Pregnancy outcome and follow-up of offspring of donor oocytes recipient from PCOS patients.

BACKGROUND: The use of donated oocytes (DO) for in vitro fertilization(IVF) treatment in patients with infertility is generally recognized, and females with polycystic ovarian syndrome (PCOS) can participate in oocyte donation programs as donor patients. However, the pregnancy outcomes and offspring follow-up in patients with PCOS as the recipients are unclear. This study was to compare the pregnancy outcomes and follow-up of offspring in PCOS and non-PCOS receptor. METHODS: This was a retrospective cohort study of 62 patients undergoing the oocyte reception program were separated into 2 groups: Group I, PCOS oocyte recipients (n = 30); Group II, non-PCOS recipients (n = 32). Medical records were reviewed, and rates of fertilization, cleavage, high-quality embryos and blastocysts were compared between PCOS and non-PCOS groups. Rates of implantation, pregnancy, ectopic pregnancy, early abortion, multiple pregnancies, and offspring outcomes were calculated using the first single vitrified-warmed blastocyst transfer (SVBT) analysis between the groups. RESULTS: The average recipient age and body mass index (BMI) of PCOS and non-PCOS patients was (36.3 ± 2.6 vs. 36.2 ± 2.8, and 23.4 ± 3.9 vs. 23.7 ± 4.0), respectively (P > 0.05). The fertilization, cleavage, high-quality embryos and blastocyst rates were not significantly different between the PCOS and non-PCOS groups. Rates of implantation, pregnancy, ectopic pregnancy, early abortion, and multiple pregnancies were not significantly different in SVBT between the PCOS and non-PCOS groups. The incidence of complications, such as pre-eclampsia or gestational diabetes, between PCOS and non-PCOS groups was similar (11.8% vs.11.1%, 5.9% vs.5.5%; P > 0.05). Preterm births were also similar (11.8% vs.16.7%, P > 0.05). Donor oocytes are more likely to be delivered via cesarean Sect. (80.0% vs. 86.7%: P > 0.05). The mean gestational age, birth weight, and height were comparable between the 2 groups during full-term delivery. CONCLUSION: There was no difference in the pregnancy outcomes and follow-up of the offspring between the PCOS and non-PCOS groups.

Accumulation of Smooth Muscle 22α Protein Accelerates Senescence of Vascular Smooth Muscle Cells via Stabilization of p53 In Vitro and In Vivo.

OBJECTIVE: Smooth muscle (SM) 22α, an actin-binding protein, displays an upregulated expression as a marker during cellular senescence. However, the causal relationship between SM22α and senescence is poorly understood. This study aimed to investigate the role of SM22α in angiotensin II (Ang II)-induced senescence of vascular smooth muscle cells (VSMCs). APPROACH AND RESULTS: We prepared a model of VSMC senescence induced by Ang II and found that the expression of SM22α in VSMCs was increased in response to chronic Ang II treatment. Overexpression of SM22α promoted Ang II-induced VSMC senescence, whereas knockdown of SM22α suppressed this process. Moreover, this effect of SM22α was p53 dependent. Increased SM22α protein obstructed ubiquitination and degradation of p53 and subsequently improved its stability. Furthermore, SM22α inhibited phosphorylation of Mdm2 (mouse double minute 2 homolog), an E3 ubiquitin-protein ligase, accompanied by a decreased interaction between Mdm2 and p53. Using LY294002, a PI3K/Akt inhibitor, we found that PI3K/Akt-mediated Mdm2 phosphorylation and activation was inhibited in senescent or SM22α-overexpressed VSMCs, in parallel with decreased p53 ubiquitination. We further found that SM22α inhibited activation of PI3K/Akt/Mdm2 pathway via strengthening actin cytoskeleton. In the in vivo study, we showed that the disruption of SM22α reduced the increase of blood pressure induced by Ang II, associated with decreased VSMC senescence through a mechanism similar to that in VSMCs in vitro. CONCLUSIONS: In conclusion, these findings suggest that the accumulation of SM22α promotes Ang II-induced senescence via the suppression of Mdm2-mediated ubiquitination and degradation of p53 in VSMCs in vitro and in vivo.

