Inhibition of CIRBP represses the proliferation and migration of vascular smooth muscle cells via inhibiting Rheb/mTORC1 axis.

The excessive migration and proliferation of vascular smooth muscle cells (VSMCs) plays a vital role in vascular intimal hyperplasia. CIRBP is involved in the proliferation of various cancer cells. This study was aimed to explore the role of CIRBP in the proliferation and migration of VSMCs. Adenovirus was used to interfere with cold-inducible RNA-binding protein (CIRBP) expression, while lentivirus was used to overexpress Ras homolog enriched in brain (Rheb). Western blotting and qRT-PCR were used to evaluate the expression of CIRBP, Rheb, and mechanistic target of rapamycin complex 1 (mTORC1) activity. The cell proliferation was determined by Ki67 immunofluorescence staining and CCK-8 assay. The wound healing assay was performed to assess cell migration. Additionally, immunohistochemistry was conducted to explore the role of CIRBP in intimal hyperplasia after vascular injury. We found that silencing CIRBP inhibited the proliferation and migration of VSMCs, decreased the expression of Rheb and mTORC1 activity. Restoration of mTORC1 activity via insulin or overexpression of Rheb via lentiviral transfection both attenuated the inhibitory effects of silencing CIRBP on the proliferation and migration of VSMCs. Moreover, Rheb overexpression abolished the inhibitory effect of silencing CIRBP on mTORC1 activity in VSMCs. CIRBP was upregulated in the injured carotid artery. Silencing CIRBP ameliorated intimal hyperplasia after vascular injury. In the summary, silencing CIRBP attenuates mTORC1 activity via reducing Rheb expression, thereby supressing the proliferation and migration of VSMCs and intimal hyperplasia after vascular injury.

Role of Nuclear Receptor Subfamily 1 Group D Member 1 in the Proliferation, Migration of Vascular Smooth Muscle Cell, and Vascular Intimal Hyperplasia.

ABSTRACT: Excessive proliferation and migration of vascular smooth muscle cells (VSMCs) cause neointimal hyperplasia after percutaneous vascular interventions. Nuclear receptor subfamily 1 group D member 1 (NR1D1), a crucial member of circadian clock, is involved in the regulation of atherosclerosis and cellular proliferation. However, whether NR1D1 affects vascular neointimal hyperplasia remains unclear. In this study, we found that activating NR1D1 reduced injury-induced vascular neointimal hyperplasia. Overexpression of NR1D1 reduced the number of Ki-67-positive VSMCs and migrated VSMCs after platelet-derived growth factor (PDGF)-BB treatment. Mechanistically, NR1D1 suppressed the phosphorylation of AKT and 2 main effectors of the mammalian target of rapamycin complex 1 (mTORC1), S6, and 4EBP1 in PDGF-BB-challenged VSMCs. Re-activation of mTORC1 by Tuberous sclerosis 1 siRNA (si Tsc1 ) and re-activation of AKT by SC-79 abolished NR1D1-mediated inhibitory effects on proliferation and migration of VSMCs. Moreover, decreased mTORC1 activity induced by NR1D1 was also reversed by SC-79. Simultaneously, Tsc1 knockdown abolished the vascular protective effects of NR1D1 in vivo. In conclusion, NR1D1 reduces vascular neointimal hyperplasia by suppressing proliferation and migration of VSMCs in an AKT/mTORC1-dependent manner.

Nuclear receptor subfamily 1 group D member 1 suppresses the proliferation, migration of adventitial fibroblasts, and vascular intimal hyperplasia via mammalian target of rapamycin complex 1/ß-catenin pathway.

