Metabolomic Profiling of Long-Chain Polyunsaturated Fatty Acid Oxidation in Adults with Retinal Vein Occlusion: A Case-Control Study.

BACKGROUND: Long-chain polyunsaturated fatty acids (LCPUFAs) and their metabolites are closely related to neovascular eye diseases. However, the clinical significance of their oxylipins in retinal vein occlusion (RVO) remains inconclusive. OBJECTIVES: This case-control study aimed to explore metabolomic profiles of LCPUFA oxidation in RVO and to identify potential indicators for diagnosis and pathologic progression. METHODS: The plasma concentrations of ω-3 (n-3) and ω-6 (n-6) LCPUFA and their oxylipins in 44 adults with RVO and 36 normal controls were analyzed using ultraperformance liquid chromatography tandem mass spectrometry. Univariate analysis combined with principal component and orthogonal projections to latent structure discriminant analysis was used to screen differential metabolites. Aortic ring and choroidal explant sprouting assays were used to investigate the effects of 5-oxo-eicosatetraenoic acids (ETE) on angiogenesis ex vivo. Tubule formation and wound healing assays were performed to verify its effects on human retinal microvascular endothelial cell functions. RESULTS: Higher ω-6 and lower ω-3 LCPUFA plasma concentrations were measured in the adults with RVO compared with control (odds ratio [OR]: 2.34; 95% confidence interval [CI]: 1.42, 3.86; P < 0.001; OR: 0.28; 95% CI: 0.15, 0.51; P < 0.001). Metabolomic analysis revealed 20 LCPUFA and their oxylipins dysregulated in RVO, including increased arachidonic acid (ω-6, OR: 1.85; 95% CI: 1.18, 2.90; P < 0.001) and its lipoxygenase product 5-oxo-ETE (OR: 11.76; 95% CI: 3.73, 37.11; P < 0.001), as well as decreased docosahexaenoic acid (ω-3, OR: 0.13; 95% CI: 0.05, 0.33; P < 0.001). Interestingly, 5-oxo-ETE was downregulated in ischemic compared with nonischemic central RVO. Exogenous 5-oxo-ETE attenuated aortic ring and choroidal explant sprouting and inhibited tubule formation and migration of human retinal microvascular endothelial cells in a dose-dependent manner, possibly via suppressing the vascular endothelial growth factor signaling pathway. CONCLUSIONS: The plasma concentrations of ω-6 and ω-3 LCPUFA and their oxylipins were associated with RVO. The ω-6 LCPUFA-derived metabolite 5-oxo-ETE was a potential marker of RVO development and progression.

An improved procedure for isolating adult mouse cardiomyocytes for epicardial activation mapping.

Cardiovascular disease is a leading cause of death and disability worldwide. Although genetically modified mouse models offer great potential for robust research in vivo, in vitro studies using isolated cardiomyocytes also provide an important approach for investigating the mechanisms underlying cardiovascular disease pathogenesis and drug actions. Currently, isolation of mouse adult cardiomyocytes often relies on aortic retrograde intubation under a stereoscopic microscope, which poses considerable technical barriers and requires extensive training. Although a simplified, Langendorff-free method has been used to isolate viable cardiomyocytes from the adult mouse heart, the system requires enzymatic digestions and continuous manual technical operation. This study established an optimized approach that allows isolation of adult mouse cardiomyocytes and epicardial activation mapping of mouse hearts using a Langendorff device. We used retrograde puncture through the abdominal aorta in vivo and enzymatic digestion on the Langendorff perfusion device to isolate adult mouse cardiomyocytes without using a microscope. The yields of isolated cardiomyocytes were amenable to patch clamp techniques. Furthermore, this approach allowed epicardial activation mapping. We used a novel, simplified method to isolate viable cardiomyocytes from adult mouse hearts and to map epicardial activation. This novel approach could be beneficial in more extensive research in the cardiac field.

Loganin Inhibits Angiotensin II-Induced Cardiac Hypertrophy Through the JAK2/STAT3 and NF-κB Signaling Pathways.

Loganin is an iridoid glycoside extracted from Cornus officinalis, which is a traditional oriental medicine, and many biological properties of loganin have been reported. Nevertheless, it is not clear whether loganin has therapeutic effect on cardiovascular diseases. Hence, the aim of the present study was to investigate the effect of loganin on Ang II-induced cardiac hypertrophy. In the present study, we reported for the first time that loganin inhibits Ang II-provoked cardiac hypertrophy and cardiac damages in H9C2 cells and in mice. Furthermore, loganin can achieve cardioprotective effects through attenuating cardiac fibrosis, decreasing pro-inflammatory cytokine secretion, and suppressing the phosphorylation of critical proteins such as JAK2, STAT3, p65, and IκBα. Besides, the outstanding findings of the present study were to prove that loganin has no significant toxicity or side effects on normal cells and organs. Based on these results, we conclude that loganin mitigates Ang II-induced cardiac hypertrophy at least partially through inhibiting the JAK2/STAT3 and NF-κB signaling pathways. Accordingly, the natural product, loganin, might be a novel effective agent for the treatment of cardiac hypertrophy and heart failure.

