The lysine methyltransferase SMYD2 facilitates neointimal hyperplasia by regulating the HDAC3-SRF axis.

Coronary restenosis is an important cause of poor long-term prognosis in patients with coronary heart disease. Here, we show that lysine methyltransferase SMYD2 expression in the nucleus is significantly elevated in serum- and PDGF-BB-induced vascular smooth muscle cells (VSMCs), and in tissues of carotid artery injury-induced neointimal hyperplasia. Smyd2 overexpression in VSMCs (Smyd2-vTg) facilitates, but treatment with its specific inhibitor LLY-507 or SMYD2 knockdown significantly inhibits VSMC phenotypic switching and carotid artery injury-induced neointima formation in mice. Transcriptome sequencing revealed that SMYD2 knockdown represses the expression of serum response factor (SRF) target genes and that SRF overexpression largely reverses the inhibitory effect of SMYD2 knockdown on VSMC proliferation. HDAC3 directly interacts with and deacetylates SRF, which enhances SRF transcriptional activity in VSMCs. Moreover, SMYD2 promotes HDAC3 expression via tri-methylation of H3K36 at its promoter. RGFP966, a specific inhibitor of HDAC3, not only counteracts the pro-proliferation effect of SMYD2 overexpression on VSMCs, but also inhibits carotid artery injury-induced neointima formation in mice. HDAC3 partially abolishes the inhibitory effect of SMYD2 knockdown on VSMC proliferation in a deacetylase activity-dependent manner. Our results reveal that the SMYD2-HDAC3-SRF axis constitutes a novel and critical epigenetic mechanism that regulates VSMC phenotypic switching and neointimal hyperplasia.

Free-moving-state microscopic imaging of cerebral oxygenation and hemodynamics with a photoacoustic fiberscope.

We report the development of a head-mounted photoacoustic fiberscope for cerebral imaging in a freely behaving mouse. The 4.5-gram imaging probe has a 9-µm lateral resolution and 0.2-Hz frame rate over a 1.2-mm wide area. The probe can continuously monitor cerebral oxygenation and hemodynamic responses at single-vessel resolution, showing significantly different cerebrovascular responses to external stimuli under anesthesia and in the freely moving state. For example, when subjected to high-concentration CO2 respiration, enhanced oxygenation to compensate for hypercapnia can be visualized due to cerebral regulation in the freely moving state. Comparative studies exhibit significantly weakened compensation capabilities in obese rodents. This new imaging modality can be used for investigating both normal and pathological cerebrovascular functions and shows great promise for studying cerebral activity, disorders and their treatments.

BRD4770 inhibits vascular smooth muscle cell proliferation via SUV39H2, but not EHMT2 to protect against neointima formation.

The behavior of vascular smooth muscle cells (VSMCs) contributes to the formation of neointima. We previously found that EHMT2 suppressed autophagy activation in VSMCs. BRD4770, an inhibitor of EHMT2/G9a, plays a critical role in several kinds of cancers. However, whether and how BRD4770 regulates the behavior of VSMCs remain unknown. In this study, we evaluate the cellular effect of BRD4770 on VSMCs by series of experiments in vivo and ex vivo. We demonstrated that BRD4770 inhibited VSMCs' growth by blockage in G2/M phase in VSMCs. Moreover, our results demonstrated that the inhibition of proliferation was independent on autophagy or EHMT2 suppression which we previous reported. Mechanistically, BRD4770 exhibited an off-target effect from EHMT2 and our further study reveal that the proliferation inhibitory effect by BRD4770 was associated with suppressing on SUV39H2/KTM1B. In vivo, BRD4770 was also verified to rescue VIH. Thus, BRD4770 function as a crucial negative regulator of VSMC proliferation via SUV39H2 and G2/M cell cycle arrest and BRD4770 could be a molecule for the therapy of vascular restenosis.

In vivo endoscopic ultrasound imaging with a rotational-scanning, all-optical ultrasound probe.

All-optical ultrasound manipulates ultrasound waves based on laser and photonics technologies, providing an alternative approach for pulse-echo ultrasound imaging. However, its endoscopic imaging capability is limited ex vivo by the multifiber connection between the endoscopic probe and the console. Here, we report on all-optical ultrasound for in vivo endoscopic imaging using a rotational-scanning probe that relies on a small laser sensor to detect echo ultrasound waves. The acoustically induced lasing frequency change is measured via heterodyne detection by beating the two orthogonally polarized laser modes, enabling a stable output of ultrasonic responses and immunity to low-frequency thermal and mechanical disturbances. We miniaturize its optical driving and signal interrogation unit and synchronously rotate it with the imaging probe. This specialized design leaves a single-fiber connection to the proximal end and allows fast rotational scanning of the probe. As a result, we used a flexible, miniature all-optical ultrasound probe for in vivo rectal imaging with a B-scan rate of 1 Hz and a pullback range of ∼7 cm. This can visualize the gastrointestinal and extraluminal structures of a small animal. This imaging modality offers an imaging depth of 2 cm at a central frequency of ∼20 MHz, showing promise for high-frequency ultrasound imaging applications in gastroenterology and cardiology.

