Human Smooth Muscle Cell Proliferation Research Articles

Early-life microRNA signatures in cord blood associated with allergic rhinitis and asthma development.

MicroRNAs (miRNAs) are involved in the biological regulation of asthma and allergies. To investigate the association between cord blood miRNAs and the development of allergic rhinitis and early childhood asthma. miRNAs were sequenced from cord blood of subjects participating in the Vitamin D Antenatal Asthma Reduction Trial. Multivariable miRNA differential expression analyses were performed to examine their association with physician diagnosed asthma and allergic rhinitis by age 3, as well as active asthma status at age 6 years. miRNA signatures were further investigated for their ability to induce human airway smooth muscle cell (HASMC) proliferation in vitro. In a cohort of 389 subjects, elevated cord blood expression of miR-149-5p was associated with both age 3 allergic rhinitis and asthma (log2FC: 1.87 and 1.42, respectively, FDR<0.001), as well as age 6 active asthma status (log2FC: 2.26, FDR<0.001). Higher expressions of miR-99b-5p, miR-125a-5p, and miR-200c-3p were also associated with both diagnosis of allergic rhinitis at age of 3 years and active asthma status at age of 6 (allergic rhinitis: log2FC: 0.6, 0.62, and 1.06, respectively, FDR<0.001; active asthma: log2FC: 0.55, 0.60, 1.10, respectively, FDR<0.001, respectively). Higher expression of miR-145-5p was associated with both new onset asthma after age 3 and active asthma status at age 6 (log2FC: 0.73 and 0.40, FDR<0.001, respectively). These five miRNA signatures target key hubs in the interactome module of 71 genes associated with allergic rhinitis and asthma. Transfection of miR-125a-5p and miR-145-5p into HASMC induced cell proliferation. The dysregulation of cord blood miRNAs at birth are associated with allergic rhinitis and early childhood asthma. The miRNAs that regulate post-embryonic development and immune response may serve as potential biomarkers and preventive targets for asthma.

Functional screening identifies miRNAs with a novel function inhibiting vascular smooth muscle cell proliferation.

Proliferation of vascular smooth muscle cells (vSMCs) is a crucial contributor to pathological vascular remodeling. MicroRNAs (miRNAs) are powerful gene regulators and attractive therapeutic agents. Here, we aimed to systematically identify and characterize miRNAs with therapeutic potential in targeting vSMC proliferation. Using high-throughput screening, we assessed the impact of 2,042 human miRNA mimics on vSMC proliferation and identified seven miRNAs with novel vSMC anti-proliferative function: miR-323a-3p, miR-449b-5p, miR-491-3p, miR-892b, miR-1827, miR-4774-3p, and miR-5681b. miRNA-mimic treatment affects proliferation of vSMCs from different vascular beds. Focusing on vein graft failure, where miRNA-based therapeutics can be applied to the graft exvivo, we showed that these miRNAs reduced human saphenous vein smooth muscle cell (HSVSMC) proliferation without toxic effect. HSVSMC transcriptomics revealed a distinct set of targets for each miRNA, leading to the common downregulation of a cell-cycle gene network for all miRNAs. For miR-449b-5p, we showed that its candidate target, CCND1, contributes to HSVSMC proliferation. In contrast to HSVSMCs, miRNA overexpression in endothelial cells led to a limited response in terms of proliferation and transcriptomics. In an exvivo vein organ model, overexpression of miR-323a-3p and miR-449b-5p reduced medial proliferation. Collectively, the results of our study show the therapeutic potential of seven miRNAs to target pathological vascular remodeling.

Death-associated protein kinase 1 prevents hypoxia-induced metabolic shift and pulmonary arterial smooth muscle cell proliferation in PAH