BACKGROUND: In-stent restenosis hardly limits the therapeutic effect of the percutaneous vascular intervention. Although the restenosis is significantly ameliorated after the application of new drug-eluting stents, the incidence of restenosis remains at a high level. OBJECTIVE: Vascular adventitial fibroblasts (AFs) play an important role in intimal hyperplasia and subsequent restenosis. The current study was aimed to investigate the role of nuclear receptor subfamily 1, group D, member 1 (NR1D1) in the vascular intimal hyperplasia. METHODS AND RESULTS: We observed increased expression of NR1D1 after the transduction of adenovirus carrying Nr1d1 gene (Ad-Nr1d1) in AFs. Ad-Nr1d1 transduction significantly reduced the numbers of total AFs, Ki-67-positive AFs, and the migration rate of AFs. NR1D1 overexpression decreased the expression level of ß-catenin and attenuated the phosphorylation of the effectors of mammalian target of rapamycin complex 1 (mTORC1), including mammalian target of rapamycin (mTOR) and 4E binding protein 1 (4EBP1). Restoration of ß-catenin by SKL2001 abolished the inhibitory effects of NR1D1 overexpression on the proliferation and migration of AFs. Surprisingly, the restoration of mTORC1 activity by insulin could also reverse the decreased expression of ß-catenin, attenuated proliferation, and migration in AFs induced by NR1D1 overexpression. In vivo, we found that SR9009 (an agonist of NR1D1) ameliorated the intimal hyperplasia at days 28 after injury of carotid artery. We further observed that SR9009 attenuated the increased Ki-67-positive AFs, an essential part of vascular restenosis at days 7 after injury to the carotid artery. CONCLUSION: These data suggest that NR1D1 inhibits intimal hyperplasia by suppressing the proliferation and migration of AFs in a mTORC1/ß-catenin-dependent manner.

Inhibition of PIKfyve Ameliorates the Proliferation and Migration of Vascular Smooth Muscle Cells and Vascular Intima Hyperplasia By Reducing mTORC1 Activity.

ABSTRACT: This study was designed to investigate the role and mechanism of PIKfyve in the proliferation and migration of vascular smooth muscle cells (VSMCs) and vascular intima hyperplasia. We first observed increased protein levels of PIKfyve, phospho (p)-S6 Ribosomal Protein (S6)Ser235/236, p-4EBP1Thr37/46 in VSMCs after 24 hours of platelet-derived growth factor (PDGF)-BB treatment. By using cell counting kit-8 assay, Ki-67 immunofluorescence staining and wound healing assay, we found that PIKfyve inhibition ameliorated the enhanced activity of VSMC proliferation and migration induced by PDGF-BB. Silencing PIKfyve also suppressed the phosphorylation of S6 and 4EBP1 (2 major effectors of mammalian target of rapamycin complex 1), glucose consumption, activity of hexokinase, and LDH in PDGF-BB-challenged VSMCs. After rescuing the phosphorylation of S6 and 4EBP1 by silencing Tsc1, the suppressive effects of PIKfyve inhibition on glucose utilization, proliferation, and migration in VSMCs were abolished. The animal model of vascular restenosis was established in C57BL/6J mice by wire injury. We found the expression of PIKfyve was increased in carotid artery at day 28 after injury. Reducing the activity of PIKfyve alleviated vascular neointima hyperplasia after injury. In conclusion, targeting PIKfyve might be a novel effective method to reduce the proliferation and migration of VSMCs and vascular restenosis by affecting mammalian target of rapamycin complex 1-mediated glucose utilization.

TRPV1 Protect against Hyperglycemia and Hyperlipidemia Induced Liver Injury via OPA1 in Diabetes.

Type 2 diabetes mellitus (T2DM)-associated mitochondrial impairment may a key factor leading to liver injury. Transient receptor potential receptor vanilloid 1 (TRPV1) regulates the energy expenditure and cholesterol metabolism in hepatocytes and protects against oxidative toxicity. Optic atrophy 1 (OPA1) is involved in the protection of TRPV1 on cardiac microvascular and lung injury. The aim of this study is to identify the role of TRPV1 in redox signals and liver protection via OPA1. TRPV1 knockout (TRPV1-/-) mice were used. And T2DM associated liver injury was induced by high glucose and high fatty acid (HG/HF) treatment. Mechanisms were studied by TUNEL staining, transmission electron microscope (TEM) analysis, reverse transcription polymerase chain reaction (RT-PCR) and Western blotting in vivo and in vitro. We determined that HG/HF treatment increased TRPV1 expression in liver tissues and AML12 cells. The knockout of TRPV1 increased the apoptotic hepatocytes rate. The inhibition of TRPV1 by 5'-iRTX in HG/HF group elevated the reactive oxygen species (ROS) levels, whereas TRPV1 agonist capsaicin reduced ROS. Our studies also showed that the OPA1 expression was lower in livers from HG/HF treated mice than the control, and genetic ablation of TRPV1 decreased OPA1 expression to a greater extent than the HG/HF mice. The protective effects of TRPV1 on mitochondrial were blocked by OPA1 siRNA. In conclusion, our study showed that the identified regulation of TRPV1 to OPA1 has important implication to the pathogenesis of T2DM-associated liver injury. Targeting the action of TRPV1 and OPA1 presents a potential therapeutic intervention.