Methyltransferase-like 3 suppresses phenotypic switching of vascular smooth muscle cells by activating autophagosome formation.

Prevention of neointima formation is the key to improving long-term outcomes after stenting or coronary artery bypass grafting. RNA N6 -methyladenosine (m6 A) methylation has been reported to be involved in the development of various cardiovascular diseases, but whether it has a regulatory effect on neointima formation is unknown. Herein, we revealed that methyltransferase-like 3 (METTL3), the major methyltransferase of m6 A methylation, was downregulated during vascular smooth muscle cell (VSMC) proliferation and neointima formation. Knockdown of METTL3 facilitated, while overexpression of METTL3 suppressed the proliferation of human aortic smooth muscle cells (HASMCs) by arresting HASMCs at G2/M checkpoint and the phosphorylation of CDC2 (p-CDC2) was inactivated by METTL3. On the other hand, the migration and synthetic phenotype of HASMCs were enhanced by METTL3 knockdown, but inhibited by METTL3 overexpression. The protein levels of matrix metalloproteinase 2 (MMP2), MMP7 and MMP9 were reduced, while the expression level of tissue inhibitor of metalloproteinase 3 was increased in HASMCs with METTL3 overexpression. Moreover, METTL3 promoted the autophagosome formation by upregulating the expression of ATG5 (autophagy-related 5) and ATG7. Knockdown of either ATG5 or ATG7 largely reversed the regulatory effects of METTL3 overexpression on phenotypic switching of HASMCs, as evidenced by increased proliferation and migration, and predisposed to synthetic phenotype. These results indicate that METTL3 inhibits the phenotypic switching of VSMCs by positively regulating ATG5-mediated and ATG7-mediated autophagosome formation. Thus, enhancing the level of RNA m6 A or the formation of autophagosomes is the promising strategy to delay neointima formation.

The Latest Breakthroughs in Immunotherapy for Acute Myeloid Leukemia, with a Special Focus on NKG2D Ligands.

Acute myeloid leukemia (AML) is a hematological malignancy characterized by clonal expansion of stem and myeloid progenitor cells. Immunotherapy has revolutionized the care for other cancers such as solid tumors and lymphomas, and has the potential to effectively treat AML. There has been substantial progress in the developments of immunotherapeutic approaches for AML over the last several years, including the development of antibodies that further increase the innate immunogenicity of leukemia cells by the inhibition of NKG2D ligand-particularly MICA and MICB-shedding, chimeric proteins such as IL-15 superagonist that expand natural killer (NK) cells, blockers of immunologic checkpoints such as NKG2A, and chemicals that indirectly increase expression of immune stimulatory proteins in leukemia stem cells. Furthermore, cellular therapies have been designed to enable alloreactive immunity by allogeneic NK cells or target leukemia antigens such as mutated NPM1. These immunotherapeutic approaches have demonstrated remarkable efficacies in preclinical studies and have successfully transitioned to early phase clinical trials, to establish safety and initial signal of clinical activity. Here, we briefly discuss some of the most recent and impactful developments in the AML immunotherapy field and provide our perspectives for the future directions of this exciting and new therapeutic opportunity.

Targeting Ferroptosis as a Novel Approach to Alleviate Aortic Dissection.

A variety of programmed cell death types have been shown to participate in the loss of smooth muscle cells (SMCs) during the development of aortic dissection (AD), but it is still largely unclear whether ferroptosis is involved in the development of AD. In the present study, we found that the expression of key ferroptosis regulatory proteins, solute carrier family 7 member 11 (SLC7A11), ferroptosis suppressor protein 1 (FSP1) and glutathione peroxidase 4 (GPX4) were downregulated in aortas of Stanford type A AD (TAAD) patients, and liproxstatin-1, a specific inhibitor of ferroptosis, obviously abolished the ß-aminopropionitrile (BAPN)-induced development and rupture of AD in mice. Furthermore, the expression of methyltransferase-like 3 (METTL3), a major methyltransferase of RNA m6A, was remarkably upregulated in the aortas of TAAD patients, and the protein levels of METTL3 were negatively correlated with SLC7A11 and FSP1 levels in human aortas. Overexpression of METTL3 in human aortic SMCs (HASMCs) inhibited, while METTL3 knockdown promoted SLC7A11 and FSP1 expression. More importantly, overexpression of METTL3 facilitated imidazole ketone erastin- and cystine deprivation-induced ferroptosis, while knockdown of METTL3 repressed ferroptosis of HASMCs. Overexpression of either SLC7A11 or FSP1 largely abrogated the effect of METTL3 on HASMC ferroptosis. Therefore, we have revealed that ferroptosis is a critical cause of AD in both humans and mice and that METTL3 promotes ferroptosis of HASMCs by inhibiting the expression of SLC7A11 and FSP1. Thus, targeting ferroptosis or m6A RNA methylation is a potential novel strategy for the treatment of AD.

