Arterial relaxation is coupled to inhibition of mitochondrial fission in arterial smooth muscle cells:comparison of vasorelaxant effects of verapamil and phentolamine

Mitochondria are morphologically dynamic organelles which undergo fission and fusion processes. Our previous study found that arterial constriction was always accompanied by increased mitochondrial fission in smooth muscle cells, whereas inhibition of mitochondrial fission in smooth muscle cells was associated with arterial relaxation. Here, we used the typical vasorelaxants, verapamil and phentolamine, to further confirm the coupling between arterial constriction and mitochondrial fission in rat aorta. Results showed that phentolamine but not verapamil induced vasorelaxation in phenylephrine(PE)-induced rat thoracic aorta constriction. Verapamil, but not phentolamine, induced vasorelaxation in high K^+(KPSS)-induced rat thoracic aorta constriction. Pre-treatment with phentolamine prevented PEbut not KPSS-induced aorta constriction and pre-treatment with verapamil prevented both PE-and KPSSinduced aorta constriction. Transmission electron microscopy(TEM) results showed that verapamil but not phentolamine inhibited KPSS-induced excessive mitochondrial fission in aortic smooth muscle cells,and verapamil prevented both PE-and KPSS-induced excessive mitochondrial fission in aortic smoothmuscle cells. Verapamil inhibited KPSS-induced excessive mitochondrial fission in cultured vascular smooth muscle cells(A10). These results further demonstrate that arterial relaxation is coupled to inhibition of mitochondrial fission in arterial smooth muscle cells.

LncDACH1 promotes mitochondrial oxidative stress of cardiomyocytes by interacting with sirtuin3 and aggravates diabetic cardiomyopathy

Diabetic cardiomyopathy(DCM)is a common complication in diabetic patients.The molecular mechanisms of DCM remain to be fully elucidated.The intronic long noncoding RNA of DACH1(lnc DACH1)has been demonstrated to be closely associated with heart failure and cardiac regeneration.In this study,we investigated the role of lnc DACH1 in DCM and the underlying molecular mechanisms.The expression of lnc DACH1 was increased in DCM hearts and in high glucose-treated cardiomyocytes.Knockout of lnc DACH1 reduced mitochondrial oxidative stress,cell apoptosis,cardiac fibrosis and hypertrophy,and improved cardiac function in DCM mice.Overexpression of lnc DACH1 exacerbated mitochondria-derived reactive oxygen species(ROS)level and apoptosis,decreased activity of manganese superoxide dismutase(Mn-SOD);while silencing of lnc DACH1 attenuated ROS production,mitochondrial dysfunction,cell apoptosis,and increased the activity of Mn-SOD in cardiomyocytes treated with high glucose.Lnc DACH1 directly bound to sirtuin3(SIRT3)and facilitated its degradation by ubiquitination,therefore promoting mitochondrial oxidative injury and cell apoptosis in mouse hearts.In addition,SIRT3 silencing abrogated the protective effects of lnc DACH1 deficiency in cardiomyocytes.In summary,lnc DACH1 aggravates DCM by promoting mitochondrial oxidative stress and cell apoptosis via increasing ubiquitination-mediated SIRT3 degradation in mouse hearts.Inhibition of lnc DACH1 represents a novel therapeutic strategy for the intervention of diabetic cardiomyopathy.

Exosomes secreted from cardiomyocytes suppress the sensitivity of tumor ferroptosis in ischemic heart failure