Inhibition of GPR35 Preserves Mitochondrial Function After Myocardial Infarction by Targeting Calpain 1/2.

Ischemia and anoxia-induced mitochondrial impairment may be a key factor leading to heart injury during myocardial infarction (MI). Calpain 1 and 2 are involved in the MI-induced mitochondria injury. G protein-coupled receptor 35 (GPR35) could be triggered by hypoxia. Whether or not GPR35 regulates calpain 1/2 in the pathogenesis of MI is still unclear. In this study, we determined that MI increases GPR35 expression in myocardial tissue. Suppression of GPR35 protects heart from MI injury in mice through reduction of reactive oxygen species activity and mitochondria-dependent apoptosis. Further studies show that GPR35 regulates calpain 1/2. Suppression of GPR35 reduces the expression and activity of calpain 1/2, and alleviates calpain 1/2-associated mitochondrial injury to preserve cardiac function. Based on these data, we conclude that a functional inhibition of GPR35 downregulates calpain 1/2 and contributes to maintenance of cardiac function under pathologic conditions with mitochondrial disorder. In conclusion, our study showed that the identified regulation by GPR35 of calpain 1/2 has important implications for the pathogenesis of MI. Targeting the action of GPR35 and calpain 1/2 in mitochondria presents a potential therapeutic intervention for MI.

Role of polypyrimidine tract-binding protein 1/yin yang 2 signaling in regulating vascular smooth muscle cell proliferation and neointima hyperplasia.

Abnormal proliferation of vascular smooth muscle cells (VSMCs) is a hallmark of vascular restenosis. We investigated whether polypyrimidine tract-binding protein 1 (PTBP1), a novel regulator of cell proliferation and differentiation, is implicated in VSMC proliferation and neointima hyperplasia responding to injury. C57BL/6 J mice of 10-12 weeks old were randomly divided into sham and carotid artery injury group. Primary VSMCs obtained from thoracic aortas of 10- to 12-week-old mice were treated with physiological saline and platelet derived growth factor-BB (PDGF-BB). Adenovirus expressing shCon, shPTBP1 or shYY2 were transfected into the injured common carotid artery or VSMCs. qRT-PCR and immunoblotting were used to determine the mRNA and protein expression levels, respectively. Immunohistochemical staining of H&E and Ki-67 were used to evaluate restenosis of vessels. Cell counting kit-8 assay and Ki-67 immunofluorescent staining were utilized to evaluate the rate of VSMC proliferation. The expression of PTBP1 were upregulated both in injured arteries and in PDGF-BB-treated VSMCs. PTBP1 inhibition significantly attenuated neointima hyperplasia and Ki-67 positive area induced by injury. Knockdown of PTBP1 in vitro also suppressed VSMC proliferation after PDGF-BB treatment. The effects of PTBP1 inhibition mentioned above were all abolished by knockdown of YY2. Finally, we identified four cell cycle regulators (p53, p21, Cdkn1c, Cdkn2b) that were regulated by PTBP1/YY2 axis both in vitro and in vivo. These findings demonstrated that PTBP1 is a critical regulator of VSMC proliferation and neointima hyperplasia via modulating the expression of YY2.

Cardiac resynchronization therapy improves myocardial conduction.

