Four weeks after ascending TAC operation, the animals from the sham and TAC groups were subjected to echocardiography examination. Briefly, the mice were anaesthetized by 3% isoflurane using an anesthesia machine. The hair on the left chest was carefully removed, and cardiac geometry was determined from the parasternal long axis view with a probe frequency of 30 MHz using a small animal color ultrasonic diagnostic apparatus (Vevo 2100, VisualSonics, Toronto, Canada). The images of the left ventricular area were captured using M-type echocardiography. The interventricular septum (IVS) thickness and left ventricular posterior wall (LVPW) thickness were measured.
2.7. Evaluation of Cardiac Index
After assessment by echocardiography, the animals were sacrificed by an overdose of 5% isoflurane. The heart was immediately dissected and rinsed with ice-cold saline to remove blood clots. After draining the heart tissue on sterile paper, the whole weight of the heart was measured using a digital balance. The left ventricular weight (LVW) was determined by removing the atrium and right ventricle from the whole heart. The heart mass index (HMI) and left ventricular mass index (LVMI) were calculated as follows: ; . The length of the medial malleolar distance on the right hindlimb to the tibial plateau edge was defined as the tibia length (TL). The ratios of LVW to TL were used as an index of cardiac hypertrophy.
2.8. Hematoxylin and Eosin (H&E) Staining
After animals were sacrificed by an overdose of 5% isoflurane, a part of the heart tissue was fixed with 4% paraformaldehyde and embedded in paraffin. The paraffin-embedded heart tissue was sectioned into 5 μm sections and stained by hematoxylin and eosin. The stained sections were examined under a light microscope (Nikon, Tokyo, Japan).
2.9. Transmission Electron Microscopy (TEM)
The mitochondria in the heart tissue were evaluated by TEM. Briefly, the heart tissue was sectioned into 1 mm³ pieces, which were fixed with 4% glutaraldehyde and 1% osmic acid. Then, the tissue was dehydrated with acetone, embedded in Epon 821, and cut into 70 nm sections. Then, the sections were double stained with uranyl acetate and lead citrate. The mitochondria were examined using TEM (JEM-1230, Tokyo, Japan). Mitochondrial volume and mitochondrial number were evaluated based on the TEM images.
2.10. Rat Cardiomyocyte Culture
Neonatal Sprague-Dawley rats (1-2 days old) were sacrificed by cervical dislocation, and the heart was immediately dissected under sterile conditions. Ventricular tissue was isolated from the atria and digested in Hanks balanced salt solution containing 0.25% trypsin (Sigma-Aldrich, St. Louis, USA) at 37°C for 5 min, and the digestion cycle was repeated 10 times. After digestion, the supernatants were pooled and mixed with an equal volume of DMEM supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, USA). After centrifugation at for 5 min, the supernatant was discarded, and the cell pellet was resuspended in DMEM supplemented with 10% FBS. After incubation for 4 h at 37°C in a humidified 5% CO2 incubator, cardiomyocytes were collected from the medium. Cardiac fibroblasts adhered to the walls of the dishes. Cardiomyocytes were cultured in 6-well plates for 24 h and in fresh DMEM supplemented with 10% FBS for 2-3 days before in vitro assays.
2.11. Construction of the Ndusf1 siRNA and Overexpression Vectors and Ang II Treatment
The siRNAs targeting Ndusf1 (si-Ndusf1) and the corresponding scrambled siRNAs were designed and synthesized by RiboBio (Guangzhou, China). The vector for Ndusf1 overexpression was constructed by cloning the full-length Ndusf1 sequence into the pcDNA3.1 vector, and the empty pcDNA3.1 vector was used as the corresponding negative control. All plasmids were purchased from RiboBio. For transfections, rat cardiomyocytes were seeded in 12-well plates and cultured for 24 h; then, cardiomyocytes were transfected with various plasmids or siRNAs by using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, USA) according to the manufacturer’s protocol. Cardiomyocytes were collected for the experiments 24 h after the transfection. For angiotensin II (Ang II; Sigma-Aldrich) treatment, cardiomyocytes were seeded in 12-well plates and cultured for 24 h; then, cardiomyocytes were treated with 100 nM Ang II for 24 h and harvested for subsequent experiments.
2.12. Quantitative Real-Time PCR (qRT-PCR)
Total RNA from cardiomyocytes and heart tissue was extracted using TRIzol reagent (Invitrogen, Carlsbad, USA) according to the manufacturer’s protocol. RNA was reverse transcribed using a PrimerScript RT kit with gDNA eraser (Takara, Dalian, USA). Real-time PCR was performed using a SYBR Premix Ex Taq II kit (Takara) on an ABI7900 instrument (Applied Biosystems, Foster City, USA). The parameters for thermal cycling were as follows: 95°C for 15 s, 55°C for 15 s, and 72°C for 15 s for 40 cycles. The relative mRNA expression levels were determined by the comparative Ct method, and β-actin was used as the internal control.
2.13. Western Blot Assay
Proteins from cardiomyocytes or heart tissue were isolated using RIPA buffer supplemented with proteinase inhibitors (Sigma-Aldrich). The concentrations of the protein samples were measured by the BCA method. Equal amounts of proteins (50 μg) were resolved by gel electrophoresis and transferred to a polyvinylidene difluoride (PVDF) membrane. After blocking with 5% nonfat milk at room temperature for 1 h, the membranes were incubated with primary antibodies against NDUFS1 (1 : 1,000; CST, Danvers, USA), atrial natriuretic peptide (ANP; 1 : 1,000; CST), brain natriuretic peptide (BNP; 1 : 1,000; CST), myosin heavy chain beta (β-MHC; 1 : 1,000; CST), and β-actin (1 : 2,000; CST) at 4°C overnight. Then, the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies (1 : 2,000; CST) for 2 h at room temperature. The immunoreactive bands were analyzed by using a chemiluminescence system (Bio-Rad).
