Smooth Muscle Cell Injury Research Articles

Exploring the roles and therapeutic implications of melatonin-mediated KLF6 in the development of intracranial aneurysm

Background Intracranial aneurysm (IA) is a cerebrovascular disease with a high mortality rate due to ruptured subarachnoid hemorrhage. While Krüppel-like factor 6 (KLF6) dysregulation has been implicated in cancer and cardiovascular diseases, its role in IA remains unclear. Materials and methods The GSE122897 and GSE15629 datasets were downloaded from the Gene Expression Omnibus database. Immune cell infiltration and hypoxia analysis were performed to explore the effects of KLF6 on IA. Weighted gene co-expression network analysis was used to identify hub genes related to KLF6 expression for subsequent analyses. Hypoxia-related genes were identified. Drug prediction was performed for IA. Samples from healthy individuals and patients with IA were collected to detect the expression of endothelin-1 (ET-1), vascular hematoma factor (vWF), and KLF6. A model of H2O2-induced human brain vascular smooth muscle cells (HBVSMC) injury was constructed to explore the effects of KLF6 and melatonin to treat IA. Results T cells CD4 memory resting and monocytes were significantly different in the KLF6 high and low expression groups. Four hypoxia-related gene sets were significantly enriched in the KLF6 high-expression group. Six hypoxia-related hub genes were obtained, which were significantly associated with KLF6. Drug prediction showed that melatonin may be a potential drug for IA. The levels of ET-1, vWF, and KLF6 were significantly upregulated in patients with IA. KLF6 exacerbates H2O2-induced injury in HBVSMC, ameliorated by melatonin. Conclusion KLF6 may be a potential target for IA treatment, with melatonin-mediated KLF6 effects playing a crucial role in the development of IA.

Esaxerenone Attenuates Aldosterone-Induced Mitochondrial Damage-Mediated Pyroptosis in Mouse Aorta and Rat Vascular Smooth Muscle Cells.

Vascular smooth muscle cell (VSMC) injury caused by the inflammatory response plays a key role in cardiovascular disease (CVD), and the vasoprotective effects of mineralocorticoid receptor blockers (MRBs) support the role of mineralocorticoid receptor (MR) activation. C57BL/6 mice and VSMCs isolated from rats were treated with aldosterone and esaxerenone. Caspase-1, GSDMD-N, IL-1β, and NR3C2 expression and DNA damage in aortic VSMCs were detected using immunohistochemistry, Western blotting, and TUNEL staining. Mitochondrial changes were detected by transmission electron microscopy (TEM). Reactive oxygen species (ROS), MitoTracker, JC-I, mitochondrial respiratory chain complexes I-V, and NR3C2 were detected using immunofluorescence and flow cytometry. Pyroptosis was detected with scanning electron microscopy (SEM). After aldosterone treatment, the number of TUNEL-positive cells increased significantly, and the expression of caspase-1, GSDMD-N, and IL-1β increased. TEM revealed mitochondrial damage, and SEM revealed specific pyroptotic changes, such as cell membrane pore changes and cytoplasmic extravasation. Increased ROS levels and nuclear translocation of NR3C2 were also observed. These pyroptosis-related changes were reversed by esaxerenone. Aldosterone activates the MR and mediates mitochondrial damage, thereby inducing pyroptosis in VSMCs via the NLRP3/caspase-1 pathway. Esaxerenone inhibits MR activation and reduces mitochondrial damage and oxidative stress, thereby inhibiting pyroptosis.

Effects of S100A12 reduction on H2O2-induced injury of human vascular smooth muscle cells (HVSMCs) [Retraction

[This retracts the article on p. 11617 in vol. 10, PMID: 31966519.].

Knockdown of circ_0113656 assuages oxidized low-density lipoprotein-induced vascular smooth muscle cell injury through the miR-188-3p/IGF2 pathway.

