Effects of Decamethylcyclopentasiloxane on Reproductive Systems in Female Rats.

The female reproductive system becomes fertile through the action of hormones involved in the hypothalamic-pituitary-ovarian axis. On the other hand, estrogen-like endocrine disruptors released into the environment come into contact with humans by various routes and affect the reproductive system. Exposure to these chemicals can cause problems with the reproductive process, from egg ovulation to implantation, or cause female reproductive diseases. These reproductive problems cause infertility. Decamethylcyclopentasiloxane (D5) is used for lubrication in silicone polymers, households, and personal care products. In the case of D5, it is discharged through factory wastewater and can bioaccumulate. Therefore, it accumulates in the human body. In this study, D5 was administered orally for four weeks to determine the effects of D5 on the reproductive process. As a result, D5 increases the number of follicles in the ovary and suppresses the expression of genes related to the growth of follicles. In addition, it increases the gonadotropin hormone, inducing estradiol enhancement and progesterone reduction. Because of these changes in the reproductive system when exposed to D5, the industry should reconsider using D5.

Effects of Sodium Arsenite on the Myocardial Differentiation in Mouse Embryonic Bodies.

Arsenic in inorganic form is a known human carcinogen; even low levels of arsenic can interfere with the endocrine system. In mammalian development, arsenic exposure can cause a malformation of fetuses and be lethal. This study examined the effects of sodium arsenite (SA) as the inorganic form of arsenic in embryonic bodies (EBs) with three germ layers in the developmental stage. This condition is closer to the physiological condition than a 2D cell culture. The SA treatment inhibited EBs from differentiating into cardiomyocytes. A treatment with 1 µM SA delayed the initiation of beating, presenting successful cardiomyocyte differentiation. In particular, mitochondria function analysis showed that SA downregulated the transcription level of the Complex IV gene. SA increased the fission form of mitochondrion identified by the mitochondria number and length. In addition, a treatment with D-penicillamine, an arsenic chelator, restored the beat of EBs against SA, but not mitochondrial dysfunction. These findings suggest that SA is a toxicant that induces mitochondrial damage and interferes with myocardial differentiation and embryogenesis. This study suggests that more awareness of SA exposure during pregnancy is required because even minuscule amounts have irreversible adverse effects on embryogenesis through mitochondria dysfunction.

Pre-validation of an alternative test method for prediction of developmental neurotoxicity.

Exposure to neurodevelopmental toxicants can cause permanent brain injury. Hance, determining the neurotoxicity of unknown substances is essential for the safety of substance. As an alternative method to animal studies, developmental neurotoxicity test (DNT) and the first discriminant function (DF) were established in previous study. This study aimed to increase the predictability of the DNT method and perform a mobility test. Two endpoints of 29 newly investigated substances were used to establish a second-generation DF (2nd GDF). As two endpoints, the half-inhibitory concentration of the cell viability (IC50) was determined using a cell counting kit-8 assay. The half-inhibitory concentration of differentiation (ID50) was determined by measuring the green fluorescent protein (GFP) intensity in 46C cells. The substances were treated dose-dependently to measure IC50 and ID50. The 2nd GDF classified 29 chemicals accurately as toxic and non-toxic. Four participants of three independent laboratories were enrolled to test the mobility. The results of the test set were highly accurate in reproducibility (100% of accuracy, sensitivity, and specificity) and mobility (accuracy 93.33%, sensitivity 90.91%, and specificity 100%). In conclusion, the protocol is transferable, reproducible, and accurate. Therefore, this could be a standardizing method for determining a neurotoxicant as an alternative for animal experiments.

Endocrine-Disrupting Chemicals and Their Adverse Effects on the Endoplasmic Reticulum.

There is growing concern regarding the health and safety issues of endocrine-disrupting chemicals (EDCs). Long-term exposure to EDCs has serious adverse health effects through both hormone-direct and hormone-indirect ways. Accordingly, some EDCs can be a pathogen and an inducer to the susceptibility of disease, even if they have a very low affinity on the estrogen receptor, or no estrogenic effect. Endoplasmic reticulum (ER) stress recently attracted attention in this research area. Because ER and ER stress could be key regulators of the EDC's adverse effects, such as the malfunction of the organ, as well as the death, apoptosis, and proliferation of a cell. In this review, we focused on finding evidence which shows that EDCs could be a trigger for ER stress and provide specific examples of EDCs, which are known to cause ER stress currently.