2.14. Assessment of mtDNA Copy Number
The mtDNA/nDNA ratio was evaluated by using the qRT-PCR assay as described previously. The primers were designed to target mtDNA (NADH dehydrogenase: 1,5-AAACGCCCTAACAACCAT-3 and 5-GGATAGGATGC TCGGATT-3) and nDNA (β-actin: 5-ATGGTGGGAATGGGTCAGAA-3 and 5-CTTTTCACG GTTGGCCTTAG-3). The relative mtDNA copy number was calculated by normalizing the mtDNA content to the expression of the β-actin gene.
2.15. Assessment of Mitochondrial Membrane Potential (MMP)
MMP of cardiomyocytes was evaluated using a JC-1 mitochondria staining kit (Thermo Fisher Scientific). Briefly, cardiomyocytes ( cells/well) were plated in 96-well plates, treated for 24, and incubated with JC-1 fluorescent dye for 20 min at room temperature in the dark. The fluorescent staining by JC-1 was evaluated by fluorescence microscopy. JC-1 monomers were imaged at excitation and emission wavelengths of 490 nm and 530 nm, respectively; JC-1 aggregates were imaged at excitation and emission wavelengths of 525 nm and 590 nm, respectively.
2.16. Detection of Mitochondrial ROS
The production of mitochondrial ROS was determined by using a MitoSOX fluorescent staining kit (Thermo Fisher Scientific, Waltham, USA) according to the manufacturer’s protocol. Confocal laser scanning microscopy was used to capture fluorescent images, which were further analyzed using ImageJ software.
2.17. Flow Cytometry Analysis of ROS-Positive Cells
ROS production in cardiomyocytes was evaluated using the 2,7-dichlorofluorescein diacetate (DCF-DA) staining assay (Thermo Fisher Scientific). Briefly, the cells were incubated with DCF-DA for 30 min at 37°C in the dark, washed, resuspended in PBS, and maintained on ice for immediate assay by flow cytometry (BD Biosciences). The data were analyzed using FACSDiva software (BD) to calculate the number of ROS-positive cardiomyocytes.
2.18. Mitochondrial Mass Analysis Using MitoTracker Red Staining
MitoTracker Red staining was performed to assess mitochondrial mass. Briefly, cardiomyocytes were incubated with 100 nM MitoTracker Red for 30 min at 37°C. Fluorescence was detected at excitation and emission wavelengths of 490 and 516 nm, respectively, using an ELx-800 microplate reader (BioTek; Winooski, VT, USA).
2.19. Statistical Analysis
The data are presented as the . All data analyses were performed using GraphPad Prism software (version 8; GraphPad Software, La Jolla, USA). Statistical significance of differences between various treatment groups was assessed using unpaired Student’s -test or one-way ANOVA followed by the Bonferroni multiple comparison test. indicated statistical significance.
3. Results
3.1. scRNA-seq Clustering by the Seurat Package and Functional Enrichment Analysis
The number of principal components was set as 12, and cardiomyocytes were classified into six clusters based on UMAP visualization after batch correction (Figure 1(a)). A total of 3,408 highly variable genes were detected after normalization. Consequently, a total of 288 markers were identified by the Wilcoxon signed-rank test. These markers were then subjected to GO enrichment analysis. As shown in Figure 1(b) and Table S1, the genes were significantly enriched in “ribonucleotide metabolic process” (, ), “mitochondrion organization” (, ), “muscle cell development” (, ), “heart contraction” (, ), and “proton transmembrane transport” (, ) in the biological process category. Additionally, DE genes were significantly enriched in “mitochondrial protein complex” (, ), “myelin sheath” (, ), “respiratory chain” (, ), “intercalated disc” (, ), and “chaperone complex” (, ) in the cellular component category. For molecular function, the terms “structural constituent of ribosome” (, ), “electron transfer activity” (, ), “proton transmembrane transporter activity” (, ), “coenzyme binding” (, ), and “ubiquitin protein ligase binding” (, ) were also enriched.
(a)
Background:
Aortic dissection is one of the most detrimental cardiovascular diseases with a high risk of mortality and morbidity. This study aimed to examine the key genes and microRNAs associated with Stanford type A aortic dissection (AAD).
Methods:
The expression data of AAD and healthy samples were downloaded from two microarray datasets in the Gene Expression Omnibus (GEO) database to identify highly preserved modules by weighted gene co-expression network analysis (WGCNA). Differentially expressed genes (DEGs) and differentially expressed miRNAs (DEmiRNAs) were selected and functionally annotated. The predicted interactions between the DEGs and DEmiRNAs were further illustrated.
Results:
In two highly preserved modules, 459 DEGs were identified. These DEGs were functionally enriched in the HIF1, Notch, and PI3K/Akt pathways. Furthermore, 6 DEmiRNAs that were enriched in the regulation of vasculature development and HIF1 pathway, were predicted to target 23 DEGs.
Conclusions:
Our study presented several promising modulators, both DEGs and DEmiRNAs, as well as possible pathological pathways for AAD, which narrows the scope for further fundamental research.
Objective:
Abdominal aortic aneurysm (AAA) is a pathological condition of permanent vessel dilatation that predisposes to the potentially fatal consequence of aortic rupture. SGLT-2 (sodium-glucose cotransporter 2) inhibitors have emerged as powerful pharmacological tools for type 2 diabetes mellitus treatment. Beyond their glucose-lowering effects, recent studies have shown that SGLT-2 inhibitors reduce cardiovascular events and have beneficial effects on several vascular diseases such as atherosclerosis; however, the potential effects of SGLT-2 inhibition on AAA remain unknown. This study evaluates the effect of oral chronic treatment with empagliflozin-an SGLT-2 inhibitor-on dissecting AAA induced by Ang II (angiotensin II) infusion in apoE (apolipoprotein E)-/- mice. Approach and Results: Empagliflozin treatment significantly reduced the Ang II-induced increase in maximal suprarenal aortic diameter in apoE-/- mice independently of blood pressure effects. Immunohistochemistry analysis revealed that empagliflozin diminished Ang II-induced elastin degradation, neovessel formation, and macrophage infiltration at the AAA lesion. Furthermore, Ang II infusion resulted in a marked increase in the expression of chemokines (CCL-2 [chemokine (C-C motif) ligand 2] and CCL-5 [chemokine (C-C motif) ligand 5]), VEGF (vascular endothelial growth factor), and MMP (matrix metalloproteinase)-2 and MMP-9 in suprarenal aortic walls of apoE-/- mice, and all were reduced by empagliflozin cotreatment. Western blot analysis revealed that p38 MAPK (p38 mitogen-activated protein kinase) and NF-κB (nuclear factor-κB) activation was also reduced in the suprarenal aortas of apoE-/- mice cotreated with empagliflozin. Finally, in vitro studies in human aortic endothelial cells and macrophages showed that empagliflozin inhibited leukocyte-endothelial cell interactions and release of proinflammatory chemokines.
