Riboflavin protects against heart failure via SCAD-dependent DJ-1-Keap1-Nrf2 signalling pathway.

BACKGROUND AND PURPOSE: Our recent studies have shown that flavin adenine dinucleotide (FAD) exerts cardiovascular protective effects by supplementing short-chain acyl-CoA dehydrogenase (SCAD). The current study aimed to elucidate whether riboflavin (the precursor of FAD) could improve heart failure via activating SCAD and the DJ-1-Keap1-Nrf2 signalling pathway. EXPERIMENTAL APPROACH: Riboflavin treatment was given to the mouse transverse aortic constriction (TAC)-induced heart failure model. Cardiac structure and function, energy metabolism and apoptosis index were assessed, and relevant signalling proteins were analysed. The mechanisms underlying the cardioprotection by riboflavin were analysed in the cell apoptosis model induced by tert-butyl hydroperoxide (tBHP). KEY RESULTS: In vivo, riboflavin ameliorated myocardial fibrosis and energy metabolism, improved cardiac dysfunction and inhibited oxidative stress and cardiomyocyte apoptosis in TAC-induced heart failure. In vitro, riboflavin ameliorated cell apoptosis in H9C2 cardiomyocytes by decreasing reactive oxygen species (ROS). At the molecular level, riboflavin significantly restored FAD content, SCAD expression and enzymatic activity, activated DJ-1 and inhibited the Keap1-Nrf2/HO1 signalling pathway in vivo and in vitro. SCAD knockdown exaggerated the tBHP-induced DJ-1 decrease and Keap1-Nrf2/HO1 signalling pathway activation in H9C2 cardiomyocytes. The knockdown of SCAD abolished the anti-apoptotic effects of riboflavin on H9C2 cardiomyocytes. DJ-1 knockdown hindered SCAD overexpression anti-apoptotic effects and regulation on Keap1-Nrf2/HO1 signalling pathway in H9C2 cardiomyocytes. CONCLUSIONS AND IMPLICATIONS: Riboflavin exerts cardioprotective effects on heart failure by improving oxidative stress and cardiomyocyte apoptosis via FAD to stimulate SCAD and then activates the DJ-1-Keap1-Nrf2 signalling pathway.

Riboflavin ameliorates pathological cardiac hypertrophy and fibrosis through the activation of short-chain acyl-CoA dehydrogenase.

Short-chain acyl-CoA dehydrogenase (SCAD), the rate-limiting enzyme for fatty acid ß-oxidation, has a negative regulatory effect on pathological cardiac hypertrophy and fibrosis. FAD, a coenzyme of SCAD, participates in the electron transfer of SCAD-catalyzed fatty acid ß-oxidation, which plays a crucial role in maintaining the balance of myocardial energy metabolism. Insufficient riboflavin intake can lead to symptoms similar to short-chain acyl-CoA dehydrogenase (SCAD) deficiency or flavin adenine dinucleotide (FAD) gene abnormality, which can be alleviated by riboflavin supplementation. However, whether riboflavin can inhibit pathological cardiac hypertrophy and fibrosis remains unclear. Therefore, we observed the effect of riboflavin on pathological cardiac hypertrophy and fibrosis. In vitro experiments, riboflavin increased SCAD expression and the content of ATP, decreased the free fatty acids content and improved PE-induced cardiomyocytes hypertrophy and AngⅡ-induced cardiac fibroblasts proliferation by increasing the content of FAD, which were attenuated by knocking down the expression of SCAD using small interfering RNA. In vivo experiments, riboflavin significantly increased the expression of SCAD and the energy metabolism of the heart to improve TAC induced pathological myocardial hypertrophy and fibrosis in mice. The results demonstrate that riboflavin improves pathological cardiac hypertrophy and fibrosis by increasing the content of FAD to activate SCAD, which may be a new strategy for treating pathological cardiac hypertrophy and fibrosis.