HINT1 (Histidine Triad Nucleotide-Binding Protein 1) Attenuates Cardiac Hypertrophy Via Suppressing HOXA5 (Homeobox A5) Expression.

BACKGROUND: Cardiac hypertrophy is an important prepathology of, and will ultimately lead to, heart failure. However, the mechanisms underlying pathological cardiac hypertrophy remain largely unknown. This study aims to elucidate the effects and mechanisms of HINT1 (histidine triad nucleotide-binding protein 1) in cardiac hypertrophy and heart failure. METHODS: HINT1 was downregulated in human hypertrophic heart samples compared with nonhypertrophic samples by mass spectrometry analysis. Hint1 knockout mice were challenged with transverse aortic constriction surgery. Cardiac-specific overexpression of HINT1 mice by intravenous injection of adeno-associated virus 9 (AAV9)-encoding Hint1 under the cTnT (cardiac troponin T) promoter were subjected to transverse aortic construction. Unbiased transcriptional analyses were used to identify the downstream targets of HINT1. AAV9 bearing shRNA against Hoxa5 (homeobox A5) was administrated to investigate whether the effects of HINT1 on cardiac hypertrophy were HOXA5-dependent. RNA sequencing analysis was performed to recapitulate possible changes in transcriptome profile.Coimmunoprecipitation assays and cellular fractionation analyses were conducted to examine the mechanism by which HINT1 regulates the expression of HOXA5. RESULTS: The reduction of HINT1 expression was observed in the hearts of hypertrophic patients and pressure overloaded-induced hypertrophic mice, respectively. In Hint1-deficient mice, cardiac hypertrophy deteriorated after transverse aortic construction. Conversely, cardiac-specific overexpression of HINT1 alleviated cardiac hypertrophy and dysfunction. Unbiased profiler polymerase chain reaction array showed HOXA5 is 1 target for HINT1, and the cardioprotective role of HINT1 was abolished by HOXA5 knockdown in vivo. Hoxa5 was identified to affect hypertrophy through the TGF-ß (transforming growth factor ß) signal pathway. Mechanically, HINT1 inhibited PKCß1 (protein kinase C ß type 1) membrane translocation and phosphorylation via direct interaction, attenuating the MEK/ERK/YY1 (mitogen-activated protein kinase/extracellular signal-regulated kinase kinase/yin yang 1) signal pathway, downregulating HOXA5 expression, and eventually attenuating cardiac hypertrophy. CONCLUSIONS: HINT1 protects against cardiac hypertrophy through suppressing HOXA5 expression. These findings indicate that HINT1 may be a potential target for therapeutic interventions in cardiac hypertrophy and heart failure.

Characterization and Management of Arrhythmic Events in Young Patients With Brugada Syndrome.

BACKGROUND: Information on young patients with Brugada syndrome (BrS) and arrhythmic events (AEs) is limited. OBJECTIVES: The purpose of this study was to describe their characteristics and management as well as risk factors for AE recurrence. METHODS: A total of 57 patients (age ≤20 years), all with BrS and AEs, were divided into pediatric (age ≤12 years; n = 26) and adolescents (age 13 to 20 years; n = 31). RESULTS: Patients' median age at time of first AE was 14 years, with a majority of males (74%), Caucasians (70%), and probands (79%) who presented as aborted cardiac arrest (84%). A significant proportion of patients (28%) exhibited fever-related AE. Family history of sudden cardiac death (SCD), prior syncope, spontaneous type 1 Brugada electrocardiogram (ECG), inducible ventricular fibrillation at electrophysiological study, and SCN5A mutations were present in 26%, 49%, 65%, 28%, and 58% of patients, respectively. The pediatric group differed from the adolescents, with a greater proportion of females, Caucasians, fever-related AEs, and spontaneous type-1 ECG. During follow-up, 68% of pediatric and 64% of adolescents had recurrent AE, with median time of 9.9 and 27.0 months, respectively. Approximately one-third of recurrent AEs occurred on quinidine therapy, and among the pediatric group, 60% of recurrent AEs were fever-related. Risk factors for recurrent AE included sinus node dysfunction, atrial arrhythmias, intraventricular conduction delay, or large S-wave on ECG lead I in the pediatric group and the presence of SCN5A mutation among adolescents. CONCLUSIONS: Young BrS patients with AE represent a very arrhythmogenic group. Current management after first arrhythmia episode is associated with high recurrence rate. Alternative therapies, besides defibrillator implantation, should be considered.