Conclusions:
Pharmacological inhibition of SGLT-2 by empagliflozin inhibits AAA formation. SGLT-2 inhibition might represent a novel promising therapeutic strategy to prevent AAA progression ( Visual Overview ).
Metabolic syndrome (MS), which includes metabolic disorders such as protein disorder, glucose disorder, lipid disorder, and carbohydrate disorder, has been growing rapidly around the world. Glycolipid disorders are a main type of metabolic syndrome and are characterized by abdominal obesity and abnormal metabolic disorders of lipid, glucose, and carbohydrate utilization, which can cause cardiovascular and cerebrovascular diseases. Glycolipid disorders are closely related to intestinal flora and its metabolites. However, studies about the biological mechanisms of the intestinal flora and its metabolites with glycolipid disorders have not been clear. When glycolipid disorders are treated with drugs, a challenging problem is side effects. Traditional Chinese medicine (TCM) and dietary supplements have fewer side effects to treat it. Numerous basic and clinical studies have confirmed that TCM decoctions, Chinese medicine monomers, or compounds can treat glycolipid disorders and reduce the incidence of cardiovascular disease. In this study, we reviewed the relationship between the intestinal flora and its metabolites in glycolipid metabolic disorders and the effect of TCM in treating glycolipid metabolic disorders through the intestinal flora and its metabolites. This review provides new perspectives and strategies for future glycolipid disorders research and treatment.
Chronic activation of the renin‐angiotensin‐aldosterone system (RAAS) promotes and perpetuates the syndromes of congestive heart failure, systemic hypertension, and chronic kidney disease. Excessive circulating and tissue angiotensin II (AngII) and aldosterone levels lead to a pro‐fibrotic, ‐inflammatory, and ‐hypertrophic milieu that causes remodeling and dysfunction in cardiovascular and renal tissues. Understanding of the role of the RAAS in this abnormal pathologic remodeling has grown over the past few decades and numerous medical therapies aimed at suppressing the RAAS have been developed. Despite this, morbidity from these diseases remains high. Continued investigation into the complexities of the RAAS should help clinicians modulate (suppress or enhance) components of this system and improve quality of life and survival. This review focuses on updates in our understanding of the RAAS and the pathophysiology of AngII and aldosterone excess, reviewing what is known about its suppression in cardiovascular and renal diseases, especially in the cat and dog.
The WHO 2016 report indicates that worldwide obesity is rising, with over 600 million people in the obese range (BMI>30). The recommended daily calorie intake for adults is 2000 kcal and 2500 kcal for women and men respectively. The average American consumes 3770 kcal/day and the average person in the UK consumes 3400 kcal/day. With such increased caloric intake, there is an increased load on metabolic pathways, in particular glucose metabolism. Such metabolism requires micronutrients as enzyme co-factors. The recommended daily allowance (RDA) for thiamine is 1.3 mg/day and 0.5 mg thiamine is required to process 1000 kilocalories (kcal). Therefore, despite the appearance of being overfed, there is now increasing evidence that the obese population may nutritionally depleted of essential micronutrients. Thiamine deficiency has been reported to be in the region of 16–47% among patients undergoing bariatric surgery for obesity. Thiamine, in turn, requires magnesium to be in its active form thiamine diphosphate, (TDP). TDP also requires magnesium to achieve activation of TDP dependent enzymes, including transketolase (TK), pyruvate dehydrogenase (PDH) and alpha-keto glutaric acid dehydrogenase (AKGDH), during metabolism of glucose. Thiamine and magnesium therefore play a critical role in glucose metabolism and their deficiency may result in the accumulation of anaerobic metabolites including lactate due to a mismatch between caloric burden and function of thiamine dependent enzymes. It may therefore be postulated that thiamine and magnesium deficiency are under-recognized in obesity and may be important in the progress of obesity and obesity related chronic disease states. The aim of the present systematic review was to examine the role of thiamine dependent enzymes in obesity and obesity related chronic disease states.
The vascular smooth muscle cell (VSMC) phenotypic switch is considered to be the key pathophysiological change in various cardiovascular diseases, such as aortic dissection, atherosclerosis, and hypertension. The results in this study showed that TGF-β1 promotes the proliferation, migration and morphological changes of VSMC.TGF-β1 promoted the expressions of PI3K, P-PI3K, AKT, P-AKT, ID2, and OPN protein and suppressed the expressions of α-SMA and SM22α protein; the opposite results were observed for TGF-β1 inhibitor group, AKT inhibitor group and Combined inhibitors group. After the stimulation of TGF-β1 signaling, the mRNA levels of PI3K, AKT, ID2, and OPN were the highest, while the mRNA levels of α-SMA and SM22α were the lowest; the opposite results were found in the same groups above. These results suggested the PI3K/AKT/ID2 signaling pathway is involved in TGF-β1-mediated human aortic VSMC phenotypic switching, that is from a contractile to synthetic phenotype, and Combined inhibitors was more effective in inhibiting the phenotypic switch than a single inhibitor. The Combined inhibitors experiments may provide new avenues for the prevention and treatment of thoracic aortic dissection (TAD) that are based on the pathological effects of phenotypic switching.