Short-chain acyl-CoA dehydrogenase is a potential target for the treatment of vascular remodelling.

OBJECTIVES: Short-chain acyl-CoA dehydrogenase (SCAD), a key enzyme in the fatty acid oxidation process, is not only involved in ATP synthesis but also regulates the production of mitochondrial reactive oxygen species (ROS) and nitric oxide synthesis. The purpose of this study was to investigate the possible role of SCAD in hypertension-associated vascular remodelling. METHODS: In-vivo experiments were performed on spontaneously hypertensive rats (SHRs, ages of 4 weeks to 20 months) and SCAD knockout mice. The aorta sections of hypertensive patients were used for measurement of SCAD expression. In-vitro experiments with t-butylhydroperoxide (tBHP), SCAD siRNA, adenovirus-SCAD (MOI 90) or shear stress (4, 15 dynes/cm 2 ) were performed using human umbilical vein endothelial cells (HUVECs). RESULTS: Compared with age-matched Wistar rats, aortic SCAD expression decreased gradually in SHRs with age. In addition, aerobic exercise training for 8 weeks could significantly increase SCAD expression and enzyme activity in the aortas of SHRs while decreasing vascular remodelling in SHRs. SCAD knockout mice also exhibited aggravated vascular remodelling and cardiovascular dysfunction. Likewise, SCAD expression was also decreased in tBHP-induced endothelial cell apoptosis models and the aortas of hypertensive patients. SCAD siRNA caused HUVEC apoptosis in vitro , whereas adenovirus-mediated SCAD overexpression (Ad-SCAD) protected against HUVEC apoptosis. Furthermore, SCAD expression was decreased in HUVECs exposed to low shear stress (4 dynes/cm 2 ) and increased in HUVECs exposed to 15 dynes/cm 2 compared with those under static conditions. CONCLUSION: SCAD is a negative regulator of vascular remodelling and may represent a novel therapeutic target for vascular remodelling.

Association of circulating MtDNA with CVD in hemodialysis patients and in vitro effect of exogenous MtDNA on cardiac microvascular inflammation.

BACKGROUND: Chronic kidney disease (CKD) patients sustain a fairly high prevalence of cardiovascular disease (CVD). Microvascular inflammation is an early manifestation of CVD, and the released mitochondrial DNA (MtDNA) has been proposed to be a crucial integrator of inflammatory signals. Herein, the aim of this study was to determine the relationship between CVD, microvessel, and circulating MtDNA in the settings of uremia. METHODS: Forty-two maintenance hemodialysis (MHD) patients and 36 health controls were enrolled in this study. Plasma cell-free MtDNA was detected by TaqMan-based qPCR assay. CVD risk markers including high-sensitive C-reactive protein (Hs-CRP), monocyte chemoattractant protein-1 (MCP-1), fibrinogen, and erythrocyte sedimentation rate (ESR) were measured by standard assays. Ten-year CVD risk was calculated from the Framingham risk score (FRS) model. In vitro study, human cardiac microvascular endothelial cells (HCMECs) were incubated with normal or uremic serum, with or without exogenous MtDNA. Intracellular toll-like receptor 9 (TLR9), adhesion molecule 1 (ICAM-1), MCP-1 and tumor necrosis factor-α (TNF-α) and cytosolic MtDNA were detected by qPCR. RESULTS: Plasma MtDNA in MHD patients was significantly higher than healthy controls (4.74 vs. 2.41 × 105 copies/mL; p = 0.000). Subsequently, the MHD patients were classified into two groups based on the MtDNA median (4.34 × 105 copies/mL). In stratified analyses, the levels of Hs-CRP (5.02 vs. 3.73 mg/L; p = 0.042) and MCP-l (99.97 vs. 64.72 pg/mL; p = 0.008) and FRS (21.80 vs. 16.52; p = 0.016) in the high plasma MtDNA group were higher than those in the low plasma MtDNA group. In vitro study, we found that exogenous MtDNA aggravated uremic serum-induced microvascular inflammation (ICAM-1 and TNF-α) in HCMECs (all p < 0.05). Besides, the addition of MtDNA to the medium resulted in a further increase in cytosolic MtDNA and TLR9 levels in uremic serum-treated cells (all p < 0.05). In patients with MHD, MtDNA levels in plasma were significantly reduced after a single routine hemodialysis (pre 4.47 vs. post 3.45 × 105 copies/mL; p = 0.001) or hemodiafiltration (pre 4.85 vs. post 4.09 × 105 copies/mL; p = 0.001). These two approaches seem similar in terms of MtDNA clearance rate (21.26% vs. 11.94%; p = 0.172). CONCLUSIONS: Overall, the present study suggests that MtDNA released into the circulation under the uremic toxin environment may adversely affect the cardiovascular system by exacerbating microvascular inflammation, and that reducing circulating MtDNA might be a future therapeutic strategy for the prevention of MHD-related CVD.

