Cell Phenotypic Modulation Research Articles

Identification of biological significance of different stages of varicose vein development based on mRNA sequencing.

Normal veins could develop to varicose vein (VV) by some risk factors, and might further progress to shallow vein thrombosis (SVT). However, the molecular mechanism of key genes associated with the progression and regression of VV are still not thorough enough. In this study, the healthy control (HC), VV, and SVT vascular samples were collected for transcriptome sequencing. The differentially expressed genes (DEGs) were screened by "DESeq2", including DEGs1 (HC vs. VV), DEGs2 (HC vs. SVT) and DEGs3 (VV vs. SVT). And their functional enrichment analyses were conducted by "ClusterProfiler". The receiver operating characteristic (ROC) curve was used to obtain the key genes (KGs) of the pathogenesis of VV and SVT. The qRT-PCR assay was performed to validate the expressions of KGs. Immune cell infiltration analyses were conducted based on ssGSEA method. The competitive endogenous RNAs (ceRNAs) regulatory network was constructed. The target drugs of KGs were predicted using DrugBank database. The biofunctions of DACT3 were further investigated through a series of experiments in vitro. All of these DEGs were associated with inflammation and immunity related functions. Immune cell infiltration was significantly different between VV and SVT. Six key genes including PLP2, DACT3, LRRC25, PILRA, MSX1 and APOD that were associated with the progression and regression of VV were screened. The expression of LRRC25 and PILRA was significantly negatively associated with central memory T cell, and significantly positively associated with B cell. Besides, XIST was the critical regulator of multiple KGs. Cimetidine was potential drug for VV and SVT therapy. Overexpression of DACT3 significantly inhibited the proliferation and migration of vascular smooth muscle cells (VSMCs), and affected their cell cycle and phenotypic transition. This study identified six key genes associated with the progression and regression of VV. Among them, DACT3 was proved to hinder VV progression. These findings may help to deepen understanding its underlying mechanisms.

P15-09 Immunometabolism and phenotype modulation of human dendritic cells exposed to Nickel and Cobalt

P15-09 Immunometabolism and phenotype modulation of human dendritic cells exposed to Nickel and Cobalt

PTPN14 aggravates neointimal hyperplasia via boosting PDGFRβ signaling in smooth muscle cells

Smooth muscle cell (SMC) phenotypic modulation, primarily driven by PDGFRβ signaling, is implicated in occlusive cardiovascular diseases. However, the promotive and restrictive regulation mechanism of PDGFRβ and the role of protein tyrosine phosphatase non-receptor type 14 (PTPN14) in neointimal hyperplasia remain unclear. Our study observes a marked upregulation of PTPN14 in SMCs during neointimal hyperplasia. PTPN14 overexpression exacerbates neointimal hyperplasia in a phosphatase activity-dependent manner, while SMC-specific deficiency of PTPN14 mitigates this process in mice. RNA-seq indicates that PTPN14 deficiency inhibits PDGFRβ signaling-induced SMC phenotypic modulation. Moreover, PTPN14 interacts with intracellular region of PDGFRβ and mediates its dephosphorylation on Y692 site. Phosphorylation of PDGFRβY692 negatively regulates PDGFRβ signaling activation. The levels of both PTPN14 and phospho-PDGFRβY692 are correlated with the degree of stenosis in human coronary arteries. Our findings suggest that PTPN14 serves as a critical modulator of SMCs, promoting neointimal hyperplasia. PDGFRβY692, dephosphorylated by PTPN14, acts as a self-inhibitory site for controlling PDGFRβ activation.

Epsins oversee smooth muscle cell reprograming by influencing master regulators KLF4 and OCT4.

Smooth muscle cells in major arteries play a crucial role in regulating coronary artery disease. Conversion of smooth muscle cells into other adverse cell types in the artery propels the pathogenesis of the disease. Curtailing artery plaque buildup by modulating smooth muscle cell reprograming presents us a new opportunity to thwart coronary artery disease. Here, our report how Epsins, a family of endocytic adaptor proteins oversee the smooth muscle cell reprograming by influencing master regulators OCT4 and KLF4. Using single-cell RNA sequencing, we characterized the phenotype of modulated smooth muscle cells in mouse atherosclerotic plaque and found that smooth muscle cells lacking epsins undergo profound reprogramming into not only beneficial myofibroblasts but also endothelial cells for injury repair of diseased endothelium. Our work lays concrete groundwork to explore an uncharted territory as we show that depleting Epsins bolsters smooth muscle cells reprograming to endothelial cells by augmenting OCT4 activity but restrain them from reprograming to harmful foam cells by destabilizing KLF4, a master regulator of adverse reprograming of smooth muscle cells. Moreover, the expression of Epsins in smooth muscle cells positively correlates with the severity of both human and mouse coronary artery disease. Integrating our scRNA-seq data with human Genome-Wide Association Studies (GWAS) identifies pivotal roles Epsins play in smooth muscle cells in the pathological process leading to coronary artery disease. Our findings reveal a previously unexplored direction for smooth muscle cell phenotypic modulation in the development and progression of coronary artery disease and unveil Epsins and their downstream new targets as promising novel therapeutic targets for mitigating metabolic disorders.