BACKGROUND: Cardiac resynchronization therapy (CRT) reverses left ventricular remodeling and improves left ventricular systolic function. However, little is known about whether CRT improves ventricular conduction. OBJECTIVE: To determine the relationship between CRT response and QRS narrowing. METHODS: The study included consecutive patients who underwent CRT with defibrillator (CRT-D) implantation between January 2002 and December 2012 and had subsequent generator replacement (GR). At the time of GR, super-responder was defined as those who had left ventricular ejection fraction (LVEF) of 50% or more; patients who had an LVEF of 36-49% with an increase of more than 5% were considered responders, and the others were nonresponders. All patients were assessed 6 months after CRT-D implantation and at the time of GR. RESULTS: Of 114 study patients, 58 (50.9%) were nonresponders, 29 (25.4%) responders, and 27 (23.7%) super-responders. QRS narrowing at 6 months was significant in super-responders (175.4 ± 21.4 to 159.7 ± 20.7 ms, P = 0.001) and responders (169.0 ± 21.3 to 157.7 ± 17.6 ms, P = 0.008). At time of GR, only super-responders had further QRS narrowing (159.7 ± 20.7 to 146.3 ± 19.2 ms, P < 0.001). QRS duration change after 6 months was independently associated with super-response (hazard ratio: 1.20 for every 5 ms QRS duration narrowing; 95% confidence interval, 1.06-1.38; P = 0.005), and QRS narrowing of 10 ms or more was associated with lower risk of all-cause mortality after GR. CONCLUSION: Continuous QRS narrowing after CRT, as an electrical marker, is associated with a super-response to CRT. Further QRS narrowing 6 months after CRT implantation was associated with lower risk of mortality after GR.

Overexpression of SARAF Ameliorates Pressure Overload-Induced Cardiac Hypertrophy Through Suppressing STIM1-Orai1 in Mice.

BACKGROUND/AIMS: Activation of stromal interaction molecule 1 (STIM1) and Orai1 participates in the development of cardiac hypertrophy. Store-operated Ca2+ entry-associated regulatory factor (SARAF) is an intrinsic inhibitor of STIM1-Orai1 interaction. Thus, we hypothesized that SARAF could prevent cardiac hypertrophy. METHODS: Male C57BL/6 mice, aged 8 weeks, were randomly divided into sham and abdominal aortic constriction surgery groups and were infected with lentiviruses expressing SARAF and GFP (Lenti-SARAF) or GFP alone (Lenti-GFP) via intramyocardial injection. At 4 weeks after aortic constriction, left ventricular structure and function were assessed by echocardiography and hemodynamic assays. The gene and protein expressions of SARAF, STIM1, and Orai1 were measured by quantitative PCR and Western blot, respectively. RESULTS: Gene and protein expressions of SARAF were significantly decreased, while STIM1 and Orai1 were increased in the heart tissue compared with sham group. Overexpression of SARAF in the heart prevented the upregulation of STIM1 and Orai1, and importantly, attenuated aortic constriction-induced decrease in maximal rate of left ventricular pressure decay and increases in thickness of interventricular septum and left ventricular posterior wall, heart weight/body weight ratio, and size of cardiomyocytes. Blood pressure detected through the carotid artery and left ventricular systolic function were not affected by SARAF overexpression. In addition, overexpression of SARAF also attenuated angiotensin II-induced upregulation of STIM1 and Orai1 and hypertrophy of cultured cardiomyocytes. CONCLUSION: Overexpression of SARAF in the heart prevents cardiac hypertrophy, probably through suppressing the upregulation of STIM1/Orai1.

Activation of transient receptor potential vanilloid 1 accelerates re-endothelialization and inhibits neointimal formation after vascular injury.

OBJECTIVE: Transient receptor potential vanilloid 1 (TRPV1) is an important regulator of endothelial function, but the effects of TRPV1 on endothelial recovery and neointimal formation after vascular injury remain elusive. We tested the effects of activating TRPV1 using capsaicin on re-endothelialization and neointimal formation after wire-induced injury of the carotid artery in mice. METHODS: The human umbilical vein endothelial cells (HUVECs) were treated with the TRPV1 agonist capsaicin, its antagonist capsazepine, intracellular calcium chelator BAPTA, or mitofusin 2 (Mfn2)-specific short interfering RNA (siRNA). The migration, proliferation, mitochondrial morphology, membrane potential, and adenosine triphosphate production were measured. The carotid artery wire injury procedure was performed in male TRPV1 knockout mice and C57BL/6J wild-type (WT) mice that were then treated with or without Mfn2 siRNA. The re-endothelialization and neointimal formation were evaluated. RESULTS: Capsaicin significantly enhanced the migration and proliferation of HUVECs. Both capsazepine and BAPTA abolished capsaicin-induced migration and proliferation of HUVECs. In addition, capsaicin stimulated the formation of reticular mitochondria, augmented mitochondrial membrane potential, increased adenosine triphosphate production, and upregulated Mfn2. However, these effects were attenuated by knockdown of Mfn2 with specific siRNA. Dietary capsaicin markedly accelerated re-endothelialization and inhibited neointimal formation in WT mice but not in TRPV1 knockout mice. Moreover, Mfn2 siRNA also attenuated capsaicin-induced enhancement of endothelial recovery and suppression of neointimal hyperplasia in WT mice. CONCLUSIONS: Activation of TRPV1 with capsaicin attenuates neointimal formation by accelerating re-endothelialization through upregulation of Mfn2.

