Deletion of endothelial IGFBP5 protects against ischaemic hindlimb injury by promoting angiogenesis.

BACKGROUND: Angiogenesis is critical for forming new blood vessels from antedating vascular vessels. The endothelium is essential for angiogenesis, vascular remodelling and minimisation of functional deficits following ischaemia. The insulin-like growth factor (IGF) family is crucial for angiogenesis. Insulin-like growth factor-binding protein 5 (IGFBP5), a binding protein of the IGF family, may have places in angiogenesis, but the mechanisms are not yet completely understood. We sought to probe whether IGFBP5 is involved in pathological angiogenesis and uncover the molecular mechanisms behind it. METHODS AND RESULTS: IGFBP5 expression was elevated in the vascular endothelium of gastrocnemius muscle from critical limb ischaemia patients and hindlimb ischaemic (HLI) mice and hypoxic human umbilical vein endothelial cells (HUVECs). In vivo, loss of endothelial IGFBP5 (IGFBP5EKO) facilitated the recovery of blood vessel function and limb necrosis in HLI mice. Moreover, skin damage healing and aortic ring sprouting were faster in IGFBP5EKO mice than in control mice. In vitro, the genetic inhibition of IGFBP5 in HUVECs significantly promoted tube formation, cell proliferation and migration by mediating the phosphorylation of IGF1R, Erk1/2 and Akt. Intriguingly, pharmacological treatment of HUVECs with recombinant human IGFBP5 ensued a contrasting effect on angiogenesis by inhibiting the IGF1 or IGF2 function. Genetic inhibition of IGFBP5 promoted cellular oxygen consumption and extracellular acidification rates via IGF1R-mediated glycolytic adenosine triphosphate (ATP) metabolism. Mechanistically, IGFBP5 exerted its role via E3 ubiquitin ligase Von Hippel-Lindau (VHL)-regulated HIF1α stability. Furthermore, the knockdown of the endothelial IGF1R partially abolished the reformative effect of IGFBP5EKO mice post-HLI. CONCLUSION: Our findings demonstrate that IGFBP5 ablation enhances angiogenesis by promoting ATP metabolism and stabilising HIF1α, implying IGFBP5 is a novel therapeutic target for treating abnormal angiogenesis-related conditions.

NMDARs activation regulates endothelial ferroptosis via the PP2A-AMPK-HMGB1 axis.

N-methyl-D-aspartate receptors (NMDARs) are ligand-gated, voltage-dependent channels of the ionotropic glutamate receptor family. The present study explored whether NMDAR activation induced ferroptosis in vascular endothelial cells and its complicated mechanisms in vivo and in vitro. Various detection approaches were used to determine the ferroptosis-related cellular iron content, lipid reactive oxygen species (LOS), siRNA molecules, RNA-sequence, MDA, GSH, and western blotting. The AMPK activator Acadesine (AICAR), HMGB1 inhibitor glycyrrhizin (GLY), PP2A inhibitor LB-100, and NMDAR inhibitor MK801 were used to investigate the involved in vivo and in vitro pathways. The activation of NMDAR with L-glutamic acid (GLU) or NMDA significantly promoted cellular ferroptosis, iron content, MDA, and the PTGS2 expression, while decreasing GPX4 expression and GSH concentration in human umbilical vein endothelial cells (HUVECs), which was reversed by ferroptosis inhibitors Ferrostatin-1(Fer-1), Liproxstatin-1 (Lip-1), or Deferoxamine (DFO). RNA-seq revealed that ferroptosis and SLC7A11 participate in NMDA or GLU-mediated NMDAR activation. The PP2A-AMPK-HMGB1 pathway was majorly associated with NMDAR activation-induced ferroptosis, validated using the PP2A inhibitor LB-100, AMPK activator AICAR, or HMGB1 siRNA. The role of NMDAR in ferroptosis was validated in HUVECs induced with the ferroptosis activator errasin or RSL3 and counteracted by the NMDAR inhibitor MK-801. The in vivo results showed that NMDA- or GLU-induced ferroptosis and LOS production was reversed by MK-801, LB-100, AICAR, MK-801, and GLY, confirming that the PP2A-AMPK-HMGB1 pathway is involved in NMDAR activation-induced vascular endothelium ferroptosis. In conclusion, the present study demonstrated a novel role of NMDAR in endothelial cell injury by regulating ferroptosis via the PP2A-AMPK-HMGB1 pathway.