TRAF6-Mediated SM22α K21 Ubiquitination Promotes G6PD Activation and NADPH Production, Contributing to GSH Homeostasis and VSMC Survival In Vitro and In Vivo.

RATIONALE: Vascular smooth muscle cell (VSMC) survival under stressful conditions is integral to promoting vascular repair, but facilitates plaque stability during the development of atherosclerosis. The cytoskeleton-associated smooth muscle (SM) 22α protein is involved in the regulation of VSMC phenotypes, whereas the pentose phosphate pathway plays an essential role in cell proliferation through the production of dihydronicotinamide adenine dinucleotide phosphate. OBJECTIVE: To identify the relationship between dihydronicotinamide adenine dinucleotide phosphate production and SM22α activity in the development and progression of vascular diseases. METHODS AND RESULTS: We showed that the expression and activity of glucose-6-phosphate dehydrogenase (G6PD) are promoted in platelet-derived growth factor (PDGF)-BB-induced proliferative VSMCs. PDGF-BB induced G6PD membrane translocation and activation in an SM22α K21 ubiquitination-dependent manner. Specifically, the ubiquitinated SM22α interacted with G6PD and mediated G6PD membrane translocation. Furthermore, we found that tumor necrosis factor receptor-associated factor (TRAF) 6 mediated SM22α K21 ubiquitination in a K63-linked manner on PDGF-BB stimulation. Knockdown of TRAF6 decreased the membrane translocation and activity of G6PD, in parallel with reduced SM22α K21 ubiquitination. Elevated levels of activated G6PD consequent to PDGF-BB induction led to increased dihydronicotinamide adenine dinucleotide phosphate generation through stimulation of the pentose phosphate pathway, which enhanced VSMC viability and reduced apoptosis in vivo and in vitro via glutathione homeostasis. CONCLUSIONS: We provide evidence that TRAF6-induced SM22α ubiquitination maintains VSMC survival through increased G6PD activity and dihydronicotinamide adenine dinucleotide phosphate production. The TRAF6-SM22α-G6PD pathway is a novel mechanism underlying the association between glucose metabolism and VSMC survival, which is beneficial for vascular repair after injury but facilitates atherosclerotic plaque stability.

SM22α inhibits vascular inflammation via stabilization of IκBα in vascular smooth muscle cells.

Smooth muscle (SM) 22α, an actin-binding protein, is down-regulated in atherosclerotic arteries. Disruption of SM22α promotes arterial inflammation through activation of reactive oxygen species (ROS)-mediated nuclear factor (NF)-κB pathways. This study aimed to investigate the mechanisms by which SM22α regulates vascular inflammatory response. The ligation injury model of SM22α(-/-) mice displayed up-regulation of inflammatory molecules MCP-1, VCAM-1, and ICAM-1 in the carotid arteries. Similar results were discovered in human atherosclerotic samples. In vitro studies, overexpression of SM22α attenuated TNF-α-induced IκBα phosphorylation and degradation, accompanied by decreased NF-κB activity and reduced inflammatory molecule expression. Using coimmunoprecipitation, we found that SM22α interacted with and stabilized IκBα in quiescent VSMCs. Upon TNF-α stimulation, SM22α was phosphorylated by casein kinase (CK) II at Thr139, leading to dissociation of SM22α from IκBα, followed by IκBα degradation and NF-κB activation. Our findings demonstrate that SM22α is a phosphorylation-regulated suppressor of IKK-IκBα-NF-κB signaling cascades. SM22α may be a novel therapeutic target for human vascular diseases and other inflammatory conditions.

Phosphorylation of smooth muscle 22α facilitates angiotensin II-induced ROS production via activation of the PKCδ-P47phox axis through release of PKCδ and actin dynamics and is associated with hypertrophy and hyperplasia of vascular smooth muscle cells in vitro and in vivo.

