Haijian Sun

Endothelial dysfunction and cardiometabolic diseases: Role of long non-coding RNAs.

The vascular endothelium is recognized as a barrier between blood and blood vessel wall. The abnormality of vascular endothelium is critical for atherosclerosis, hypertension and diabetes. Oxidative stress, inflammation, obesity, hyperlipidemia and insulin resistance are major contributors to endothelial dysfunction in cardiovascular disorders. Therapeutic strategies against endothelial dysfunction are developed to prevent and treat vascular lesions. In recent years, long non-coding RNAs (lncRNAs) are emerged as novel modulators in the proliferation and differentiation of various cell types. LncRNAs have attracted considerable attention due to their multiple biological roles in the prognostic prediction, diagnosis and treatment of cancers. LncRNAs are also involved in pathogenesis of cardiovascular diseases. However, the correlations between lncRNAs and endothelial dysfunction are still largely obscure. In this review, we will highlight recent updates associated to the importance of lncRNAs in the pathogenesis of endothelial dysfunction in cardiovascular disorders, and the basic molecular mechanisms of lncRNAs in regulation of endothelial function are also discussed. LncRNAs may become promising therapeutic targets in endothelial dysfunction-related diseases.

Hypaphorine Attenuates Lipopolysaccharide-Induced Endothelial Inflammation via Regulation of TLR4 and PPAR-γ Dependent on PI3K/Akt/mTOR Signal Pathway

Endothelial lesion response to injurious stimuli is a necessary step for initiating inflammatory cascades in blood vessels. Hypaphorine (Hy) from different marine sources is shown to exhibit anti-inflammatory properties. However, the potential roles and possible molecular mechanisms of Hy in endothelial inflammation have yet to be fully clarified. We showed that Hy significantly inhibited the positive effects of lipopolysaccharide (LPS) on pro-inflammatory cytokines expressions, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), monocyte chemoattractant protein 1 (MCP-1) and vascular cellular adhesion molecule-1 (VCAM-1), as well as induction of the phosphorylation of Akt and mTOR in HMEC-1 cells. The downregulated peroxisome proliferator-activated receptor γ (PPAR-γ) and upregulated toll-like receptor 4 (TLR4) expressions in LPS-challenged endothelial cells were prevented by Hy. Inhibition of both PI3K and mTOR reversed LPS-stimulated increases in TLR4 expressions and decreases in PPAR-γ levels. Genetic silencing of TLR4 or PPAR-γ agonist pioglitazone obviously abrogated the levels of pro-inflammatory cytokines in LPS-treated HMEC-1 cells. These results suggest that Hy may exert anti-inflammatory actions through the regulation of TLR4 and PPAR-γ dependent on PI3K/Akt/mTOR signal pathways. Hy may be considered as a therapeutic agent that can potentially relieve or ameliorate endothelial inflammation-associated diseases.

C1q/TNF-Related Protein-9 Ameliorates Ox-LDL-Induced Endothelial Dysfunction via PGC-1α/AMPK-Mediated Antioxidant Enzyme Induction

Oxidized low-density lipoprotein (ox-LDL) accumulation is one of the critical determinants in endothelial dysfunction in many cardiovascular diseases such as atherosclerosis. C1q/TNF-related protein 9 (CTRP9) is identified to be an adipocytokine with cardioprotective properties. However, the potential roles of CTRP9 in endothelial function remain largely elusive. In the present study, the effects of CTRP9 on the proliferation, apoptosis, migration, angiogenesis, nitric oxide (NO) production and oxidative stress in human umbilical vein endothelial cells (HUVECs) exposed to ox-LDL were investigated. We observed that treatment with ox-LDL inhibited the proliferation, migration, angiogenesis and the generation of NO, while stimulated the apoptosis and reactive oxygen species (ROS) production in HUVECs. Incubation of HUVECs with CTRP9 rescued ox-LDL-induced endothelial injury. CTRP9 treatment reversed ox-LDL-evoked decreases in antioxidant enzymes including heme oxygenase-1 (HO-1), nicotinamide adenine dinucleotide phosphate (NAD(P)H) dehydrogenase quinone 1, and glutamate-cysteine ligase (GCL), as well as endothelial nitric oxide synthase (eNOS). Furthermore, CTRP9 induced activation of peroxisome proliferator-activated receptor γ co-activator 1α (PGC1-α) and phosphorylation of adenosine monophosphate-activated protein kinase (AMPK). Of interest, AMPK inhibition or PGC1-α silencing abolished CTRP9-mediated antioxidant enzymes levels, eNOS expressions, and endothelial protective effects. Collectively, we provided the first evidence that CTRP9 attenuated ox-LDL-induced endothelial injury by antioxidant enzyme inductions dependent on PGC-1α/AMPK activation.