In Vivo Stimulation of α- and ß-Adrenoceptors in Mice Differentially Alters Small RNA Content of Circulating Extracellular Vesicles.

When myocardial function is compromised as in heart failure (HF), there is activation of the sympathetic nervous system with elevated circulating catecholamine levels. These catecholamines activate cardiac and extra-cardiac adrenergic receptors (ARs). Interest in secreted extracellular vesicles (EVs) from the heart is growing and in HF, it is not known whether excessive activation of α- or ß-adrenergic receptors (ARs) could induce specific changes in EV content. In this study, we have evaluated, by next generation sequencing, the small RNA content, including micro-RNAs (miRs), of circulating EVs of mice exposed to chronic selective α- or ß- AR stimulation. EVs from mouse blood were purified by differential ultracentrifugation resulting in EVs with an average size of 116.6 ± 4.8 nm that by immunoblotting included protein markers of EVs. We identified the presence of miRs in blood EVs using miR-21-5p and -16-5p real-time PCR as known constituents of blood exosomes that make up a portion of EVs. We next performed next generation sequencing (NGS) of small non-coding RNAs found in blood EVs from mice following 7 days of chronic treatment with isoproterenol (ISO) or phenylephrine (PE) to stimulate α- or ß-ARs, respectively. PE increased the percent of genomic repeat region reads and decreased the percent of miR reads. In miR expression analysis, PE and ISO displayed specific patterns of miR expression that suggests differential pathway regulation. The top 20 KEGG pathways predicted by differential expressed miRs show that PE and ISO share 11 of 20 pathways analyzed and reveal also key differences including three synapse relative pathways induced by ISO relative to PE treatment. Both α-and ß-AR agonists can alter small RNA content of circulating blood EVs/exosomes including differential expression and loading of miRs that indicate regulation of distinct pathways. This study provides novel insight into chronic sympathetic nervous system activation in HF where excessive catecholamines may not only participate in pathological remodeling of the heart but alter other organs due to secretion of EVs with altered miR content.

Characterization of ßARKct engineered cellular extracellular vesicles and model specific cardioprotection.

Recent data supporting any benefit of stem cell therapy for ischemic heart disease have suggested paracrine-based mechanisms via extracellular vesicles (EVs) including exosomes. We have previously engineered cardiac-derived progenitor cells (CDCs) to express a peptide inhibitor, ßARKct, of G protein-coupled receptor kinase 2, leading to improvements in cell proliferation, survival, and metabolism. In this study, we tested whether ßARKct-CDC EVs would be efficacious when applied to stressed myocytes in vitro and in vivo. When isolated EVs from ßARKct-CDCs and control GFP-CDCs were added to cardiomyocytes in culture, they both protected against hypoxia-induced apoptosis. We tested whether these EVs could protect the mouse heart in vivo, following exposure either to myocardial infarction (MI) or acute catecholamine toxicity. Both types of EVs significantly protected against ischemic injury and improved cardiac function after MI compared with mice treated with EVs from mouse embryonic fibroblasts; however, ßARKct EVs treated mice did display some unique beneficial properties including significantly altered pro- and anti-inflammatory cytokines. Importantly, in a catecholamine toxicity model of heart failure (HF), myocardial injections of ßARKct-containing EVs were superior at preventing HF compared with control EVs, and this catecholamine toxicity protection was recapitulated in vitro. Therefore, introduction of the ßARKct into cellular EVs can have improved reparative properties in the heart especially against catecholamine damage, which is significant as sympathetic nervous system activity is increased in HF.NEW & NOTEWORTHY ßARKct, the peptide inhibitor of GRK2, improves survival and metabolic functions of cardiac-derived progenitor cells. As any benefit of stem cells in the ischemic and injured heart suggests paracrine mechanisms via secreted EVs, we investigated whether CDC-ßARKct engineered EVs would show any benefit over control CDC-EVs. Compared with control EVs, ßARKct-containing EVs displayed some unique beneficial properties that may be due to altered pro- and anti-inflammatory cytokines within the vesicles.

Fimasartan for Remodeling after Myocardial Infarction.