Pulmonary hypertension (pH) is a general term used to describe high blood pressure in the lungs from any cause. Pulmonary arterial hypertension (PAH) is a progressive, and fatal disease that causes the walls of the pulmonary arteries to tighten and stiffen. One of the major characteristics of PAH is the hyperproliferation and resistance to apoptosis of vascular cells, which trigger excessive pulmonary vascular remodeling and vasoconstriction. The death-associated protein DAP-kinase (DAPK) is a tumor suppressor and Ser/Thr protein kinase, which was previously shown to regulate the hypoxia inducible factor (HIF)-1α. Against this background, we now show that DAPK1 regulates human pulmonary smooth muscle cell (hPASMC) proliferation and energy metabolism in a HIF-dependent manner. DAPK1 expression is downregulated in pulmonary vessels and PASMCs of human and experimental PH lungs. Reduced expression of DAPK1 in hypoxia and non-hypoxia PAH-PASMCs correlates with increased expression of HIF-1/2α. RNA interference-mediated depletion of DAPK1 leads to fundamental metabolic changes, including a significantly decreased rate of oxidative phosphorylation associated with enhanced expression of both HIF-1α and HIF-2α and glycolytic enzymes, as hexokinase 2 (HK2), lactate dehydrogenase A (LDHA), and an integrator between the glycolysis and citric acid cycle, pyruvate dehydrogenase kinase 1 (PDK1). DAPK1 ablation in healthy donor hPASMCs leads to an increase in proliferation, while its overexpression provides the opposite effects. Together our data indicate that DAPK1 serves as a new inhibitor of the pro-proliferative and glycolytic phenotype of PH in PASMCs acting via HIF-signaling pathway.

MicroRNA-637/661 ameliorate hypoxic-induced pulmonary arterial hypertension by targeting TRIM29 signaling pathway

The pathogenesis of pulmonary arterial hypertension (PAH) is closely linked to the abnormal proliferation of pulmonary artery smooth muscle cells. Studies have demonstrated that microRNAs play pivotal roles in the progression of pulmonary hypertension. We found that microRNA-637 (miR-637) and microRNA-661 (miR-661) are expressed at low levels in the serum of PAH patients. Moreover, the overexpression of miR-637 or miR-661 inhibited human pulmonary artery smooth muscle cell (HPASMC) proliferation and migration in hypoxic culture. Mechanistically, we overexpressed these two microRNAs in HPASMCs, and the RNA-sequencing (RNA-seq) results demonstrated that TRIM29 mRNA was suppressed, indicating that TRIM29 is a substrate. TRIM29 accumulates in the serum of patients with PAH and promotes cell proliferation and migration by activating AKT/mTOR signalling. In addition, overexpression of miR-637 or miR-661 reversed TRIM29-mediated HPASMC proliferation and migration. This study revealed that miR-637 and miR-661 are able to inhibit the proliferation ability of HPASMCs under hypoxic conditions through targeting TRIM29, suggesting that the microRNA-637/661/TRIM29 axis may act as a target for PAH treatment.

CircNAP1L4 regulates pulmonary artery smooth muscle cell proliferation via the NAP1L4-mediated super-enhancer-driven glycolysis gene hexokinase II (HK II) in pulmonary hypertension.

Glycolysis is a major determinant of pulmonary artery smooth muscle cell (PASMC) proliferation in pulmonary hypertension (PH). Circular RNAs (circRNAs) are powerful regulators of glycolysis in multiple diseases; however, the role of circRNAs in glycolysis in PH has been poorly characterized. The aim of this study was to uncover the regulatory mechanism of a new circRNA, circNAP1L4, in human pulmonary artery smooth muscle cell (HPASMC) proliferation through the host protein NAP1L4 to regulate the super-enhancer-driven glycolysis gene hexokinase II (HK II). CircNAP1L4 was downregulated in hypoxic HPASMCs and plasma of PH patients. Functionally, circNAP1L4 overexpression inhibited glycolysis and proliferation in hypoxic HPASMCs. Mechanistically, circNAP1L4 directly bound to its host protein NAP1L4 and affected the ability of NAP1L4 to move into the nucleus to regulate the epigenomic signals of the super-enhancer of HK II. Intriguingly, circNAP1L4 overexpression inhibited the proliferation but not the migration of human pulmonary arterial endothelial cells (HPAECs) cocultured with HPASMCs. Furthermore, pre-mRNA-processing-splicing Factor 8 (PRP8) was found to regulate the production ratio of circNAP1L4 and linear NAP1L4. Invivo, targeting circNAP1L4 alleviates SU5416 combined with hypoxia (SuHx)-induced PH. Overall, these findings reveal a new circRNA that inhibits PASMC proliferation and serves as a therapeutic target for PH.

