Cytosolic DNA initiates a vicious circle of aging-related endothelial inflammation and mitochondrial dysfunction via STING: the inhibitory effect of Cilostazol.

Endothelial senescence, aging-related inflammation, and mitochondrial dysfunction are prominent features of vascular aging and contribute to the development of aging-associated vascular disease. Accumulating evidence indicates that DNA damage occurs in aging vascular cells, especially in endothelial cells (ECs). However, the mechanism of EC senescence has not been completely elucidated, and so far, there is no specific drug in the clinic to treat EC senescence and vascular aging. Here we show that various aging stimuli induce nuclear DNA and mitochondrial damage in ECs, thus facilitating the release of cytoplasmic free DNA (cfDNA), which activates the DNA-sensing adapter protein STING. STING activation led to a senescence-associated secretory phenotype (SASP), thereby releasing pro-aging cytokines and cfDNA to further exacerbate mitochondrial damage and EC senescence, thus forming a vicious circle, all of which can be suppressed by STING knockdown or inhibition. Using next-generation RNA sequencing, we demonstrate that STING activation stimulates, whereas STING inhibition disrupts pathways associated with cell senescence and SASP. In vivo studies unravel that endothelial-specific Sting deficiency alleviates aging-related endothelial inflammation and mitochondrial dysfunction and prevents the development of atherosclerosis in mice. By screening FDA-approved vasoprotective drugs, we identified Cilostazol as a new STING inhibitor that attenuates aging-related endothelial inflammation both in vitro and in vivo. We demonstrated that Cilostazol significantly inhibited STING translocation from the ER to the Golgi apparatus during STING activation by targeting S162 and S243 residues of STING. These results disclose the deleterious effects of a cfDNA-STING-SASP-cfDNA vicious circle on EC senescence and atherogenesis and suggest that the STING pathway is a promising therapeutic target for vascular aging-related diseases. A proposed model illustrates the central role of STING in mediating a vicious circle of cfDNA-STING-SASP-cfDNA to aggravate age-related endothelial inflammation and mitochondrial damage.

Inhibition of miR-214-3p attenuates ferroptosis in myocardial infarction via regulating ME2.

Myocardial infarction (MI) contributes to an increased risk of incident heart failure and sudden death, but there is still a lack of effective treatment in clinic. Recently, growing evidence has indicated that abnormal expression of microRNAs (miRNAs) plays a crucial role in cardiovascular diseases. In this research, the involvement of miRNA-214-3p in MI was explored. A mouse model of MI was established by ligation of the left anterior descending coronary artery, and primary cultures of neonatal rat cardiomyocytes (NRCMs) were submitted to hypoxic treatment to stimulate cellular injury in vitro. Our results showed that miR-214-3p level was significantly upregulated in the infarcted region of mouse hearts and in NRCMs exposed to hypoxia, accompanying with an obvious elevation of ferroptosis. Inhibition of miR-214-3p by antagomir injection improved cardiac function, decreased infarct size, and attenuated iron accumulation and oxidant stress in myocardial tissues. MiR-214-3p could also promote ferroptosis and cellular impairments in NRCMs, while miR-214-3p inhibitor effectively protected cells from hypoxia. Furthermore, dual luciferase reporter gene assay revealed that malic enzyme 2 (ME2) is a direct target of miR-214-3p. In cardiomyocytes, overexpression of ME2 ameliorated the detrimental effects and excessive ferroptosis induced by miR-214-3p mimic, whereas ME2 depletion compromised the protective role of miR-214-3p inhibitor against hypoxic injury and ferroptosis. These findings suggest that miR-214-3p contributes to enhanced ferroptosis during MI at least partially via suppressing ME2. Inhibition of miR-214-3p may be a new approach for tackling MI.

Regulation of toll-like receptor (TLR) signaling pathways in atherosclerosis: from mechanisms to targeted therapeutics.