Circular RNA (circRNA) is involved in the pathogenesis of atherosclerosis (AS). The present work analyzed the RNA expression of circ_0113656, microRNA-188-3p (miR-188-3p), and insulin-like growth factor 2 (IGF2) by quantitative real-time polymerase chain reaction. The protein expression of proliferating cell nuclear antigen (PCNA), matrix metalloprotein 2 (MMP2), and IGF2 was detected by Western blotting. Cell viability, proliferation, invasion, and migration were analyzed using the cell counting kit-8, 5-ethynyl-2'-deoxyuridine, transwell invasion, and wound-healing assays, respectively. The interactions among circ_0113656, miR-188-3p, and IGF2 were identified by dual-luciferase reporter assay and RNA immunoprecipitation assay. The results showed that circ_0113656 and IGF2 expression were significantly upregulated, while miR-188-3p was downregulated in the blood of AS patients and oxidized low-density lipoprotein (ox-LDL)-treated HVSMCs in comparison with controls. The ox-LDL treatment induced HVSMC proliferation, migration, and invasion accompanied by increases in PCNA and MMP2 expression; however, these effects were attenuated after circ_0113656 knockdown. Circ_0113656 acted as a miR-188-3p sponge and it regulated ox-LDL-induced HVSMC disorders by binding to miR-188-3p. Besides, the regulation of miR-188-3p in ox-LDL-induced HVSMC injury involved IGF2. Further, the depletion of circ_0113656 inhibited IGF2 expression by interacting with miR-188-3p. Thus, the circ_0113656/miR-188-3p/IGF2 axis may mediate ox-LDL-induced HVSMC disorders in AS, providing a new therapeutic strategy for AS.

κ-Opioid Receptors Improve Vascular Endothelial Dysfunction in Salt-Sensitive Hypertension via PI3K/Akt/eNOS Signaling Pathway.

κ-Opioid receptors (κ-OR) are widely used to regulate the activity of the cardiovascular system. To explore the effect and mechanism of κ-OR on salt-sensitive hypertensive endothelial dysfunction, we used Dah1 rats to construct a rat model of salt-sensitive hypertension on a high-salt (HS) diet. Then, the rats were treated with κ-OR activators U50,488H (1.25 mg/kg) and inhibitor nor-BNI (2.0 mg/kg) for 4 weeks, respectively. The rat aortas were collected to detect the contents of NO, ET-1, AngII, NOS, T-AOC, SO, and NT. Protein expression was determined for NOS, Akt, and Caveolin-1. In addition, the vascular endothelial cells were extracted, and the levels of NO, TNF-α, IL-1, IL-6, IL-8, IL-10, p-Akt, and p-eNOS in cell supernatants were detected. In vivo results showed that compared with the HS group, treated with U50,488H promoted rats' vasodilation by increasing the NO content and decreasing ET-1 and AngII contents. U50,488H reduced endothelial cell apoptosis and attenuated vascular, smooth muscle cell and endothelial cell injury. U50,488H also enhanced the rats' response to oxidative stress by increasing the NOS and T-AOC contents. Moreover, U50,488H increased the eNOS, p-eNOS, Akt, and p-AKT expression and decreased the iNOS and Caveolin-1 expression. In vitro results showed that U50,488H promoted NO, IL-10, p-Akt, and p-eNOS levels in endothelial cell supernatants versus the HS group. And U50,488H reduced the adhesion of peripheral blood mononuclear cells and polymorphonuclear neutrophils to endothelial cells and the migration function of polymorphonuclear neutrophils. Our study suggested that κ-OR activation may improve vascular endothelial dysfunction in salt-sensitive hypertensive rats through the PI3K/Akt/eNOS signaling pathway. This may be a potential therapeutic approach in the treatment of hypertension.

Flow cytometric assessment of DNA double-strand break and repair kinetics in prediction of intrinsic radiosensitivity.

Objective(s): Hyperinsulinemia, secondary to insulin resistance, may lead to vascular smooth muscle cell dysfunction. In the present research, we aimed to investigate the effect of Chemokine receptor 8 (CCR8) on angiotensin II (Ang II)-induced dysfunction of vascular smooth muscle cells (VSMCs) and to explore the underlying molecular mechanism. Materials and Methods:The expression of CCR8 was analyzed in diabetics and normal people by RT-PCR and ELISA. CCK-8 assay and transwell were used to explore cell proliferation and migration, and ELISA was used to measure the content of IL-6 and TNF-α. Reactive oxygen species (ROS) kit was employed to measure ROS generation. Results:The results revealed that CCR8 was highly expressed in diabetics and Ang Ⅱ-induced VSMCs. Further studies found that interfering with the expression of CCR8 significantly reduced the production of ROS and the levels of inflammatory factors in AngⅡ-induced VSMCs. Interfering with CCR8 increased the glucose uptake induced by AngⅡ+IR. More importantly, inhibition of CCR8 alleviated Ang II-induced dysfunction of VSMCs. Inhibition of CCR8 inactivated the MAPK/NF-κB signaling pathway.Conclusion:Inhibition of CCR8 attenuates Ang II-induced VSMCs injury by inhibiting the MAPK/NF-κB pathway. CCR8 may be a new biomarker related to hypertension and insulin resistance and is a new target for the treatment of human cardiovascular diseases.