An angiotensin receptor blocker (ARB) mitigates cardiac remodeling after myocardial infarction (MI). Here, we investigated the effect of fimasartan, a new ARB, on cardiac remodeling after MI. Sprague⁻Dawley rats were assigned into 3 groups: surgery only (sham group, n = 7), MI without (MI-only group, n = 13), and MI with fimasartan treatment (MI + Fima group, n = 16). MI was induced by the permanent ligation of the left anterior descending artery. Treatment with fimasartan (10 mg/kg) was initiated 24 h after MI and continued for 7 weeks. Rats in the MI + Fima group had a higher mean ejection fraction (66.3 ± 12.5% vs. 51.3 ± 14.8%, P = 0.002) and lower left ventricular end-diastolic diameter (9.14 ± 1.11 mm vs. 9.91 ± 1.43 mm, P = 0.045) than those in the MI-only group at 7 weeks after MI. The infarct size was lower in the MI + Fima than in the MI group (P < 0.05). A microarray analysis revealed that the expression of genes related to the lipid metabolism and mitochondrial membrane ion transporters were upregulated, and those involved in fibrosis and inflammation were downregulated by fimasartan. Fimasartan attenuates cardiac remodeling and dysfunction in rats after MI and may prevent the progression to heart failure after MI.

Fibroblast Growth Factor 12 Is a Novel Regulator of Vascular Smooth Muscle Cell Plasticity and Fate.

OBJECTIVE: Vascular smooth muscle cells (VSMCs) modulate their phenotype between synthetic and contractile states in response to environmental changes; this modulation plays a crucial role in the pathogenesis of restenosis and atherosclerosis. Here, we identified fibroblast growth factor 12 (FGF12) as a novel key regulator of the VSMC phenotype switch. APPROACH AND RESULTS: Using murine models and human specimens, we found that FGF12 was highly expressed in contractile VSMCs of normal vessel walls but was downregulated in synthetic VSMCs from injured and atherosclerotic vessels. In human VSMCs, FGF12 expression was inhibited at the transcriptional level by platelet-derived growth factor-BB. Gain- and loss-of-function experiments showed that FGF12 was both necessary and sufficient for inducing and maintaining the quiescent and contractile phenotypes of VSMCs. FGF12 inhibited cell proliferation through the p53 pathway and upregulated the key factors involved in VSMC lineage differentiation, such as myocardin and serum response factor. Such FGF12-induced phenotypic change was mediated by the p38 MAPK (mitogen-activated protein kinase) pathway. Moreover, FGF12 promoted the differentiation of mouse embryonic stem cells and the transdifferentiation of human dermal fibroblasts into SMC-like cells. Furthermore, adenoviral infection of FGF12 substantially decreased neointima hyperplasia in a rat carotid artery injury model. CONCLUSIONS: In general, FGF family members induce a synthetic VSMC phenotype. Interestingly, the present study showed the unanticipated finding that FGF12 belonging to FGF family, strongly induced the quiescent and contractile VSMC phenotypes and directly promoted VSMC lineage differentiation. These novel findings suggested that FGF12 could be a new therapeutic target for treating restenosis and atherosclerosis.

The microRNA miR-34c inhibits vascular smooth muscle cell proliferation and neointimal hyperplasia by targeting stem cell factor.

The fine balance between proliferation and differentiation of vascular smooth muscle cells (VSMCs) is indispensable for the maintenance of healthy blood vessels, whereas an increase in proliferation participates in pathologic cardiovascular events such as atherosclerosis and restenosis. Here we report that microRNA-34c (miR-34c) targets stem cell factor (SCF) to inhibit VSMC proliferation and neointimal hyperplasia. In an animal model, miR-34c was significantly increased in the rat carotid artery after catheter injury. Transient transfection of miR-34c to either VSMCs or A10 cells inhibited cell survival by inducing apoptosis, which was accompanied by an increase in expression of p21, p27, and Bax. Transfection of miR-34c also attenuated VSMC migration. Bioinformatics showed that SCF is a target candidate of miR-34c. miR-34c down-regulated luciferase activity driven by a vector containing the 3'-untranslated region of SCF in a sequence-specific manner. Forced expression of SCF in A10 cells induced proliferation and migration, whereas knocking-down of SCF reduced cell survival and migration. miR-34c antagomir-induced VSMC proliferation was blocked by SCF siRNA. Delivery of miR-34c to rat carotid artery attenuated the expression of SCF and blocked neointimal hyperplasia. These results suggest that miR-34c is a new modulator of VSMC proliferation and that it inhibits neointima formation by regulating SCF.

miR-18a-5p MicroRNA Increases Vascular Smooth Muscle Cell Differentiation by Downregulating Syndecan4.