RATIONALE: We have demonstrated that smooth muscle (SM) 22α inhibits cell proliferation via blocking Ras-ERK1/2 signaling in vascular smooth muscle cells (VSMCs) and in injured arteries. The recent study indicates that SM22α disruption can independently promote arterial inflammation through activation of reactive oxygen species (ROS)-mediated NF-κB pathways. However, the mechanisms by which SM22α controls ROS production have not been characterized. OBJECTIVE: To investigate how SM22α disruption promotes ROS production and to characterize the underlying mechanisms. METHODS AND RESULTS: ROS level was measured by dihydroethidium staining for superoxide and TBA assay for malondialdehyde, respectively. We showed that downregulation and phosphorylation of SM22α were associated with angiotensin (Ang) II-induced increase in ROS production in VSMCs of rats and human. Ang II induced the phosphorylation of SM22α at Serine 181 in an Ang II type 1 receptor-PKCδ pathway-dependent manner. Phosphorylated SM22α activated the protein kinase C (PKC)δ-p47phox axis via 2 distinct pathways: (1) disassociation of PKCδ from SM22α, and in turn binding to p47phox, in the early stage of Ang II stimulation; and (2) acceleration of SM22α degradation through ubiquitin-proteasome, enhancing PKCδ membrane translocation via induction of actin cytoskeletal dynamics in later oxidative stress. Inhibition of SM22α phosphorylation abolished the Ang II-activated PKCδ-p47phox axis and inhibited the hypertrophy and hyperplasia of VSMCs in vitro and in vivo, accompanied with reduction of ROS generation. CONCLUSIONS: These findings indicate that the disruption of SM22α plays pivotal roles in vascular oxidative stress. PKCδ-mediated SM22α phosphorylation is a novel link between actin cytoskeletal remodeling and oxidative stress and may be a potential target for the development of new therapeutics for cardiovascular diseases.

miR-146a and Krüppel-like factor 4 form a feedback loop to participate in vascular smooth muscle cell proliferation.

MicroRNAs are phenotypic regulators of vascular smooth muscle cells (VSMCs). In this paper, we demonstrate that miR-146a targets the Krüppel-like factor 4 (KLF4) 3'-untranslated region and has an important role in promoting VSMC proliferation in vitro and vascular neointimal hyperplasia in vivo. Silencing of miR-146a in VSMCs increases KLF4 expression, whereas overexpression of miR-146a decreases KLF4 levels. Furthermore, we demonstrate that KLF4 competes with Krüppel-like factor 5 (KLF5) to bind to and regulate the miR-146a promoter, and that KLF4 and KLF5 exert opposing effects on the miR-146a promoter. Overexpression of KLF4 in VSMCs decreases miR-146a transcription levels. By using both gain-of-function and loss-of-function approaches, we found that miR-146a promotes VSMC proliferation in vitro. Transfection of antisense miR-146a oligonucleotide into balloon-injured rat carotid arteries markedly decreased neointimal hyperplasia. These findings suggest that miR-146a and KLF4 form a feedback loop to regulate each other's expression and VSMC proliferation.

Comparing the pregnancy outcomes of Re­ICSI and ICSI embryos in fresh ET and FET cycles.

Early rescue intracytoplasmic sperm injection (Re-ICSI) can prevent total fertilization failure (TFF) during conventional in vitro fertilization (IVF). However, the implantation rate of Re-ICSI embryos is lower than that of direct ICSI during fresh embryo transfer (ET). The aim of the present study was to investigate the effect of frozen ET (FET) after Re-ICSI. In the present retrospective study, primary infertility patients that underwent the first Re-ICSI and ICSI treatment, were studied. The clinical pregnancy rate, implantation rate, ectopic pregnancy, abortion rate and live birth rate were analyzed between the Re-ICSI and ICSI groups in fresh ET and FET cycles. The average age of patients between Re-ICSI and ICSI groups in fresh ET and FET cycles was (29.0±3.2 vs. 29.1±3.1, and 29.1±3.3 vs. 28.9±3.0), respectively (P>0.05). Compared with ICSI embryos, the clinical pregnancy, implantation and live birth rates of Re-ICSI embryos were lower in fresh ET cycles. By contrast, there were no significant differences in the pregnancy, implantation and live birth rates between the Re-ICSI and ICSI embryos during the FET cycles. Re-ICSI coupled with FET may overcome the impaired outcomes in fresh ET.