Zedoarondiol inhibits human bronchial smooth muscle cell proliferation through the CAV-1/PDGF signalling pathway

Airway remodelling in lung diseases can be treated by inhibiting excessive smooth muscle cell proliferation. Zedoarondiol (Zed) is a natural compound isolated from the Chinese herb Curcuma longa. The caveolin-1 (CAV-1) is widely expressed in lung cells and plays a key role in platelet-derived growth factor (PDGF) signalling and cell proliferation. This study aims to investigate the effect of Zed on human bronchial smooth muscle cell (HBSMC) proliferation and explore its potential molecular mechanisms. We assessed the effect of Zed on the proliferation of PDGF-stimulated HBSMCs and performed proteomic analysis to identify potential molecular targets and pathways. CAV1 siRNA was used to validate our findings in vitro. In PDGF-stimulated HBSMCs, Zed significantly inhibited excessive proliferation of HBSMCs. Proteomic analysis of zedoarondiol-treated HBSMCs revealed significant enrichment of differentially expressed proteins in cell proliferation-related pathways and biological processes. Zed inhibition of HBSMC proliferation was associated with upregulation of CAV1, regulation of the CAV-1/PDGF pathway and inhibition of MAPK and PI3K/AKT signalling pathway activation. Treatment of HBSMCs with CAV1 siRNA partly reversed the inhibitory effect of Zed on HBSMC proliferation. Thus, this study reveals that zedoarondiol potently inhibits HBSMC proliferation by upregulating CAV-1 expression, highlighting its potential value in airway remodelling and related diseases.

Deficiency of the Deubiquitinase UCHL1 Attenuates Pulmonary Arterial Hypertension.

The ubiquitin-proteasome system regulates protein degradation and the development of pulmonary arterial hypertension (PAH), but knowledge about the role of deubiquitinating enzymes in this process is limited. UCHL1 (ubiquitin carboxyl-terminal hydrolase 1), a deubiquitinase, has been shown to reduce AKT1 (AKT serine/threonine kinase 1) degradation, resulting in higher levels. Given that AKT1 is pathological in pulmonary hypertension, we hypothesized that UCHL1 deficiency attenuates PAH development by means of reductions in AKT1. Tissues from animal pulmonary hypertension models as well as human pulmonary artery endothelial cells from patients with PAH exhibited increased vascular UCHL1 staining and protein expression. Exposure to LDN57444, a UCHL1-specific inhibitor, reduced human pulmonary artery endothelial cell and smooth muscle cell proliferation. Across 3 preclinical PAH models, LDN57444-exposed animals, Uchl1 knockout rats (Uchl1-/-), and conditional Uchl1 knockout mice (Tie2Cre-Uchl1fl/fl) demonstrated reduced right ventricular hypertrophy, right ventricular systolic pressures, and obliterative vascular remodeling. Lungs and pulmonary artery endothelial cells isolated from Uchl1-/- animals exhibited reduced total and activated Akt with increased ubiquitinated Akt levels. UCHL1-silenced human pulmonary artery endothelial cells displayed reduced lysine(K)63-linked and increased K48-linked AKT1 levels. Supporting experimental data, we found that rs9321, a variant in a GC-enriched region of the UCHL1 gene, is associated with reduced methylation (n=5133), increased UCHL1 gene expression in lungs (n=815), and reduced cardiac index in patients (n=796). In addition, Gadd45α (an established demethylating gene) knockout mice (Gadd45α-/-) exhibited reduced lung vascular UCHL1 and AKT1 expression along with attenuated hypoxic pulmonary hypertension. Our findings suggest that UCHL1 deficiency results in PAH attenuation by means of reduced AKT1, highlighting a novel therapeutic pathway in PAH.

Curcumol reduces lower limb arteriosclerosis in rats by inhibiting human arterial smooth muscle cell activity.

Cardiovascular diseases, particularly those involving arterial stenosis and smooth muscle cell proliferation, pose significant health risks. This study aimed to investigate the therapeutic potential of curcumol in inhibiting platelet-derived growth factor-BB (PDGF-BB)-induced human aortic smooth muscle cell (HASMC) proliferation, migration and autophagy. Using cell viability assays, 5-ethynyl-2'-deoxyuridine (EdU) incorporation assays and Western Blot analyses, we observed that curcumol effectively attenuated PDGF-BB-induced HASMC proliferation and migration in a concentration-dependent manner. Furthermore, curcumol mitigated PDGF-BB-induced autophagy, as evidenced by the downregulation of LC3-II/LC3-I ratio and upregulation of P62. In vivo experiments using an arteriosclerosis obliterans model demonstrated that curcumol treatment significantly ameliorated arterial morphology and reduced stenosis. Additionally, curcumol inhibited the activity of the KLF5/COX2 axis, a key pathway in vascular diseases. These findings suggest that curcumol has the potential to serve as a multi-target therapeutic agent for vascular diseases.

EGR1 transcriptionally regulates SVEP1 to promote proliferation and migration in human coronary artery smooth muscle cells.