Clinical Outcomes of Cardiac Resynchronization with Epicardial Left Ventricular Lead.

BACKGROUND: Left ventricular (LV) pacing in cardiac resynchronization therapy (CRT) can be achieved via a transvenous or epicardial route. A surgically implanted epicardial LV (eLV) lead is used after a standard transvenous LV (tLV) lead implantation has failed. However, studies of clinical outcomes in patients with eLV leads and comparisons of outcome between tLV and eLV-CRT are sparse. Therefore, the purpose of this study is to compare clinical response between tLV-CRT and eLV-CRT, as well as to understand the differences within the eLV-CRT population. METHODS: Forty-four patients received eLV-CRT following unsuccessful attempts of tLV-CRT implantation between 2002 and 2013 at the University of California, San Diego (UCSD) and Mayo Clinics. These patients were matched for age, gender, and etiology of cardiomyopathy in a 1:2 ratio with a cohort of patients who received tLV-CRT during the same time period. RESULTS: During a mean follow-up of 57 months, similar clinical outcomes and survival rate were noted between tLV and eLV-CRT patients (all P > 0.05). Within the eLV-CRT group, dilated cardiomyopathy patients had significant improvement in New York Heart Association class and ejection fraction (both P < 0.05), while ischemic cardiomyopathy patients did not (both P > 0.05). eLV-CRT patients with nonanterior lead location had significantly improved survival (P < 0.001). There was also a trend for improved survival in those with nonapical lead location (P = 0.09). CONCLUSION: In this case-matched two-centered study, comparable improvements were noted in patients with tLV-CRT and eLV-CRT. Operators should target nonanterior and nonapical locations during eLV-CRT implantation. Use of eLV-CRT should be considered a viable alternative for CRT candidates.

Anisotropic hardy spaces of Musielak-Orlicz type with applications to boundedness of sublinear operators.

Let φ : ℝ(n) × [0, ∞)→[0, ∞) be a Musielak-Orlicz function and A an expansive dilation. In this paper, the authors introduce the anisotropic Hardy space of Musielak-Orlicz type, H(A)(φ)(ℝ(n)), via the grand maximal function. The authors then obtain some real-variable characterizations of H(A)(φ)(ℝ(n)) in terms of the radial, the nontangential, and the tangential maximal functions, which generalize the known results on the anisotropic Hardy space H(A)(p) (ℝ(n)) with p ∈ (0,1] and are new even for its weighted variant. Finally, the authors characterize these spaces by anisotropic atomic decompositions. The authors also obtain the finite atomic decomposition characterization of H(A)(φ)(ℝ(n)), and, as an application, the authors prove that, for a given admissible triplet (φ, q, s), if T is a sublinear operator and maps all (φ, q, s)-atoms with q < ∞ (or all continuous (φ, q, s)-atoms with q = ∞) into uniformly bounded elements of some quasi-Banach spaces ℬ, then T uniquely extends to a bounded sublinear operator from H(A)(φ)(ℝ(n)) to ℬ. These results are new even for anisotropic Orlicz-Hardy spaces on ℝ(n).

MTMR7 suppresses the phenotypic switching of vascular smooth muscle cell and vascular intimal hyperplasia after injury via regulating p62/mTORC1-mediated glucose metabolism.