Atherosclerosis, one of the life-threatening cardiovascular diseases (CVDs), has been demonstrated to be a chronic inflammatory disease, and inflammatory and immune processes are involved in the origin and development of the disease. Toll-like receptors (TLRs), a class of pattern recognition receptors that trigger innate immune responses by identifying pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs), regulate numerous acute and chronic inflammatory diseases. Recent studies reveal that TLRs have a vital role in the occurrence and development of atherosclerosis, including the initiation of endothelial dysfunction, interaction of various immune cells, and activation of a number of other inflammatory pathways. We herein summarize some other inflammatory signaling pathways, protein molecules, and cellular responses associated with TLRs, such as NLRP3, Nrf2, PCSK9, autophagy, pyroptosis and necroptosis, which are also involved in the development of AS. Targeting TLRs and their regulated inflammatory events could be a promising new strategy for the treatment of atherosclerotic CVDs. Novel drugs that exert therapeutic effects on AS through TLRs and their related pathways are increasingly being developed. In this article, we comprehensively review the current knowledge of TLR signaling pathways in atherosclerosis and actively seek potential therapeutic strategies using TLRs as a breakthrough point in the prevention and therapy of atherosclerosis.

SZC-6, a small-molecule activator of SIRT3, attenuates cardiac hypertrophy in mice.

Sirtuin3 (SIRT3), a class III histone deacetylase, is implicated in various cardiovascular diseases as a novel therapeutic target. SIRT3 has been proven to be cardioprotective in a model of Ang II-induced cardiac hypertrophy. However, a few small-molecule compounds targeting deacetylases could activate SIRT3. In this study, we generated a novel SIRT3 activator, 3-(2-bromo-4-hydroxyphenyl)-7-hydroxy-2H-chromen-2-one (SZC-6), through structural optimization of the first SIRT3 agonist C12. We demonstrated that SZC-6 directly bound to SIRT3 with Kd value of 15 µM, and increased SIRT3 deacetylation activity with EC50 value of 23.2 ± 3.3 µM. In neonatal rat cardiomyocytes (NRCMs), pretreatment with SZC-6 (10, 20, 40 µM) dose-dependently attenuated isoproterenol (ISO)-induced hypertrophic responses. Administration of SZC-6 (20, 40 and 60 mg·kg-1·d-1, s.c.) for 2 weeks starting from one week prior ISO treatment dose-dependently reversed ISO-induced impairment of diastolic and systolic cardiac function in wild-type mice, but not in SIRT3 knockdown mice. We showed that SZC-6 (10, 20, 40 µM) dose-dependently inhibited cardiac fibroblast proliferation and differentiation into myofibroblasts, which was abolished in SIRT3-knockdown mice. We further revealed that activation of SIRT3 by SZC-6 increased ATP production and rate of mitochondrial oxygen consumption, and reduced ROS, improving mitochondrial function in ISO-treated NRCMs. We also found that SZC-6 dose-dependently enhanced LKB1 phosphorylation, thereby promoting AMPK activation to inhibit Drp1-dependent mitochondrial fragmentation. Taken together, these results demonstrate that SZC-6 is a novel SIRT3 agonist with potential value in the treatment of cardiac hypertrophy partly through activation of the LKB1-AMPK pathway.

JMJD6 protects against isoproterenol-induced cardiac hypertrophy via inhibition of NF-κB activation by demethylating R149 of the p65 subunit.

Histone modification plays an important role in pathological cardiac hypertrophy and heart failure. In this study we investigated the role of a histone arginine demethylase, Jumonji C domain-containing protein 6 (JMJD6) in pathological cardiac hypertrophy. Cardiac hypertrophy was induced in rats by subcutaneous injection of isoproterenol (ISO, 1.2 mg·kg-1·d-1) for a week. At the end of the experiment, the rats underwent echocardiography, followed by euthanasia and heart collection. We found that JMJD6 levels were compensatorily increased in ISO-induced hypertrophic cardiac tissues, but reduced in patients with heart failure with reduced ejection fraction (HFrEF). Furthermore, we demonstrated that JMJD6 overexpression significantly attenuated ISO-induced hypertrophy in neonatal rat cardiomyocytes (NRCMs) evidenced by the decreased cardiomyocyte surface area and hypertrophic genes expression. Cardiac-specific JMJD6 overexpression in rats protected the hearts against ISO-induced cardiac hypertrophy and fibrosis, and rescued cardiac function. Conversely, depletion of JMJD6 by single-guide RNA (sgRNA) exacerbated ISO-induced hypertrophic responses in NRCMs. We revealed that JMJD6 interacted with NF-κB p65 in cytoplasm and reduced nuclear levels of p65 under hypertrophic stimulation in vivo and in vitro. Mechanistically, JMJD6 bound to p65 and demethylated p65 at the R149 residue to inhibit the nuclear translocation of p65, thus inactivating NF-κB signaling and protecting against pathological cardiac hypertrophy. In addition, we found that JMJD6 demethylated histone H3R8, which might be a new histone substrate of JMJD6. These results suggest that JMJD6 may be a potential target for therapeutic interventions in cardiac hypertrophy and heart failure.