A low-frequency variant of sushi, von Willebrand factor type A, EGF, and pentraxin domain-containing protein 1 (SVEP1) is associated with the risk of coronary artery disease, as determined by a genome-wide association study. SVEP1 induces vascular smooth muscle cell proliferation and an inflammatory phenotype to promote atherosclerosis. In the present study, qRT‒PCR demonstrated that the mRNA expression of SVEP1 was significantly increased in atherosclerotic plaques compared to normal tissues. Bioinformatics revealed that EGR1 was a transcription factor for SVEP1. The results of the luciferase reporter assay, siRNA interference or overexpression assay, mutational analysis and ChIP confirmed that EGR1 positively regulated the transcriptional activity of SVEP1 by directly binding to its promoter. EGR1 promoted human coronary artery smooth muscle cell (HCASMC) proliferation and migration via SVEP1 in response to oxidized low-density lipoprotein (ox-LDL) treatment. Moreover, the expression level of EGR1 was increased in atherosclerotic plaques and showed a strong linear correlation with the expression of SVEP1. Our findings indicated that EGR1 binding to the promoter region drive SVEP1 transcription to promote HCASMC proliferation and migration.

A small-diameter vascular graft promotes rapid and benign remodeling of the neointima through dual release of nitric oxide and hydrogen sulfide

Coronary artery bypass grafting often requires autologous veins as alternatives. The limited source and problem of over-expansion are major obstacles for vein grafts, which could be solved by small-diameter artificial vascular graft. However, their clinical application is limited by thrombosis and restenosis caused by incomplete endothelialization and abnormal smooth muscle cells (SMCs) proliferation. Nitric oxide (NO) and hydrogen sulfide (H2S) are crucial signaling molecules in the cardiovascular system, known to regulate endothelial cells(ECs) and SMCs proliferation, migration, reduce oxidative stress, and inhibit inflammation to prevent intimal hyperplasia. In this study, we developed a new method to prepare a keratin based H2S donor(KAT) complexed with copper ions and electrospun it with PCL to prepare PCL/KAT-Cu small-diameter tissue-engineered vascular graft, which is capable of dual release of NO and H2S. We investigated the effects of NO and H2S release on human umbilical vein endothelial cells (HUVECs) and human umbilical arterial smooth muscle cells(HUASMCs) proliferation and migration, evaluated the graft's selectivity for HUVECs, and assessed its effects on macrophage phenotypic transitions and its protective effect on HUVECs under reactive oxygen species (ROS) conditions. Additionally, we tested the graft's ability to rapidly endothelialize under shear force using an in vitro 3D-perfusion system. After one month of in situ transplantation in rat abdominal aorta, the PCL/KAT-Cu small-diameter tissue-engineered vascular grafts demonstrated satisfactory intimal remodeling, comparable to natural blood vessels. In conclusion, our study presents a promising new strategy for remodeling small-diameter vascular grafts.

Abstract 13109: Empagliflozin Reverses Oxidized LDL-Induced RECK Suppression, MMP Activation, and Human Aortic Smooth Muscle Cell Proliferation and Migration

Empagliflozin, an FDA-approved anti-hyperglycemic agent, is a potent and highly selective inhibitor of sodium glucose co-transporter-2 (SGLT-2) in renal proximal tubule epithelial cells. It blocks glucose reabsorption and promotes glucose excretion via urine. Intriguingly, several clinical trials have reported the pleiotropic beneficial effects of SGLT-2 inhibitors in cardiovascular diseases, including antioxidant and anti-inflammatory effects. Here we investigated whether empagliflozin reverses oxidatively modified LDL (OxLDL)-induced matrix metalloproteinase activation and smooth muscle cell (SMC) migration and proliferation. Quiesced human aortic SMCs (Lonza) were treated with empagliflozin (1 uM) followed by human medium OxLDL (40 ug/mL; Kalen Biomedical). OxLDL stimulated SMC migration (1.8-fold, P&lt;0.05, n=4) and proliferation (1.5-fold, P&lt;0.05, n=4), and empagliflozin attenuated both by 67% (P&lt;0.05, n=4) and 61% (P&lt;0.05, n=4), respectively. Increased migration and proliferation by OxLDL were accompanied by elevated MMP-2 (3.2-fold, P&lt;0.05, n=5) and 9 (3.1-fold, P&lt;0.05, n=5) activities, which were also suppressed by empagliflozin by 65% and 67%, respectively (both P&lt;0.05, n=5). RECK (reversion inducing cysteine rich protein with Kazal motifs) is a membrane anchored MMP inhibitor, and we found that OxLDL dose-dependently downregulated RECK in SMCs (by 60%, P&lt;0.05, n=3) via NF-kB-dependent miR-30b induction. Intriguingly, empagliflozin restored the OxLDL downregulation of RECK to 88% of the control (n=3), as it inhibited miR-30b induction (by 49%, P&lt;0.05, n=5). Consistent with RECK-mediated effects of empagliflozin, adenoviral overexpression of RECK inhibited OxLDL-induced SMC migration (by 67%, P&lt;0.05, n=5) and proliferation (by 50%, P&lt;0.05, n=5). Together, these results suggest that the vasculoprotective effects of empagliflozin are at least in part mediated by restoring RECK, thereby attenuating SMC migration and proliferation under elevated oxidative stress condition. Our results also suggest therapeutic potential of empagliflozin or RECK overexpression in vascular proliferative diseases.