Increased level of free-circulating MtDNA in maintenance hemodialysis patients: Possible role in systemic inflammation.

BACKGROUND: Mitochondrial DNA (MtDNA) exposed to the extracellular space due to cell death and stress has immunostimulatory properties. However, the clinical significance of circulating MtDNA in maintenance hemodialysis (MHD) patients and the precise mechanism of its emergence have yet to be investigated. METHODS: This cross-sectional study consisted of 52 MHD patients and 32 age- and sex-matched healthy controls. MHD patients were further categorized into high and low circulating cell-free MtDNA (ccf-MtDNA) groups based on the median value. Copy number of MtDNA was quantified using TaqMan-based qPCR. Plasma cytokines were measured using ELISA kits. Reactive oxygen species (ROS) and mitochondrial membrane potential (Δψm) in peripheral blood mononuclear cells (PBMCs) were detected using DCFH-DA or JC-1 staining. RESULTS: The copy numbers of ccf-MtDNA in patients with MHD were higher than those in healthy controls, and these alterations were correlated with changes of cytokines TNF-α and IL-6. Adjusted model in multivariate analysis showed that the presence of anuria and longer dialysis vintage were independently associated with higher levels of ccf-MtDNA. Meanwhile, although not statistically significant, an inverse correlative trend between urinary MtDNA and ccf-MtDNA was observed in patients with residual urine. Afterward, using PBMCs as surrogates for mitochondria-rich cells, we found that patients in the high ccf-MtDNA group exhibited a significantly higher ROS production and lower Δψm in cells. CONCLUSIONS: Our data suggested that changes in ccf-MtDNA correlate with the degree of inflammatory status in MHD patients, and that the excessive MtDNA may be caused by mitochondrial dysfunction and reduced urinary MtDNA excretion.

Altered levels of circulating mitochondrial DNA in elderly people with sarcopenia: Association with mitochondrial impairment.