Significance
Excessive accumulation of oxidative stress is harmful to cancer cells. Our study demonstrates the important roles of a pentose phosphate pathway (PPP) enzyme, transketolase (TKT), in redox homeostasis in cancer development. We highlight the clinical relevance of TKT expression in cancers. We also show that TKT overexpression in cancer cells is a response of Nuclear Factor, Erythroid 2-Like 2 (NRF2) activation, a sensor to cellular oxidative stress. TKT locates at an important position that connects PPP with glycolysis to affect production of antioxidant NADPH. Our preclinical study shows that targeting TKT leads to elevation of oxidative stress, making cancer cells more vulnerable to therapeutic treatment, such as Sorafenib. Using TKT as an example, our study suggests that targeting enzymes for antioxidant production represents a direction for cancer treatment.
The survival of a cell depends on its ability to meet its energy requirements. We hypothesized that the mitochondrial reserve respiratory capacity (RRC) of a cell is a critical component of its bioenergetics that can be utilized during an increase in energy demand, thereby, enhancing viability. Our goal was to identify the elements that regulate and contribute to the development of RRC and its involvement in cell survival. The results show that activation of metabolic sensors, including pyruvate dehydrogenase and AMP-dependent kinase, increases cardiac myocyte RRC via a Sirt3-dependent mechanism. Notably, we identified mitochondrial complex II (cII) as a target of these metabolic sensors and the main source of RRC. Moreover, we show that RRC, via cII, correlates with enhanced cell survival after hypoxia. Thus, for the first time, we show that metabolic sensors via Sirt3 maximize the cellular RRC through activating cII, which enhances cell survival after hypoxia.
Ovarian cancer, the most lethal gynaecological cancer, is characterised by the shedding of epithelial cells from the ovarian surface, followed by metastasis and implantation onto the peritoneal surfaces of abdominal organs. Our proteomic studies investigating the interactions between peritoneal (LP-9) and ovarian cancer (OVCAR-5) cells found transketolase (TKT) to be regulated in the co-culture system. This study characterized TKT expression in advanced stage (III/IV) serous ovarian cancers (n = 125 primary and n = 54 peritoneal metastases), normal ovaries (n = 6) and benign serous cystadenomas (n = 10) by immunohistochemistry. In addition, we also evaluated the function of TKT in ovarian cancer cells in vitro. Nuclear TKT was present in all primary serous ovarian cancer tissues examined (median 82.0 %, range 16.5–100 %) and was significantly increased in peritoneal metastases compared with matching primary cancers (P = 0.01, Wilcoxon Rank test). Kaplan–Meier survival and Cox regression analyses showed that high nuclear TKT positivity in peritoneal metastases (>94 %) was significantly associated with reduced overall survival (P = 0.006) and a 2.8 fold increased risk of ovarian cancer death (95 % CI 1.29–5.90, P = 0.009). Knockdown of TKT by siRNAs significantly reduced SKOV-3 cell proliferation but had no effect on their motility or invasion. Oxythiamine, an inhibitor of TKT activity, significantly inhibited the proliferation of four ovarian cancer cell lines (OV-90, SKOV-3, OVCAR-3 and OVCAR-5) and primary serous ovarian cancer cells isolated from patient ascites. In conclusion, these findings indicate that TKT plays an important role in the proliferation of metastatic ovarian cancer cells and could be used as novel therapeutic target for advanced disease.
Unlike cardiac and skeletal muscle, little is known about vascular smooth muscle mitochondrial function. Therefore, this study examined mitochondrial respiratory rates in the smooth muscle of healthy human feed arteries and compared with that of healthy cardiac and skeletal muscle. Cardiac, skeletal, and smooth muscle was harvested from a total of 22 subjects (53±6 yrs) and mitochondrial respiration assessed in permeabilized fibers. Complex I+II, state 3 respiration, an index of oxidative phosphorylation capacity, fell progressively from cardiac, skeletal, to smooth muscle (54±1; 39±4; 15±1 pmol•s(-1)•mg (-1), p<0.05, respectively). Citrate synthase (CS) activity, an index of mitochondrial density, also fell progressively from cardiac, skeletal, to smooth muscle (222±13; 115±2; 48±2 umol•g(-1)•min(-1), p<0.05, respectively). Thus, when respiration rates were normalized by CS (respiration per mitochondrial content), oxidative phosphorylation capacity was no longer different between the three muscle types. Interestingly, Complex I state 2 normalized for CS activity, an index of non-phosphorylating respiration per mitochondrial content, increased progressively from cardiac, skeletal, to smooth muscle, such that the respiratory control ratio (RCR), state 3/state 2 respiration, fell progressively from cardiac, skeletal, to smooth muscle (5.3±0.7; 3.2±0.4; 1.6±0.3, pmol•s(-1)•mg (-1) p<0.05, respectively). Thus, although oxidative phosphorylation capacity per mitochondrial content in cardiac, skeletal, and smooth muscle suggest all mitochondria are created equal, the contrasting RCR and non-phosphorylating respiration highlight the existence of intrinsic functional differences between these muscle mitochondria. This likely influences the efficiency of oxidative phosphorylation and could potentially ROS production.
Protein kinase B (PKB, or Akt) plays a role in cell metabolism, growth, proliferation, and survival. Its activation is controlled by a multi-step process that involves phosphoinositide-3-kinase (PI3K).
A decline in mitochondrial function plays a key role in the aging process and increases the incidence of age-related disorders. A deeper understanding of the intricate nature of mitochondrial dynamics, which is described as the balance between mitochondrial fusion and fission, has revealed that functional and structural alterations in mitochondrial morphology are important factors in several key pathologies associated with aging. Indeed, a recent wave of studies has demonstrated the pleiotropic role of fusion and fission proteins in numerous cellular processes, including mitochondrial metabolism, redox signaling, the maintenance of mitochondrial DNA and cell death. Additionally, mitochondrial fusion and fission, together with autophagy, have been proposed to form a quality-maintenance mechanism that facilitates the removal of damaged mitochondria from the cell, a process that is particularly important to forestall aging. Thus, dysfunctional regulation of mitochondrial dynamics might be one of the intrinsic causes of mitochondrial dysfunction, which contributes to oxidative stress and cell death during the aging process. In this Commentary, we discuss recent studies that have converged at a consensus regarding the involvement of mitochondrial dynamics in key cellular processes, and introduce a possible link between abnormal mitochondrial dynamics and aging.