Targeting IL-11 system as a treatment of pulmonary arterial hypertension

IL-11 is linked to fibrotic diseases, but its role in pulmonary hypertension is unclear. We examined IL-11's involvement in idiopathic pulmonary arterial hypertension (iPAH). Using samples from control (n = 20) and iPAH (n = 6) subjects, we assessed IL-11 and IL-11Rα expression and localization through RT-qPCR, ELISA, immunohistochemistry, and immunofluorescence. A monocrotaline-induced PAH model helped evaluate the impact of siRNA-IL-11 on pulmonary artery remodeling and PH. The effects of recombinant human IL-11 and IL-11Rα on human pulmonary artery smooth muscle cell (HPASMC) proliferation, pulmonary artery endothelial cell (HPAEC) mesenchymal transition, monocyte interactions, endothelial tube formation, and precision cut lung slice (PCLS) pulmonary artery remodeling and contraction were evaluated. IL-11 and IL-11Rα were over-expressed in pulmonary arteries (3.2-fold and 75-fold respectively) and serum (1.5-fold and 2-fold respectively) of patients with iPAH. Therapeutic transient transfection with siRNA targeting IL-11 resulted in a significant reduction in pulmonary artery remodeling (by 98%), right heart hypertrophy (by 66%), and pulmonary hypertension (by 58%) in rats exposed to monocrotaline treatment. rhIL-11 and soluble rhIL-11Rα induce HPASMC proliferation and HPAEC to monocyte interactions, mesenchymal transition, and tube formation. Neutralizing monoclonal IL-11 and IL-11Rα antibodies inhibited TGFβ1 and EDN-1 induced HPAEC to mesenchymal transition and HPASMC proliferation. In 3D PCLS, rhIL-11 and soluble rhIL-11Rα do not promote pulmonary artery contraction but sensitize PCLS pulmonary artery contraction induced by EDN-1.In summary, IL-11 and IL-11Rα are more highly expressed in the pulmonary arteries of iPAH patients and contribute to pulmonary artery remodeling and the development of PH.

Empagliflozin Reverses Oxidized LDL-Induced RECK Suppression, Cardiotrophin-1 Expression, MMP Activation, and Human Aortic Smooth Muscle Cell Proliferation and Migration.

Persistent oxidative stress and inflammation contribute causally to smooth muscle cell (SMC) proliferation and migration, the characteristic features of vascular proliferative diseases. Oxidatively modified low-density lipoproteins (OxLDL) elevate oxidative stress levels, inflammatory responses, and matrix metallopeptidase (MMP) activation, resulting ultimately in SMC migration, proliferation, and phenotype change. Reversion-inducing cysteine-rich protein with Kazal motifs (RECK) is a membrane-anchored MMP inhibitor. Empagliflozin is an SGLT2 inhibitor and exerts pleiotropic cardiovascular protective effects, including antioxidant and anti-inflammatory effects. Here, we investigated (i) whether OxLDL regulates RECK expression, (ii) whether ectopic expression of RECK reverses OxLDL-induced SMC migration and proliferation, and (iii) whether pretreatment with empagliflozin reverses OxLDL-induced RECK suppression, MMP activation, and SMC migration, proliferation, and differentiation. Indeed, results show that OxLDL at pathophysiological concentration promotes SMC migration and proliferation via NF-κB/miR-30b-dependent RECK suppression. Moreover, OxLDL changed the SMC phenotype to a more pro-inflammatory type, and this effect is blunted by RECK overexpression. Further, treatment with empagliflozin reversed OxLDL-induced miR-30b induction, RECK suppression, MMP activation, SMC migration, proliferation, and proinflammatory phenotype changes. OxLDL-induced cardiotrophin (CT)-1 expression and CT-1 stimulated SMC proliferation and migration in part via leukemia inhibitory factor receptor (LIFR) and glycoprotein 130 (gp130). Ectopic expression of RECK inhibited these effects by physically associating with LIFR and gp130, as evidenced by immunoprecipitation/immunoblotting and double immunofluorescence. Importantly, empagliflozin inhibited CT-1-induced mitogenic and migratory effects. Together, these results suggest the therapeutic potential of sustaining RECK expression or empagliflozin in vascular diseases characterized by SMC proliferation and migration.