The m6A demethylase FTO promotes the osteogenesis of mesenchymal stem cells by downregulating PPARG.

N6-methyladenosine (m6A) is the most abundant posttranscriptional methylation modification that occurs in mRNA and modulates the fine-tuning of various biological processes in mammalian development and human diseases. In this study we investigated the role of m6A modification in the osteogenesis of mesenchymal stem cells (MSCs), and the possible mechanisms by which m6A modification regulated the processes of osteoporosis and bone necrosis. We performed systematic analysis of the differential gene signatures in patients with osteoporosis and bone necrosis and conducted m6A-RNA immunoprecipitation (m6A-RIP) sequencing to identify the potential regulatory genes involved in osteogenesis. We showed that fat mass and obesity (FTO), a primary m6A demethylase, was significantly downregulated in patients with osteoporosis and osteonecrosis. During the differentiation of human MSCs into osteoblasts, FTO was markedly upregulated. Both depletion of FTO and application of the FTO inhibitor FB23 or FB23-2 impaired osteogenic differentiation of human MSCs. Knockout of FTO in mice resulted in decreased bone mineral density and impaired bone formation. PPARG, a biomarker for osteoporosis, was identified as a critical downstream target of FTO. We further revealed that FTO mediated m6A demethylation in the 3'UTR of PPARG mRNA, and reduced PPARG mRNA stability in an YTHDF1-dependent manner. Overexpression of PPARG alleviated FTO-mediated osteogenic differentiation of MSCs, whereas knockdown of PPARG promoted FTO-induced expression of the osteoblast biomarkers ALPL and OPN during osteogenic differentiation. Taken together, this study demonstrates the functional significance of the FTO-PPARG axis in promoting the osteogenesis of human MSCs and sheds light on the role of m6A modification in mediating osteoporosis and osteonecrosis.

The novel STAT3 inhibitor WZ-2-033 causes regression of human triple-negative breast cancer and gastric cancer xenografts.

Hyperactive signal transducer and activator of transcription 3 (STAT3) signaling is frequently detected in human triple-negative breast cancer (TNBC) and gastric cancer, leading to uncontrolled tumor growth, resistance to chemotherapy, and poor prognosis. Thus, inhibition of STAT3 signaling is a promising therapeutic approach for both TNBC and gastric cancer, which have high incidences and mortality and limited effective therapeutic approaches. Here, we report a small molecule, WZ-2-033, capable of inhibiting STAT3 activation and dimerization and STAT3-related malignant transformation. We present in vitro evidence from surface plasmon resonance analysis that WZ-2-033 interacts with the STAT3 protein and from confocal imaging that WZ-2-033 disrupts HA-STAT3 and Flag-STAT3 dimerization in intact cells. WZ-2-033 suppresses STAT3-DNA-binding activity but has no effect on STAT5-DNA binding. WZ-2-033 inhibits the phosphorylation and nuclear accumulation of pY705-STAT3 and consequently suppresses STAT3-dependent transcriptional activity and the expression of STAT3 downstream genes. Moreover, WZ-2-033 significantly inhibited the proliferation, colony survival, migration, and invasion of TNBC cells and gastric cancer cells with aberrant STAT3 activation. Furthermore, administration of WZ-2-033 in vivo induced a significant antitumor response in mouse models of TNBC and gastric cancer that correlated with the inhibition of constitutively active STAT3 and the suppression of known STAT3 downstream genes. Thus, our study provides a novel STAT3 inhibitor with significant antitumor activity in human TNBC and gastric cancer harboring persistently active STAT3.

The novel indomethacin derivative CZ-212-3 exerts antitumor effects on castration-resistant prostate cancer by degrading androgen receptor and its variants.