BACKGROUND: Age-related chronic inflammatory process is often referred to as "inflammaging", which had been described in several human disorders, including sarcopenia. Recently, mitochondrial DNA (MtDNA) has moved into the spotlight as a "damage-associated molecular pattern" (DAMP) agent that can potentially elicit inflammation. Yet, the roles of this mitochondrial DAMP have never been investigated in sarcopenia. DESIGN: Cross-sectional study. PARTICIPANTS: From January 2021 to June 2021, elderly outpatients ≥65 years and able to finish a comprehensive geriatric assessment were recruited in our study. METHODS: Participants were divided into sarcopenia group and non-sarcopenia group according to the DXA scans and grip strength. Genomic DNA was extracted from plasma and peripheral blood mononuclear cells (PBMCs), and changes in MtDNA copies were quantified using qPCR. Plasma levels of inflammatory cytokines were measured using ELISA kits. Loss of mitochondrial membrane potential (Δψm) in PBMCs was analyzed using the fluorescent probe JC-1. RESULTS: Participants with sarcopenia were significantly older, more likely to be physically inactive, and had higher levels of circulating cell-free MtDNA (ccf-MtDNA) (all p < 0.05). After adjusting for potential confounders, ccf-MtDNA was independently associated with increased odds of sarcopenia (adjusted odds ratio (AOR), 1.576; p = 0.009). Furthermore, ROC curve analysis showed that ccf-MtDNA had an area under the curve (AUC) of 0.726 (95% CI: 0.607-0.844; p < 0.05) for distinguishing elderly subjects from sarcopenia. Compared with non-sarcopenia subjects, plasma interleukin (IL)-6 and IL-8 were significantly higher in sarcopenia subjects (both p < 0.05). By performing a correlation test, it was found that the level of IL-6 was positively correlated with ccf-MtDNA (r = 0.301; p < 0.05). Then, PBMCs were used as surrogates for mitochondria-rich cells, and the results showed that the relative amplification of MtDNA in PBMCs was significantly reduced (p < 0.05), whereas the depolarization of Δψm was significantly increased in sarcopenia subjects (p < 0.05). CONCLUSIONS: Taken together, our data suggested that circulating MtDNA might be a novel and important source of inflammatory stimuli potentially relevant for sarcopenia in elderly people, and this would provide an attractive therapeutic target to improve this disease.

Circulating Cell-Free Mitochondrial DNA: A Potential Blood-Based Biomarker for Sarcopenia in Patients Undergoing Maintenance Hemodialysis.

BACKGROUND Mitochondrial impairment and exaggerated inflammation are hallmarks of sarcopenia. Recently, cell-free mitochondrial DNA (cf-mtDNA) has been in the spotlight as an endogenous danger molecule that can potentially elicit inflammation. Yet, its actual impact on sarcopenia, especially in patients with maintenance hemodialysis (MHD), is still at an early stage of investigation. MATERIAL AND METHODS A total of 105 MHD patients were enrolled in this study. The subjects were classified into sarcopenia group (SP) and non-sarcopenia group (NSP) according to the DXA scan and grip strength. Plasma and peripheral blood mononuclear cells (PBMCs) were separated from whole blood. Circulating cf-mtDNA (ccf-mtDNA) was detected using Taq Man RT-qPCR. Cytosolic mtDNA and inflammation- and mitophagy-related genes in PBMCs were quantitated using SYBR Green RT-qPCR. ΔΨm was analyzed using the fluorescent probe JC-1. RESULTS ccf-mtDNA content was significantly higher in SP group than in NSP group. Multivariate regression analysis showed a significant correlation of ccf-mtDNA with sarcopenia after adjusting for potential confounders. A similar trend of increased mtDNA was also observed in the mitochondria-free cytoplasm of PBMCs from SP patients, together with higher expression of TLR9 and IL-6 in this group. Next, using PBMCs as surrogates for mitochondria-rich cells, we found that ΔΨm was dramatically decreased in the SP group. In parallel, the mRNA levels of mitophagy-related genes Parkin and LAMP2 were increased in the SP group. CONCLUSIONS The results obtained demonstrated that ccf-mtDNA, as a potential driver of inflammatory component, may be involved in the pathogenesis of the MHD-related sarcopenia.

Flavin adenine dinucleotide ameliorates hypertensive vascular remodeling via activating short chain acyl-CoA dehydrogenase.