The crystal structure of Saccharomyces cerevisiae transketolase, a thiamine diphosphate dependent enzyme, has been determined to 2.5 A resolution. The enzyme is a dimer with the active sites located at the interface between the two identical subunits. The cofactor, vitamin B1 derived thiamine diphosphate, is bound at the interface between the two subunits. The enzyme subunit is built up of three domains of the alpha/beta type. The diphosphate moiety of thiamine diphosphate is bound to the enzyme at the carboxyl end of the parallel beta-sheet of the N-terminal domain and interacts with the protein through a Ca2+ ion. The thiazolium ring interacts with residues from both subunits, whereas the pyrimidine ring is buried in a hydrophobic pocket of the enzyme, formed by the loops at the carboxyl end of the beta-sheet in the middle domain in the second subunit. The structure analysis identifies amino acids critical for cofactor binding and provides mechanistic insights into thiamine catalysis.
beta-Aminopropionitrile (BAPN) is a potent irreversible inhibitor of lysyl oxidase, the enzyme which initiates cross-linkage formation in elastin and collagen. The initial interaction of BAPN with aortic lysyl oxidase is competitive with elastin or alkyl amine substrates. Irreversible inhibition develops in a time- and temperature-dependent fashion upon incubation of enzyme with BAPN in the absence of substrate with a limiting inactivation rate constant of 0.16 min-1 and a KI of 6 microM at 37 degrees C. The labeled carbons of [1,2-14C]BAPN and [3-14C]BAPN covalently bind to the enzyme to equivalent extents and in parallel with the development of inactivation, negating the possibility that the nitrile moiety is eliminated from BAPN by enzymatic action. The copper content of the enzyme is not significantly altered upon interaction with BAPN. The extent of labeling by [14C]BAPN is reduced by prior treatment of the enzyme with carbonyl-modifying reagents, suggesting the possibility of enzyme-inhibitor Schiff base formation. However, BAPN is not processed to a free aldehyde product upon incubation with lysyl oxidase. A mechanism of inhibition is postulated which involves the formation of a covalent bond between an enzyme nucleophile and a ketenimine formed from BAPN by enzyme-assisted beta-proton abstraction.
Transketolases link the Embden-Meyerhof pathway to the pentose phosphate pathway. An influence of p-Akt on this metabolism was described. This study was performed to compare the expression of transketolase-like 1 (TKTL1) and p-Akt in glioblastoma multiforme (GBM) and other astrocytic gliomas (AGs, grades II and III). We analyzed 15 GBMs, 15 AGs (grade II), and 3 normal brain samples for TKTL1 expression by semiquantitative reverse transcription-polymerase chain reaction and Western blotting and 23 GBMs, 9 grade III AGs, and 7 grade II AGs immunohistochemically (TKTL1 and p-Akt). On the protein level, TKTL1 was significantly overexpressed in tumors. Immunohistochemically, the tumor grade significantly correlated with expression of TKTL1. Compared with grades II and III AGs, GBMs showed higher expression of TKTL1, more positive tumors, and a higher percentage of positive tumor cells. The percentage of positive cells for TKTL1 and p-Akt was significantly correlated. These observations could lead to additional therapeutic options targeting a specific blockade of TKTL1 enzyme activity.
Arterial remodeling is a common pathological foundation of cardiovascular diseases such as atherosclerosis, vascular restenosis, hypertension, pulmonary hypertension, aortic dissection and aneurysm. Vascular smooth muscle cells (VSMCs) are not only the main cellular components in the middle layer of the arterial wall but also the main cells involved in arterial remodeling. Dedifferentiated VSMCs lose their contractile properties and convert to synthetic, secretory, proliferative, and migratory phenotypes, which play key roles in the pathogenesis of arterial remodeling. As the main site of biological oxidation and energy transformation in eukaryotic cells, mitochondrial numbers and function are very important in maintaining the metabolic processes in VSMCs. Mitochondrial dysfunction and oxidative stress are novel triggers of the phenotypic transformation of VSMCs, leading to the onset and development of arterial remodeling. Therefore, pharmacological measures to improve mitochondrial dysfunction reverse arterial remodeling by improving VSMC metabolic disorders and phenotypic transformation, which brings new options for the treatment of cardiovascular diseases related to arterial remodeling. This review summarizes the relationship between mitochondrial dysfunction and cardiovascular diseases associated with arterial remodeling and then discusses the potential mechanism by which mitochondrial dysfunction participates in pathological arterial remodeling. Furthermore, maintaining or improving mitochondrial function can be a new intervention strategy to prevent the progression of arterial remodeling.
Aortic dissection (AD) is a disease with high mortality and lacks effective drug treatment. Recent studies have shown that the development of AD is closely related to glucose metabolism. Lactate dehydrogenase A (LDHA) is a key glycolytic enzyme and plays an important role in cardiovascular disease. However, the role of LDHA in the progression of AD remains to be elucidated. Here, we found that the level of LDHA was significantly elevated in AD patients and the mouse model established by BAPN combined with Ang II. In vitro, the knockdown of LDHA reduced the growth of human aortic vascular smooth muscle cells (HAVSMCs), glucose consumption, and lactate production induced by PDGF-BB. The overexpression of LDHA in HAVSMCs promoted the transformation of HAVSMCs from contractile phenotype to synthetic phenotype, and increased the expression of MMP2/9. Mechanistically, LDHA promoted MMP2/9 expression through the LDHA-NDRG3-ERK1/2-MMP2/9 pathway. In vivo, Oxamate, LDH and lactate inhibitor, reduced the degradation of elastic fibers and collagen deposition, inhibited the phenotypic transformation of HAVSMCs from contractile phenotype to synthetic phenotype, reduced the expression of NDRG3, p-ERK1/2, and MMP2/9, and delayed the progression of AD. To sum up, the increase of LDHA promotes the production of MMP2/9, stimulates the degradation of extracellular matrix (ECM), and promoted the transformation of HAVSMCs from contractile phenotype to synthetic phenotype. Oxamate reduced the progression of AD in mice. LDHA may be a therapeutic target for AD.