DLEU2 modulates proliferation, migration and invasion of platelet-derived growth factor-BB (PDGF-BB)-induced vascular smooth muscle cells (VSMCs) via miR-212-5p/YWHAZ axis

ABSTRACT DLEU2 has been proved to act as an oncogene in a variety of cancers, but its role in cardiovascular diseases is dearth of research. Thus, this study mainly discussed the effect and possible mechanism of DLEU2 on platelet-derived growth factor-BB (PDGF-BB)-triggered vascular smooth muscle cell (VSMC) injury. To obtain authentic results, the expressions of target genes in atherosclerosis serum were determined by reverse transcription quantitative PCR (RT-qPCR) and the protein levels were evaluated by Western blot. PDGF-BB was used to simply simulate the biological characteristics of VSMCs in vitro. The effect of DLEU2 on the biological behavior of PDGF-BB-induced VSMCs was analyzed by gain- and loss-of-function assays. Bioinformatics analysis, dual luciferase reporter assay, and Pearson correlation method were conducted to determine the relationship between target genes. The role of DLEU2/miR-212-5p/ YWHAZ (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta) axis in PDGF-BB-induced VSMCs was verified by rescue experiments. As a result, DLEU2 and YWHAZ were up-regulated, and miR-212-5p was down-regulated in atherosclerosis serum. Overexpressed DLEU2 facilitated the biological behavior of PDGF-BB-induced VSMCs, whilst siDLEU2 did the opposite. Moreover, overexpressed DLEU2 promoted proliferating cell nuclear antigen (PCNA) expression but repressed α-smooth muscle actin (α-SMA) and Calponin expressions, while it also enhanced YWHAZ expression via suppressing miR-212-5p. MiR-212-5p mimic and siYWHAZ reversed the effects of overexpressed DLEU2 on above biological characteristics and protein expressions in PDGF-BB-induced VSMCs, while the regulatory effect of miR-212-5p mimic was partially offset by overexpressed YWHAZ. Collectively, DLEU2 modulates PDGF-BB-induced VSMC injury via miR-212-5p/YWHAZ axis in atherosclerosis.

Nuclear Paraspeckle Assembly Transcript 1 Enhances Hydrogen Peroxide-Induced Human Vascular Smooth Muscle Cell Injury by Regulating miR-30d-5p/A Disintegrin and Metalloprotease 10.

Nuclear paraspeckle assembly transcript 1 (NEAT1) has been reported to be involved in the progression of many cancers; however, the role and mechanisms underlying NEAT1 in abdominal aortic aneurysm (AAA) remain unclear. The expression of NEAT1, miR-30d-5p and A disintegrin and metalloprotease 10 (ADAM10) was measured by qRT-PCR and western blot. Functional experiments were conducted by using a CCK-8 assay, EDU assay, flow cytometry, western blot, ELISA, and commercial kits. The target relation was confirmed by dual-luciferase reporter assay and the RIP assay. It was then found that NEAT1 was upregulated in peripheral blood of AAA patients ~3.46-fold, smooth muscle cells (SMCs) isolated from AAA tissues ~2.6-fold and in a hydrogen peroxide (H2O2)-induced injury model of human vascular SMC (HVSMCs) ~2.0- and 3.9-fold at 50 µmol/L and 200 µmol/L H2O2treatment, respectively. NEAT1 deletion attenuated H2O2-induced cell proliferation promotion (40.0% vs. 74.3%), apoptosis inhibition (25.0% vs. 13.5%), and reduction of inflammatory response and oxidative stress in HVSMCs. Mechanistically, NEAT1 targeted miR-30d-5p to prevent the degradation of its target, ADAM10, in HVSMCs. Further rescue experiments suggested miR-30d-5p inhibition mitigated the effects of NEAT1 deletion on H2O2-induced HVSMCs. Moreover, ADAM10 overexpression counteracted the inhibitory functions of miR-30d-5p on H2O2-evoked HVSMC injury. NEAT1 promoted H2O2-induced HVSMC injury by inducing cell apoptosis, inflammation and oxidative stress through miR-30d-5p/ADAM10 axis, indicating the possible involvement of NEAT1 in the pathogenesis of AAA.