AIMS: Flavin adenine dinucleotide (FAD), participates in fatty acid ß oxidation as a cofactor, which has been confirmed to enhance SCAD activity and expression. However, the role of FAD on hypertensive vascular remodeling is unclear. In this study, we investigated the underlying mechanisms of FAD on vascular remodeling and endothelial homeostasis. MAIN METHODS: Morphological examination of vascular remodeling were analyzed with hematoxylin and eosin (HE) staining, Verhoeff's Van Gieson (EVG) staing, Dihydroethidium (DHE) staining and Sirius red staining. HUVECs apoptotic rate was detected by flow cytometry and HUVECs reactive oxygen species (ROS) was detected by DHE-probe. Enzymatic reactions were used to detect SCAD enzyme activity. The protein level was detected by Western Blots, the mRNA level was detected by quantitative real-time PCR. KEY FINDINGS: In vivo experiments, FAD significantly decreased blood pressure and ameliorated vascular remodeling by increasing SCAD expression, Nitric Oxide (NO) production and reducing ROS production. In vitro experiments, FAD protected against the tBHP induced injury in HUVEC, by increasing the activity of SCAD, increasing the elimination of free fatty acid (FFA), scavenging ROS, reducing apoptotic rate, thereby improving endothelial cell function. SIGNIFICANCE: FAD has a new possibility for preventing and treating hypertensive vascular remodeling.

The CX3CL1/CX3CR1 axis is upregulated in chronic kidney disease and contributes to angiotensin II-induced migration of vascular smooth muscle cells.

BACKGROUND: The role of the chemokine axis, CX3CL1/CX3CR1, in the development of cardiovascular diseases has been widely speculated. Angiotensin II (Ang II) is a pivotal factor promoting cardiovascular complications in patients with chronic kidney disease (CKD). Whether there is a link between the two in CKD remains unclear. METHODS: The uremic mice were treated with losartan for 8 weeks, and the expression of aortic CX3CL1/CX3CR1 was detected. Cultured mouse aortic vascular smooth muscle cells (VSMCs) were stimulated with Ang II, and then CX3CR1 expression was assessed by western blot. After the targeted disruption of CX3CR1 by transfection with siRNA, the migration of VSMCs was detected by transwell assay. Finally, both the activation of Akt pathway and the expression of IL-6 were detected by western blot. RESULTS: Losartan treatment reduced the upregulation of aortic CX3CL1/CX3CR1 expression in uremic mice. In vitro, Ang II significantly upregulated CX3CR1 expression in VSMCs. Targeted disruption of CX3CR1 attenuated Ang II-induced migration of VSMCs. In addition, the use of CX3CR1-siRNA suppressed Akt phosphorylation and IL-6 production in VSMCs stimulated by Ang II. CONCLUSIONS: The aortic CX3CL1/CX3CR1 is upregulated by Ang II in CKD, and it contributes to Ang II-induced migration of VSMCs in vitro.

Flavine adenine dinucleotide inhibits pathological cardiac hypertrophy and fibrosis through activating short chain acyl-CoA dehydrogenase.

Short-chain acyl-CoA dehydrogenase (SCAD), the rate-limiting enzyme for fatty acid ß-oxidation, has a negative regulatory effect on pathological cardiac hypertrophy and fibrosis. Furthermore, flavin adenine dinucleotide (FAD) can enhance the expression and enzyme activity of SCAD. However, whether FAD can inhibit pathological cardiac hypertrophy and fibrosis remains unclear. Therefore, we observed the effect of FAD on pathological cardiac hypertrophy and fibrosis. FAD significantly inhibited PE-induced cardiomyocyte hypertrophy and AngII-induced cardiac fibroblast proliferation. In addition, FAD ameliorated pathological cardiac hypertrophy and fibrosis in SHR. FAD significantly increased the expression and enzyme activity of SCAD. Meanwhile, ATP content was increased, the content of free fatty acids and reactive oxygen species were decreased by FAD in vivo and in vitro. In addition, molecular dynamics simulations were also used to provide insights into the structural stability and dynamic behavior of SCAD. The results demonstrated that FAD may play an important structural role on the SCAD dimer stability and maintenance of substrate catalytic pocket to increase the expression and enzyme activity of SCAD. In conclusion, FAD can inhibit pathological cardiac hypertrophy and fibrosis through activating SCAD, which may be a novel effective treatment for pathological cardiac hypertrophy and fibrosis, thus prevent them from developing into heart failure.