KLF5 and hhLIM cooperatively promote proliferation of vascular smooth muscle cells.

Krüppel-like factor 5 (KLF5) plays an important role in cellular proliferation and differentiation. In this study, we show that adenovirus-mediated overexpression of KLF5 increased neointimal formation, while human heart LIM protein (hhLIM) decreased neointimal formation following vascular injury. Interestingly, neointimal formation was significantly increased in the animals where both hhLIM and KLF5 were introduced, suggesting that KLF5 can reverse hhLIM function in cell proliferation on the coexpression with hhLIM. These results were also confirmed the cellular level. Further mechanistic studies suggested that PDGF-BB promoted the interaction between hhLIM and KLF5 through stimulating hhLIM binding to TGF-ß control element (TCE) on the cyclin E promoter in a KLF5-dependent manner. Failure of KLF5 binding to the TCE, on the knockdown of KLF5 by transfecting siRNA, not only prevented the recruitment of hhLIM to the cyclin E promoter but also affected activation of the cyclin E promoter by KLF5. These data suggest that KLF5 reverses hhLIM function from anti-proliferation to pro-proliferation through its interaction with hhLIM on the cyclin E promoter.

Frozen blastocysts: Assessing the importance of day 5/day 6 blastocysts or blastocyst quality.

The aim of the present study was to analyze the high-quality blastocyst (HB) rate in all embryo frozen cycles and investigate the pregnancy outcomes for day 5/day 6 (D5/D6) blastocysts with respect to the blastocyst quality in programmed single vitrified-warmed blastocyst transfer (SVBT). We performed a retrospective study comparing D5/D6 HBs in in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) for all blastocyst frozen cycles. Patients were <35 years at the oocyte collection in their first fresh cycle without fresh transfer. A total of 1,560 IVF/ICSI cycles and 5,328 blastocysts were analyzed. The IVF HB rate was higher than that of ICSI (52.7% vs. 42.6%; P<0.05). The D5 HB rate was much higher than the D6 HB rate (61.6% vs. 29.4%; P<0.05). There were 22.4% (349/1,560) cycles that only had D6 blastocysts, of which IVF cycles were lower than ICSI (19.8% vs. 28.5%; P<0.05). The clinical pregnancy rate and implantation rate in the D5 group were significantly higher than these rates in the D6 group (57.4% vs. 46.2%, 58.9% vs. 47.3%; P<0.05). However, the clinical pregnancy rate and implantation rate of the D5 HBs were not significantly different from those of the D6 HBs (60% vs. 54.5%, 62% vs. 56.3%; P>0.05). In conclusion, the fertilization method (IVF/ICSI) directly influences the HB rate and blastocyst development rates. When we controlled for patient age, transfer frequency, and endometrium on day 5, it was not the development stage (D5/D6), rather the transfer blastocyst quality that played an important role in pregnancy outcomes.

Case report: Analysis of novel compound heterozygous TPP1 variants in a Chinese patient with neuronal ceroid lipofuscinosis type 2.

Neuronal ceroid lipofuscinosis type 2 (CLN2) is an autosomal recessive neurodegenerative disease caused by variants in the TPP1 gene that lead to the deficiency of the lysosomal enzyme tripeptidyl peptidase I (TPP1) activity. Herein, we report a rare case of CLN2 caused by two novel variants of TPP1. The patient presented with seizures at onset, followed by progressive cognitive impairment, motor decline, and vision loss. Novel compound heterozygous variants, c.544_545del and c.230-3C>G, in TPP1 were identified by whole-exome sequencing. The variant assessment showed that the c.544_545del is a frameshift variant mediating mRNA decay and that c.230-3C>G is a splice variant generating aberrantly spliced TPP1 mRNA, as confirmed by a Splicing Reporter Minigene assay. In conclusion, clinical history, variant assessment, and molecular analyses demonstrate that the novel compound heterozygous variants are responsible for CLN2 disease in this patient. This study expands the mutation spectrum of TPP1.

Glutamine as a Potential Noninvasive Biomarker for Human Embryo Selection.