Effect of Xiaoaiping injection on advanced hepatocellular carcinoma in patients.

OBJECTIVE: To investigate the effect of Xiaoaiping Injection (XAP) on advanced hepatocellular carcinoma (HCC) in patients. METHODS: Sixty-eight patients with advanced HCC were assigned to a control group of 36 and a treatment group of 32. The control group was treated with best supportive treatment (BST) and the treatment group was given XAP plus BST. XAP was administered daily by i.v. and the treatment course was lasted for 30 days for both groups. The immediate therapeutic efficacy, Karnofsky performance status (KPS) scores, and the changes in immunity indicators (CD3+, CD4+ and CD8+ T cells) were measured and compared before and after treatment. The progression-free survival (PFS) rate and overall survival (OS) rate in the 2 groups were analyzed. RESULTS: The immediate therapeutic efficacy and KPS of the treatment group were better than those in the control (P < 0.05). Patients in the treatment group had higher percentages of CD3 and CD4 T-lymphocytes in peripheral blood than those in the control group (P < 0.05). The median survival time was 27.0 weeks in the treatment group and 24.5 weeks in the control group. The 6-months cumulative survival rates in the treatment and control groups were 33.3% and 25.0%, respectively, with no significant difference (P > 0.05). The PFS was 18 weeks in the treatment group and 15 weeks in control group (P < 0.05). CONCLUSION: XAP enhances the quality of life (QOL) of patients with advanced HCC, improves their immunity and extends their PFS.

Studies on the anti-angiogenic effect of Marsdenia tenacissima extract in vitro and in vivo.

Marsdenia tenacissima, which is widely used as an anticancer herb in traditional Chinese medicine, has been shown to possess anticancer activities. However, the underlying molecular mechanism(s) involved in the anticancer effect of this herb are poorly understood. Angiogenesis is important in the development of cancer. The main features of angiogenesis are increased vasculature and overexpression of vascular endothelial growth factor (VEGF). In the present study, the effects of M. tenacissima extract (MTE) on human umbilical vein endothelial cell (HUVEC) proliferation, migration and capillary-like tube formation were investigated in vitro and using the chick embryo chorioallantoic membrane (CAM) assay in vivo. It was observed that MTE inhibited the proliferation of HUVECs by blocking the cell cycle progression from G1 to S. In addition, MTE inhibited the migration and tube formation of HUVECs. MTE treatment decreased the VEGF-A expression in human hepatoma cells (HepG2), as well as the expression of VEGF-A and VEGF receptor (VEGFR)-2 in HUVECs. MTE exposure in the CAM was able to reduce the formation of blood vessels in chick embryos. Overall, the present data suggest that extracts of M. tenacissima may serve as potential anti-angiogenesis agents.

Xiao Jin Wan, a traditional Chinese herbal formula, inhibits proliferation via arresting cell cycle progression at the G2/M phase and promoting apoptosis via activating the mitochondrial­dependent pathway in U-2OS human osteosarcoma cells.

Xiao Jin Wan (XJW) is a well-known traditional Chinese folk-medicine, which is commonly used for the treatment of various types of diseases including cancers. However, the mechanism of the anticancer activity of XJW against U-2OS human osteosarcoma cells, have not yet been reported. In the present study, we investigated the cellular effects of the XJW on the U-2OS human osteosarcoma cell line. Our results showed that XJW induced cell morphological changes, reduced cell viability in a dose- and time-dependent manner and arrested in the G2/M phase of the cell cycle suggesting that XJW inhibited the proliferation of U-2OS cells. Hoechst 33258 staining and Annexin V/propidium iodide double staining exhibited the typical nuclear features of apoptosis and increased the proportion of apoptotic Annexin V-positive cells in a dose-dependent manner, respectively. In addition, XJW treatment caused loss of plasma membrane asymmetry, collapse of mitochondrial membrane potential, activation of caspase-9 and caspase-3, and increase of the ratio of pro-apoptotic Bax to anti-apoptotic Bcl-2. Taken together, the results indicate that the U-2OS cell growth inhibitory activity of XJW was due to cell cycle arrested and mitochondrial-mediated apoptosis, which may partly explain the anticancer activity of Xiao Jin Wan.