BACKGROUND AND OBJECTIVES: Differentiation and de-differentiation of vascular smooth muscle cells (VSMCs) are important events in atherosclerosis and restenosis after angioplasty. MicroRNAs are considered a key regulator in cellular processes such as differentiation, proliferation, and apoptosis. Here, we report the role of new miR-18a-5p microRNA and its downstream target genes in VSMCs and in a carotid balloon injury model. MATERIALS AND METHODS: Expression of miR-18a-5p and its candidate genes was examined in VSMCs and in a carotid artery injury model by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and microRNA microarray analysis. VSMC differentiation marker genes including smooth muscle (SM) α-actin and SM22α were determined by Western blot, qRT-PCR, and a SM22α promoter study. Gene overexpression or knockdown was performed in VSMCs. RESULTS: miR-18a-5p was upregulated in the rat carotid artery at the early time after balloon injury. Transfection of the miR-18a-5p mimic promoted the VSMC differentiation markers SM α-actin and SM22α. In addition, miR-18a-5p expression was induced in differentiated VSMCs, whereas it decreased in de-differentiated VSMCs. We identified syndecan4 as a downstream target of miR-18-5p in VSMCs. Overexpression of syndecan4 decreased Smad2 expression, whereas knockdown of syndecan4 increased Smad2 expression in VSMCs. Finally, we showed that Smad2 induced the expression of VSMC differentiation marker genes in VSMCs. CONCLUSION: These results indicate that miR-18a-5p is involved in VSMC differentiation by targeting syndecan4.

Regulation of MMP/TIMP by HUVEC transplantation attenuates ventricular remodeling in response to myocardial infarction.

AIMS: We elucidated the therapeutic potential of human umbilical vein endothelial cells (HUVECs) for ameliorating progressive heart failure in a myocardial infarction (MI) rat model. MAIN METHODS: MI was induced by ligation of left anterior descending artery, and HUVEC was transplanted 1week after MI. Cardiac function was evaluated by echocardiography, and histological analyses were performed. KEY FINDINGS: Phosphate-buffered saline (MI-V, n=5) or HUVEC (MI-HV, n=5) were injected into the border zone and infarcted area 7days after ligation of the left coronary artery in rats. The MI-HV group showed attenuation of left ventricular (LV) remodeling compared with the MI-V group. In the infarcted myocardium, a few of injected HUVEC was retained up to 28days. The ratios of matrix metalloproteinase (MMP)-2 or MMP-9 to tissue inhibitor of metalloproteinase (TIMP)-1 or TIMP-3 were decreased in the MI-HV group compared with the MI-V group. In vivo zymography analysis showed that HUVEC transplantation decreased the activities of MMP-2 and MMP-9. In immunohistochemistry, decreased MMP-2 and increased TIMP-1 and TIMP-3 expression were observed at 48h after HUVEC transplantation. These effects on MMP/TIMP balance were inhibited by L-NAME administration (an eNOS inhibitor, 10mg/kg). NOS inhibition decreased the protein expressions of TIMP-1 and TIMP-3 but did not change the protein expressions of MMP-2 and MMP-9. SIGNIFICANCE: Our data suggest that altered balance between MMP and TIMP by HUVEC transplantation contributed to attenuation of ventricular remodeling after MI via eNOS.

Protective role of 5-azacytidine on myocardial infarction is associated with modulation of macrophage phenotype and inhibition of fibrosis.