NMDARs antagonist MK801 suppresses LPS-induced apoptosis and mitochondrial dysfunction by regulating subunits of NMDARs via the CaM/CaMKII/ERK pathway.

Lipopolysaccharide (LPS) displays a robust immunostimulatory ability upon Toll-like receptor 4 (TLR4) recognition. N-methyl-D-aspartate receptors (NMDARs) are highly compartmentalized in most cells and implicated in various inflammatory disorders. However, the relationship between TLR4 and NMDARs has not been explored deeply. This study aimed to examine the role of NMDARs and its specific inhibitor MK801 in LPS-treated endothelial cell dysfunction and the related mechanism in vivo and in vitro. The results showed that pre-treatment with MK801 significantly decreased LPS-induced cell death, cellular Ca2+, cellular reactive oxygen species, and glutamate efflux. Moreover, MK801 restrained LPS-induced mitochondrial dysfunction by regulating mitochondrial membrane potential and mitochondrial Ca2+ uptake. The oxygen consumption, basal and maximal respiration rate, and ATP production in LPS-treated HUVECs were reversed by MK801 via regulating ATP synthesis-related protein SDHB2, MTCO1, and ATP5A. The molecular pathway involved in MK801-regulated LPS injury was mediated by phosphorylation of CaMKII and ERK and the expression of MCU, MCUR1, and TLR4. LPS-decreased permeability in HUVECs was improved by MK801 via the Erk/ZO-1/occluding/Cx43 axis. Co-immunoprecipitation assay and western blotting showed three subtypes of NMDARs, NMDAζ1, NMDAε2, and NMDAε4 were bound explicitly to TLR4, suppressed by LPS, and promoted by MK801. Deficiency of NMDAζ1, NMDAε2, or NMDAε4 induced cell apoptosis, Ca2+ uptake, ROS production, and decreased basal and maximal respiration rate, and ATP production, suggesting that NMDARs integrity is vital for cell and mitochondrial function. In vivo investigation showed MK801 improved impairment of vascular permeability, especially in the lung and mesentery in LPS-injured mice. Our study displayed a novel mechanism and utilization of MK801 in LPS-induced ECs injury and permeability.

Heart-targeting exosomes from human cardiosphere-derived cells improve the therapeutic effect on cardiac hypertrophy.

Exosomes of human cardiosphere-derived cells (CDCs) are very promising for treating cardiovascular disorders. However, the current challenge is inconvenient delivery methods of exosomes for clinical application. The present study aims to explore the potential to enhance the therapeutic effect of exosome (EXO) from human CDCs to myocardial hypertrophy. A heart homing peptide (HHP) was displayed on the surface of exosomes derived from CDCs that were forced to express the HHP fused on the N-terminus of the lysosomal-associated membrane protein 2b (LAMP2b). The cardiomyocyte-targeting capability of exosomes were analyzed and their therapeutic effects were evaluated in a mouse model of myocardial hypertrophy induced by transverse aorta constriction (TAC). The molecular mechanisms of the therapeutic effects were dissected in angiotensin II-induced neonatal rat cardiomyocyte (NRCMs) hypertrophy model using a combination of biochemistry, immunohistochemistry and molecular biology techniques. We found that HHP-exosomes (HHP-EXO) accumulated more in mouse hearts after intravenous delivery and in cultured NRCMs than control exosomes (CON-EXO). Cardiac function of TAC mice was significantly improved with intravenous HHP-EXO administration. Left ventricular hypertrophy was reduced more by HHP-EXO than CON-EXO via inhibition of ß-MHC, BNP, GP130, p-STAT3, p-ERK1/2, and p-AKT. Similar results were obtained in angiotensin II-induced hypertrophy of NRCMs, in which the beneficial effects of HHP-EXO were abolished by miRNA-148a inhibition. Our results indicate that HHP-EXO preferentially target the heart and improve the therapeutic effect of CDCs-exosomes on cardiac hypertrophy. The beneficial therapeutic effect is most likely attributed to miRNA-148a-mediated suppression of GP130, which in turn inhibits STAT3/ERK1/2/AKT signaling pathway, leading to improved cardiac function and remodeling.

