SP2509 functions as a novel ferroptosis inhibitor by reducing intracellular iron level in vascular smooth muscle cells.

Previous studies have shown that ferroptosis of vascular smooth muscle cells (VSMCs) is involved in the development of aortic dissection (AD) and that histone methylation regulates this process. SP2509 acts as a specific inhibitor of lysine-specific demethylase 1 (LSD1), which governs a variety of biological processes. However, the effect of SP2509 on VSMC ferroptosis and AD remains to be elucidated. This aim of this study was to investigate the role and underlying mechanism of SP2509-mediated histone methylation on VSMC ferroptosis. Here, a mouse model of AD was established, and significantly reduced levels of H3K4me1 and H3K4me2 (target of SP2509) were found in the aortas of AD mice. In VSMCs, SP2509 treatment led to a dose-dependent increase in H3K4me2 levels. Furthermore, we found that SP2509 provided equivalent protection to ferrostatin-1 against VSMC ferroptosis, as evidenced by increased cell viability, decreased cell death and lipid peroxidation. RNA-sequencing analysis and subsequent experiments revealed that SP2509 counteracted cystine deficiency-induced response to inflammation and oxidative stress. More importantly, we demonstrated that SP2509 inhibited the expression of TFR and ferritin to reduce intracellular iron levels, thereby effectively blocking the process of ferroptosis. Therefore, our findings indicate that SP2509 protects VSMCs from multiple stimulus-induced ferroptosis by reducing intracellular iron levels, thereby preventing lipid peroxidation and cell death. These findings suggest that SP2509 may be a promising drug to alleviate AD by reducing iron deposition and VSMC ferroptosis.

Knockdown of TRPV2 inhibits the migration of RAW264.7 cells toward low fluid shear stress region.

Our previous studies have demonstrated that macrophages (RAW264.7) have a special ability for sensing the gradient of fluid shear stress (FSS) and migrate toward the low-FSS region. However, the molecular mechanism regulating this phenomenon is still unclear. In this study, we examined the transcriptome genes in RAW264.7 cells, MC3T3-E1 osteoblasts, mesenchymal stem cells, canine renal epithelial cells, and periodontal ligament cells. The expression levels of genes related to cell migration, force transfer, and force sensitivity in the Ca2+ signaling pathway were analyzed. We observed that the transient receptor potential cation channel type 2 (TRPV2) was highly expressed in RAW264.7 cells. Furthermore, we used lentiviral transfection to knockdown TRPV2 expression in RAW264.7 cells and studied the effect of TRPV2 on the migration of RAW264.7 cells under a gradient FSS field. The results showed that compared with normal cells, TRPV2-knockdown cells had impaired ability for sensing FSS gradient to migrate toward the low-FSS region and lower intracellular calcium response to FSS stimulation. This study may reveal the molecular mechanism of regulating the directional migration of macrophages under a gradient FSS field.

Histone acetyltransferase P300 deficiency promotes ferroptosis of vascular smooth muscle cells by activating the HIF-1α/HMOX1 axis.