LILRB4 knockdown inhibits aortic dissection development by regulating pyroptosis and the JAK2/STAT3 signaling pathway

Aortic dissection (AD) is a life-threatening condition with a high mortality rate and without effective pharmacological therapies. Our previous study illustrated that leukocyte immunoglobulin-like receptor B4 (LILRB4) knockdown promoted the contractile phenotypic switch and apoptosis of AD cells. This study aimed to further investigate the role of LILRB4 in animal models of AD and elucidate its underlying molecular mechanisms. Animal models of AD were established using 0.1% beta-aminopropionitrile and angiotensin II and an in vitro model was developed using platelet-derived growth factor BB (PDGF-BB). The effects of LILRB4 knockdown on histopathological changes, pyroptosis, phenotype transition, extracellular matrix (ECM), and Janus kinase 2 (JAK2)/signal transducers and activators of transcription 3 (STAT3) pathways were assessed using a series of in vivo and in vitro assays. The effects of the JAK2 inhibitor AG490 on AD cell function, phenotypic transition, and ECM were explored. LILRB4 was highly expressed in AD and its knockdown increased survival rate, reduced AD incidence, and alleviated histopathological changes in the AD mouse model. Furthermore, LILRB4 knockdown promoted contractile phenotype switch, stabilized the ECM, and inhibited pyroptosis. Mechanistically, LILRB4 knockdown inhibited the JAK2/STAT3 signaling pathway. JAK2 inhibitor AG490 inhibited cell viability and migration, enhanced apoptosis, induced G0/G1 cell cycle arrest, and suppressed S-phase progression in PDGF-BB-stimulated human aortic smooth muscle cells. LILRB4 knockdown suppresses AD development by inhibiting pyroptosis and the JAK2/STAT3 signaling pathway.

Macrophages and HLA-Class II Alleles in Multiple Sclerosis: Insights in Therapeutic Dynamics.

Antigen presentation is a crucial mechanism that drives the T cell-mediated immune response and the development of Multiple Sclerosis (MS). Genetic alterations within the highly variable Major Histocompatibility Complex Class II (MHC II) have been proven to result in significant changes in the molecular basis of antigen presentation and the clinical course of patients with both Adult-Onset MS (AOMS) and Pediatric-Onset MS (POMS). Among the numerous polymorphisms of the Human Leucocyte Antigens (HLA), within MHC II complex, HLA-DRB1*15:01 has been labeled, in Caucasian ethnic groups, as a high-risk allele for MS due to the ability of its structure to increase affinity to Myelin Basic Protein (MBP) epitopes. This characteristic, among others, in the context of the trimolecular complex or immunological synapsis, provides the foundation for autoimmunity triggered by environmental or endogenous factors. As with all professional antigen presenting cells, macrophages are characterized by the expression of MHC II and are often implicated in the formation of MS lesions. Increased presence of M1 macrophages in MS patients has been associated both with progression and onset of the disease, each involving separate but similar mechanisms. In this critical narrative review, we focus on macrophages, discussing how HLA genetic alterations can promote dysregulation of this population's homeostasis in the periphery and the Central Nervous System (CNS). We also explore the potential interconnection in observed pathological macrophage mechanisms and the function of the diverse structure of HLA alleles in neurodegenerative CNS, seen in MS, by comparing available clinical with molecular data through the prism of HLA-immunogenetics. Finally, we discuss available and experimental pharmacological approaches for MS targeting the trimolecular complex that are based on cell phenotype modulation and HLA genotype involvement and try to reveal fertile ground for the potential development of novel drugs.