We examined whether a shift in macrophage phenotype could be therapeutic for myocardial infarction (MI). The mouse macrophage cell line RAW264.7 was stimulated with peptidoglycan (PGN), with or without 5-azacytidine (5AZ) treatment. MI was induced by ligation of the left anterior descending coronary artery in rats, and the rats were divided into two groups; a saline-injection group and a 5AZ-injection group (2.5 mg/kg/day, intraperitoneal injection). LV function was evaluated and immunohistochemical analyses were performed 2 weeks after MI. Cardiac fibrosis was induced by angiotensin II (AngII) infusion with or without 5AZ (5 mg/kg/day) in mice. Nitric oxide was produced by PGN, which was reduced by 77.87% after 5AZ treatment. Both induction of inducible nitric oxide synthase (iNOS) and iNOS promoter activity by PGN were inhibited by 5AZ. Ejection fraction (59.00 ± 8.03% versus 42.52 ± 2.58%), contractility (LV dP/dt-max, 8299.76 ± 411.56 mmHg versus 6610.36 ± 282.37 mmHg) and relaxation indices (LV dP/dt-min, -4661.37 ± 210.73 mmHg versus -4219.50 ± 162.98 mmHg) were improved after 5AZ administration. Cardiac fibrosis in the MI+5AZ was 8.14 ± 1.00%, compared with 14.93 ± 2.98% in the MI group (P < 0.05). Arginase-1(+)CD68(+) macrophages with anti-inflammatory phenotype were predominant in the infarct border zone of the MI+5AZ group, in comparison with the MI group. AngII-induced cardiac fibrosis was also attenuated after 5AZ administration. In cardiac fibroblasts, pro-fibrotic mediators and cell proliferation were increased by AngII, and these increases were attenuated after 5AZ treatment. 5AZ exerts its cardiac protective role through modulation of macrophages and cardiac fibroblasts.

Restoration of angiogenic capacity of diabetes-insulted mesenchymal stem cells by oxytocin.

BACKGROUND: Angiogenesis is the main therapeutic mechanism of cell therapy for cardiovascular diseases, but diabetes is reported to reduce the function and number of progenitor cells. Therefore, we studied the effect of streptozotocin-induced diabetes on the bone marrow-mesenchymal stem cell (MSC) function, and examined whether diabetes-impaired MSC could be rescued by pretreatment with oxytocin. RESULTS: MSCs were isolated and cultured from diabetic (DM) or non-diabetic (non-DM) rat, and proliferation rate was compared. DM-MSC was pretreated with oxytocin and compared with non-DM-MSC. Angiogenic capacity was estimated by tube formation and Matrigel plug assay, and therapeutic efficacy was studied in rat myocardial infarction (MI) model.The proliferation and angiogenic activity of DM-MSC were severely impaired but significantly improved by pretreatment with oxytocin. Krüppel-like factor 2 (KLF2), a critical angiogenic factor, was dramatically reduced in DM-MSC and significantly restored by oxytocin. In the Matrigel plug assay, vessel formation of DM-BMSCs was attenuated but was recovered by oxytocin. In rat MI model, DM-MSC injection did not ameliorate cardiac injury, whereas oxytocin-pretreated DM-MSC improved cardiac function and reduced fibrosis. CONCLUSIONS: Our results show that diabetes influenced MSC by reducing angiogenic capacity and therapeutic potential. We demonstrate the striking effect of oxytocin on stem cell dysfunction and suggest the use of oxytocin as a priming reagent in autologous stem cell therapy.

The microRNA miR-132 targets Lrrfip1 to block vascular smooth muscle cell proliferation and neointimal hyperplasia.

OBJECTIVE: The proliferation and remodeling of vascular smooth muscle cells (VSMCs) is an important pathological event in atherosclerosis and restenosis. Here we report that microRNA-132 (miR-132) blocks vascular smooth muscle cells (VSMC) proliferation by inhibiting the expression of LRRFIP1 [leucine-rich repeat (in Flightless 1) interacting protein-1]. METHODS AND RESULTS: MicroRNA microarray revealed that miR-132 was upregulated in the rat carotid artery after catheter injury, which was further confirmed by quantitative real-time RT-PCR. Transfection of a miR-132 mimic significantly inhibited the proliferation of VSMCs, whereas transfection of a miR-132 antagomir increased it. miR-132 mimic inhibited VSMC migration and induced apoptosis. miR-132 mimic increased the protein amounts of both p27 and smooth muscle (SM) α-actin, whereas it decreased SM α-actin and Bcl2. Bioinformatics showed that LRRFIP1 is a target candidate of miR-132. miR-132 down-regulated luciferase activity driven by a vector containing the 3'-untranslated region of Lrrfip1 in a sequence-specific manner. LRRFIP1 induced VSMC proliferation and increased phosphorylation of ERK. Immunohistochemical analysis revealed that Lrrfip1 was clearly expressed along with the basal laminar area of smooth muscle, and its expression pattern was disrupted 7 days after arterial injury. LRRFIP1 mRNA was decreased 14 days after injury. Delivery of miR-132 to rat carotid artery reduced LRRFIP1 expression and attenuated neointimal proliferation in carotid artery injury models. CONCLUSIONS: Our results suggest that miR-132 is a novel regulator of VSMC proliferation that represses neointimal formation by inhibiting LRRFIP1 expression.