BRD4770 functions as a novel ferroptosis inhibitor to protect against aortic dissection.

Smooth muscle cell (SMC) loss is the characteristic feature in the pathogenesis of aortic dissection (AD), and ferroptosis is a novel iron-dependent regulated cell death driven by the excessive lipid peroxidation accumulation. However, whether targeting ferroptosis is an effective approach for SMC loss and AD treatment remains unclear. Here, we found that the iron level, ferroptosis-related molecules TFR, HOMX1, ferritin and the lipid peroxidation product 4-hydroxynonenal were increased in the aorta of AD. Then, we screened several inhibitors of histone methyltransferases and found that BRD4770 had a protective effect on cystine deprivation-, imidazole ketone erastin- or RSL3-induced ferroptosis of SMCs. The classic ferroptosis pathways, System Xc--GPX4, FSP1-CoQ10 and GCH1-BH4 pathways which were inhibited by ferroptosis inducers, were re-activated by BRD4770 via inhibiting mono-, di- and tri- methylated histone H3 at lysine 9 (H3K9me1/2/3). RNA-sequencing analysis revealed that there was a positive feedback regulation between ferroptosis and inflammatory response, and BRD4770 can reverse the effects of inflammation activation on ferroptosis. More importantly, treatment with BRD4770 attenuated aortic dilation and decreased morbidity and mortality in a ß-Aminopropionitrile monofumarate-induced mouse AD model via inhibiting the inflammatory response, lipid peroxidation and ferroptosis. Taken together, our findings demonstrate that ferroptosis is a novel and critical pathological mechanism that is involved in SMC loss and AD development. BRD4770 is a novel ferroptosis inhibitor and has equivalent protective effect to Ferrostatin-1 at the optimal concentration. Translating insights into the anti-ferroptosis effects of BRD4770 may reveal a potential therapeutic approach for targeting SMC ferroptosis in AD.

Resurgent regenerated fiber Bragg gratings and thermal annealing techniques for ultra-high temperature sensing beyond 1400°C.

We report for the first time the resurgence of regenerated fiber Bragg gratings (RFBGs) useful for ultra-high temperature measurements exceeding 1400 °C. A detailed study of the dynamics associated with grating regeneration in six-hole microstructured optical fibers (SHMOFs) and single mode fibers (SMFs) was conducted. Rapid heating and rapid cooling techniques appeared to have a significant impact on the thermal sustainability of the RFBGs in both types of optical fibers reaching temperature regimes exceeding 1400 °C. The presence of air holes sheds new light in understanding the thermal response of RFBGs and the stresses associated with them, which governs the variation in the Bragg wavelength.

EHMT2/G9a Inhibits Aortic Smooth Muscle Cell Death by Suppressing Autophagy Activation.

Although EHMT2 (also known as G9a) plays a critical role in several kinds of cancers and cardiac remodeling, its function in vascular smooth muscle cells (VSMCs) remains unknown. In the present study, we revealed a novel function of EHMT2 in regulating autophagic cell death (ACD) of VSMC. Inhibition of EHMT2 by BIX01294 or knockdown of EHMT2 resulted in reduced VSMC numbers which were independent of proliferation and apoptosis. Interestingly, EHMT2 protein levels were significantly decreased in VSMCs treated with autophagic inducers. Moreover, more autophagic vacuoles and accumulated LC3II were detected in VSMCs treated with BIX01294 or lenti-shEHMT2 than their counterparts. Furthermore, we found that EHMT2 inhibited the ACD of VSMCs by suppressing autophagosome formation. Mechanistically, the pro-autophagic effect elicited by EHMT2 inhibition was associated with SQSTM1 and BECN1 overexpression. Moreover, these detrimental effects were largely nullified by SQSTM1 or BECN1 knockdown. More importantly, similar results were observed in primary human aortic VSMCs. Overall, these findings suggest that EHMT2 functions as a crucial negative regulator of ACD via decreasing SQSTM1 or BECN1 expression and that EHMT2 could be a potent therapeutic target for cardiovascular diseases (e.g., aortic dissection).