Quality of Root Filling after Obturation with Gutta-percha and 3 Different Sealers of Minimally Instrumented Root canals of the Maxillary First Molar.

INTRODUCTION: The aim of this study was to compare the quality of root fillings completed by a modified single-cone (MSC) technique with 3 different sealers after minimal instrumentation and multisonic cleaning of root canals of maxillary first molars. METHODS: Root canals of 18 maxillary first molars were instrumented to size 15/.04 taper using rotary files. Sodium hypochlorite 5.25% was used during instrumentation; the final cleaning was performed by the GentleWave System (Sonendo Inc, Laguna Hills, CA). The specimens were allocated into 3 groups and root filled by the MSC technique using a size fitted gutta-percha master cone and GuttaFlow Bioseal (Coltene Whaledent GmBH + Co KG, Langenau, Switzerland), GuttaFlow 2 (Coltene Whaledent GmBH + Co KG), and MTA Fillapex (Angelus Industria de Produtos Odontológicos S/A, Londrina, PR, Brazil) sealers. Micro-computed tomographic scans were obtained before and after instrumentation, post-GentleWave, and after obturation. Reconstructed images were analyzed for the volumetric percentage of filling materials. Mesiobuccal roots of the selected teeth were sectioned at 0.5-mm increments starting at the apex of the root. The cross sections were further examined using a light microscope. RESULTS: The 3 groups had 90%-99% of the canal space filled with the root filling material. The mean volume of the filling material was higher in the GuttaFlow Bioseal and GuttaFlow 2 groups than in the MTA Fillapex group (P < .05). There was no significant difference among the apical, middle, and coronal thirds. The cross-sectional images showed no obvious gaps or voids in the GuttaFlow groups. After instrumentation, 49 of the 189 canal thirds (25.9%) had hard tissue debris in the root canal system. After GentleWave cleaning, only 4 of 63 canals (6.3%) and 4 of the 189 canal thirds (2.1%) still had debris. CONCLUSIONS: The MSC method with GuttaFlow 2 and GuttaFlow Bioseal sealers after multisonic cleaning of minimally instrumented molar canals resulted in high-quality root fillings. Multisonic cleaning of minimally instrumented molars seems to be effective in debris removal.

UCP2 alleviates tubular epithelial cell apoptosis in lipopolysaccharide-induced acute kidney injury by decreasing ROS production.

Uncoupling protein 2 (UCP2), an anion transporter, modulates the production of mitochondrial reactive oxygen species (ROS) and plays an important role in protecting against cell apoptosis. However, the role of UCP2 in sepsis-associated AKI remains unclear. In the present study, we investigated the role of UCP2 in LPS-induced AKI in vitro and in vivo. UCP2 expression was increased in tubular epithelial cells (TECs) treated with LPS. Accordingly, UCP2 expression was distinctly upregulated in renal tissues from the animals with LPS-induced AKI. Furthermore, UCP2 silencing dramatically aggravated LPS-induced apoptosis, accompanied by increased ROS production in renal tubular epithelial cell. Additionally, the inhibition of UCP2 by genipin, a specific UCP2 inhibitor, exacerbated the kidney injury of animals with LPS-induced AKI. Moreover, NAC (N-acetylcysteine), a potent ROS scavenger, obviously suppressed apoptosis induced by UCP2 silencing, which suggests that the increased ROS levels were associated with tubular epithelial cell apoptosis induced by UCP2 silencing. Therefore, UCP2 exerts a protective effect on the LPS-induced apoptosis of tubular epithelial cells by reducing excess ROS production. In conclusion, our findings highlight the renoprotective actions of UCP2 on inhibiting the production of apoptotic factors and oxidative stress to improve tubular cell survival in the LPS-induced AKI model.