Androgen receptor (AR) serves as a main therapeutic target for prostate cancer (PCa). However, resistance to anti-androgen therapy (SAT) inevitably occurs. Indomethacin is a nonsteroidal anti-inflammatory drug that exhibits activity against prostate cancer. Recently, we designed and synthesized a series of new indomethacin derivatives (CZ compounds) via Pd (II)-catalyzed synthesis of substituted N-benzoylindole. In this study, we evaluated the antitumor effect of these novel indomethacin derivatives in castration-resistant prostate cancer (CRPC). Upon employing CCK-8 cell viability assays and colony formation assays, we found that these derivatives had high efficacy against CRPC tumor growth in vitro. Among these derivatives, CZ-212-3 exhibited the most potent efficacy against CRPC cell survival and on apoptosis induction. Mechanistically, CZ-212-3 significantly suppressed the expression of AR target gene networks by degrading AR and its variants. Consistently, CZ-212-3 significantly inhibited tumor growth in CRPC cell line-based xenograft and PDX models in vivo. Taken together, the data show that the indomethacin derivative CZ-212-3 significantly inhibited CRPC tumor growth by degrading AR and its variants and could be a promising agent for CRPC therapy.

Diagnostic value of ultrasound and contrast-enhanced ultrasound in septic acute kidney injury.

PURPOSE: This study aimed to explore the clinical value of ultrasonic Doppler examination and contrast-enhanced ultrasound (US) in the circulation of septic acute kidney injury (AKI). METHODS: Patients with intensive care unit-related infection were divided into AKI group and control groups. The AKI group was divided into three subgroups according to the serum creatinine value: stage 1, stage 2, and stage 3. Relevant parameters and blood flow of the renal artery were measured, and further contrast-enhanced US was performed and time-intensity curve was analyzed. RESULTS: The renal blood flow (RBF) and time-averaged velocity decreased significantly in the AKI group compared with the control group (p = .021 and p = .001). The peak value decreased and time to peak (TTP) prolonged in the AKI group (p < .001). With the aggravation of the disease, the RBF decreased slightly among subgroups (p = 0.124). However, the peak value gradually decreased and the TTP prolonged (all p < .05). The multiple linear regression model showed that only PI, RI, and TTP were independently and linearly correlated with the serum creatinine value. CONCLUSIONS: Doppler US and contrast-enhanced US are of great help in the detection of condition changes and prognosis of patients with sepsis-induced AKI.

MicroRNA-214 contributes to Ang II-induced cardiac hypertrophy by targeting SIRT3 to provoke mitochondrial malfunction.

Reduction of expression and activity of sirtuin 3 (SIRT3) contributes to the pathogenesis of cardiomyopathy via inducing mitochondrial injury and energy metabolism disorder. However, development of effective ways and agents to modulate SIRT3 remains a big challenge. In this study we explored the upstream suppressor of SIRT3 in angiotensin II (Ang II)-induced cardiac hypertrophy in mice. We first found that SIRT3 deficiency exacerbated Ang II-induced cardiac hypertrophy, and resulted in the development of spontaneous heart failure. Since miRNAs play crucial roles in the pathogenesis of cardiac hypertrophy, we performed miRNA sequencing on myocardium tissues from Ang II-infused Sirt3-/- and wild type mice, and identified microRNA-214 (miR-214) was significantly up-regulated in Ang II-infused mice. Similar results were also obtained in Ang II-treated neonatal mouse cardiomyocytes (NMCMs). Using dual-luciferase reporter assay we demonstrated that SIRT3 was a direct target of miR-214. Overexpression of miR-214 in vitro and in vivo decreased the expression of SIRT3, which resulted in extensive mitochondrial damages, thereby facilitating the onset of hypertrophy. In contrast, knockdown of miR-214 counteracted Ang II-induced detrimental effects via restoring SIRT3, and ameliorated mitochondrial morphology and respiratory activity. Collectively, these results demonstrate that miR-214 participates in Ang II-induced cardiac hypertrophy by directly suppressing SIRT3, and subsequently leading to mitochondrial malfunction, suggesting the potential of miR-214 as a promising intervention target for antihypertrophic therapy.