Tongxinluo Prevents Endothelial Dysfunction Induced by Homocysteine Thiolactone In Vivo via Suppression of Oxidative Stress.

Aim. To explore whether Chinese traditional medicine, tongxinluo (TXL), exerts beneficial effects on endothelial dysfunction induced by homocysteine thiolactone (HTL) and to investigate the potential mechanisms. Methods and Results. Incubation of cultured human umbilical vein endothelial cells with HTL (1 mM) for 24 hours significantly reduced cell viabilities assayed by MTT, and enhanced productions of reactive oxygen species. Pretreatment of cells with TXL (100, 200, and 400 µg/mL) for 1 hour reversed these effects induced by HTL. Further, coincubation with GW9662 (0.01, 0.1 mM) abolished the protective effects of TXL on HTL-treated cells. In ex vivo experiments, exposure of isolated aortic rings from rats to HTL (1 mM) for 1 hour dramatically impaired acetylcholine-induced endothelium-dependent relaxation, reduced SOD activity, and increased malondialdehyde content in aortic tissues. Preincubation of aortic rings with TXL (100, 200, and 400 µg/mL) normalized the disorders induced by HTL. Importantly, all effects induced by TXL were reversed by GW9662. In vivo analysis indicated that the administration of TXL (1.0 g/kg/d) remarkably suppressed oxidative stress and prevented endothelial dysfunction in rats fed with HTL (50 mg/kg/d) for 8 weeks. Conclusions. TXL improves endothelial functions in rats fed with HTL, which is related to PPARγ-dependent suppression of oxidative stress.

Androgen receptor promotes esophageal cancer cell migration and proliferation via matrix metalloproteinase 2.

Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies worldwide. Androgen receptor (AR) plays an important role in many kinds of cancers. However, the molecular mechanisms of AR in ESCC are poorly characterized. In the present study, Western blot analysis and real-time quantitative PCR were performed to identify differentially expressed AR in 40 ESCC tissue samples, which revealed that the messenger RNA (mRNA) and protein expression of AR is upregulated in the ESCC tissue samples. AR overexpression induced increases in ESCC cell invasion and proliferation in vitro. Silencing of AR inhibited the proliferation of KYSE450 cells which have a relatively high level of AR, and the invasion of KYSE450 cells was distinctly suppressed. Furthermore, AR knockdown led to substantial reductions in matrix metalloproteinase 2 (MMP2) and p-AKT levels in ESCC cell lines, but no significant change in AKT and MMP9 expression. These results suggest that AR is involved in tumor progression, and thus, AR could represent selective targets for the molecularly targeted treatments of ESCC.

Resistance to cetuximab in EGFR-overexpressing esophageal squamous cell carcinoma xenografts due to FGFR2 amplification and overexpression.

Esophageal carcinoma is one of the most virulent malignant diseases and a major cause of cancer-related deaths worldwide. Despite improvements in surgical techniques and perioperative management and surgery combined with chemotherapy and/or radiotherapy, the prognosis of esophageal squamous cell carcinoma (ESCC) at an advanced stage remains poor. ESCC shows a relatively high incidence of EGFR (50% - 70%), and the humanized monoclonal antibody (mAb) cetuximab against EGFR has been undergoing clinical development. However, all responding patients eventually developed acquired resistance to cetuximab. In the current study, we described a cetuximab-sensitive ESCC xeongraft model that developed resistance to cetuximab as a result of FGFR2 gene amplification and overexpression. Inhibition of FGFR2 signaling in this xenograft model restored its sensitivity to cetuximab. The antitumor effect may be induced by inhibition of AKT phosphorylation. These findings suggest that combination therapyincluding cetuximab and FGFR2 inhibition may be a promising strategy to treat ESCC.

Nicotine upregulates microRNA-21 and promotes TGF-ß-dependent epithelial-mesenchymal transition of esophageal cancer cells.

A consistent positive association between cigarette smoking and the human esophageal cancer has been confirmed all over the world. However, details in the association need to be more focused on and be identified. Recently, aberrantly expressed microRNAs (miRNAs) have been shown to be promising biomarkers for understanding the tumorigenesis of a wide array of human cancers, including the esophageal cancer, and the deregulation on the epithelial to mesenchymal transition (EMT) by miRNAs is involved in the tumorigenesis. In present study, we were going to identify the role of nicotine-induced miR-21 in the EMT of esophageal cells. We found that there was an overexpression of miR-21 in esophageal specimens, having an association with cigarette smoking, and the upregulation of miR-21 was also induced by nicotine in esophageal carcinoma cell line, EC9706. Moreover, the upregulated miR-21 by nicotine promoted EMT transforming growth factor beta (TGF-ß) dependently. Thus, the present study reveals a novel oncogenic role of nicotine in human esophageal cancer.