Hsa_circ_0031891 targets miR-579-3p to enhance HMGB1 expression and regulate PDGF-BB-induced human aortic vascular smooth muscle cell proliferation, migration, and dedifferentiation.

Atherosclerosis (AS) is an underlying cause of the majority of coronary artery disease (CAD), in which proliferation, migration, and dedifferentiation of vascular smooth muscle cells (VSMCs) exert vital roles. It has been reported that circular RNAs (circRNAs) are associated with the VSMCs function. Here, we undertook to explore the biological function and mechanism of hsa_circ_0031891 in a platelet-derived growth factor-BB (PDGF-BB)-induced AS cell model. Hsa_circ_0031891 and microRNA-579-3p (miR-579-3p) levels were detected by real-time quantitative polymerase chain reaction (RT-qPCR). Cell proliferation and migration were detected using Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), wound healing, and transwell assay. Protein levels of alpha-smooth muscle actin (α-SMA), smooth muscle protein 22-α (SM22-α), Osteopontin, and High mobility group box-1 (HMGB1) were determined using western blot assay. After predicting via a variety of bioinformatics software, the binding between miR-579-3p and hsa_circ_0031891 or HMGB1 was validated using dual-luciferase reporter and RNA pull-down assays. Increased hsa_circ_0031891 and HMGB1 and reduced miR-579-3p were found in CAD patients and PDGF-BB-induced human aortic vascular smooth muscle cells (HA-VSMCs). Moreover, hsa_circ_0031891 deficiency relieved PDGF-BB-mediated HA-VSMC proliferation, migration, and dedifferentiation. Mechanically, hsa_circ_0031891 modulated HMGB1 expression via sponging miR-579-3p. Hsa_circ_0031891 boosted PDGF-BB-induced proliferation, migration, and dedifferentiation partly by regulating the miR-579-3p/HMGB1 axis, hinting at a feasible therapeutic strategy for AS.

Kisspeptin regulates airway hyperresponsiveness and remodeling in a mouse model of asthma.

Asthma is a multifactorial disease of origin characterized by airway hyperresponsiveness (AHR) and airway remodeling. Several pieces of evidence from other pathologies suggest that Kisspeptins (Kp) regulate cell proliferation, migration, and invasion, mechanisms that are highly relevant to asthma. Our recent in vitro studies show Kp-10 (active peptide of Kp), via its receptor, KISS1R, inhibits human airway smooth muscle cell proliferation. Here, we hypothesize a crucial role for Kp-10 in regulating AHR and airway remodeling in vivo. Utilizing C57BL/6J mice, we assessed the effect of chronic intranasal Kp-10 exposure on mixed allergen (MA)-induced mouse model of asthma. MA-challenged mice showed significant deterioration of lung function compared to those exposed to vehicle (DPBS); Kp-10 treatment significantly improved the MA-altered lung functions. Mice treated with Kp-10 alone did not show any notable changes in lung functions. MA-exposed mice showed a significant reduction in KISS1R expression as compared to vehicle alone. MA-challenged mice showed significant alterations in immune cell infiltration in the airways and remodeling changes. Proinflammatory cytokines were significantly increased upon MA exposure, an effect abrogated by Kp-10 treatment. Furthermore, biochemical and histological studies showed Kp-10 exposure significantly reduced MA-induced smooth muscle mass and soluble collagen in the lung. Overall, our findings highlight the effect of chronic Kp-10 exposure in regulating MA-induced AHR and remodeling. © 2023 The Pathological Society of Great Britain and Ireland.