Angiopoietin-2 induces angiogenesis via exosomes in human hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most common primary liver cancer and is a highly vascularized solid tumor. Angiopoietin-2 (ANGPT2) has been described as an attractive target for antiangiogenic therapy. Exosomes are small extracellular vesicles secreted by most cell types and contribute to cell-to-cell communication by delivering functional cargo to recipient cells. The expression of ANGPT2 in tumor-derived exosomes remains unknown. METHODS: We detected the ANGPT2 expression in HCC-derived exosomes by immunoblotting, enzyme-linked immunosorbent assay and immunogold labeling, then observed exosomal ANGPT2 internalization and recycling by confocal laser scanning microscopy, co-immunoprecipitation and immunoblotting. We used two HCC cell lines (Hep3B and MHCC97H) to overexpress ANGPT2 by lentivirus infection or knockdown ANGPT2 by the CRISPR/Cas system, then isolated exosomes to coculture with human umbilical vein endothelial cells (HUVECs) and observed the angiogenesis by Matrigel microtubule formation assay, transwell migration assay, wound healing assay, cell counting kit-8 assay, immunoblotting and in vivo tumorigenesis assay. RESULTS: We found that HCC-derived exosomes carried ANGPT2 and delivered it into HUVECs by exosome endocytosis, this delivery led to a notable increase in angiogenesis by a Tie2-independent pathway. Concomitantly, we observed that HCC cell-secreted exosomal ANGPT2 was recycled by recipient HUVECs and might be reused. In addition, the CRISPR-Cas systems to knock down ANGPT2 significantly inhibited the angiogenesis induced by HCC cell-secreted exosomal ANGPT2, and obviously suppressed the epithelial-mesenchymal transition activation in HCC. CONCLUSIONS: Taken together, these results reveal a novel pathway of tumor angiogenesis induced by HCC cell-secreted exosomal ANGPT2 that is different from the classic ANGPT2/Tie2 pathway. This way may be a potential therapeutic target for antiangiogenic therapy. Video Abstract.

Pharmacological and cardiovascular perspectives on the treatment of COVID-19 with chloroquine derivatives.

The novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19) and an ongoing severe pandemic. Curative drugs specific for COVID-19 are currently lacking. Chloroquine phosphate and its derivative hydroxychloroquine, which have been used in the treatment and prevention of malaria and autoimmune diseases for decades, were found to inhibit SARS-CoV-2 infection with high potency in vitro and have shown clinical and virologic benefits in COVID-19 patients. Therefore, chloroquine phosphate was first used in the treatment of COVID-19 in China. Later, under a limited emergency-use authorization from the FDA, hydroxychloroquine in combination with azithromycin was used to treat COVID-19 patients in the USA, although the mechanisms of the anti-COVID-19 effects remain unclear. Preliminary outcomes from clinical trials in several countries have generated controversial results. The desperation to control the pandemic overrode the concerns regarding the serious adverse effects of chloroquine derivatives and combination drugs, including lethal arrhythmias and cardiomyopathy. The risks of these treatments have become more complex as a result of findings that COVID-19 is actually a multisystem disease. While respiratory symptoms are the major clinical manifestations, cardiovascular abnormalities, including arrhythmias, myocarditis, heart failure, and ischemic stroke, have been reported in a significant number of COVID-19 patients. Patients with preexisting cardiovascular conditions (hypertension, arrhythmias, etc.) are at increased risk of severe COVID-19 and death. From pharmacological and cardiovascular perspectives, therefore, the treatment of COVID-19 with chloroquine and its derivatives should be systematically evaluated, and patients should be routinely monitored for cardiovascular conditions to prevent lethal adverse events.

sFRP1 protects H9c2 cardiac myoblasts from doxorubicin-induced apoptosis by inhibiting the Wnt/PCP-JNK pathway.

Doxorubicin (Dox) is an effective chemotherapy drug against a wide range of cancers, including both hematological and solid tumors. However, the serious cardiotoxic effect restricted its clinical application. We previously have illuminated the protective role of canonical Wnt/ß-catenin signaling in Dox-induced cardiotoxicity. Secreted frizzled-related protein 1 (sFRP1) is one of the endogenous inhibitors of both canonical and noncanonical Wnt signaling. In this study, we investigated the relationship between sFRP1 and noncanonical Wnt/PCP-JNK (Wnt/planar cell polarity-c-Jun N-terminal kinase) pathway in Dox-induced cardiotoxicity in vitro and in vivo. We showed that treatment of H9c2 cardiac myoblasts with Dox (1 µM) time-dependently suppressed cell viability accompanied by significantly decreased sFRP1 protein level and increased Wnt/PCP-JNK signaling. Pretreatment with SP600125, the Wnt/PCP-JNK signaling inhibitor, attenuated Dox-induced apoptosis of H9c2 cells. Overexpression of sFRP1 protected H9c2 cells from Dox-induced apoptosis by inhibiting the Wnt/PCP-JNK pathway. After intraperitoneal injection of a cumulative dose of 15 mg/kg Dox, rats displayed significant cardiac dysfunction; their heart showed inhibited Wnt/ß-catenin signaling and activated Wnt/PCP-JNK signaling. These results suggest that sFRP1 may be a novel target for Dox-induced cardiotoxicity.