BACKGROUND: E1A-associated 300-kDa protein (P300), an endogenous histone acetyltransferase, contributes to modifications of the chromatin landscape of genes involved in multiple cardiovascular diseases. Ferroptosis of vascular smooth muscle cells (VSMCs) is a novel pathological mechanism of aortic dissection. However, whether P300 regulates VSMC ferroptosis remains unknown. METHODS: Cystine deprivation (CD) and imidazole ketone erastin (IKE) were used to induce VSMC ferroptosis. Two different knockdown plasmids targeting P300 and A-485 (a specific inhibitor of P300) were used to investigate the function of P300 in the ferroptosis of human aortic smooth muscle cells (HASMCs). Cell counting kit-8, lactate dehydrogenase and flow cytometry with propidium iodide staining were performed to assess the cell viability and death under the treatment of CD and IKE. BODIPY-C11 assay, immunofluorescence staining of 4-hydroxynonenal and malondialdehyde assay were conducted to detect the level of lipid peroxidation. Furthermore, co-immunoprecipitation was utilized to explore the interaction between P300 and HIF-1α, HIF-1α and P53. RESULTS: Compared with normal control, the protein level of P300 was significantly decreased in HASMCs treated with CD and IKE, which was largely nullified by the ferroptosis inhibitor ferrostatin-1 but not by the autophagy inhibitor or apoptosis inhibitor. Knockdown of P300 by short-hairpin RNA or inhibition of P300 activity by A-485 promoted CD- and IKE-induced HASMC ferroptosis, as evidenced by a reduction in cell viability and aggravation of lipid peroxidation of HASMCs. Furthermore, we found that hypoxia-inducible factor-1α (HIF-1α)/heme oxygenase 1 (HMOX1) pathway was responsible for the impacts of P300 on ferroptosis of HASMCs. The results of co-immunoprecipitation demonstrated that P300 and P53 competitively bound HIF-1α to regulate the expression of HMOX1. Under normal conditions, P300 interacted with HIF-1α to inhibit HMOX1 expression, while reduced expression of P300 induced by ferroptosis inducers would favor HIF-1α binding to P53 to trigger HMOX1 overexpression. Furthermore, the aggravated effects of P300 knockdown on HASMC ferroptosis were largely nullified by HIF-1α knockdown or the HIF-1α inhibitor BAY87-2243. CONCLUSION: Thus, our results revealed that P300 deficiency or inactivation facilitated CD- and IKE-induced VSMC ferroptosis by activating the HIF-1α/HMOX1 axis, which may contribute to the development of diseases related to VSMC ferroptosis.

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.

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.

Synergistic effects of fluid shear stress and adhesion morphology on the apoptosis and osteogenesis of mesenchymal stem cells.

Mechanical microenvironments, such as characteristics defining mechanical environments and fluid flow play an important role in steering the fate of mesenchymal stem cells (MSCs). However, the synergistic effect of adhesion morphology and fluid flow on the biological behavior of MSCs is seldom investigated. In this article, 0.5 or 0.8 Pa fluid shear stress (FSS) was applied to the MSCs on micropatterned substrates, and the apoptosis and osteogenic differentiation of MSCs were measured by double fluorescent staining. Results showed that the cellular adhesion patterns with low circularity and large area are beneficial to the osteogenic differentiation of individual MSCs. Meanwhile, FSS facilitated osteogenic differentiation of MSCs, as shown by the expression of alkaline phosphatase, osteocalcin, and collagen I. In addition, nuclear transfer of Yes-associated protein, a transcriptional regulator in MSCs, was enhanced after being exposed to FSS. These results demonstrated the synergistic effects of FSS and adhesion morphology in directing the fate of MSCs, and these effects may be adopted to design bio-functional substrates for cell transplantation in tissue engineering.

Comprehensive analysis identified a reduction in ATP1A2 mediated by ARID3A in abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is characterized by abdominal aorta dilatation and progressive structural impairment and is usually an asymptomatic and potentially lethal disease with a risk of rupture. To investigate the underlying mechanisms of AAA initiation and progression, seven AAA datasets related to human and mice were downloaded from the GEO database and reanalysed in the present study. After comprehensive bioinformatics analysis, we identified the enriched pathways associated with inflammation responses, vascular smooth muscle cell (VSMC) phenotype switching and cytokine secretion in AAA. Most importantly, we identified ATPase Na+ /K+ transporting subunit alpha 2 (ATP1A2) as a key gene that was significantly decreased in AAA samples of both human and mice; meanwhile, its reduction mainly occurred in VSMCs of the aorta; this finding was validated by immunostaining and Western blot in human and mouse AAA samples. Furthermore, we explored the potential upstream transcription factors (TFs) that regulate ATP1A2 expression. We found that the TF AT-rich interaction domain 3A (ARID3A) bound the promoter of ATP1A2 to suppress its expression. Our present study identified the ARID3A-ATP1A2 axis as a novel pathway in the pathological processes of AAA, further elucidating the molecular mechanism of AAA and providing potential therapeutic targets for AAA.

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.

Effect of trachea stiffness on tumor distribution in papillary thyroid microcarcinoma.