Semaphorin 7A promotes human vascular smooth muscle cell proliferation and migration through the β-catenin signaling pathway

Background: Vascular smooth muscle cells (VSMCs) undergo a conversion from a contractile phenotype to a proliferative synthetic phenotype, contributing to the pathogenesis of cardiovascular diseases. Semaphorin 7A (SEMA7A) is a glycosylphosphatidylinositol-anchored membrane protein that plays an important role in vascular homeostasis by regulating endothelial cell behaviors. However, the expression and role of SEMA7A in VSMCs remain unclear.Methods: In this study, we screened for VSMC-regulating genes in publicly available datasets and analyzed the expression of SEMA7A in human coronary artery smooth muscle cells (hCASMCs) treated with platelet-derived growth factor-BB (PDGF-BB). The effects of SEMA7A overexpression and knockdown on hCASMC proliferation and migration were examined. The signaling pathways involved in the action of SEMA7A in hCASMCs were determined.Results: Bioinformatic analysis showed that SEMA7A was significantly dysregulated in VSMCs treated with oxidized low-density lipoprotein or overexpressing progerin, a pro-atherogenic gene. The PDGF-BB stimulation led to a concentration- and time-dependent induction of SEMA7A. Depletion of SEMA7A attenuated PDGF-BB-induced hCASMC proliferation and migration. Conversely, overexpression of SEMA7A enhanced hCASMC proliferation and migration. Mechanistically, SEMA7A stimulated the activation of the β-catenin pathway and upregulated c-Myc, CCND1, and MMP7. Knockdown of β-catenin impaired SEMA7A-induced hCASMC proliferation and migration.Conclusions: SEMA7A triggers phenotype switching in VSMCs through the β-catenin signaling pathway and may serve as a potential therapeutic target for cardiovascular diseases.

The extract from Hyssopus cuspidatus Boriss. Prevents bronchial airway remodeling by inhibiting mouse bronchial wall thickening and hASMC proliferation and migration

Ethnopharmacological relevanceBronchial asthma, a non-communicable chronic respiratory disease, affects people of all ages. An important pathological feature of bronchial asthma is airway remodeling. Hyssopus cuspidatus Boriss. has been used to treat bronchial asthma for over 100 years in Uygur medicine. The ethanol extract of Hyssopus cuspidatus Boriss.(JAX2) can improve airway inflammation in asthma. However, the anti-asthmatic airway-remodeling effect of JAX2 is unclear. Aim of the studyThe current study investigated the anti-airway remodeling effect of JAX2 and elucidated its mechanism of action. Materials and methodsThe present study established an ovalbumin-induced mouse model of asthma and platelet-derived growth factor-BB-induced human airway smooth muscle cells (hASMCs) proliferation model, with dexamethasone (DEX) and feining tablets (FNP) designated as positive control drugs. Pathological changes in lung tissues were observed using hematoxylin and eosin staining. Interleukin (IL)-5, IL-10, IL-13, and IL-33 levels in the bronchoalveolar lavage fluid (BALF) and serum of mice were determined using enzyme-linked immunosorbent assay (ELISA). Changes in the expression and distribution of TGF-β1, p-ERK1/2, Smad2/3, and p-Smad3 in lung tissues were determined using immunohistochemistry. Western blotting (WB) was used to determine the protein levels of p-ERK1/2 in lung tissues and cells. MTS assay was used to determine the effects of JAX2 on cell proliferation. IL-5, IL-10, IL-13, MMP-2, and MMP-9 levels in the cell supernatant were determined using ELISA. HASMCs migration was observed using the scratch and transwell methods. The effect of JAX2 on the hASMCs cycle was determined using flow cytometry. ResultsJAX2 significantly improved the pathological status of lung tissues in asthmatic mice. It could also significantly reduce IL-5, IL-13, and IL-33 levels in the BALF and serum of asthmatic mice in a dose-dependent manner and significantly increase IL-10 levels. TGF-β1, p-ERK1/2, Smad2/3, and p-Smad3 expression in lung tissues were decreased in a dose-dependent manner. The protein level of p-ERK1/2 in lung tissues was also reduced. JAX2 could significantly inhibit the proliferation and migration of PDGF-BB-induced hASMCs. IL-5, IL-13, MMP-9, and MMP-2 levels decreased significantly, and IL-10 levels increased significantly in a dose-dependent manner in the cell supernatant. JAX2 could block hASMCs in the G0/G1 phase, thereby inhibiting cell proliferation. p-ERK1/2 protein levels were found to decrease in a dose-dependent manner. ConclusionsJAX2 significantly inhibits airway remodeling in asthma. Its mechanism of action may be inhibiting the proliferation and migration of hASMCs, releasing inflammatory factors and metalloproteinases, activating the ERK1/2 signal pathway, and promoting the secretion of anti-inflammatory factors.

Bempedoic acid, an ATP citrate lyase inhibitor, reduces intimal hyperplasia via activation of AMPKα signaling pathway.