[Change of short-chain acyl-CoA dehydrogenase in heart failure after myocardial infarction in rats and the intervention of aerobic exercise].

OBJECTIVE: To Study the changes of short-chain acyl-CoA dehydrogenase (SCAD) in heart failure (HF) after myocardial infarction (MI), and the effect of aerobic exercise on SCAD. METHODS: Healthy male Sprague-Dawley (SD) rats were divided into sham operation group (Sham group), sham operation swimming group (Sham+swim group), HF model group (LAD group) and HF swimming group (LAD+swim group) by random number table method, with 9 rats in each group. The left anterior descending branch of coronary artery (LAD) was ligated to establish a rat model of HF after MI. In Sham group, only one loose knot was threaded under the left coronary artery, and the rest operations were the same as those in LAD group. Rats in Sham+swim group and LAD+swim group were given swimming test for 1 week after operation (from 15 minutes on the 1st day to 60 minutes on the 5th day). Then they were given swimming endurance training (from the 2nd week onwards, 60 minutes daily, 6 times weekly, 10 weeks in a row). Tail artery systolic pressure (SBP) was measured before swimming endurance training and every 2 weeks until the end of the 10th week. Ten weeks after swimming training, echocardiography was performed to measure cardiac output (CO), stroke volume (SV), left ventricular ejection fraction (LVEF), shortening fraction (FS), left ventricular end-systolic diameter (LVESD), left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic volume (LVESV), and left ventricular end-diastolic volume (LVEDV). Morphological changes of heart were observed by Masson staining. Apoptosis of myocardial cells was detected by transferase-mediated deoxyuridine triphosphate-biotin nick end labeling stain (TUNEL) and apoptosis index (AI) was calculated. Reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot were used to detect the mRNA and protein expression of myocardial SCAD respectively. In addition, the enzyme activity of SCAD, the content of adenosine triphosphate (ATP) and free fatty acid (FFA) in serum and myocardium were detected according to the kit instruction steps. RESULTS: Compared with Sham group, Sham+swim group showed SBP did not change significantly, with obvious eccentric hypertrophy and increased myocardial contractility, and LAD group showed persistent hypotension, obvious MI, thinning of left ventricle, and decreased myocardial systolic/diastolic function. Compared with LAD group, SBP, systolic/diastolic function and MI in LAD+swim group were significantly improved [SBP (mmHg, 1 mmHg = 0.133 kPa): 119.5±4.4 vs. 113.2±4.5 at 4 weeks, 120.3±4.0 vs. 106.5±3.7 at 6 weeks, 117.4±1.3 vs. 111.0±2.3 at 8 weeks, 126.1±1.6 vs. 119.4±1.9 at 10 weeks; CO (mL/min): 59.10±6.31 vs. 33.19±4.76, SV (µL): 139.42±17.32 vs. 84.02±14.26, LVEF: 0.523±0.039 vs. 0.309±0.011, FS: (28.17±2.57)% vs. (15.93±3.64)%, LVEDD (mm): 8.80±0.19 vs. 9.35±0.30, LVESD (mm): 5.90±0.77 vs. 7.97±0.60, LVEDV (µL): 426.57±20.84 vs. 476.24±25.18, LVESV (µL): 209.50±25.18 vs. 318.60±16.10; AI: (20.4±1.4)% vs. (31.2±4.6)%; all P < 0.05]. Compared with Sham group, the mRNA and protein expression of myocardium SCAD, the activity of SCAD in Sham+swim group were significantly increased, the content of ATP was slightly increased, the content of serum FFA was significantly decreased, and the content of myocardial FFA was slightly decreased; conversely, the mRNA and protein expression of myocardium SCAD, the activity of SCAD and the content of ATP in LAD group were significantly decreased, the content of serum and myocardial FFA were significantly increased. Compared with LAD group, the mRNA and protein expression of myocardium SCAD, the content of ATP were significantly increased in LAD+swim group [SCAD mRNA (2-ΔΔCt): 0.52±0.16 vs. 0.15±0.01, SCAD/GAPDH (fold increase from Sham group): 0.94±0.08 vs. 0.60±0.11, ATP content (µmol/g): 52.8±10.1 vs. 14.7±6.1, all P < 0.05], the content of serum and myocardial FFA were significantly decreased [serum FFA (nmol/L): 0.11±0.03 vs. 0.29±0.04, myocardial FFA (nmol/g): 32.7±8.2 vs. 59.7±10.7, both P < 0.05], and the activity of SCAD was slightly increased (kU/g: 12.3±4.3 vs. 8.9±5.8, P > 0.05). CONCLUSIONS: The expression of SCAD in HF was significantly down-regulated, and the expression was significantly up-regulated after aerobic exercise intervention, indicating that swimming may improve the severity of HF by up-regulating the expression of SCAD.