PARP1 interacts with HMGB1 and promotes its nuclear export in pathological myocardial hypertrophy.

High-mobility group box 1 (HMGB1) exhibits various functions according to its subcellular location, which is finely conditioned by diverse post-translational modifications, such as acetylation. The nuclear HMGB1 may prevent from cardiac hypertrophy, whereas its exogenous protein is proven to induce hypertrophic response. This present study sought to investigate the regulatory relationships between poly(ADP-ribose) polymerase 1 (PARP1) and HMGB1 in the process of pathological myocardial hypertrophy. Primary-cultured neonatal rat cardiomyocytes (NRCMs) were respectively incubated with three cardiac hypertrophic stimulants, including angiotensin II (Ang II), phenylephrine (PE), and isoproterenol (ISO), and cell surface area and the mRNA expression of hypertrophic biomarkers were measured. the catalytic activity of PARP1 was remarkably enhanced, meanwhile HMGB1 excluded from the nucleus. PARP1 overexpression by infecting with adenovirus PARP1 (Ad-PARP1) promoted the nuclear export of HMGB1, facilitated its secretion outside the cell, aggravated cardiomyocyte hypertrophy, which could be alleviated by HMGB1 overexpression. PE treatment led to the similar results, while that effect was widely depressed by PARP1 silencing or its specific inhibitor AG14361. Moreover, SD rats were intraperitoneally injected with 3-aminobenzamide (3AB, 20 mg/kg every day, a well-established PARP1 inhibitor) 7 days after abdominal aortic constriction (AAC) surgery for 6 weeks, echocardiography and morphometry of the hearts were measured. Pre-treatment of 3AB relieved AAC-caused the translocation of nuclear HMGB1 protein, cardiac hypertrophy, and heart dysfunction. Our research offers a novel evidence that PARP1 combines with HMGB1 and accelerates its translocation from nucleus to cytoplasm, and the course finally causes cardiac hypertrophy.

Histone methyltransferase NSD2 mediates the survival and invasion of triple-negative breast cancer cells via stimulating ADAM9-EGFR-AKT signaling.

Triple-negative breast cancer (TNBC) is a heterogeneous disease with a poor prognosis due to the lack of an effective targeted therapy. Histone lysine methyltransferases (KMTs) have emerged as attractive drug targets for cancer therapy. However, the function of the majority of KMTs in TNBC has remained largely unknown. In the current study, we found that KMT nuclear receptor binding SET domain protein 2 (NSD2) is overexpressed in TNBC tumors and that its overexpression is associated with poor survival of TNBC patients. NSD2 regulates TNBC cell survival and invasion and is required for tumorigenesis and tumor growth. Mechanistically, NSD2 directly controls the expression of EGFR and ADAM9, a member of the ADAM (a disintegrin and metalloproteinase) family that mediates the release of growth factors, such as HB-EGF. Through its methylase activity, NSD2 overexpression stimulates EGFR-AKT signaling and promotes TNBC cell resistance to the EGFR inhibitor gefitinib. Together, our results identify NSD2 as a major epigenetic regulator in TNBC and provide a rationale for targeting NSD2 alone or in combination with EGFR inhibitors as a targeted therapy for TNBC.

Salvia miltiorrhizaBurge (Danshen): a golden herbal medicine in cardiovascular therapeutics.