New concepts regarding the diagnosis, classification, and treatment of aortic dissection have been recently developed. The aim of this paper is to describe the current state of knowledge on this subject and discuss any controversies surrounding it. Novel findings in the pathomechanisms of aortic dissection have evolved focusing on the indications for preventive surgery, biomarkers, and four-dimensional (4D)-flow magnetic resonance imaging. New classifications of aortic dissections have been proposed (TEM, STS/SVS). Finally, recent treatment improvements in aortic dissection treatment options have been presented, i.e., the frozen elephant trunk approach, thoracic endovascular repair, and the endo-Bentall concept as a future option.
Aims:
Aortic aneurysm/dissection (AAD) is a life-threatening disorder lacking effective pharmacotherapeutic remedies. Aldehyde dehydrogenase 2 (ALDH2) polymorphism is tied with various risk factors for AAD including hypertension, atherosclerosis, and hypercholesterolaemia although direct correlation between the two remains elusive.
Methods and results:
Two independent case-control studies were conducted involving 307 AAD patients and 399 healthy controls in two geographically distinct areas in China. Our data revealed that subjects carrying mutant ALDH2 gene possessed a ∼50% reduced risk of AAD compared with wild-type (WT) alleles. Using 3-aminopropionitrile fumarate (BAPN)- and angiotensin II (Ang II)-induced AAD animal models, inhibition of ALDH2 was found to retard development of AAD. Mechanistically, ALDH2 inhibition ablated pathological vascular smooth muscle cell (VSMC) phenotypical switch through interaction with myocardin, a determinant of VSMC contractile phenotype. Using microarray and bioinformatics analyses, ALDH2 deficiency was found to down-regulate miR-31-5p, which further altered myocardin mRNA level. Gain-of-function and loss-of-function studies verified that miR-31-5p significantly repressed myocardin level and aggravated pathological VSMC phenotypical switch and AAD, an effect that was blunted by ALDH2 inhibition. We next noted that ALDH2 deficiency increased Max expression and decreased miR-31-5p level. Moreover, ALDH2 mutation or inhibition down-regulated levels of miR-31-5p while promoting myocardin downstream contractile genes in the face of Ang II in primary human VSMCs.
Conclusions:
ALDH2 deficiency is associated with a lower risk of AAD in patients and mice, possibly via suppressing VSMC phenotypical switch in a miR-31-5p-myocardin-dependent manner. These findings favour a role for ALDH2 and miR-31-5p as novel targets for AAD therapy.
Obesity has recently become a prevalent health threat worldwide. Although emerging evidence has suggested a strong link between the pentose phosphate pathway (PPP) and obesity, the role of transketolase (TKT), an enzyme in the non-oxidative branch of the PPP which connects PPP and glycolysis, remains obscure in adipose tissues. In this study, we specifically delete TKT in mouse adipocytes and find no obvious phenotype upon normal diet feeding. However, adipocyte TKT abrogation attenuates high fat diet (HFD)-induced obesity, reduces hepatic steatosis, improves glucose tolerance, alleviates insulin resistance and increases energy expenditure. Mechanistically, TKT deficiency accumulates non-oxidative PPP metabolites, decreases glycolysis and pyruvate input into the mitochondria, leading to increased lipolytic enzyme gene expression and enhanced lipolysis, fatty acid oxidation and mitochondrial respiration. Therefore, our data not only identify a novel role of TKT in regulating lipolysis and obesity, but also suggest limiting glucose-derived carbon into the mitochondria induces lipid catabolism and energy expenditure.
Factors causing the weakness that underlies thoracic aorta aneurysms and dissections are not well known. Based on the findings of apoptosis and ischemic-like necrosis, we hypothesized a possible role for mitochondrial disturbances in the pathogenesis of these diseases. To evaluate if mitochondria at the aortic medial layer are damaged, samples of ascending aortas with aneurysms (n = 6), acute dissections (n = 5), and hypertensive (n = 9) and normotensive controls (n = 7) were analyzed by transmission electron microscopy. Number of mitochondria, areas of cytoplasm, and areas of mitochondria were measured, and area percentage of the cytoplasm corresponding to mitochondria, their number by unit of area, and their mean area were calculated in randomly taken photographs. Data were compared using one-way analysis of variance or Kruskal-Wallis tests. Significant differences (P ≤ 0.05) were found in the number of mitochondria and their mean area, showing opposite results: the number increased and the mean area decreased from normotensive controls to hypertensive controls to acute dissections to aneurysms, although post hoc tests showed that only the differences between the aneurysms and either both controls (number of mitochondria/mm2: 10.37 in normotensive controls, 15.61 in hypertensive controls, and 43.67 in aneurysms) or normotensive controls only (mean area: 2800.15 in normotensive controls vs 894.91 μm2 in aneurysms) were significant. In conclusion, there are more, smaller mitochondria in ascending aorta aneurysms. This pattern possibly corresponds to dysfunctional mitochondria, indicating that alterations in the dynamics of these organelles may play a role in the pathogenesis of thoracic aorta aneurysms and dissections.
Decades of research reveal that MDM2 participates in cellular processes ranging from macro-molecular metabolism to cancer signaling mechanisms. Two recent studies uncovered a new role for MDM2 in mitochondrial bioenergetics. Through the negative regulation of NDUFS1 (NADH:ubiquinone oxidoreductase 75 kDa Fe-S protein 1) and MT-ND6 (NADH dehydrogenase 6), MDM2 decreases the function and efficiency of Complex I (CI). These observations propose several important questions: (1) Where does MDM2 affect CI activity? (2) What are the cellular consequences of MDM2-mediated regulation of CI? (3) What are the physiological implications of these interactions? Here, we will address these questions and position these observations within the MDM2 literature.