To determine whether glutamine consumption is associated with embryo quality and aneuploidy, a retrospective study was conducted in an in vitro fertilization center. Spent embryo culture media from patients undergoing assisted reproduction treatment and preimplantation genetic testing (PGT) were obtained on day 3 of in vitro culture. Embryo quality was assessed for cell number and fragmentation rate. PGT for aneuploidy was performed using whole genome amplification and DNA sequencing. Glutamine levels in spent embryo culture media were analyzed by gas chromatography-mass spectrometry. The results demonstrated that glutamine was a primary contributor to the classification of the good-quality and poor-quality embryos based on the orthogonal partial least-squares discriminant analysis model. Glutamine consumption in the poor-quality embryos was significantly higher than that in the good-quality embryos (P < 0.05). A significant increase in glutamine consumption was observed from aneuploid embryos compared with that from euploid embryos (P < 0.01). The Pearson correlation coefficients between embryo quality and glutamine consumption, and between aneuploidy and glutamine consumption, were 0.430 and 0.757, respectively. The area under the ROC curve was 0.938 (95% CI: 0.902-0.975) for identifying aneuploidy. Animal experiments demonstrate that increased glutamine consumption may be a compensatory mechanism to mitigate oxidative stress. Our data suggest that glutamine consumption is associated with embryo quality and aneuploidy. Glutamine may serve as a molecular indicator for embryo assessment and aneuploidy testing.

Blockade of the Ras-extracellular signal-regulated kinase 1/2 pathway is involved in smooth muscle 22 alpha-mediated suppression of vascular smooth muscle cell proliferation and neointima hyperplasia.

OBJECTIVE: Vascular smooth muscle cells (VSMCs) can switch between differentiated and dedifferentiated phenotypes, and this phenotype switch is believed to be essential for repair of vascular injury. We studied the inhibitory effect of smooth muscle 22 alpha (SM22 alpha) on VSMC proliferation in vitro and in vivo and explored the potential molecular mechanisms of this effect. METHODS AND RESULTS: By using coimmunoprecipitation and glutathione S-transferase pull-down assays, we have shown that SM22 alpha binds to Ras in SM22 alpha-overexpressed VSMCs in the presence or absence of platelet-derived growth factor-BB stimulation. SM22 alpha arrested cell cycle progression through segregation of Ras with Raf-1 and downregulation of the Raf-1-MEK1/2-extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase signaling cascade. The inhibitory effect of SM22 alpha on VSMC proliferation was verified in vivo. The infection of rat carotid arteries with recombinant adenovirus encoding SM22 alpha inhibited neointimal hyperplasia via suppression of the Raf-1-MEK1/2-extracellular signal-regulated kinase 1/2 signaling pathway. CONCLUSIONS: These findings suggest that high expression of SM22 alpha inhibits cell proliferation via reduction of the response to mitogen stimuli in VSMCs and provide a novel mechanism by which VSMCs maintain their contractile phenotype and resist mitogenic stimuli in an SM22 alpha-dependent manner.

Flavonoids from Inula britannica reduces oxidative stress through inhibiting expression and phosphorylation of p47(phox) in VSMCs.

CONTEXT: Inula britanica Linn. (Compositae) is a traditional Chinese medicinal herb that has been used to treat bronchitis and inflammation. The total flavonoid extracts (TFEs) isolated from its flowers can inhibit neointimal formation induced by balloon injury in vivo. OBJECTIVE: To investigate the mechanism by which TFE suppresses oxidative stress generation and the subsequent inflammation response in vitro. MATERIALS AND METHODS: The cultured vascular smooth muscle cells (VSMCs) form rats were exposed to oxidative stress following pretreatment with or without TFE at different concentration. Then, fluorescence staining was used to detect superoxide anion (O2(˙-)) production, and the lever of maleic dialdehyde (MDA) and superoxide dismutase (SOD) was measured at the same time. Furthermore, tumor necrosis factor-α (TNF-α) was measured by enzyme linked immunosorbent assay (ELISA), reverse transcription-PCR and western blot were performed to detect the expression activity of p47(phox) gene, and immunoprecipitation was used to test the level of p47(phox) phosphorylation. RESULTS: TFE inhibited the production of O2(˙-) induced by H2O2 in VSMCs, with decrease in secretion of TNF-α; elevated the activity of SOD in the medium, similar to the effect of quercetin; reduced the level of MDA in culture medium of VSMCs. The pretreatment with TFE resulted in decrease the level of p47(phox) mRNA and protein, and even p47(phox) phosphorylation in VSMCs, compared with H2O2 control. DISCUSSION AND CONCLUSION: These findings demonstrate that TFE is capable of attenuating the oxidative stress generation and the subsequent inflammation response via preventing the overexpression and activation of p47(phox) and the increased TNF-α secretion in VSMCs in vitro.