B cell translocation gene, a direct target of miR-142-5p, inhibits vascular smooth muscle cell proliferation by down-regulating cell cycle progression.

Vascular smooth muscle cell (VSMC) proliferation plays a key role in neointimal hyperplasia and restenosis. Here we report the role of the microRNA miR-142-5p and its downstream target genes on the proliferation of cultured VSMCs. miR-142-5p promoted VSMC proliferation by down-regulating B cell translocation gene 3 (BTG3). We found that BTG3 inhibited the expression of cell cycle regulatory genes and cell growth. As shown by luciferase reporter assay, miR-142-5p bound directly to the 3'-untranslated region of BTG3. Overexpression of miR-142-5p induced expression of cell cycle regulatory genes. Thus, BTG3, a novel, direct target of miR-142-5p, negatively regulates VSMC proliferation.

Cilostazol protects vessels against hyperglycemic injury and accelerates healing after implantation of drug-eluting stent in a type 1 diabetes mellitus rat aorta stent model.

OBJECTIVE: Cilostazol, a selective phosphodiesterase-3 (PDE-3) inhibitor, can effectively suppress platelet activation and attenuate the increase in carotid intima-media thickness in diabetes mellitus (DM) patients. Therefore, we investigated whether cilostazol had effects on the healing process after implantation of a drug-eluting stent (DES) in a rat model of type 1 DM. METHODS AND RESULTS: Streptozotocin-induced DM rats were divided into 2 groups in which cilostazol (30 mg/kg/day; DM-Cilostazol) or vehicle (DM-Vehicle) was orally administered. Age-matched rats treated with the vehicle were used as a control group (NDM-Vehicle). After 4 weeks, cilostazol changed the expression of vascular cell adhesion molecule and intercellular adhesion molecule and the apoptotic cell ratio of the media (DM-Vehicle: 53.5 ± 9.8%, DM-Cilostazol: 26.4 ± 8.3%, p < 0.05) in the aortic wall. Also, in a modified aortic ring test, cilostazol preserved the angiogenic potential of the aorta ([height of the sprouting tubes] DM-Vehicle: 0 ± 0 µm, DM-Cilostazol: 344.6 ± 236.8 µm, p < 0.05). After implantation of paclitaxel-eluting stents (PES) in rats treated with cilostazol or vehicle, thrombus formation, deposition of fibrin, and infiltration of inflammatory cells were attenuated by cilostazol. In particular, the re-endothelialization by von Willebrand factor expression in the DM-PES-Cilostazol group was enhanced compared with that in the DM-PES-Vehicle group. CONCLUSION: Cilostazol has potential for protecting vessels against hyperglycemic injury and for accelerating the healing process after implantation of DES.

Sulforaphane inhibits restenosis by suppressing inflammation and the proliferation of vascular smooth muscle cells.

OBJECTIVE: Sulforaphane, a naturally occurring organosulfur compound in broccoli, has chemopreventive properties in cancer. However, the effects of sulforaphane in vascular diseases have not been examined. We therefore aimed to investigate the effects of sulforaphane on vascular smooth muscle cell (VSMC) proliferation and neointimal formation and the related mechanisms. METHODS: The expression of vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) was examined in VSMCs. The nuclear translocation of nuclear factor-κB (NF-κB) and GATA6 expression was examined in VSMCs and in a carotid artery injury model by Western blot and immunohistochemistry. We also investigated whether local delivery of sulforaphane affected neointimal formation. RESULTS: Sulforaphane inhibited the mRNA and protein expression of VCAM-1 induced by tumor necrosis factor (TNF)-α in VSMCs. Treatment of VSMCs with sulforaphane blocked TNF-α-induced IκBα degradation and NF-κB p65 and GATA6 expression. Furthermore, NF-κB p65 and GATA6 expression were reduced in sulforaphane-treated carotid injury sections. Notably, binding of GATA6 to the VCAM-1 promoter was dramatically reduced by sulforaphane. The MTT, BrdU incorporation, and in vitro scratch assays revealed that the proliferation and migration of VSMCs were reduced by sulforaphane. Furthermore, local administration of sulforaphane significantly reduced neointima formation 14 days after vascular injury in rats. CONCLUSIONS: Our results indicate that sulforaphane inhibits neointima formation via targeting of adhesion molecules through the suppression of NF-κB/GATA6. Furthermore, sulforaphane regulates migration and proliferation in VSMCs. Sulforaphane may be a potential therapeutic agent for preventing restenosis after vascular injury.