Heart failure(HF)patients in general have a higher risk of developing cancer.Several animal studies have indicated that cardiac remodeling and HF remarkably accelerate tumor progression,highlighting a cause-and-effect relationship between these two disease entities.Targeting ferroptosis,a prevailing form of non-apoptotic cell death,has been considered a promising therapeutic strategy for human cancers.Exosomes critically contribute to proximal and distant organ-organ communications and play crucial roles in regulating diseases in a paracrine manner.However,whether exosomes control the sensitivity of cancer to ferroptosis via regulating the cardiomyocyte-tumor cell crosstalk in ischemic HF has not yet been explored.Here,we demonstrate that myocardial infarction(MI)decreased the sensitivity of cancer cells to the canonical ferroptosis activator erastin or imidazole ketone erastin in a mouse model of xenograft tumor.Post-MI plasma exosomes potently blunted the sensitivity of tumor cells to ferroptosis inducers both in vitro in mouse Lewis lung carcinoma cell line LLC and osteosarcoma cell line K7M2 and in vivo with xenograft tumorigenesis model.The expression of miR-22-3p in cardiomyocytes and plasma-exosomes was significantly upregulated in the failing hearts of mice with chronic MI and of HF patients as well.Incubation of tumor cells with the exosomes isolated from post-MI mouse plasma or overexpression of miR-22-3p alone abrogated erastin-induced ferroptotic cell death in vitro.Cardiomyocyte-enriched miR-22-3p was packaged in exosomes and transferred into tumor cells.Inhibition of cardiomyocyte-specific miR-22-3p by AAV9 sponge increased the sensitivity of cancer cells to ferroptosis.ACSL4,a pro-ferroptotic gene,was experimentally established as a target of miR-22-3p in tumor cells.Taken together,our findings uncovered for the first time that MI suppresses erastin-induced ferroptosis through releasing miR-22-3p-enriched exosomes derived from cardiomyocytes.Therefore,targeting exosome-mediated cardiomyocyte/tumor pathological communication may offer a novel approach for the ferroptosis-based antitumor therapy.

Mitochondrial uncoupler BAM15 inhibits artery constriction and potently activates AMPK in vascular smooth muscle cells

Our previous studies found that mitochondrial uncouplers CCCP and niclosamide inhibited artery constriction and the mechanism involved AMPK activation in vascular smooth muscle cells. BAM15 is a novel type of mitochondrial uncoupler. The aim of the present study is to identify the vasoactivity of BAM15 and characterize the BAM15-induced AMPK activation in vascular smooth muscle cells(A10 cells). BAM15 relaxed phenylephrine(PE)-induced constricted rat mesenteric arteries with intact and denuded endothelium. Pretreatment with BAM15 inhibited PEinduced constriction of rat mesenteric arteries with intact and denuded endothelium. BAM15, CCCP,and niclosamide had the comparable IC50 value of vasorelaxation in PE-induced constriction of rat mesenteric arteries. BAM15 was less cytotoxic in A10 cells compared with CCCP and niclosamide.BAM15 depolarized mitochondrial membrane potential, induced mitochondrial fission, increased mitochondrial ROS production, and increased mitochondrial oxygen consumption rate in A10 cells.BAM15 potently activated AMPK in A10 cells and the efficacy of BAM15 was stronger than that of CCCP, niclosamide, and AMPK positive activators metformin and AICAR. In conclusion, BAM15 activates AMPK in vascular smooth muscle cells with higher potency than that of CCCP, niclosamide and the known AMPK activators metformin and AICAR. The present work indicates that BAM15 is a potent AMPK activator.

HYD-PEP06 suppresses hepatocellular carcinoma metastasis,epithelial-mesenchymal transition and cancer stem cell-like properties by inhibiting PI3K/AKT and WNT/β-catenin signaling activation

HYD-PEP06,an endostatin-modified polypeptide,has been shown to produce effective anticolorectal carcinoma effects through inhibiting epithelial-mesenchymal transition(EMT).However,whether HYD-PEP06 has similar suppressive effect on hepatocellular carcinoma(HCC) remained unknown.In this study,HYD-PEP06 inhibited metastasis and EMT but not proliferation in vitro.Cignal finder pathway reporter array and Western blot analysis revealed that HYD-PEP06 suppressed HCCLM3 cell metastasis and EMT by inhibiting the PI3 K/AKT pathway.Moreover,HYD-PEP06 exerted antimetastasis effects in HepG2 cancer stem-like cells(CSCs) via suppressing the WNT/β-catenin signaling pathway.Finally,in HCCLM3 tumor-bearing BALB/c nu/nu nude mice,HYD-PEP06 substantially suppressed tumor growth,lung metastasis and HCC progress.Our results suggest that HYD-PEP06 inhibits the metastasis and EMT of HCC and CSCs as well,and thus has the potential as an agent for HCC treatment.