Salusin-β mediates high glucose-induced endothelial injury via disruption of AMPK signaling pathway

The dysregulated proliferation, migration, apoptosis and angiogenesis of endothelial cells are involved in diabetic endothelial dysfunction. The circulating salusin-β levels were increased in diabetic patients, and salusin-β contributes to diabetic cardiomyopathy in rats. However, the roles of salusin-β in diabetes mellitus-induced endothelial dysfunction are not fully understood. Herein, we demonstrated the increased expressions of salusin-β in human umbilical vein endothelial cells (HUVECs) cultured in HG medium. Exposure of HUVECs to HG inhibited the proliferation, migration, and angiogenesis, retarded cell cycle progression of endothelial cells, which were rescued by knockdown of salusin-β. We also established that silencing of salusin-β with adenoviruse-mediated shRNA reduced high glucose-induced apoptosis by up-regulating Bcl-2 expression and down-regulating Bax and caspase-3 expressions. Blockade of salusin-β ameliorated HG-induced suppression of adenosine monophosphate-activated protein kinase (AMPK) signaling pathway. Of note, pretreatment with AMPK inhibitor Compound C abolished salusin-β silencing-mediated endothelial protective effects. In summary, our results highlighted the involvement of salusin-β in HG-related endothelial dysfunction, and salusin-β contributed high glucose-induced endothelial injury via inactivation of AMPK signaling pathway.

Interactions of TLR4 and PPARγ, Dependent on AMPK Signalling Pathway Contribute to Anti-Inflammatory Effects of Vaccariae Hypaphorine in Endothelial Cells

Background /Aims: Accumulating evidence indicates that endothelial inflammation is one of the critical determinants in pathogenesis of atherosclerotic cardiovascular disease. Our previous studies had demonstrated that Vaccariae prevented high glucose or oxidative stress-triggered endothelial dysfunction in vitro. Very little is known about the potential effects of hypaphorine from Vaccariae seed on inflammatory response in endothelial cells. Methods: In the present study, we evaluated the anti-inflammatory effects of Vaccariae hypaphorine (VH) on lipopolysaccharide (LPS)-challenged endothelial EA.hy926 cells. The inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), monocyte chemoattractant protein 1 (MCP-1) and vascular cellular adhesion molecule-1 (VCAM-1) were measured by real-time PCR (RT-PCR). The expressions of adenosine monophosphate-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC), toll-like receptor 4 (TLR4), peroxisome proliferator-activated receptor γ (PPARγ) were detected by Western blotting or immunofluorescence. Results: We showed that LPS stimulated the expressions of TNF-α, IL-1β, MCP-1, VCAM-1 and TLR4, but attenuated the phosphorylation of AMPK and ACC as well as PPARγ protein levels, which were reversed by VH pretreatment. Moreover, we observed that LPS-upregulated TLR4 protein expressions were inhibited by PPARγ agonist pioglitazone, and the downregulated PPARγ expressions in response to LPS were partially restored by knockdown of TLR4. The negative regulation loop between TLR4 and PPARγ response to LPS was modulated by AMPK agonist AICAR (5-Aminoimidazole-4-carboxamide riboside or acadesine) or A769662. Conclusions: Taken together, our results suggested that VH ameliorated LPS-induced inflammatory cytokines production in endothelial cells via inhibition of TLR4 and activation of PPARγ, dependent on AMPK signalling pathway.