Biomechanical factors play an important role in tumor distribution, epithelial-mesenchymal transition (EMT), invasion and other important processes. Despite fewer reports investigating biomechanical function in papillary thyroid carcinoma (PTC), a large number of PTC cases are located close to the trachea and the majority of advanced cases of PTC have been associated with invasion of the trachea. However, the effect of trachea stiffness on PTC distribution and growth remains unknown. To clarify this issue, two types of PTC cells (TPC-1 and KTC-1) were seeded on a substrate with different stiffness to observe cell proliferation and movement. To identify the effect of trachea stiffness on the thyroid, two thyroid lobes (left and right) were evenly divided into interior (close to the trachea) and lateral (away from the trachea) parts, based on the vertical line between the trachea and thyroid lateral margin with different von Mises stress values. As PTC originates from papillary thyroid microcarcinoma (PTMC) with a maximum diameter of <1 cm, the present study selected PTMC as the study subject to reflect initial PTC distribution in the thyroid. The association between the percentage of PTMC distribution in different parts of the thyroid and von Mises stress values was analyzed. Both PTC cells exhibited stronger proliferation and mobility on the stiff substrate compared with that on the soft substrate. Furthermore, the results of finite element analysis revealed that the von Mises stress values of the interior parts of the trachea were notably higher compared with that in the lateral parts. PTMC distribution in the interior trachea was notably greater compared with that in the lateral section. There was also an observed association between von Mises stress values and PTMC distribution. In addition, the results of RNA-sequencing and reverse transcription-quantitative PCR demonstrated that three biomechanical genes were overexpressed in PTMC located in the interior section compared with that in adjacent normal tissue, and the related signaling pathways were also activated in these tissues. On the whole, these results indicated that trachea stiffness may supply a suitable biomechanical environment for PTMC growth, and the related biomechanical genes may serve as novel targets for PTMC diagnosis and prognostic estimation.

Spreading area and shape regulate the apoptosis and osteogenesis of mesenchymal stem cells on circular and branched micropatterned islands.

The topography of extracellular matrix regulates the differentiation of mesenchymal stem cells (MSCs). In particular, the effect of spreading shape or area on cellular differentiation and viability of individual MSCs cultured in the confined adhesive regions is an interesting fundamental issue. In this study, the adhesive patterns with the circularity of 0.1 or 1 and the areas of 314; 628; 1,256; or 2,512 µm2 were constructed using micropatterning technology. The expression of osteogenesis marker alkaline phosphatase and the apoptosis level of individual MSCs were measured using double fluorescent staining. Results indicated that individual MSCs confined in the small area showed an apoptotic tendency, and those in the large area might enter into osteogenesis. The branched shape with small circularity increased MSC viability but reduced their pluripotency compared with the circular shape. The expression of other osteogenesis markers, such as osteocalcin and Collagen I, confirmed that large and branched pattern promoted MSC osteogenesis. In addition, the transcriptional coactivator yes-associated protein (YAP) was transferred higher in the nuclei of the large and branched cells than other micropatterned groups. This study suggested that the spreading area and shape of individual MSCs regulate their viability and osteogenesis through the YAP pathway.

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).

Spreading Shape and Area Regulate the Osteogenesis of Mesenchymal Stem Cells.

Background: Mesenchymal stem cells (MSCs) have strong self-renewal ability and multiple differentiation potential. Some studies confirmed that spreading shape and area of single MSCs influence cell differentiation, but few studies focused on the effect of the circularity of cell shape on the osteogenic differentiation of MSCs with a confined area during osteogenic process. Methods: In the present study, MSCs were seeded on a micropatterned island with a spreading area lower than that of a freely spreading area. The patterns had circularities of 1.0 or 0.4, respectively, and areas of 314, 628, or 1256 µm2. After the cells were grown on a micropatterned surface for 1 or 3 days, cell apoptosis and F-actin were stained and analyzed. In addition, the expression of ß-catenin and three osteogenic differentiation markers were immunofluorescently stained and analyzed, respectively. Results: Of these MSCs, the ones with star-like shapes and large areas promoted the expression of osteogenic differentiation markers and the survival of cells. The expression of F-actin and its cytosolic distribution or orientation also correlated with the spreading shape and area. When actin polymerization was inhibited by cytochalasin D, the shape-regulated differentiation and apoptosis of MSCs with the confined spreading area were abolished. Conclusion: This study demonstrated that a spreading shape of low circularity and a larger spreading area are beneficial to the survival and osteogenic differentiation of individual MSCs, which may be regulated through the cytosolic expression and distribution of F-actin.