Reducing Endogenous Labile Zn May Help to Reduce Smooth Muscle Cell Injury around Vascular Stents.

Vascular stent service involves complex service environments and performance requirements, among which the histocompatibility of the stent could seriously affect the therapeutic effect. In the pathology of vascular disease, the thin fiber cap is easily ruptured, exposing the necrotic core below, and triggering a series of dangerous biochemical reactions. In contrast, the thin neointima, considered an essential structure growing on the stent, may evolve into vulnerable plaque structures due to lesions induced by the stent. Therefore, the reduction of necrosis around the stent below the thin neointima is indispensable. In this work, different cell model experiments suggested that the content of endogenous labile Zn positively correlated with cell injury. Zinquin-Zn fluorescence experiments and zinc ion channels research suggested that the change in the content of endogenous labile Zn in smooth muscle cells is affected by different stent coatings. The content of endogenous labile Zn in cells negatively correlated with cell viability. Animal experiments indirectly verified the increase in endogenous labile Zn by detecting the expression of Zn regulatory protein (metallothionein) in the necrotic tissues. Reducing the content of endogenous labile Zn may favor a reduction in smooth muscle cell injury and necrosis. This biochemical mechanism is effective in improving the therapeutic effect of vascular stents.

Capsaicin Attenuates Arterial Calcification Through Promoting SIRT6-Mediated Deacetylation and Degradation of Hif1α (Hypoxic-Inducible Factor-1 Alpha).

Sustained Hif1α (hypoxic-inducible factor-1 alpha) accumulation plays a central role in osteogenic transdifferentiation and subsequent calcification. Capsaicin, the potent agonist of TRPV1 (transient receptor potential vanilloid type 1), was found to mitigate hypoxic-related injury and reverse phenotypic switch of vascular smooth muscle cells. However, its role in arterial calcification and the underlying mechanisms remain unexplored. We used data from Multi-Ethnic Study of Atherosclerosis to examine the association of coronary artery calcification and chili consumption. Chronic kidney disease mice and high phosphate-induced vascular smooth muscle cells calcification models were established to investigate the anticalcification effect of capsaicin, evaluated by calcium deposition and changes in phenotype markers. Chili consumption was negatively correlated with coronary artery calcification and conferred a smaller progression burden during follow-up. Capsaicin reduced calcium deposition and osteogenic transdifferentiation both in vivo and in vitro. Using siTRPV1 (small interfering RNA and the antagonist of TRPV1), the anticalcification effect of capsaicin was abrogated. Hif1α was increased in phosphate-treated vascular smooth muscle cells and its degradation was accelerated by capsaicin. Retaining Hif1α stability using cobalt chloride (CoCl2) or MG132 abolished the protective effect of capsaicin. We further identified an increased expression of SIRT6 (Sirtuin 6) in response to capsaicin and confirmed the physical interaction between SIRT6 and Hif1α. Acetylated Hif1α was decreased, whereas hydroxylated Hif1α was increased under capsaicin treatment. Using immunohistochemistry analysis, we observed increased SIRT6 and reduced Hif1α in both SIRT6 transgenic and capsaicin-treated chronic kidney disease mice. Capsaicin facilitates deacetylation and degradation of Hif1α by upregulating SIRT6, which inhibits osteogenic transdifferentiation and protects against arterial calcification. These data highlight a promising therapeutic target for the management of arterial calcification.

Engagement of the CXCL12-CXCR4 Axis in the Interaction of Endothelial Progenitor Cell and Smooth Muscle Cell to Promote Phenotype Control and Guard Vascular Homeostasis.