FGF-2-mediated FGFR1 signaling in human microvascular endothelial cells is activated by vaccarin to promote angiogenesis

Angiogenesis is a complex physiological process involving the growth of new capillaries. The impaired angiogenesis plays important roles in chronic wounds and ischaemic heart disease. Fibroblast growth factor 2 (FGF-2) exerts pro-angiogenic actions via activation of fibroblast growth factor receptor 1 (FGFR-1). We have identified that vaccarin increased the angiogenic activity of endothelial cells. In this study, we investigated whether FGF-2-mediated FGFR1 signaling pathway participated in vaccarin-mediated neovascularization formation. Human microvascular endothelial cells (HMEC)-1 were incubated with various doses of vaccarin. Our results showed that vaccarin dose-dependently up-regulated FGF-2 levels and phosphorylation of FGFR-1. Neutralization of FGF-2 with anti-FGF-2 antibody also abolished the proliferation, migration and tube formation of HMEC-1 cells induced by vaccarin. Both FGFR-1 inhibitor SU5402 and FGFR-1 siRNA blocked vaccarin-induced cell cycle progression and angiogenesis. The mouse Matrigel model study further unveiled that vaccarin stimulated the neovascularization and microvessel density in vivo, which was prevented by FGFR-1 inhibitor SU5402. Taken together, our results demonstrated for the first time that vaccarin was a novel inducer for FGF-2 expression, followed by phosphorylation of FGFR-1 and subsequent angiogenic behaviors in endothelial cells. Vaccarin may be a promising candidate of angiogenesis activator for neurovascular repair or therapy.

Chicoric acid prevents PDGF-BB-induced VSMC dedifferentiation, proliferation and migration by suppressing ROS/NFκB/mTOR/P70S6K signaling cascade

Phenotypic switch of vascular smooth muscle cells (VSMCs) is characterized by increased expressions of VSMC synthetic markers and decreased levels of VSMC contractile markers, which is an important step for VSMC proliferation and migration during the development and progression of cardiovascular diseases including atherosclerosis. Chicoric acid (CA) is identified to exert powerful cardiovascular protective effects. However, little is known about the effects of CA on VSMC biology. Herein, in cultured VSMCs, we showed that pretreatment with CA dose-dependently suppressed platelet-derived growth factor type BB (PDGF-BB)-induced VSMC phenotypic alteration, proliferation and migration. Mechanistically, PDGF-BB-treated VSMCs exhibited higher mammalian target of rapamycin (mTOR) and P70S6K phosphorylation, which was attenuated by CA pretreatment, diphenyleneiodonium chloride (DPI), reactive oxygen species (ROS) scavenger N-acetyl-l-cysteine (NAC) and nuclear factor-κB (NFκB) inhibitor Bay117082. PDGF-BB-triggered ROS production and p65-NFκB activation were inhibited by CA. In addition, both NAC and DPI abolished PDGF-BB-evoked p65-NFκB nuclear translocation, phosphorylation and degradation of Inhibitor κBα (IκBα). Of note, blockade of ROS/NFκB/mTOR/P70S6K signaling cascade prevented PDGF-BB-evoked VSMC phenotypic transformation, proliferation and migration. CA treatment prevented intimal hyperplasia and vascular remodeling in rat models of carotid artery ligation in vivo. These results suggest that CA impedes PDGF-BB-induced VSMC phenotypic switching, proliferation, migration and neointima formation via inhibition of ROS/NFκB/mTOR/P70S6K signaling cascade.

Vaccaria hypaphorine alleviates lipopolysaccharide-induced inflammation via inactivation of NFκB and ERK pathways in Raw 264.7 cells