One of the common pathophysiological basis of atherosclerosis is intimal hyperplasia. ATP citrate lyase (ACLY) has been reported as a promising therapeutic target for treatment of dyslipidemia and atherosclerosis. However, the role of ACLY in intimal hyperplasia has yet to be clarified. The current investigation studies the molecular effects of ACLY and bempedoic acid, an ACLY inhibitor, on platelet-derived growth factor (PDGF)-induced primary human aortic smooth muscle cells (HASMCs) proliferation in vitro and on femoral arterial wire-injured neointimal hyperplasia in mouse in vivo. The role of ACLY in intimal hyperplasia was further investigated in mice treated with bempedoic acid. Cell proliferation was measured by CCK8 and BrdU assays. We explored further mechanisms using western blot, qPCR and immunofluorescence. We found that ACLY was significantly increased in dedifferentiated VSMC in vitro and vivo. Bempedoic acid which can inhibit ACLY expression effectively blocked PDGF-induced VSMC proliferation and dedifferentiation by activating AMPK/ACC signaling pathway. Moreover, bempedoic acid also attenuated VSMC proliferation and inhibited VSMC dedifferentiation in the wire-injured mouse femoral arteries, resulting in reduced neointima formation. We demonstrates that bempedoic acid reduces ACLY expression to restrain VSMC proliferation and dedifferentiation by activating AMPK/ACC signaling pathway, which may provide a potential therapeutic strategy for diseases associated with intimal hyperplasia including restenosis and atherosclerosis.

IL-11 system participates in pulmonary artery remodeling and hypertension in pulmonary fibrosis

BackgroundPulmonary hypertension (PH) associated to idiopathic pulmonary fibrosis (IPF) portends a poor prognosis. IL-11 has been implicated in fibrotic diseases, but their role on pulmonary vessels is unknown. Here we analyzed the contribution of IL-11 to PH in patients with IPF and the potential mechanism implicated.MethodsPulmonary arteries, lung tissue and serum of control subjects (n = 20), IPF (n = 20) and PH associated to IPF (n = 20) were used to study the expression and localization of IL-11 and IL-11Rα. Two models of IL-11 and bleomycin-induced lung fibrosis associated to PH were used in Tie2-GFP transgenic mice to evaluate the contribution of IL-11 and endothelial cells to pulmonary artery remodeling. The effect of IL-11 and soluble IL-11Rα on human pulmonary artery endothelial cells and smooth muscle cell transformations and proliferation were analyzed.ResultsIL-11 and IL-11Rα were over-expressed in pulmonary arteries and serum of patients with PH associated to IPF vs IPF patients without PH. Recombinant mice (rm)IL-11 induced lung fibrosis and PH in Tie2-GFP mice, activating in vivo EnMT as a contributor of pulmonary artery remodeling and lung fibrosis. Transient transfection of siRNA-IL-11 reduced lung fibrosis and PH in Tie2-GFP bleomycin model. Human (h)rIL-11 and soluble hrIL-11Rα induced endothelial to mesenchymal transition (EnMT) and pulmonary artery smooth muscle cell to myofibroblast-like transformation, cell proliferation and senescence in vitro.ConclusionsIL-11 and IL-11Rα are overexpressed in pulmonary arteries of PH associated to IPF patients, and contributes to pulmonary artery remodeling and PH.

Study on Thyroxine Preventing Coronary Atherosclerosis through TGFBR1 Signaling Pathway

To explore the mechanism of Thyroxine inhibiting Human Aortic Vascular Smooth Muscle Cell (HASMC) proliferation, migration and vascular intimal hyperplasia through the transforming growth factor b receptor 1(TGFBR1). 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2H-tetrazolium (MTS), 5-ethynyl-2' -deoxyuridine (EdU) staining, Transwell, cell scratch test and Western bloting were performed sequentially to detect the effects of Thyroxine on HASMC and the TGFBR1 signaling pathway. A cell model of TGFBR1 overexpression was constructed to further explore the mechanism of TGFBR1 in Thyroxine inhibiting the activation of HASMC. Thyroxine can inhibit HASMC proliferation and migration, and the activation of TGFBR1 signaling pathway,both in a concentration-dependent manner. Following the overexpression of TGFBR1, the effect of Thyroxine on inhibiting HASMC proliferation, migration and activation of TGFBR1 signaling pathway was partially inhibited. Thyroxine can inhibit the proliferation, migration and vascular intimal hyperplasia of HASMC by inhibiting the activation of TGFBR1 signaling pathway, thereby playing a role in preventing and treating vascular restenosis and related diseases.