Endothelial progenitor cells (EPCs) are involved in vascular repair and modulate properties of smooth muscle cells (SMCs) relevant for their contribution to neointima formation following injury. Considering the relevant role of the CXCL12–CXCR4 axis in vascular homeostasis and the potential of EPCs and SMCs to release CXCL12 and express CXCR4, we analyzed the engagement of the CXCL12–CXCR4 axis in various modes of EPC–SMC interaction relevant for injury- and lipid-induced atherosclerosis. We now demonstrate that the expression and release of CXCL12 is synergistically increased in a CXCR4-dependent mechanism following EPC–SMC interaction during co-cultivation or in response to recombinant CXCL12, thus establishing an amplifying feedback loop Additionally, mechanical injury of SMCs induces increased release of CXCL12, resulting in enhanced CXCR4-dependent recruitment of EPCs to SMCs. The CXCL12–CXCR4 axis is crucially engaged in the EPC-triggered augmentation of SMC migration and the attenuation of SMC apoptosis but not in the EPC-mediated increase in SMC proliferation. Compared to EPCs alone, the alliance of EPC–SMC is superior in promoting the CXCR4-dependent proliferation and migration of endothelial cells. When direct cell–cell contact is established, EPCs protect the contractile phenotype of SMCs via CXCL12–CXCR4 and reverse cholesterol-induced transdifferentiation toward a synthetic, macrophage-like phenotype. In conclusion we show that the interaction of EPCs and SMCs unleashes a CXCL12–CXCR4-based autoregulatory feedback loop promoting regenerative processes and mediating SMC phenotype control to potentially guard vascular homeostasis.

Downregulation of microRNA-512-3p enhances the viability and suppresses the apoptosis of vascular endothelial cells, alleviates autophagy and endoplasmic reticulum stress as well as represses atherosclerotic lesions in atherosclerosis by adjusting spliced/unspliced ratio of X-box binding protein 1 (XBP-1S/XBP-1U)

ABSTRACT AS is an important pathological basis of cardiovascular disease. miRNAs are involved in almost all steps of AS, including the injury and dysfunction of endothelial cells and vascular smooth muscle cells. This work elucidated the biological functions of miR-512-3p in AS and probed into the underlying molecular mechanism. In the present work, ox-LDL-treated HUVECs served as the in vitro model of AS and ApoE-/- mice were nourished with a high-fat diet to establish an in vivo model of AS. Proliferation, apoptosis, and migration of HUVECs were evaluated by CCK-8, TUNEL staining, Western blot, and transwell assays. Immunofluorescence examined LC3 expression and levels of autophagy-related and ER stress-related proteins were determined by Western blot assay. In addition, starBase predicted the complementary binding sites of XBP-1 to miR-512-3p and luciferase reporter assay confirmed the interaction between miR-512-3p and XBP-1. Moreover, H&E staining was employed to evaluate atherosclerotic lesions in AS model mice. Results revealed that ox-LDL treatment decreased the proliferative and migrative activities and promoted the apoptosis of HUVECs as well as induced autophagy and ER stress, which were abrogated by miR-512-3p silencing. Importantly, ox-LDL treatment elevated miR-512-3p expression and XBP-1 was a direct target of miR-512-3p. Mechanistically, knockdown of miR-512-3p enhanced the viability, suppressed the apoptosis, and promoted the migration of ox-LDL-treated HUVECs, alleviated atherosclerotic lesions in AS model mice as well as repressed autophagy and ER stress by targeting XBP-1 to manipulate the ratio of XBP-1S/XBP-1 U.

Sex-Specific MicroRNAs in Neurovascular Units in Ischemic Stroke.

Accumulating evidence pinpoints sex differences in stroke incidence, etiology and outcome. Therefore, more understanding of the sex-specific mechanisms that lead to ischemic stroke and aggravation of secondary damage after stroke is needed. Our current mechanistic understanding of cerebral ischemia states that endothelial quiescence in neurovascular units (NVUs) is a major physiological parameter affecting the cellular response to neuron, astrocyte and vascular smooth muscle cell (VSMC) injury. Although a hallmark of the response to injury in these cells is transcriptional activation, noncoding RNAs such as microRNAs exhibit cell-type and context dependent regulation of gene expression at the post-transcriptional level. This review assesses whether sex-specific microRNA expression (either derived from X-chromosome loci following incomplete X-chromosome inactivation or regulated by estrogen in their biogenesis) in these cells controls NVU quiescence, and as such, could differentiate stroke pathophysiology in women compared to men. Their adverse expression was found to decrease tight junction affinity in endothelial cells and activate VSMC proliferation, while their regulation of paracrine astrocyte signaling was shown to neutralize sex-specific apoptotic pathways in neurons. As such, these microRNAs have cell type-specific functions in astrocytes and vascular cells which act on one another, thereby affecting the cell viability of neurons. Furthermore, these microRNAs display actual and potential clinical implications as diagnostic and prognostic biomarkers in ischemic stroke and in predicting therapeutic response to antiplatelet therapy. In conclusion, this review improves the current mechanistic understanding of the molecular mechanisms leading to ischemic stroke in women and highlights the clinical promise of sex-specific microRNAs as novel diagnostic biomarkers for (silent) ischemic stroke.