BackgroundActivation of macrophage is involved in many inflammation diseases. Lipopolysaccharide (LPS) is a powerful inflammatory signal contributing to monocytes/macrophages activation associated with increased proinflammatory cytokines expressions. We recently identified that vaccarin was expected to protect endothelial cells from injury. Hypaphorine was abundantly found in vaccaria semen. However, the potential roles and underlying mechanisms of vaccaria hypaphorine on macrophage inflammation have been poorly defined.MethodsThis study was designed to determine the effects of vaccaria hypaphorine on LPS-mediated inflammation in RAW 264.7 cells.ResultsIn this study, we demonstrated that vaccaria hypaphorine dramatically ameliorated LPS-induced nitric oxide (NO) release and productions of proinflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, IL-10, monocyte chemoattractant protein 1 (MCP-1) and prostaglandin E2 (PGE2) in RAW 264.7 cells. LPS-stimulated expressions of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) were down-regulated by vaccaria hypaphorine. Furthermore, vaccaria hypaphorine retarded LPS-induced phosphorylation of ERK, nuclear factor kappa beta (NFκB), NFκB inhibitor IκBα, and IKKβ. Immunofluorescence staining revealed that vaccaria hypaphorine eliminated the nuclear translocation of NFκB in LPS-treated RAW 264.7 cells.ConclusionIt was seen that vaccaria hypaphorine counteracted inflammation via inhibition of ERK or/and NFκB signaling pathways. Collectively, we concluded that vaccaria hypaphorine can be served as an anti-inflammatory candidate.

Vaccarin administration ameliorates hypertension and cardiovascular remodeling in renovascular hypertensive rats

Sympathetic overdrive, activation of renin angiotensin systems (RAS), and oxidative stress are vitally involved in the pathogenesis of hypertension and cardiovascular remodeling. We recently identified that vaccarin protected endothelial cell function from oxidative stress or high glucose. In this study, we aimed to investigate whether vaccarin attenuated hypertension and cardiovascular remodeling. Two‐kidney one‐clip (2K1C) model rats were used, and low dose of vaccarin (10 mg/kg), high dose of vaccarin (30 mg/kg), captopril (30 mg/kg) were intraperitoneally administrated. Herein, we showed that 2K1C rats exhibited higher systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP), left ventricular mass/body weight ratio, myocardial hypertrophy or fibrosis, media thickness, and media thickness to lumen diameter, which were obviously alleviated by vaccarin and captopril. In addition, both vaccarin and captopril abrogated the increased plasma renin, angiotensin II (Ang II), norepinephrine (NE), and the basal sympathetic activity. The AT1R protein expressions, NADPH oxidase subunit NOX‐2 protein levels and malondialdehyde (MDA) content were significantly increased, whereas superoxide dismutase (SOD) and catalase (CAT) activities were decreased in myocardium, aorta, and mesenteric artery of 2K1C rats, both vaccarin and captopril treatment counteracted these changes in renovascular hypertensive rats. Collectively, we concluded that vaccarin may be a novel complementary therapeutic medicine for the prevention and treatment of hypertension. The mechanisms for antihypertensive effects of vaccarin may be associated with inhibition of sympathetic activity, RAS, and oxidative stress.

Vaccarin protects human microvascular endothelial cells from apoptosis via attenuation of HDAC1 and oxidative stress

Abstract Vaccarin (VAC), an active flavonoid glycoside from vaccariae semen, exhibits extensive biological activities including vascular endothelial cell protective effects. Histone deacetylase1 (HDAC1) is an epigenetic regulator in cellular apoptosis. In this study, we evaluated the protective effects of VAC on high glucose (HG)‐induced cell apoptosis in human microvascular endothelial cells (HMEC‐1). The levels of reactive oxygen species, activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH‐Px) were measured. Expressions of HDAC1, Bax, Bcl‐2, caspase‐3 and cleaved caspase‐3 were detected with western blot. Flow cytometry was used to determine cell apoptosis and cell cycle. We found that HG treatment decreased cell vitality, upregulated HDAC1 protein level, promoted reactive oxygen species production, induced cell cycle arrest and cell apoptosis in HMEC‐1 cells, which were all rectified by VAC. Both scavenging reactive oxygen species and inhibition of HDAC1 alleviated the apoptosis of HMEC‐1 cells in response to HG. Pretreatment with VAC prevented HG‐stimulated reactive oxygen species generation and HDAC1 expression in HMEC‐1 cells. Taken together, these data suggested that VAC protected against HG‐induced endothelial cell apoptosis via inhibition of reactive oxygen species accumulation and HDAC1 expression. VAC may be a potential therapeutic agent for treatment of diabetes mellitus (DM)‐related endothelial dysfunction. Graphical abstract Schematic model delineating the pathway wherein VAC protects against HG‐induced endothelial cell apoptosis. Figure. No Caption available.