Calcium response in bone cells at different osteogenic stages under unidirectional or oscillatory flow.

It was found that preosteoblast MC3T3-E1 cells were less responsive in calcium signaling than mature osteocyte MLO-Y4 cells when a steady fluid flow was exerted on a micropatterned cell network. However, the effect of fluid flow on the calcium response in preosteocyte MLO-A5 was seldom investigated. In the present study, MLO-A5 as well as MC3T3-E1 and MLO-Y4 cells were cultured on a regular substrate with high or low density under unidirectional or oscillatory fluid flow. The results showed that calcium oscillation in the cells during late osteogenesis was significantly stronger than during early osteogenesis regardless of the fluid flow type or the presence of a physical cell-cell connection. Calcium oscillation produced by the oscillatory flow in the three types of cells was stronger than that produced by the unidirectional flow, but MC3T3-E1 and MLO-A5 cells exhibited limited potential for calcium oscillation compared with MLO-Y4 cells. After suramin was used to block the binding of extracellular adenosine triphosphate (ATP) to the membrane P2 receptor, the calcium oscillation in the three types of bone cells with or without physical connections was significantly suppressed as a single responsive peak under unidirectional flow. For the ATP-blocking group of low-density cells under oscillatory flow, the number of oscillation peaks in three types of cells was still more than two. It indicates that besides the ATP pathway, other mechanosensitive calcium pathways may exist under oscillatory flow. The present study provided further evidence for the osteogenic stage-dependent calcium response of bone cells under unidirectional or oscillatory fluid flow.

Size-matching hierarchical micropillar arrays for detecting circulating tumor cells in breast cancer patients' whole blood.

Circulating tumor cells (CTCs) are important markers for cancer diagnosis and treatment, but it is still a challenge to recognize and isolate CTCs because they are very rare in the blood. To selectively recognize CTCs and improve the capture efficiency, micro/nanostructured substrates have been fabricated for this application; however the size of CTCs is often ignored in designing and engineering micro/nanostructured substrates. Herein, a spiky polymer micropillar array is fabricated for capturing CTCs with high efficiency. The surface of the micropillar is cactus-like, and is composed of nanospikes. This hierarchical polymer array is designed according to the size of CTCs, which allows for more interactions of the CTCs with the array by setting the size of gaps among the micropillars to match with the CTCs. This polymer array is created by molding on an ordered silicon array, and then it is coated with an antiepithelial cell adhesion molecule antibody (anti-EpCAM). After co-culture with MCF-7 cells for 45 min, the capture efficiency of this array for CTCs is up to 91% ± 2%. Moreover, the anti-EpCAM modified polymer micropillar arrays present an excellent capacity to isolate CTCs from the whole blood samples of breast cancer patients. This study may provide a new concept for capturing target cells by designing and engineering micro/nanostructured substrates according to the size of target cells.

Extracts and components of Ficus carica leaves suppress survival, cell cycle, and migration of triple-negative breast cancer MDA-MB-231 cells.

BACKGROUND: Products from Ficus carica have been used in traditional medicine to treat many diseases. This study aimed to analyze anticancer effects of extracts of F. carica leaves on the triple-negative breast cancer cell line MDA-MB-231. MATERIALS AND METHODS: The human breast cancer cell line MDA-MB-231 was used to evaluate effects of F. carica extracts. Effects of F. carica on cell viability were evaluated using MTT assays. Cell-cycle distribution was examined using cell-cycle analysis. Wound-healing assays were used to evaluate migration of MDA-MB-231. Quantitative reverse-transcription polymerase chain reaction was used to detect levels of Bax, p53, p21, GATA3, ELF5, cyclin-dependent kinases, MMP2, and tissue inhibitors of metalloproteinase. RESULTS: We investigated the mechanism of anti-growth effects, and found that the expressions of genes that promote apoptosis were increased. In addition, the treated cells illustrated increased portion at S phase and changed expression of cyclin-dependent kinases, demonstrating cell-cycle arrest at the S phase. Furthermore, treated cells showed decreased cell mobility, which is essential for metastasis. Two of the active components of F. carica leaves, bergapten and psoralen, had similar anticancer effects as F. carica leaf extracts, indicating that these two components might play important roles in anticancer effects of F. carica leaves. CONCLUSION: Our findings suggest that F. carica leaves might be a good source to develop drugs for suppressing cancer-cell growth and migration to treat triple-negative breast cancers.