BACKGROUND AND AIMS: Myotubularin-related protein 7 (MTMR7) suppresses proliferation in various cell types and is associated with cardiovascular and cerebrovascular diseases. However, whether MTMR7 regulates vascular smooth muscle cell (VSMC) and vascular intimal hyperplasia remains unclear. We explored the role of MTMR7 in phenotypic switching of VSMC and vascular intimal hyperplasia after injury. METHODS AND RESULTS: MTMR7 expression was significantly downregulated in injured arteries. Compared to wild type (WT) mice, Mtmr7-transgenic (Mtmr7-Tg) mice showed reduced intima/media ratio, decreased percentage of Ki-67-positive cells within neointima, and increased Calponin expression in injured artery. In vitro, upregulating MTMR7 by Len-Mtmr7 transfection inhibited platelet derived growth factor (PDGF)-BB-induced proliferation, migration of VSMC and reversed PDGF-BB-induced decrease in expression of Calponin and SM-MHC. Microarray, single cell sequence, and other bioinformatics analysis revealed that MTMR7 is highly related to glucose metabolism and mammalian target of rapamycin complex 1 (mTORC1). Further experiments confirmed that MTMR7 markedly repressed glycolysis and mTORC1 activity in PDGF-BB-challenged VSMC in vitro. Restoring mTORC1 activity abolished MTMR7-mediated suppression of glycolysis, phenotypic shift in VSMC in vitro and protection against vascular intimal hyperplasia in vivo. Furthermore, upregulating MTMR7 in vitro led to dephosphorylation and dissociation of p62 from mTORC1 in VSMC. External expression of p62 in vitro also abrogated the inhibitory effects of MTMR7 on glycolysis and phenotypic switching in PDGF-BB-stimulated VSMC. CONCLUSIONS: Our study demonstrates that MTMR7 inhibits injury-induced vascular intimal hyperplasia and phenotypic switching of VSMC. Mechanistically, the beneficial effects of MTMR7 are conducted via suppressing p62/mTORC1-mediated glycolysis.

Acipimox attenuates atherosclerosis and enhances plaque stability in ApoE-deficient mice fed a palmitate-rich diet.

Saturated fatty acids (FA) have been linked to an increased risk of cardiovascular disease. The effects of acipimox, a FA-lowering agent, on palmitate- (an important saturated fatty acid) stimulated atherosclerosis remains to be elucidated. We investigated the effects of acipimox on atherosclerosis in ApoE(-/-) mice fed a palmitate-rich diet. Male ApoE(-/-) mice, 6-8 weeks of age, were randomized into three groups. The animals were fed a normal chow diet in the control group, a diet containing 5% palmitic acid in the palmitate group, and a diet containing 5% palmitic acid and 0.02% acipimox in the acipimox group. The plasma lipid profiles, aortic lesions, plaque collagen content and the expression of matrix metalloproteinase (MMP)-2, MMP-3, MMP-9, and MMP-14 and the tissue inhibitor of MMP (TIMP)-1, and TIMP-2 were determined after a 12-week treatment. The palmitate-rich diet significantly increased plasma FA concentrations (P<0.01), enhanced atherosclerotic lesions (P<0.01), decreased plaque collagen content (P<0.01) and upregulated MMP-2 (P<0.05) in the aorta. Additionally, all of these harmful effects were significantly attenuated by co-treatment with acipimox (P<0.05 or P<0.01). The present study suggests that acipimox attenuates atherosclerosis and enhances plaque stability in ApoE(-/-) mice fed a palmitate-rich diet.

Inhibition of uncoupling protein 2 with genipin exacerbates palmitate-induced hepatic steatosis.

BACKGROUND: Uncoupling protein 2 (UCP2) was reported to be involved in lipid metabolism through regulating the production of superoxide anion. However, the role of UCP2 in hepatocytes steatosis has not been determined. We hypothesized that UCP2 might regulate hepatic steatosis via suppressing oxidative stress. RESULTS: We tested this hypothesis in an in vitro model of hepatocytic steatosis in HepG2 cell lines induced by palmitic acid (PA). We found that treatment with PA induced an obvious lipid accumulation in HepG2 cells and a significant increase in intracellular triglyceride content. Moreover, the specific inhibition of UCP2 by genipin remarkably exacerbated PA-induced hepatocytes steatosis. Interestingly, the PA-induced superoxide overproduction can also be enhanced by incubation with genipin. In addition, administration with the antioxidant tempol abolished genipin-induced increase in intracellular lipid deposition. We further found that genipin significantly increased the protein expression of fatty acid translocase (FAT)/CD36. CONCLUSIONS: These findings suggest that UCP2 plays a protective role in PA-induced hepatocytic steatosis through ameliorating oxidative stress.