Acacetin ameliorates cardiac hypertrophy by activating Sirt1/AMPK/PGC-1α pathway.

Cardiac hypertrophy is a major risk factor for developing heart failure. This study investigates the effects of the natural flavone acacetin on myocardial hypertrophy in cellular level and whole animals. In cardiomyocytes from neonatal rat with hypertrophy induced by angiotensin II (Ang II), acacetin at 0.3, 1, and 3 µM reduced the increased myocyte surface area, brain natriuretic peptide (BNP), and ROS production by upregulating anti-oxidative molecules (i.e. Nrf2, SOD1, SOD2, HO-1), anti-apoptotic protein Bcl-2, and downregulating the pro-apoptotic protein Bax and the inflammatory cytokine IL-6 in a concentration-dependent manner. In addition, acacetin rescued Ang II-induced impairment of PGC-1α, PPARα and pAMPK. These beneficial effects of acacetin were mediated by activation of Sirt1, which was confirmed in cardiac hypertrophy induced by abdominal aorta constriction (AAC) in SD rats. Acacetin prodrug (10 mg/kg, s.c., b.i.d.) treatment reduced the elevated artery blood pressure, improved the increased heart size and thickness of left ventricular wall and the ventricular fibrosis associated with inhibiting myocardial fibrosis and BNP, and reversed the impaired protective signal molecules including PGC-1α, Nrf2, PPARα, pAMPK and Sirt1 of left ventricular tissue. Our results demonstrate the novel pharmacological effect that acacetin ameliorates cardiac hypertrophy via Sirt1-mediated activation of AMPK/PGC-1α signal molecules followed by reducing oxidation, inflammation and apoptosis.

The roles of epicardial adipose tissue in heart failure.

Heart failure is a growing health problem resulting in the decreased life expectancy of patients and severely increased the healthcare burden. Penetrating research on the pathogenesis and regulation mechanism of heart failure is important for treatment of heart failure. Epicardial adipose tissue (EAT) has been demonstrated as not only a dynamic organ with biological functions but also an inert lipid store with regulating systemic metabolism. EAT mediates physiological and pathophysiological processes of heart failure by regulating adipogenesis, cardiac remodeling, insulin resistance, cardiac output, and renin angiotensin aldosterone system (RAAS). Moreover, EAT secretes a wide range of adipokines, adrenomedullin, adiponectin, and miRNAs through paracrine, endocrine, and vasocrine pathways, which involve in various extracellular and intracellular mechanism of cardiac-related cells in the progress of cardiovascular disease especially in heart failure. Nevertheless, mechanisms and roles of EAT on heart failure are barely summarized. Understanding the regulating mechanisms of EAT on heart failure may give rise to novel therapeutic targets and will open up innovative strategies to myocardial injury as well as in heart failure.

Cardiac senescence is alleviated by the natural flavone acacetin via enhancing mitophagy.