Salvia miltiorrhiza Burge (Danshen) is an eminent medicinal herb that possesses broad cardiovascular and cerebrovascular protective actions and has been used in Asian countries for many centuries. Accumulating evidence suggests that Danshen and its components prevent vascular diseases, in particular, atherosclerosis and cardiac diseases, including myocardial infarction, myocardial ischemia/reperfusion injury, arrhythmia, cardiac hypertrophy and cardiac fibrosis. The published literature indicates that lipophilic constituents (tanshinone I, tanshinone IIa, tanshinone IIb, cryptotanshinone, dihydrotanshinone, etc) as well as hydrophilic constituents (danshensu, salvianolic acid A and B, protocatechuic aldehyde, etc) contribute to the cardiovascular protective actions of Danshen, suggesting a potential synergism among these constituents. Herein, we provide a systematic up-to-date review on the cardiovascular actions and therapeutic potential of major pharmacologically active constituents of Danshen. These bioactive compounds will serve as excellent drug candidates in small-molecule cardiovascular drug discovery. This article also provides a scientific rationale for understanding the traditional use of Danshen in cardiovascular therapeutics.

G3BP2 is involved in isoproterenol-induced cardiac hypertrophy through activating the NF-κB signaling pathway.

The RasGAP SH3 domain-binding proteins (G3BPs) are a family of RNA-binding proteins that can co-ordinate signal transduction and post-transcriptional gene regulation. G3BPs have been shown to be involved in mediating a great diversity of cellular processes such as cell survival, growth, proliferation and apoptosis. But the potential roles of G3BPs in the pathogenesis and progression of cardiovascular diseases remain to be clarified. In the present study, we provide the first evidence that suggests the participation of G3BP2 in cardiac hypertrophy. In cultured neonatal rat cardiomyocytes (NRCMs), treatment with isoproterenol (ISO, 0.1-100 µmol/L) significantly elevated the mRNA and protein levels of G3BP2. Similar results were observed in the hearts of rats subjected to 7D-injection of ISO, accompanied by obvious heart hypertrophy and elevated the expression of hypertrophy marker genes ANF, BNP and ß-MHC in heart tissues. Overexpression of G3BP2 in NRCMs led to hypertrophic responses evidenced by increased cellular surface area and the expression of hypertrophy marker genes, whereas knockdown of G3BP2 significantly attenuated ISO-induced hypertrophy of NRCMs. We further showed that G3BP2 directly interacted with IκBα and promoted the aggregation of the NF-κB subunit p65 in the nucleus and increased NF-κB-dependent transcriptional activity. NF-κB inhibition with PDTC (50 µmol/L) or p65 knockdown significantly decreased the hypertrophic responses in NRCMs induced by ISO or G3BP2 overexpression. These results give new insight into the functions of G3BP2 and may help further elucidate the molecular mechanisms underlying cardiac hypertrophy.

mTORC2 facilitates endothelial cell senescence by suppressing Nrf2 expression via the Akt/GSK-3ß/C/EBPα signaling pathway.

Vascular endothelial cell senescence is a leading cause of age-associated and vascular diseases. Mammalian target of rapamycin complex 2 (mTORC2) is a conserved serine/threonine (Ser/Thr) protein kinase that plays an important regulatory role in various cellular processes. However, its impact on endothelial senescence remains controversial. In this study we investigated the role and molecular mechanisms of mTORC2 in endothelial senescence. A replicative senescence model and H2O2-induced premature senescence model were established in primary cultured human umbilical vein endothelial cells (HUVECs). In these senescence models, the formation and activation of mTORC2 were significantly increased, evidenced by the increases in binding of Rictor (the essential component of mTORC2) to mTOR, phosphorylation of mTOR at Ser2481 and phosphorylation of Akt (the effector of mTORC2) at Ser473. Knockdown of Rictor or treatment with the Akt inhibitor MK-2206 attenuated senescence-associated ß-galactosidase (ß-gal) staining and expression of p53 and p21 proteins in the senescent endothelial cells, suggesting that mTORC2/Akt facilitates endothelial senescence. The effect of mTORC2/Akt on endothelial senescence was due to suppression of nuclear factor erythroid 2-related factor 2 (Nrf2) at the transcriptional level, since knockdown of Rictor reversed the reduction of Nrf2 mRNA expression in endothelial senescence. Furthermore, mTORC2 suppressed the expression of Nrf2 via the Akt/GSK-3ß/C/EBPα signaling pathway. These results suggest that the mTORC2/Akt/GSK-3ß/C/EBPα/Nrf2 signaling pathway is involved in both replicative and inducible endothelial senescence. The deleterious role of mTORC2 in endothelial cell senescence suggests therapeutic strategies (targeting mTORC2) for aging-associated diseases and vascular diseases.