ORMDL3 regulates cigarette smoke–induced endoplasmic reticulum stress in airway smooth muscle cells

Orosomucoid 1-like protein 3 (ORMDL3), a transmembrane protein localized in the endoplasmic reticulum (ER), has been genetically associated with chronic obstructive pulmonary disease (COPD), in addition to childhood-onset asthma. However, the functional role of ORMDL3 in the pathogenesis of COPD is still unknown. Because cigarette smoke is the major risk factor for COPD, we aimed to investigate the role of ORMDL3 in cigarette smoke-induced human airway smooth muscle cell (HASMC) injury. The mRNA and protein expression of ORMDL3 was examined in HASMCs from nonsmokers and smokers without or with COPD. Knockdown of ORMDL3 in primary healthy HASMCs was performed using small interfering RNA before exposure to cigarette smoke medium (CSM) for 24 hours. Inflammatory, proliferative/apoptotic, ER stress, and mitochondrial markers were evaluated. Elevation of ORMDL3 mRNA and protein expression was observed in HASMCs of smokers without or with COPD. CSM caused significant upregulation of ORMDL3 expression in healthy nonsmokers. ORMDL3 knockdown regulated CSM-induced inflammation, cell proliferation, and apoptosis. Silencing ORMDL3 led to reduction of CSM-induced ER stress via inhibition of unfolded protein response pathways such as activating transcription factor 6 and protein kinase RNA-like ER kinase. ORMDL3 was also involved in CSM-induced mitochondrial dysfunction via the mitochondrial fission process. We report the induction of ORMDL3 in HASMCs after cigarette smoke exposure. ORMDL3 may mediate cigarette smoke-induced activation of unfolded protein response pathways during airway smooth muscle cell injury.

Abnormal TPM2 expression is involved in regulation of atherosclerosis progression via mediating RhoA signaling in vitro.

Ox-LDL (oxidized low-density lipoprotein)-induced endothelial cell injury and dysfunction of vascular smooth muscle cells play critical roles in the development of atherosclerosis (AS). Tropomyosin 2 (TPM2) has been implicated in cardiac diseases, but its critical role and regulatory mechanism in AS progression have not yet been elucidated. The expression of TPM2 was investigated in AS tissues. Ox-LDL was used to construct an AS in vitro model based on endothelial and vascular smooth muscle cells (HAECs and VSMCs). An overexpression assay was performed to evaluate the role of TPM2 in AS. Meanwhile, the involvement of the RhoA pathway in TPM2-mediated AS progression was evaluated using narciclasine. Tropomyosin 2 was dramatically upregulated in both AS tissues and ox-LDL-induced HAECs. Overexpression of TPM2 attenuated ox-LDL-stimulated cell growth depression, inflammatory and adhesive responses in HAECs, as well as oxidative stress and mitochondrial dysfunction. Additionally, VSMCs, impacted by TPM2-overexpressed HAECs, showed alleviated cellular processes which were abnormally activated by ox-LDL. Furthermore, depressed activation of the RhoA pathway was found in TPM2-overexpressed HAECs and activating the signaling rescued these effects of TPM2 exerted on ox-LDL-stimulated HAECs and VSMCs. TPM2 had an advantageous impact on ox-LDL-induced AS progression in vitro by mediating the RhoA pathway. This evidence might contribute to the therapy of AS.

Endothelial Progenitor Cells Modulate the Phenotype of Smooth Muscle Cells and Increase Their Neointimal Accumulation Following Vascular Injury