Ethyl acetate extraction from a Chinese herbal formula, Jiedu Xiaozheng Yin, inhibits the proliferation of hepatocellular carcinoma cells via induction of G0/G1 phase arrest in vivo and in vitro.

Jiedu Xiaozheng Yin (JXY), a polyherbal formula of traditional Chinese medicine (TCM), has been used to treat various kinds of cancer in China. However, the mechanism of its anticancer activity has yet to be elucidated. Air-dried herbs were extracted with reagents of different polarity. HepG2 cells were treated with different doses of ethyl acetate extract (EE-JXY) and chloroform extract (CE-JXY) for 24 h. Cell viability was detected by MTT assay. Colony formation ability was also evaluated. Cell cycle was evaluated by FACS. Tumor bearing BALB/c nude mice was treated with EE-JXY (0.06 g/kg) for 20 days. Tumor volume and weight were monitored. The percentage of PCNA-positive cells and the level of G1 phase proteins [cyclin-dependent kinase2 (CDK2), cyclin­dependent kinase4 (CDK4), cyclin D and cyclin E and G2 phase proteins [cyclin-dependent kinase1 (CDK1), cyclin A and cyclin B] were detected by immunohistochemistry and western blotting. EE-JXY and CE-JXY dose-dependently inhibited the growth of HepG2 cells (P<0.01 for both). Furthermore, EE-JXY inhibited the formation of cell colonies and blocked the cell cycle to G1 phase in a dose-dependent manner (P<0.01 for all). EE-JXY showed an obviously antitumor effect in vivo (P<0.05). Further investigation showed that EE-JXY decreased the proliferation index of tumors (P<0.01) through increasing the expression of G1-related proteins (cyclin D and cyclin E, P<0.05 and P<0.01). These results suggested that JXY inhibits the growth of HepG2 cells at least via arresting the cell cycle at the G0/G1 phase.

Jiedu Xiaozheng Yin, a Chinese herbal formula, inhibits tumor angiogenesis via downregulation of VEGF-A and VEGFR-2 expression in vivo and in vitro.

Angiogenesis is crucial for cancer growth and metastasis and inhibition of angiogenesis has been recognized to be a promising strategy for the treatment of cancer. Traditional Chinese medicine (TCM) has been used for thousands of years to treat cancer. Jiedu Xiaozheng Yin (JXY), a polyherbal formula of TCM, has been used to treat various tumors in China. However, the mechanism of its anticancer activity has yet to be fully elucidated. Using human umbilical vein endothelial cells (HUVECs), chick chorioallantoic membrane (CAM) and a hepatoma mouse xenograft model, we investigated the underlying molecular mechanisms of ethanol extract of Jiedu Xiaozheng Yin (EE-JXY). EE-JXY treatment significantly inhibited tumor cell growth both in vitro and in the mouse xenograft model (P<0.05). Moreover, EE-JXY reduced tube formation of HUVECs and angiogenesis in the CAM (P<0.01) and microvessel density (MVD) of tumor in vivo (P<0.05). Further studies showed that EE-JXY was able to suppress the expression of vascular endothelial growth factor A (VEGF-A) and vascular endothelial growth factor receptor 2 (VEGFR-2) in both HepG2 cells and HUVECs (P<0.01) and in tumor (P<0.01). Thus, JXY suppressed tumor growth at least by inhibiting angiogenesis.

Effect of didrovaltrate on I-calcium current in rabbit ventricular myocytes.

OBJECTIVE: To investigate the effect of didrovaltrate on L-type calcium current (I(Ca-L)) in rabbit ventricular myocytes. METHODS: We used the whole cell patch clamp recording technique. RESULTS: Didrovaltrate at concentrations of 30 microg/ L and 100 microg/L significantly decreased peak I(Ca-L) (I(Ca-Lmax)) from (6.01 +/- 0.48) pA/pF to (3.45 +/- 0.27) pA/pF and (2.16 +/- 0.19) pA/pF (42.6% and 64.1%, n=8, P< 0.01), respectively. Didrovaltrate shifted upwards the current-voltage curves of I(Ca-L) without changing their active, peak and reverse potentials. Didrovaltrate affected the steady-state inactivation of I(Ca-L). The half activation potential (V1/2) was significantly shifted from (-26 +/- 2) to (-36 +/- 3) mV (n=6, P<0.05), with a significant change in the slope factor (k) (from 8.8 +/- 0.8 to 11.1 +/- 0.9, n=6, P<0.05). Didrovaltrate did not affect the activation curve. CONCLUSION: Didrovaltrate blocks I(Ca-L) in a concentration-dependent manner and probably inhibits I(Ca-L) in its inactive state, which may contribute to its cardiovascular effect.