STIM1 and TRPV4 regulate fluid flow-induced calcium oscillation at early and late stages of osteoclast differentiation.

Bone resorption is mainly mediated by osteoclasts (OCs), whose formation and function are regulated by intracellular Ca2+ oscillation. Our previous studies demonstrated that fluid shear stress (FSS) lead to Ca2+ oscillation through mechanosensitive cation-selective channels. However, the specific channels responsible for this FSS-induced Ca2+ oscillation remain unknown. In the present study, we examined the expression of several Ca2+ channels in OCs, including STIM1, ORAI1, TRPV1, TRPV4, TRPV5, and TRPV6, by western blotting and reverse transcription-polymerase chain reaction. The results showed that STIM1 was highly expressed in early stage OCs, while TRPV4 was highly expressed in late stage OCs. We observed intracellular Ca2+ responses in OCs that were mechanically stimulated by FSS. When we blocked STIM1-dependent store-operated Ca2+ entry or inhibited TRPV4 using siRNA or drug inhibition, FSS-induced Ca2+ oscillations were almost undetectable in early and late stage OCs, respectively. These results indicate that STIM1 and TRPV4 act as mechanical transduction channels for OCs during the early and late differentiation stages, respectively, suggesting that these calcium channel could serve as markers of osteoclastogenesis or bone resorption.

EZH2 inhibits autophagic cell death of aortic vascular smooth muscle cells to affect aortic dissection.

Enhancer of zeste homolog 2 (EZH2), a methyltransferase that di- and tri-methylates lysine-27 of histone H3, largely functions as a transcriptional repressor, and plays a critical role in various kinds of cancers. Here we report a novel function of EZH2 in regulating autophagic cell death (ACD) of vascular smooth muscle cells (VSMCs) that affect aortic dissection (AD). Inhibition of EZH2 activity by UNC1999 or knockdown EZH2 resulted in VSMC loss, while overexpression of EZH2 facilitated VSMC growth, and these effects of EZH2 on VSMCs were independent of proliferation and apoptosis. Interestingly, more autophagic vacuoles and increased LC3II protein levels were identified in VSMCs with EZH2 inhibition or deficiency. Moreover, when compared with counterparts, chloroquine alone, or chloroquine with rapamycin treatment led to more LC3II accumulation in EZH2 inhibited or knockdown VSMCs, which indicated that EZH2 negatively regulated autophagosome formation. In conjunction to this, ATG5 and ATG7 protein levels were remarkably increased in EZH2 inhibited or deficient VSMCs, and ATG5 or ATG7 knockdown virtually rescued VSMC loss induced by EZH2 inhibition or knockdown. In addition, we found that the MEK-ERK1/2 signaling pathway, but not AMPKα, mTOR, or AKT pathway, is responsible for the impact of EZH2 on ACD of VSMCs. Additionally, the adverse effects of EZH2 inhibition or knockdown on VSMCs were largely reversed by PD98059, an inhibitor of MEK1. More importantly, decreased EZH2 expression levels in the aortic wall of patients with AD indicated its contribution to VSMC loss and AD occurrence. Overall, these findings revealed that EZH2 affects ACD of VSMCs and the pathologic process of AD via regulating ATG5 and ATG7 expression and MEK-ERK1/2 signaling. Our hitherto unrecognized findings indicate that EZH2 activation has therapeutic or preventive potential for AD.

Imbalance between Subpopulations of Regulatory T Cells in Patients with Acute Exacerbation of COPD.