Smooth muscle cells (SMCs) are the main driver of neointima formation and restenosis following vascular injury. In animal models, endothelial progenitor cells (EPCs) accelerate endothelial regeneration and reduce neointima formation after arterial injury; however, EPC-capture stents do not reduce target vessel failure compared with conventional stents. Here we examined the influence of EPCs on features of SMCs pivotal for their impact on injury-induced neointima formation including proliferation, migration, and phenotype switch. EPCs, their conditioned medium, and EPC-derived microparticles induced proliferation of SMCs while limiting their apoptosis. In transwell membrane experiments and scratch assays, EPCs stimulated migration of SMCs and accelerated their recovery from scratch-induced injury. Treatment of SMCs with an EPC-derived conditioned medium or microparticles triggered transformation of SMCs toward a synthetic phenotype. However, co-cultivation of EPCs and SMCs enabling direct cell-cell contacts preserved their original phenotype and protected from the transformative effect of SMC cholesterol loading. Adhesion of EPCs to SMCs was stimulated by SMC injury and reduced by blocking CXCR2 and CCR5. Interaction of EPCs with SMCs modulated their secretory products and synergistically increased the release of selected chemokines. Following carotid wire injury in athymic mice, injection of EPCs resulted not only in reduced neointima formation but also in altered cellular composition of the neointima with augmented accumulation of SMCs. EPCs stimulate proliferation and migration of SMCs and increase their neointimal accumulation following vascular injury. Furthermore, EPCs context-dependently modify the SMC phenotype with protection from the transformative effect of cholesterol when a direct cell-cell contact is established.

MALAT1 knockdown protects from bronchial/tracheal smooth muscle cell injury via regulation of microRNA-133a/ryanodine receptor 2 axis

Asthma has significant impacts on living quality particularly in children. Long noncoding RNA (lncRNA) MALAT1 plays a crucial role in neonatal respiratory diseases. Meanwhile, MALAT1 knockdown could induce viability and attenuate apoptosis of airway-related cells. However, the role of MALAT1 in neonatal asthma, asthma-related cell, and its possible mechanism is unclear. This study aims to investigate MALAT1 level in asthma and to identify the effects of MALAT1 on bronchial/tracheal smooth muscle cells (B/TSMCs). Newborn asthma modeling rat was constructed by introducing ovalbumin (OVA). MALAT1 levels in tissues or B/TSMCs were determined by RT-qPCR. Exogenous changes of MALAT1, RyR2 or miR-133a in B/TSMCs were fulfilled by cell transfection; cell apoptosis was measured by using Cell Death Detection ELISA kit and Hochest33342; IL-6, TNF-α and IL-1β level was detected by using corresponding ELISA kit; ryanodine receptor 2 (RyR2) mRNA and miR-133a level was determined by RT-qPCR; cleaved caspase-3 (c-caspase-3) and RyR2 expression was detected by Western blot; luciferase reporter assay was performed to confirm the target regulation of miR-133a on RyR2. We found that MALAT1 was significantly upregulated in tracheal tissues of newborn asthma modeling rats. In MALAT1-silenced or -overexpressed B/TSMCs, we found a synchronous change of cell apoptosis, inflammatory factor secretion (IL-6, TNF-α, and IL-1β) or RyR2 level, but a reverse change of miR-133a level with MALAT1. Besides, MALAT1 induced B/TSMCs apoptosis and inflammation increase could be partially reversed when RyR2 was silenced or when miR-133a was overexpressed. The luciferase reporter assay confirmed that RyR2 is a direct target gene of miR-133a in B/TSMCs. Finally, we conclude that MALAT1 knockdown could protect from B/TSMCs injury via regulating miR-133a/ RyR2 axis.

Study of Mesenchymal Stem Cell-Mediated Mitochondrial Transfer in In Vitro Models of Oxidant-Mediated Airway Epithelial and Smooth Muscle Cell Injury.

Mesenchymal stem cells (MSCs) have emerged as an attractive candidate for cell-based therapy. In the past decade, many animal and pilot clinical studies have demonstrated that MSCs are therapeutically beneficial for the treatment of obstructive lung diseases such as asthma and chronic obstructive pulmonary disease (COPD). However, due to the scarcity of adult human MSCs, human-induced pluripotent stem cells mesenchymal stem cells (iPSCs) are now increasingly used as a source of MSCs. iPSCs are derived by reprogramming somatic cells from a wide variety of tissues such as skin biopsies and then differentiating them into iPSC-MSCs. One of the mechanisms through which MSCs exert their protective effects is mitochondrial transfer. Specifically, transfer of mitochondria from iPSC-MSCs to lung cells was shown to protect lung cells against oxidative stress-induced mitochondrial dysfunction and apoptosis and to reduce lung injury and inflammation in in vivo models of lung disease. In this chapter, we detail our methods to visualize and quantify iPSC-MSC-mediated mitochondrial transfer and to study its effects on oxidant-induced airway epithelial and smooth muscle cell models of acute airway cell injury.