Ulinastatin attenuates LPS-induced inflammation in mouse macrophage RAW264.7 cells by inhibiting the JNK/NF-κB signaling pathway and activating the PI3K/Akt/Nrf2 pathway.

Ulinastatin (UTI) is a broad-spectrum serine protease inhibitor isolated and purified from human urine with strong anti-inflammatory and cytoprotective actions, which is widely used for the treatment of various diseases, such as pancreatitis and sepsis. Although the therapeutic effects of UTI are reported to be associated with a variety of mechanisms, the signaling pathways mediating the anti-inflammatory action of UTI remain to be elucidated. In the present study we carried out a systematic study on the anti-inflammatory and anti-oxidative mechanisms of UTI and their relationships in LPS-treated RAW264.7 cells. Pretreatment with UTI (1000 and 5000 U/mL) dose-dependently decreased the mRNA levels of pro-inflammatory cytokines (IL-1ß, IL-6, TNF-α, iNOS) and upregulated anti-inflammatory cytokines (IL-10 and TGF-ß1) in LPS-treated RAW264.7 cells. UTI pretreatment significantly inhibited the nuclear translocation of NF-κB by preventing the degradation of IκB-α. UTI pretreatment only markedly inhibited the phosphorylation of JNK at Thr183, but it did not affect the phosphorylation of JNK at Tyr185, ERK-1/2 and p38 MAPK; JNK was found to function upstream of the IκB-α/NF-κB signaling pathway. Furthermore, UTI pretreatment significantly suppressed LPS-induced ROS production by activating PI3K/Akt pathways and the nuclear translocation of Nrf2 via promotion of p62-associated Keap1 degradation. However, JNK was not involved in mediating the anti-oxidative stress effects of UTI. In summary, this study shows that UTI exerts both anti-inflammatory and anti-oxidative effects by targeting the JNK/NF-κB and PI3K/Akt/Nrf2 pathways.

Synthesis of the novel PARP-1 inhibitor AG-690/11026014 and its protective effects on angiotensin II-induced mouse cardiac remodeling.

We previously identified AG-690/11026014 (6014) as a novel poly(ADP-ribose) polymerase-1 (PARP-1) inhibitor that effectively prevented angiotensin II (Ang II)-induced cardiomyocyte hypertrophy. In the present study, we reported a new synthesis route for 6014, and investigated its protective effects on Ang II-induced cardiac remodeling and cardiac dysfunction and the underlying mechanisms in mice. We designed a new synthesis route to obtain a sufficient quantity of 6014 for this in vivo study. C57BL/6J mice were infused with Ang II and treated with 6014 (10, 30, 90 mg·kg-1·d-1, ig) for 4 weeks. Then two-dimensional echocardiography was performed to assess the cardiac function and structure. Histological changes of the hearts were examined with HE staining and Masson's trichrome staining. The protein expression was evaluated by Western blot, immunohistochemistry and immunofluorescence assays. The activities of sirtuin-1 (SIRT-1) and the content of NAD+ were detected with the corresponding test kits. Treatment with 6014 dose-dependently improved cardiac function, including LVEF, CO and SV and reversed the changes of cardiac structure in Ang II-infused mice: it significantly ameliorated Ang II-induced cardiac hypertrophy evidenced by attenuating the enlargement of cardiomyocytes, decreased HW/BW and LVW/BW, and decreased expression of hypertrophic markers ANF, BNP and ß-MHC; it also prevented Ang II-induced cardiac fibrosis, as implied by the decrease in excess accumulation of extracellular matrix (ECM) components collagen I, collagen III and FN. Further studies revealed that treatment with 6014 did not affect the expression levels of PARP-1, but dose-dependently inhibited the activity of PARP-1 and subsequently restored the activity of SIRT-1 in heart tissues due to the decreased consumption of NAD+ and attenuated Poly-ADP-ribosylation (PARylation) of SIRT-1. In conclusion, the novel PARP-1 inhibitor 6014 effectively protects mice against AngII-induced cardiac remodeling and improves cardiac function. Thus, 6014 might be a potential therapeutic agent for heart diseases..