There are many differences in arterial diseases between men and women, including prevalence, clinical manifestations, treatments, and prognosis. The new policy of the National Institutes of Health, which requires the inclusion of sex as a biological variable for preclinical studies, aims to foster new mechanistic insights and to enhance our understanding of sex differences in human diseases. The purpose of this statement is to suggest guidelines for designing and reporting sex as a biological variable in animal models of atherosclerosis, thoracic and abdominal aortic aneurysms, and peripheral arterial disease. We briefly review sex differences of these human diseases and their animal models, followed by suggestions on experimental design and reporting of animal studies for these vascular pathologies.
Purpose:
To test the hypothesis that diabetes-related factors (metabolic syndrome [MetS], glucose, insulin, and leptin) are inversely associated with abdominal aortic aneurysm (AAA) risk.
Methods:
We followed 13,736 participants, aged 45-64 years, without prior AAA surgery at baseline (1987-1989), for AAA occurrence through 2011. Hazard ratios (HRs) and their 95% confidence intervals (CIs) of AAA were calculated using Cox regression.
Results:
During 275,054 person-years of follow-up, we identified 518 AAA events. Fasting serum glucose was associated inversely with AAA risk (HR [95% CI] per one unit increment in log2(glucose), 0.54 [0.36-0.80]), but fasting insulin was not associated with AAA. Plasma leptin was also associated inversely with AAA occurrence (HR [95% CI] per one unit increment in log2(leptin), 0.83 [0.71-0.98]). Compared with individuals without MetS, those with MetS had increased risk of AAA (HR [95% CI], 1.24 [1.04-1.48]). Among individuals with or without diabetes, the HRs increased monotonically with a greater number of non-glucose MetS components.
Conclusions:
Diabetes, fasting glucose, and plasma leptin were inversely associated with risk of AAA. In contrast, the MetS was associated with increased risk of AAA, due to the influence of the non-glucose MetS components.
Objective:
Smooth muscle (SM) 22α, an actin-binding protein, displays an upregulated expression as a marker during cellular senescence. However, the causal relationship between SM22α and senescence is poorly understood. This study aimed to investigate the role of SM22α in angiotensin II (Ang II)-induced senescence of vascular smooth muscle cells (VSMCs).
Approach and results:
We prepared a model of VSMC senescence induced by Ang II and found that the expression of SM22α in VSMCs was increased in response to chronic Ang II treatment. Overexpression of SM22α promoted Ang II-induced VSMC senescence, whereas knockdown of SM22α suppressed this process. Moreover, this effect of SM22α was p53 dependent. Increased SM22α protein obstructed ubiquitination and degradation of p53 and subsequently improved its stability. Furthermore, SM22α inhibited phosphorylation of Mdm2 (mouse double minute 2 homolog), an E3 ubiquitin-protein ligase, accompanied by a decreased interaction between Mdm2 and p53. Using LY294002, a PI3K/Akt inhibitor, we found that PI3K/Akt-mediated Mdm2 phosphorylation and activation was inhibited in senescent or SM22α-overexpressed VSMCs, in parallel with decreased p53 ubiquitination. We further found that SM22α inhibited activation of PI3K/Akt/Mdm2 pathway via strengthening actin cytoskeleton. In the in vivo study, we showed that the disruption of SM22α reduced the increase of blood pressure induced by Ang II, associated with decreased VSMC senescence through a mechanism similar to that in VSMCs in vitro.
Conclusions:
In conclusion, these findings suggest that the accumulation of SM22α promotes Ang II-induced senescence via the suppression of Mdm2-mediated ubiquitination and degradation of p53 in VSMCs in vitro and in vivo.
Background:
The development of thoracic aortic dissection (TAD) is attributed to a broad range of degenerative, genetic, structural, oxidative, apoptotic, and acquired disease states. In this study, we examined the role of the disturbed p53-MDM2 feed-back loop in the formation of TAD, and one of a potential feed-back loop regulator, TRIM-25.
Methods:
Surgical specimens of the aorta from TAD patients (n=10) and controls (n=10) were tested for α-smooth muscle actin (α-SMA),p53,murine double minute2(MDM2) and tripartite motif protein-25(TRIM-25) by western blot, immunohistochemical staining and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) , respectively.
Results:
When compared with controls, western blot shows that the protein levels of p53,MDM2 and TRIM25 was increased significantly in the aortic media of thoracic aortic dissection patients. qRT-PCR further verified the mRNA expression of MDM2 and TRIM25 were also increased 6 and 4 folds respectively in the TAD media of the aortic wall. Immunohistochemistry results showed significantly decreased staining of α-SMA,smoth muscle cells and more collagen deposition in the media of the aortic wall from patients with TAD.
Conclusions:
This study provided a new insight into the disturbed p53-MDM2 feedback loop in the pathogenesis of TAD, and this may be because of the TRIM25 overexpression.
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Acute aortic dissection (AAD) is a relatively rare but much-feared clinical presentation which has a high mortality, particularly if definitive treatment is delayed. A combination of aortic wall stress and abnormalities of the medial layer of the aortic wall leads to disruption of the media and an intimal tear with subsequent penetration of blood, which splits the aortic wall layers. This creates a cavity within the medial layer, the so-called false lumen (FL), which is separated from the native true lumen (TL) by the dissection membrane.1 This process can result in disruption of the adventitia (aortic rupture) or in a second tear in the dissection membrane, which allows blood to re-enter the TL. If AAD occurs within the ascending aorta (AA), 40% of patients die immediately and mortality is 1%–2% for each hour afterwards resulting in a 48-hour mortality of approximately 50% (see figure 1).2
Figure 1
Spontaneous prognosis of acute aortic dissection (adapted from Kirklin et al ).51
The most important risk factor for the development of AAD is poorly controlled hypertension. Men are more often affected and the risk increases with age. Other important pathogenic factors are positive family history and genetic history (connective tissue disease, Ehlers-Danlos syndrome, Marfan syndrome), aortic disease and aortic valve (AV) diseases, history of cardiac surgery and previous trauma.3 ,4 Although dilatation of the aorta (aortic aneurysms) increases the risk through greater wall stress, AAD can as well occur in patients with …
Background:
Individually, diabetes mellitus, hypertension, and dyslipidemia have been shown to increase the risk of cardiovascular disease. While traditional management of Type 2 diabetes has focused mainly on glycemic control, robust evidence supports the integration of hypertension and dyslipidemia management to reduce the risk of cardiovascular disease. The primary objective of this study was to assess the level of control of blood glucose, blood pressure, and blood lipids (3Bs) among patients with type 2 diabetes. An additional objective was to investigate the impact of hospital type, physician specialty, treatment pattern, and patient profile on clinical outcomes.