Cardiac senescence is associated with cardiomyopathy which is a degenerative disease in the aging process of the elderly. The present study investigates using multiple experimental approaches whether the natural flavone acacetin could attenuate myocardial senescence in C57/BL6 mice and H9C2 rat cardiac cells induced by D-galactose. We found that the impaired heart function in D-galactose-induced accelerated aging mice was improved by oral acacetin treatment in a dose-dependent manner. Acacetin significantly countered the increased serum advanced glycation end products, the myocardial telomere length shortening, the increased cellular senescence marker proteins p21 and p53, and the reduced mitophagy signaling proteins PINK1/Parkin and Sirt6 expression in aging mice. In H9C2 rat cardiac cells, acacetin alleviated cell senescence induced by D-galactose in a concentration-dependent manner. Acacetin decreased p21 and p53 expression, up-regulated PINK1/Parkin, LC3II/LC3I ratio, pLKB1, pAMPK and Sirt6, and reversed the depolarized mitochondrial membrane potential in aging cardiac cells. Mitophagy inhibition with 3-methyladenine or silencing Sirt6 abolished the protective effects of acacetin against cardiac senescence. Further analysis revealed that acacetin effect on Sirt6 was mediated by Sirt1 activation and increase of NAD+/NADH ratio. These results demonstrate that acacetin significantly inhibits in vivo and in vitro cardiac senescence induced by D-galactose via Sirt1-mediated activation of Sirt6/AMPK signaling pathway, thereby enhancing mitophagy and preserving mitochondrial function, which suggests that acacetin may be a drug candidate for treating cardiovascular disorders related to aging.

Doxorubicin cardiomyopathy is ameliorated by acacetin via Sirt1-mediated activation of AMPK/Nrf2 signal molecules.

Doxorubicin cardiotoxicity is frequently reported in patients undergoing chemotherapy. The present study investigates whether cardiomyopathy induced by doxorubicin can be improved by the natural flavone acacetin in a mouse model and uncovers the potential molecular mechanism using cultured rat cardiomyoblasts. It was found that the cardiac dysfunction and myocardial fibrosis induced by doxorubicin were significantly improved by acacetin in mice with impaired Nrf2/HO-1 and Sirt1/pAMPK molecules, which is reversed by acacetin treatment. Doxorubicin decreased cell viability and increased ROS production in rat cardiomyoblasts; these effects are significantly countered by acacetin (0.3-3 µM) in a concentration-dependent manner via activating Sirt1/pAMPK signals and enhancing antioxidation (Nrf2/HO-1 and SOD1/SOD2) and anti-apoptosis. These protective effects were abolished in cells with silencing Sirt1. The results demonstrate for the first time that doxorubicin cardiotoxicity is antagonized by acacetin via Sirt1-mediated activation of AMPK/Nrf2 signal molecules, indicating that acacetin may be a drug candidate used clinically for protecting against doxorubicin cardiomyopathy.

The Natural Flavone Acacetin Confers Cardiomyocyte Protection Against Hypoxia/Reoxygenation Injury via AMPK-Mediated Activation of Nrf2 Signaling Pathway.

The present study investigates the potential signal pathway of acacetin in cardioprotection against ischemia/reperfusion injury using an in vitro hypoxia/reoxygenation model in primary cultured neonatal rat cardiomyocytes and H9C2 cardiomyoblasts. It was found that acacetin (0.3-3 µM) significantly decreased the apoptosis and reactive oxygen species production induced by hypoxia/reoxygenation injury in cardiomyocytes and H9C2 cardiomyoblasts via reducing the pro-apoptotic proteins Bax and cleaved-caspase-3 and increasing the anti-apoptotic protein Bcl-2. In addition, acacetin not only suppressed the release of pro-inflammatory cytokines TLR-4 and IL-6 induced by hypoxia/reoxygenation injury, but also increased the secretion of anti-inflammatory cytokine IL-10. Moreover, acacetin increased Nrf2 and HO-1 in a concentration-dependent manner, and rescued SOD1 and SOD2 reduction induced by hypoxia/reoxygenation insult. These beneficial effects of acacetin disappeared in cells with silenced Nrf2, suggesting that Nrf2 activation participates in the cardioprotective effect of acacetin against hypoxia/reoxygenation insult. However, acacetin-induced Nrf2 activation was not observed in cells with silenced AMPK and in ventricular tissues of rat hearts treated with the AMPK inhibitor Compound C and subjected to ischemia/reperfusion injury. Our results demonstrate for the first time that AMPK-mediated Nrf2 activation is involved in the cardiomyocytes protection of acacetin against hypoxia/reoxygenation injury by activating a series of intracellular signals involved in anti-oxidation, anti-inflammation, and anti-apoptosis.