Mitochondrial uncoupler triclosan induces vasorelaxation of rat arteries

Our previous studies found that mitochondrial uncouplers induced vasodilation. Triclosan, the broad spectrum antibacterial agent, is the active ingredient in soaps and toothpastes. It was reported that triclosan induced mitochondrial uncoupling, so we aim to investigate the effects of triclosan on vascular function of rat mesenteric arteries and aorta. The isometric tension of rat mesenteric artery and thoracic aorta was recorded by multi-wire myograph system. The cytosolic [Ca^(2+)]_i, mitochondrial reactive oxygen species(mitoROS), and mitochondrial membrane potential of smooth muscle cells(A10 cells) were measured using laser scanning confocal microscopy. Triclosan treatment relaxed phenylephrine(PE)-and high K^(+)(KPSS)-induced constriction, and pre-treatment with triclosan inhibited PE-and KPSS-induced constriction of rat mesenteric arteries. In rat thoracic aorta, triclosan also relaxed PE-and KPSS-induced constriction. Triclosan induces vasorelaxation without involving KATPchannel activation in smooth muscle cells of arteries.Triclosan treatment increased cytosolic [Ca^(2+)]_i, mitochondrial ROS production and depolarized mitochondrial membrane potential in A10 cells. In conclusion, triclosan induces mitochondrial uncoupling in vascular smooth muscle cells and relaxes the constricted rat mesenteric arteries and aorta of rats. The present results suggest that triclosan would indicate vasodilation effect if absorbed excessively in vivo.

Anthelmintics nitazoxanide protects against experimental hyperlipidemia and hepatic steatosis in hamsters and mice

Lipid metabolism disorders contribute to hyperlipidemia and hepatic steatosis.It is ideal to develop drugs simultaneous improving both hyperlipidemia and hepatic steatosis.Nitazoxanide is an FDA-approved oral antiprotozoal drug with excellent pharmacokinetic and safety profile.We found that nitazoxanide and its metabolite tizoxanide induced mild mitochondrial uncoupling and subsequently activated AMPK in Hep G2 cells.Gavage administration of nitazoxanide inhibited high-fat diet(HFD)-induced increases of liver weight,blood and liver lipids,and ameliorated HFD-induced renal lipid accumulation in hamsters.Nitazoxanide significantly improved HFD-induced histopathologic changes of hamster livers.In the hamsters with pre-existing hyperlipidemia and hepatic steatosis,nitazoxanide also showed therapeutic effect.Gavage administration of nitazoxanide improved HFD-induced hepatic steatosis in C57BL/6J mice and western diet(WD)-induced hepatic steatosis in Apoe-/-mice.The present study suggests that repurposing nitazoxanide as a drug for hyperlipidemia and hepatic steatosis treatment is promising.

PEP06 polypeptide 30 is a novel clusterdissociating agent inhibiting αv integrin/FAK/Src signaling in oral squamous cell carcinoma cells

Collectively migrating tumor cells have been recently implicated in enhanced metastasis of epithelial malignancies.In oral squamous cell carcinoma(OSCC),av integrin is a crucial mediator of multicellular clustering and collective movement in vitro;however,its contribution to metastatic spread remains to be addressed.According to the emerging therapeutic concept,dissociation of tumor clusters into single cells could significantly suppress metastasis-seeding ability of carcinomas.This study aimed to investigate the anti-OSCC potential of novel endostatin-derived polypeptide PEP06 as a clusterdissociating therapeutic agent in vitro.Firstly,we found marked enrichment ofαv integrin in collectivelyinvading multicellular clusters in human OSCCs.Our study revealed that metastatic progression of OSCC was associated with augmented immunostaining of av integrin in cancerous lesions.Following PEP06treatment,cell clustering on fibronectin,migration,multicellular aggregation,anchorage-independent survival and colony formation of OSCC were significantly inhibited.Moreover,PEP06 suppressed av integrin/FAK/Sre signaling in OSCC cells.PEP06-induced loss of active Src and E-cadherin from cell-cell contacts contributed to diminished collective migration of OSCC in vitro.Overall,these results suggest that PEP06 polypeptide 30 inhibiting av integrin/FAK/Src signaling and disrupting E-cadherin-based intercellular junctions possesses anti-metastatic potential in OSCC by acting as a cluster-dissociating therapeutic agent.