Methods:
This was a cross-sectional, multicenter observational study. A nationally representative sample of outpatients with established type 2 diabetes were enrolled at hospitals representative of geographic regions, tiers, and physician specialties in China. Main clinical measurements were the levels of glycosylated hemoglobin (HbA1c), blood pressure, and total serum cholesterol in reference to target goals.
Results:
A total of 25,817 adults with type 2 diabetes (mean age 62.6 years, 47% male) were enrolled at 104 hospitals. Seventy-two percent reported comorbid hypertension, dyslipidemia, or both. Patients with concurrent type 2 diabetes, hypertension, and dyslipidemia were 6 times more likely to report a prior history of cardiovascular disease compared with those with type 2 diabetes alone. The mean HbA1c level was 7.6%. While 47.7%, 28.4%, and 36.1% of patients achieved the individual target goals for control of blood glucose (HbA1c <7%), blood pressure (systolic blood pressure <130 mm Hg, diastolic blood pressure <80 mm Hg), and blood lipids (total cholesterol <4.5 mmol/L), respectively, only 5.6% achieved all 3 target goals. Lower body mass index (<24 kg/m(2)), no active smoking or drinking, higher education, and diabetes duration <5 years were independent predictors of better cardiovascular disease risk control.
Conclusion:
Achieving adequate control of risk factors for cardiovascular disease in patients with type 2 diabetes remains a clinical challenge. Interventions to achieve control of 3Bs coupled with modification of additional cardiovascular disease predictors are crucial for optimization of clinical outcomes in patients with type 2 diabetes.
Vascular smooth muscle metabolism is characterized by substantial production of lactic acid even under fully oxygenated conditions. The role the aerobic production of lactate plays in the energetics of smooth muscle is obscure and was investigated in this study. Helical strips of porcine carotid arteries were incubated in medium containing 1 mM dichloroacetate (DCA), an agent that stimulates pyruvate dehydrogenase and promotes the oxidation of glucose. Lactate production in resting muscle was decreased in the presence of DCA (0.033 +/- 0.006 vs. 0.111 +/- 0.014 mumol.g-1.min-1, P < 0.02), indicating diversion of glucose metabolism from lactate production to enhanced glucose oxidation. This was associated with reduction in the level of ATP+phosphocreatine (PCr) (0.99 +/- 0.01 vs. 1.40 +/- 0.09 mumol/g, P < 0.05) and cataplerosis of the tricarboxylic acid (TCA) cycle. Contraction by KCl was also associated with reduced lactate production in the presence of DCA (0.086 +/- 0.017 vs. 0.20 +/- 0.002 mumol.g-1.min-1, P < 0.01), but ATP+PCr normalized, and there was anaplerosis of the TCA cycle. Glycogen in control arteries declined by approximately 1.3 mumol/g over 30 min K+ contraction but was unchanged in the presence of DCA. By calculation, the glycogen spared could be accounted for by the quantity of glucose diverted from lactate production to glucose oxidation during contraction. It is concluded that the aerobic production of lactate is a mechanism affording optimal coordination and modulation of glucose supply and oxidative energy production with energy demand.
The accumulation and proliferation of vascular smooth muscle cells (VSMC) within the vessel wall is an important pathogenic feature in the development of atherosclerosis. Glucose metabolism has been implicated to play an important role in this cellular mechanism. To further elucidate the role of glucose metabolism in atherogenesis, glycolysis and its regulation have been investigated in proliferating VSMC. Platelet derived growth factor (PDGF BB)-induced proliferation of VSMCs significantly stimulated glucose flux through glycolysis. Further evaluating the enzymatic regulation of this pathway, the analysis of flux:metabolite co-responses revealed that anaerobic glycolytic flux is controlled at different sites of gycolysis in proliferating VSMCs, being consistent with the concept of multisite modulation. These findings indicate that regulation of glycolytic flux in proliferating VSMCs differs from traditional concepts of metabolic control of the Embden-Meyerhof pathway.
Most risk factors are similar for abdominal aortic aneurysm (AAA) and atherosclerosis, e.g. smoking, male gender, age, high blood pressure, hyperlipidemia. Diabetes mellitus however, is a risk factor for atherosclerosis, but diabetic patients seldom develop AAA. The reason for this discrepancy is unknown. Increased aortic wall stress seems to be an etiologic factor in the formation, growth and rupture of AAA in man. The aim of our study was to study the wall stress in the abdominal aorta in diabetic patients compared with healthy controls.
39 patients with diabetes mellitus and 46 age - and sex matched healthy subjects were examined with B-mode ultrasound to determine the lumen diameter (LD) and intima-media thickness (IMT) in the abdominal aorta (AA) and the common carotid artery (CCA). Diastolic blood pressure (DBP) was measured non-invasively in the brachial artery. LaPlace law was used to calculate circumferential wall stress.
Age, DBP, and LD in the abdominal aorta were not significantly different in the diabetic patients compared to controls. IMT in the AA was larger in the diabetic patients, 0.89+/-0.17 vs 0.73+/-0.11 mm (p<.001). Accordingly aortic wall stress was reduced in the diabetics, 7.8+/-1.7 x 10(5) vs 9.7+/-1.9 x 10(5)dynes/cm(2) (p<.001).
Wall stress in the abdominal aorta is reduced in diabetes mellitus. This is mainly due to a thicker aortic wall compared to healthy controls. The reduced aortic wall stress coincides with the fact that epidemiological studies have shown a decreased risk of aneurysm development in diabetic patients.