Lower growth arrest-specific 5 level in endometrium is related to endometriosis via promoting cell proliferation and angiogenesis.

Long noncoding RNAs are a group of more than 200 nt, nonprotein coding RNAs, some of which are dysregulated in many pathophysiological processes including endometriosis. This study aims to clarify the roles of dysregulated growth arrest-specific 5 (GAS5) in patients with endometriosis, and unveil the underlying mechanisms. We obtained endometrium samples from 37 patients with endometriosis and 23 controls without endometriosis. Primary endometrial stromal cells (ESCs) and endothelial cells were separated from the endometrium. Levels of GAS5 were quantified using quantitative real-time polymerase chain reaction, and levels of p27, cleaved caspase-3, cleaved poly (ADP-Ribose) polymerase 1, vascular endothelial growth factor A, tissue inhibitor of metalloproteinases 3 (TIMP3), and trypsin-modified soy protein 10 were assessed by immunoblotting. Cell viability was examined using MTT assays, and the cell cycle and apoptosis were analyzed by flow cytometry. Endothelial cell tube formation capacity was assayed in vitro. GAS5 and p27 levels were found lower in the endometrium samples from patients with endometriosis. Primary ESCs from patients with endometriosis had increased viability, reduced apoptosis, and a relatively uncontrolled cell cycle. Gain- and loss-of-function studies confirmed that GAS5 regulated p27 expression in ESCs. Furthermore, GAS5 level was relatively low in primary endothelial cells from patients with endometriosis and GAS5 acted as an angiogenesis inhibitor by regulating the miR-181c-TIMP3 axis. Thus, lower GAS5 level in endometrium might be related to endometriosis by regulating cell proliferation, apoptosis, cell cycle, and angiogenesis.

Krüppel-like factor 4 inhibits proliferation by platelet-derived growth factor receptor beta-mediated, not by retinoic acid receptor alpha-mediated, phosphatidylinositol 3-kinase and ERK signaling in vascular smooth muscle cells.

Proliferation inhibition of vascular smooth muscle cells (VSMCs) is governed by the activity of a transcription factor network. Krüppel-like factor 4 (Klf4), retinoic acid receptor (RAR alpha), and platelet-derived growth factor receptor (PDGFR) are expressed in VSMCs and are components of such a network. However, the relationship among them in the regulation of VSMC proliferation remains unknown. Here, we investigated the mechanisms whereby Klf4 mediates the growth inhibitory effects in VSMCs through RAR alpha and PDGFR beta. We demonstrated that Klf4 directly binds to the 5' regulatory region of RAR alpha, down-regulates RAR alpha expression, and specifically inhibits RAR alpha-mediated phosphatidylinositol 3-kinase (PI3K) and ERK signaling in cultured VSMCs induced by the synthetic retinoid Am80. Of particular interest, Klf4 inhibits RAR alpha and PDGFR beta expression while blocking PI3K and ERK signaling induced by Am80 and PDGF-BB, respectively. The anti-proliferative effects of Klf4 on neointimal formation depend largely on PDGFR-mediated PI3K signaling without involvement of the RAR alpha-activated signaling pathways. These findings provide a novel mechanism for signal suppression and growth inhibitory effects of Klf4 in VSMCs. Moreover, the results of this study suggest that Klf4 is one of the key mediators of retinoid actions in VSMCs.

Smooth muscle SIRT1 reprograms endothelial cells to suppress angiogenesis after ischemia.