JAK2/STAT3/BMP-2 axis and NF-κB pathway are involved in erythropoietin-induced calcification in rat vascular smooth muscle cells.

BACKGROUND: Vascular calcification is common in chronic kidney disease (CKD) patients, while erythropoietin (EPO) is widely used in the treatment of renal anemia in CKD patients, whether there is a link between the two is still not clear. METHODS: The primary rat vascular smooth muscle cells (VSMCs) and CKD rats were treated with EPO and the calcium deposition was observed by alizarin red staining, von Kossa staining and calcium quantification. Activation of JAK2/STAT3/BMP-2 axis and NF-κB signaling pathways was investigated by Western blotting. RESULTS: EPO-induced calcium deposition in VSMCs and significantly potentiated calcification in CKD rats. Furthermore, EPO activated JAK2/STAT3/BMP-2 axis, NF-κB pathway and the pro-calcification effect of EPO was partially blocked by the STAT3 inhibitor (Cryptotanshinone) or NF-κB inhibitor (BAY 11-7082), respectively, in vitro. CONCLUSION: EPO could promote VSMCs calcification in vitro and in vivo and this effect may be achieved through the JAK2/STAT3/BMP-2 axis and NF-κB pathway.

CX3CL1/CX3CR1 Axis Contributes to Angiotensin II-Induced Vascular Smooth Muscle Cell Proliferation and Inflammatory Cytokine Production.

Angiotensin II (Ang II) dysregulation has been determined in many diseases. The CX3CL1/CX3CR1 axis, which has a key role in cardiovascular diseases, is involved in the proliferation and inflammatory cytokine production of vascular smooth muscle cells (VSMCs). In this study, we aim to explore whether Ang II has a role in the expression of CX3CL1/CX3CR1, thus contributing to the proliferation and pro-inflammatory status of VSMCs. Cultured mouse aortic VSMCs were stimulated with 100 nmol/L of Ang II, and the expression of CX3CR1 was assessed by western blot. The results demonstrated that Ang II significantly up-regulated CX3CR1 expression in VSMCs and induced the production of reactive oxygen species (ROS) and the phosphorylation of p38 MAPK. Inhibitors of NADPH oxidase, ROS, and AT1 receptor significantly reduced Ang II-induced CX3CR1 expression. Targeted disruption of CX3CR1 by transfection with siRNA significantly attenuated Ang II-induced VSMC proliferation as well as down-regulated the expression of proliferating cell nuclear antigen (PCNA). Furthermore, CX3CR1-siRNA suppressed the effect of Ang II on stimulating Akt phosphorylation. Besides, the use of CX3CR1-siRNA decreased inflammatory cytokine production induced by Ang II treatment. Our results indicate that Ang II up-regulates CX3CR1 expression in VSMCs via NADPH oxidase/ROS/p38 MAPK pathway and that CX3CL1/CX3CR1 axis contributes to the proliferative and pro-inflammatory effects of Ang II in VSMCs.