Positive regulation of endothelial Tom70 by metformin as a new mechanism against cardiac microvascular injury in diabetes.

Microvascular protection is the main mechanism of metformin against diabetic complications. Cardiac microvascular endothelial cells (CMECs) are the basic component of cardiac microvessels, and they suffer from oxidative stress and mitochondrial dysfunction under type 2 diabetes mellitus (T2DM). Translocase of the outer mitochondrial membrane 70 (Tom70) improves mitochondrial dysfunction, but its role in the hearts of T2DM patients remains unclear. The purpose of this study was to demonstrate the protective effect of metformin on diabetic cardiac microvascular injury and to identify the role of Tom70 in this effect. T2DM mice were established by multiple intraperitoneal injections of low-dose streptozotocin and 12-week high-fat feeding. CMECs were isolated and cultured with normal glucose (NG), high glucose (HG), and HG plus high fat (HG-HF) media. The results indicated that long-term metformin treatment partly reversed cardiovascular complication and mitigated cardiac microvascular injury in T2DM. In addition, exposure to HG-HF led to CMEC damage, aggravated oxidative stress, aggravated mitochondrial dysfunction, and reduced mitochondrial Tom70 expression, whereas upregulation of Tom70 significantly ameliorated these injuries. Furthermore, metformin treatment promoted Tom70 expression and effectively reversed CMEC injury induced by HG-HF. However, all of these effects were interrupted after Tom70 was knocked down. In conclusion, T2DM damages microvascular integrity by activating a cycle of decreased Tom70 expression, mitochondrial dysfunction, and reactive oxygen species (ROS) overload in CMECs. However, metformin suppresses oxidative stress, relieves mitochondrial dysfunction, and promotes the expression of Tom70, ultimately ameliorating diabetic microvascular injury and heart complications.

Inhibition of VRK1 suppresses proliferation and migration of vascular smooth muscle cells and intima hyperplasia after injury via mTORC1/ß-catenin axis.

Characterized by abnormal proliferation and migration of vascular smooth muscle cells (VSMCs), neointima hyperplasia is a hallmark of vascular restenosis after percutaneous vascular interventions. Vaccinia-related kinase 1 (VRK1) is a stress adaptionassociated ser/thr protein kinase that can induce the proliferation of various types of cells. However, the role of VRK1 in the proliferation and migration of VSMCs and neointima hyperplasia after vascular injury remains unknown. We observed increased expression of VRK1 in VSMCs subjected to platelet-derived growth factor (PDGF)-BB by western blotting. Silencing VRK1 by shVrk1 reduced the number of Ki-67-positive VSMCs and attenuated the migration of VSMCs. Mechanistically, we found that relative expression levels of ß-catenin and effectors of mTOR complex 1 (mTORC1) such as phospho (p)-mammalian target of rapamycin (mTOR), p-S6, and p-4EBP1 were decreased after silencing VRK1. Restoration of ß-catenin expression by SKL2001 and re-activation of mTORC1 by Tuberous sclerosis 1 siRNA (siTsc1) both abolished shVrk1-mediated inhibitory effect on VSMC proliferation and migration. siTsc1 also rescued the reduced expression of ß-catenin caused by VRK1 inhibition. Furthermore, mTORC1 re-activation failed to recover the attenuated proliferation and migration of VSMC resulting from shVrk1 after silencing ß-catenin. We also found that the vascular expression of VRK1 was increased after injury. VRK1 inactivation in vivo inhibited vascular injury-induced neointima hyperplasia in a ß-catenin-dependent manner. These results demonstrate that inhibition of VRK1 can suppress the proliferation and migration of VSMC and neointima hyperplasia after vascular injury via mTORC1/ß-catenin pathway. [BMB Reports 2022; 55(5): 244-249].

Plin5 inhibits proliferation and migration of vascular smooth muscle cell through interacting with PGC-1α following vascular injury.