Objective: Vascular smooth muscle cells (VSMCs) undergo the phenotypic changes from contractile to synthetic state during vascular remodeling after ischemia. SIRT1 protects against stress-induced vascular remodeling via maintaining VSMC differentiated phenotype. However, the effect of smooth muscle SIRT1 on the functions of endothelial cells (ECs) has not been well clarified. Here, we explored the role of smooth muscle SIRT1 in endothelial angiogenesis after ischemia and the underlying mechanisms. Methods: We performed a femoral artery ligation model using VSMC specific human SIRT1 transgenic (SIRT1-Tg) and knockout (KO) mice. Angiogenesis was assessed in in vivo by quantification of the total number of capillaries, wound healing and matrigel plug assays, and in vitro ECs by tube formation, proliferation and migration assays. The interaction of HIF1α with circRNA was examined by using RNA immunoprecipitation, RNA pull-down and in situ hybridization assays. Results: The blood flow recovery was significantly attenuated in SIRT1-Tg mice, and markedly improved in SIRT1-Tg mice treated with SIRT1 inhibitor EX527 and in SIRT1-KO mice. The density of capillaries significantly decreased in the ischemic gastrocnemius of SIRT1-Tg mice compared with SIRT1-KO and WT mice, with reduced expression of VEGFA, which resulted in decreased number of arterioles. We identified that the phenotypic switching of SIRT1-Tg VSMCs was attenuated in response to hypoxia, with high levels of contractile proteins and reduced expression of the synthetic markers and NG2, compared with SIRT1-KO and WT VSMCs. Mechanistically, SIRT1-Tg VSMCs inhibited endothelial angiogenic activity induced by hypoxia via the exosome cZFP609. The cZFP609 was delivered into ECs, and detained HIF1α in the cytoplasm via its interaction with HIF1α, thereby inhibiting VEGFA expression and endothelial angiogenic functions. Meantime, the high cZFP609 expression was observed in the plasma of the patients with atherosclerotic or diabetic lower extremity peripheral artery disease, associated with reduced ankle-brachial index. Knockdown of cZFP609 improved blood flow recovery after hindlimb ischemia in SIRT1-Tg mice. Conclusions: Our findings demonstrate that SIRT1 may impair the plasticity of VSMCs. cZFP609 mediates VSMCs to reprogram endothelial functions, and serves as a valuable indicator to assess the prognosis and clinical outcomes of ischemic diseases.

Insulin-independent GLUT4 translocation in proliferative vascular smooth muscle cells involves SM22α.

The insulin-sensitive glucose transporter 4 (GLUT4) is a predominant facilitative glucose transporter in vascular smooth muscle cells (VSMCs) and is significantly upregulated in rabbit neointima. This study investigated the role of GLUT4 in VSMC proliferation, the cellular mechanism underlying PDGF-BB-stimulated GLUT4 translocation, and effects of SM22α, an actin-binding protein, on this process. Chronic treatment of VSMCs with PDGF-BB significantly elevated GLUT4 expression and glucose uptake. PDGF-BB-induced VSMC proliferation was dependent on GLUT4-mediated glucose uptake. Meanwhile, the response of GLUT4 to insulin decreased in PDGF-BB-stimulated VSMCs. PDGF-BB-induced GLUT4 translocation partially rescued glucose utilization in insulin-resistant cells. Immunofluorescence and western blot analysis revealed that PDGF-BB induced GLUT4 translocation in an actin dynamics-dependent manner. SM22α disruption facilitated GLUT4 translocation and glucose uptake by promoting actin dynamics and cortical actin polymerization. Similar results were observed in VSMCs of SM22α -/- mice. The in vivo experiments showed that the glucose level in the neointima induced by ligation was significantly increased in SM22α -/- mice, accompanied by increased neointimal thickness, compared with those in wild-type mice. These findings suggest that GLUT4-mediated glucose uptake is involved in VSMC proliferation, and provide a novel link between SM22α and glucose utilization in PDGF-BB-triggered proliferation. KEY MESSAGES: • GLUT4-mediated glucose uptake is required for the VSMC proliferation. • PDGF-BB-induced GLUT4 translocation partially rescues glucose uptake in insulin resistance. • SM22α disruption enhances PDGF-BB-induced GLUT4 translocation. • Glucose level in injured vascular tissue is positively correlated with neointimal hyperplasia.