Human regulatory T cells (Tregs) have been reported to be not significantly different in the peripheral blood of patients with chronic obstructive pulmonary disease (COPD) and healthy controls. Recent research has identified some new markers for Tregs and indicated that Tregs are composed of distinct subpopulations. The aim of the study was to describe the changing patterns of circulating Treg subpopulations in patients with acute exacerbation of COPD (AECOPD) and healthy controls, and to explore their potential roles in AECOPD pathogenesis. Blood samples were obtained from 30 never-smokers with normal lung function and 30 patients with COPD before and after they had an exacerbation. The proportions of Treg subpopulations were evaluated using flow cytometry. In the peripheral blood, decreased proportions of CD4+CD25+CD127low Tregs, CD4+CD25+CD45RA+ Tregs, and CD4+CD25+CD62L+ Tregs and an increased proportion of CD4+CD25+CD45RO+ Tregs were found in patients with stable COPD compared with non-smokers with normal lung function. The patients showed further changes in Treg subpopulations when they had an AECOPD, with an overall decrease in a suppressive subset, indicating that the immune negative regulatory population of Tregs did not play an effective role. Immune homeostasis favored inflammation, and a negative correlation between the circulating tumor necrosis factor-alpha and the proportions of CD4+CD25+CD62L+ cells (r = -0.698, p < 0.05) in patients with AECOPD was found. The imbalance between the suppressive subsets and the proinflammatory subset of Tregs and the decline of Treg subpopulations with immunosuppressive activity may play important roles in AECOPD progression.

LSD1 knockdown reveals novel histone lysine methylation in human breast cancer MCF-7 cells.

Histone lysine methylation, which plays an important role in the regulation of gene expression, genome stability, chromosome conformation and cell differentiation, is a dynamic process that is collaboratively regulated by lysine methyltransferases (KMTs) and lysine demethylases (KDMs). LSD1, the first identified KDMs, catalyzes the demethylation of mono- and di-methylated H3K4 and H3K9. Here, we systematically investigated the effects of LSD1 knockdown on histone methylations. Surprisingly, in addition to H3K4 and H3K9, the methylation level on other histone lysines, such as H3K27, H3K36 and H3K79, are also increased. The expression of SOX2, E-cadherin and FoxA2 are increased upon LSD1 knockdown, and the methylation level of H3K4, H3K27 and H3K36 in the promoter region of these genes are all changed after LSD1 knockdown. Our results show that LSD1 knockdown has a broad effect on histone lysine methylation, which indicates that LSD1 regulates histone lysine methylation in collaboration with other KMTs and KDMs.

LSD1 collaborates with EZH2 to regulate expression of interferon-stimulated genes.

Histone methylation is a complicate and dynamic epigenetic modification that regulates gene transcription, chromosomal structure and cell differentiation. Here, we discovered the interaction between the H3K4 demethylase, lysine specific demethylase 1 (LSD1, an important component of CoREST repressor complex) and the H3K27 methyltransferase, enhancer of zeste homolog 2 (EZH2, an essential component of PRC2). Immuno-precipitation and GST-pull down assay were performed to observe the interaction between the proteins. The MCF-7 cells were cultured and transfected with the siRNA. The mRNA and proteins were examined by using the real-time polymerase chain reaction (RT-PCR) and western blot assay, respectively. HPLC and LC-MS/MS analysis were performed to purify the proteins. RT-PCR-based quantitative ChIP analysis were performed. LSD1 interacts with histone modification protein EZH2 in MCF-7 cells. LSD1 and EZH2 target a few common genes. LSD1 knockdown and EZH2 knockdown affect protein expression. LSD1 knockdown and EZH2 knockdown affect the proteins involving in IFN signaling pathway. LSD1 and EZH2 modify histone methylation at IRF9 gene locus. We systematically analyzed the proteins that are affected by either LSD1 or EZH2 knockdown with proteomic approaches and identified that the interferon pathway and some other pathways are commonly affected. The interaction between LSD1 and EZH2 stabilizes the binding of LSD1 to the promoter region of IRF9, which is a key transcription factor of the interferon pathway. In conclusion, our study revealed that the coordination between histone demethylases and methyl-transferases might serve as a double lock system to suppress the expression of interferon stimulated genes.