Abnormal proliferation and migration of vascular smooth muscle cell (VSMC) is a hallmark of vascular neointima hyperplasia. Perilipin 5 (Plin5), a regulator of lipid metabolism, is also confirmed to be involved in vascular disorders, such as microvascular endothelial dysfunction and atherosclerosis. To investigate the regulation and function of plin5 in the phenotypic alteration of VSMC, -an animal model of vascular intima hyperplasia was established in C57BL/6 J and Plin5 knockdown (Plin5±) mice by wire injure. Immunohistochemical staining was used to analyze neointima hyperplasia in artery. Ki-67, dihydroethidium immunofluorescence staining and wound healing assay were used to measure proliferation, reactive oxygen species (ROS) generation and migration of VSMC, respectively. Plin5 was downregulated in artery subjected to vascular injury and in VSMC subjected to platelet-derived growth factor (PDGF)-BB. Plin5 knockdown led to accelerated neointima hyperplasia, excessive proliferation and migration of VSMC after injury. In vitro, we observed increased ROS content in VSMC isolated from Plin5± mice. Antioxidative N-acetylcysteine (NAC) inhibited VSMC proliferation and migration induced by PDGF-BB or plin5 knockdown. More importantly, plin5-peroxlsome proliferator-activated receptor-γ coactivator (PGC)-1α interaction was also attenuated in VSMC after knockdown of plin5. Overexpression of PGC-1α suppressed PDGF-BB-induced ROS generation, proliferation, and migration in VSMC isolated from Plin5± mice. These data suggest that plin5 serves as a potent regulator of VSMC proliferation, migration, and neointima hyperplasia by interacting with PGC-1α and affecting ROS generation.

Angiotensin II receptor blocker telmisartan enhances running endurance of skeletal muscle through activation of the PPAR-δ/AMPK pathway.

Clinical trials have shown that angiotensin II receptor blockers reduce the new onset of diabetes in hypertensives; however, the underlying mechanisms remain unknown. We investigated the effects of telmisartan on peroxisome proliferator activated receptor γ (PPAR-δ) and the adenosine monophosphate (AMP)-activated protein kinase (AMPK) pathway in cultured myotubes, as well as on the running endurance of wild-type and PPAR-δ-deficient mice. Administration of telmisartan up-regulated levels of PPAR-δ and phospho-AMPKα in cultured myotubes. However, PPAR-δ gene deficiency completely abolished the telmisartan effect on phospho-AMPKαin vitro. Chronic administration of telmisartan remarkably prevented weight gain, enhanced running endurance and post-exercise oxygen consumption, and increased slow-twitch skeletal muscle fibres in wild-type mice, but these effects were absent in PPAR-δ-deficient mice. The mechanism is involved in PPAR-δ-mediated stimulation of the AMPK pathway. Compared to the control mice, phospho-AMPKα level in skeletal muscle was up-regulated in mice treated with telmisartan. In contrast, phospho-AMPKα expression in skeletal muscle was unchanged in PPAR-δ-deficient mice treated with telmisartan. These findings highlight the ability of telmisartan to improve skeletal muscle function, and they implicate PPAR-δ as a potential therapeutic target for the prevention of type 2 diabetes.

Cardiac extracellular matrix tenascin-C deposition during fibronectin degradation.

Tenascin-C (TN-C) might aggravate left ventricular remodeling after myocardial infarction (MI). Our previous study demonstrated that ventricular remodeling after MI is linked with the degradation of fibronectin (FN). The aim of the present study was to determine whether cardiac extracellular matrix TN-C deposition after MI requires FN degradation. We found that treatment with angiotensin (ANG) II significantly down-regulated FN while remarkably up-regulated TN-C in co-cultured cardiomyocytes and fibroblasts. Inhibitors of matrix metalloproteinase (MMP)-2, MMP-3 or MMP-9 significantly attenuated ANG II-induced loss of FN and obviously blunted ANG II-induced re-expression of TN-C in co-cultured cells. Moreover, FN fragments dose-dependently induced the deposition of TN-C. In addition, MI induced a significant reduction of FN protein expression and a marked elevation of TN-C expression level at day 7 after MI compared with the sham group. The present findings suggest that cardiac TN-C matrix deposition after MI is induced by FN degradation, which is dependent on the activation of MMPs. These findings might contribute to gain mechanistic insights into the regulation of TN-C formation after MI.