CKII-SIRT1-SM22α loop evokes a self-limited inflammatory response in vascular smooth muscle cells.

AIMS: Sirtuin 1 (SIRT1) inhibits nuclear factor kappa B (NF-κB) activity in response to the inflammatory cytokine tumour necrosis factor alpha (TNF-α). Smooth muscle (SM) 22α is a phosphorylation-regulated suppressor of IKK-IκBα-NF-κB signalling cascades in vascular smooth muscle cells (VSMCs). Sm22α knockout results in increased expression of pro-inflammatory genes in the aortas which are controlled by NF-κB. This study aimed to investigate the relationship between SM22α and SIRT1 in the control of vascular inflammation. METHODS AND RESULTS: The ligation injury model of Sirt1-Tg/Sm22α-/- mice displayed an increased level of the inflammatory molecules in the carotid arteries compared with Sirt1-Tg mice, accompanied with aggravating neointimal hyperplasia. In the in vitro study, on the one hand, we showed that TNF-α induced the epigenetic silencing of SM22α transcription via EZH2-mediated H3K27 methylation in the SM22α promoter region, contributing to inflammatory response. On the other hand, TNF-α simultaneously induced SIRT1 phosphorylation via CKII and thereby protected against inflammation. Phosphorylated SIRT1 interacted with and deacetylated EZH2 and, subsequently, promoted SM22α transcription by inhibiting EZH2 activity. Increased SM22α in turn facilitated the phosphorylation and activation of SIRT1 via recruitment of CKII to SIRT1, which amplified the anti-inflammatory effect of SIRT1. CONCLUSION: Our findings demonstrate that, in response to TNF-α stimulation, CKII-SIRT1-SM22α acts in a loop to reinforce the expression of SM22α, which limits the inflammatory response in VSMCs in vivo and in vitro. The anti-inflammatory effect of SIRT1 may be dependent on SM22α to some extent. Our data point to targeted activation of SIRT1 in VSMCs as a promising therapeutic avenue in preventing cardiovascular diseases.

Smooth muscle 22α facilitates angiotensin II-induced signaling and vascular contraction.

UNLABELLED: Smooth muscle 22α (SM22α) is involved in stress fiber formation and enhances contractility in vascular smooth muscle cells (VSMCs). In many cases, SM22α acts as an adapter protein to assemble signaling complexes and regulate signaling, but whether SM22α regulates contractile signaling induced by angiotensin II (AngII) remains unclear. To address this issue, we established a hypertension model of Sm22α(-/-) mice, and demonstrated that hypertension induced by AngII was attenuated in Sm22α(-/-) mice. A decreased vasoconstriction was observed in aortic rings from Sm22α(-/-) mice. Furthermore, loss of SM22α resulted in a reduced contractile response to AngII in VSMCs in vitro. The phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) induced by AngII was impaired following depletion of SM22α, in parallel with a reduced contractility. The decay of ERK1/2 activity was associated with increased expression of mitogen-activated protein kinase phosphatase 3 (MKP3). Inhibition of MKP3 activity rescued ERK1/2 activity. SM22α depletion caused an enhanced interaction of MKP3 with ERK1/2, and a reduced ubiquitination and degradation of MKP3. Knockdown of SM22α extended the half-life of MKP3. In conclusion, SM22α promotes AngII-induced contraction by maintenance of ERK1/2 signaling cascades through facilitating ubiquitination and degradation of MKP3. KEY MESSAGE: The vasoconstriction is attenuated in aortic rings from Sm22α(-/-) mice. MKP3 mediates dephosphorylation of ERK1/2 in AngII-induced VSMC contraction. SM22α inhibits the interaction of ERK1/2 with MKP3. SM22α promotes ubiquitination and degradation of MKP3. SM22α facilitates AngII-induced contraction by maintenance of ERK1/2 signaling.