Hsa_circ_0029589 knockdown inhibits the proliferation, migration and invasion of vascular smooth muscle cells via regulating miR-214-3p and STIM1

AimsCircular RNAs (circRNAs) are relevant to atherosclerosis progression. However, the role and mechanism of circRNA hsa_circ_0029589 (circ_0029589) in atherosclerosis are not fully understood. This research aims to explore the function and mechanism of circ_0029589 in oxidized low-density lipoprotein (ox-LDL)-caused vascular smooth muscle cells (VSMCs) injury in vitro. Main methodsVSMCs were challenged via ox-LDL to mimic atherosclerosis-like injury in vitro. Circ_0029589, microRNA-214-3p (miR-214-3p) and stromal interaction molecule 1 (STIM1) abundances were detected via quantitative reverse transcription polymerase chain reaction or western blot. Cell proliferation was investigated via cell viability, cycle, apoptosis and proliferation-associated protein levels. Cell migration and invasion were assessed via transwell analysis. The relationship between miR-214-3p and circ_0029589 or STIM1 was tested via dual-luciferase reporter analysis and RNA immunoprecipitation. Key findingsCirc_0029589 level was enhanced in ox-LDL-challenged VSMCs. Circ_0029589 interference constrained cell proliferation, migration and invasion in ox-LDL-challenged VSMCs. miR-214-3p was targeted by circ_0029589 and miR-214-3p knockdown weakened the suppressive function of circ_0029589 silence on VSMCs proliferation, migration and invasion. STIM1 was targeted via miR-214-3p and miR-214-3p could suppress VSMCs proliferation, migration and invasion via decreasing STIM1. Moreover, circ_0029589 modulated STIM1 level by miR-214-3p. SignificanceCirc_0029589 knockdown repressed proliferation, migration and invasion of VSMCs challenged via ox-LDL by regulating miR-214-3p and STIM1, indicating that circ_0029589 might play important role in atherosclerosis.

Effect of total flavones of Clematis filamentosa Dunn on the oxLDL-induced injury of vascular smooth muscle cells by regulating miR-455-5p

This experiment was conducted to investigate whether total flavones of Clematis filamentosa Dunn affect the inflammatory response and apoptosis of vascular smooth muscle cells induced by oxidized low-density lipoprotein (oxLDL) by regulating microRNA-455-5p (miR-455-5p). 50 mg/mL oxLDL was performed to stimulate the injury of vascular smooth muscle cells, and the total flavones of Clematis filamentosa Dunn were added at concentrations of 75, 150, and 300 μg/mL. The expressions of inflammatory factors IL-1β and TNF-α were analyzed by ELISA, the apoptosis was evaluated by flow cytometry, the expression of Bcl-2 and Bax was determined by western blot, and the real-time fluorescence quantitative PCR (qRT-PCR) was applied to detect miR-455-5p expression. MiR-455-5p mimic was transfected into vascular smooth muscle cells and then induced injury with oxLDL; miR-455-5p inhibitor was transfected into vascular smooth muscle cells and treated with oxLDL and 300 μg/mL total flavones of Clematis filamentosa Dunn. The above methods were employed to investigate the inflammatory response and apoptosis of cells. The total flavones of Clematis filamentosa Dunn significantly inhibited the expression of IL-1β, TNF-α, apoptosis rate, Bax protein expression of oxLDL induced vascular smooth muscle cells, and remarkably promoted the expression of Bcl-2 protein and miR-455-5p, which all showed concentration dependence (p<0.05). Overexpression of miR-455-5p reduced IL-1β, TNF-α expression, apoptosis rate, Bax protein expression, and greatly increased Bcl-2 protein expression in oxLDL injured vascular smooth muscle cells (p<0.05). After interfering with the expression of miR-455-5p, the inhibitory effect of total flavones of Clematis filamentosa Dunn on the expression of IL-1β, TNF-α, apoptosis, Bax protein expression of oxLDL-induced vascular smooth muscle cells was reversed, and its promotion effect on Bcl-2 protein expression was also reversed. Total flavones of Clematis filamentosa Dunn can reduce oxLDL-induced vascular smooth muscle cell inflammation and inhibit its apoptosis. The mechanism of action is related to the up-regulation of miR-455-5p expression.