Curcumin reduces the cardiac ischemia-reperfusion injury: involvement of the toll-like receptor 2 in cardiomyocytes.

Curcumin, a polyphenolic compound derived from turmeric, has protective effects on myocardial injury through attenuation of oxidative stress and inflammation. Toll-like receptor 2 (TLR2), a key mediator of the innate immune system, is involved in myocardial infarction and examined if controlled by curcumin. Rat cardiomyocytes (CMs) were stimulated with tumor necrosis factor (TNF)-α, peptidoglycan (PGN) or hypoxia/reoxygenation (H/R) with or without curcumin pretreatment. Sprague-Dawley rats were fed curcumin (300 mg/kg/day) 1 week before cardiac ischemia/reperfusion (I/R) injury. The expression level of TLR2 and cardiac function were assessed. Both mRNA and protein of TLR2 were up-regulated in infarcted myocardium, while TLR4 remained unchanged. In CMs, TLR2 and monocyte chemoattractant protein (MCP)-1 mRNAs were increased by TNF-α, PGN or H/R, whereas they were blunted by curcumin. Immunofluorescence staining of CMs also showed that TLR2 and MCP-1 were increased after H/R, whereas curcumin-pretreated CMs were not. In animal study, 2 weeks after I/R, TLR2 was increased in the infarct zone, whereas it stayed unchanged in the Cur+I/R group. Macrophage infiltration (CD68), high-mobility group box 1 and fibrosis were increased in the I/R group, whereas they were decreased in the Cur+I/R group. Connexin 43 was reduced in the I/R group, while it recovered significantly in the Cur+I/R group. Cardiac contractility in the Cur+I/R group was also improved compared with that in the I/R group (max dp/dt in Cur+I/R group: 9660±612 vs. I/R group: 8119±366, P<.05). These results suggest that selective inhibition of TLR2 by curcumin could be preventive and therapeutic for myocardial infarction.

Enhancer of polycomb1 lessens neointima formation by potentiation of myocardin-induced smooth muscle differentiation.

OBJECTIVE: Previously, we reported that enhancer of polycomb1 (Epc1) induces skeletal muscle differentiation through the serum response factor (SRF). Considering that SRF plays a critical role in vascular smooth muscle cell (VSMC) differentiation, we expected that Epc1 also works in VSMCs. Here we examined the effect of Epc1 on neointima formation after arterial balloon injury and the underlying mechanism. METHODS: Epc1 expression was examined in carotid artery injury or VSMC models. Interaction with myocardin (Myocd), a master regulator of smooth muscle differentiation, was examined by immunoprecipitation or promoter analysis with smooth muscle (SM) 22α promoter. Finally, we investigated whether local delivery of Epc1 regulated neointimal formation after injury. RESULTS: Epc1 expression was down-regulated during proliferation induced by platelet-derived growth factor BB, whereas it was upregulated during differentiation in VSMCs. Forced expression of Epc1 induced VSMC differentiation. Epc1 physically interacted with Myocd to synergistically activate SM22α promoter activity. Transient transfection of Epc1 enhanced the physical interaction between Myocd and SRF, whereas that interaction was reduced when A10 cells were treated with siRNA for Epc1. Local delivery of Epc1 significantly reduced neointima formation induced by balloon injury. CONCLUSIONS: Our results indicate that Epc1 induces VSMC differentiation by interacting with Myocd to induce SRF-dependent smooth muscle genes. We propose that Epc1 acts as a novel negative regulator of neointima formation after carotid injury.