Inhibition of tartrate-resistant acid phosphatase 5 can prevent cardiac fibrosis after myocardial infarction

BackgroundMyocardial infarction (MI) leads to enhanced activity of cardiac fibroblasts (CFs) and abnormal deposition of extracellular matrix proteins, resulting in cardiac fibrosis. Tartrate-resistant acid phosphatase 5 (ACP5) has been shown to promote cell proliferation and phenotypic transition. However, it remains unclear whether ACP5 is involved in the development of cardiac fibrosis after MI. The present study aimed to investigate the role of ACP5 in post-MI fibrosis and its potential underlying mechanisms.MethodsClinical blood samples were collected to detect ACP5 concentration. Myocardial fibrosis was induced by ligation of the left anterior descending coronary artery. The ACP5 inhibitor, AubipyOMe, was administered by intraperitoneal injection. Cardiac function and morphological changes were observed on Day 28 after injury. Cardiac CFs from neonatal mice were extracted to elucidate the underlying mechanism in vitro. The expression of ACP5 was silenced by small interfering RNA (siRNA) and overexpressed by adeno-associated viruses to evaluate its effect on CF activation.ResultsThe expression of ACP5 was increased in patients with MI, mice with MI, and mice with Ang II-induced fibrosis in vitro. AubipyOMe inhibited cardiac fibrosis and improved cardiac function in mice after MI. ACP5 inhibition reduced cell proliferation, migration, and phenotypic changes in CFs in vitro, while adenovirus-mediated ACP5 overexpression had the opposite effect. Mechanistically, the classical profibrotic pathway of glycogen synthase kinase-3β (GSK3β)/β-catenin was changed with ACP5 modulation, which indicated that ACP5 had a positive regulatory effect. Furthermore, the inhibitory effect of ACP5 deficiency on the GSK3β/β-catenin pathway was counteracted by an ERK activator, which indicated that ACP5 regulated GSK3β activity through ERK-mediated phosphorylation, thereby affecting β-catenin degradation.ConclusionACP5 may influence the proliferation, migration, and phenotypic transition of CFs, leading to the development of myocardial fibrosis after MI through modulating the ERK/GSK3β/β-catenin signaling pathway.

Stress, Vascular Smooth Muscle Cell Phenotype and Atherosclerosis: Novel Insight into Smooth Muscle Cell Phenotypic Transition in Atherosclerosis.

Our work is to establish more distinct association between specific stress and vascular smooth muscle cells (VSMCs) phenotypes to alleviate atherosclerotic plaque burden and delay atherosclerosis (AS) progression. In recent years, VSMCs phenotypic transition has received significant interests. Different stresses were found to be associated with VSMCs phenotypic transition. However, the explicit correlation between VSMCs phenotype and specific stress has not been elucidated clearly yet. We discover that VSMCs phenotypic transition, which is widely involved in the progression of AS, is associated with specific stress. We discuss approaches targeting stresses to intervene VSMCs phenotypic transition, which may contribute to develop innovative therapies for AS.

Somatic PDGFRB activating variants promote smooth muscle cell phenotype modulation in intracranial fusiform aneurysm

BackgroundThe fusiform aneurysm is a nonsaccular dilatation affecting the entire vessel wall over a short distance. Although PDGFRB somatic variants have been identified in fusiform intracranial aneurysms, the molecular and cellular mechanisms driving fusiform intracranial aneurysms due to PDGFRB somatic variants remain poorly understood.MethodsIn this study, single-cell sequencing and immunofluorescence were employed to investigate the phenotypic changes in smooth muscle cells within fusiform intracranial aneurysms. Whole-exome sequencing revealed the presence of PDGFRB gene mutations in fusiform intracranial aneurysms. Subsequent immunoprecipitation experiments further explored the functional alterations of these mutated PDGFRB proteins. For the common c.1684 mutation site of PDGFRβ, we established mutant smooth muscle cell lines and zebrafish models. These models allowed us to simulate the effects of PDGFRB mutations. We explored the major downstream cellular pathways affected by PDGFRBY562D mutations and evaluated the potential therapeutic effects of Ruxolitinib.ResultsSingle-cell sequencing of two fusiform intracranial aneurysms sample revealed downregulated smooth muscle cell markers and overexpression of inflammation-related markers in vascular smooth muscle cells, which was validated by immunofluorescence staining, indicating smooth muscle cell phenotype modulation is involved in fusiform aneurysm. Whole-exome sequencing was performed on seven intracranial aneurysms (six fusiform and one saccular) and PDGFRB somatic mutations were detected in four fusiform aneurysms. Laser microdissection and Sanger sequencing results indicated that the PDGFRB mutations were present in smooth muscle layer. For the c.1684 (chr5: 149505131) site mutation reported many times, further cell experiments showed that PDGFRBY562D mutations promoted inflammatory-related vascular smooth muscle cell phenotype and JAK-STAT pathway played a crucial role in the process. Notably, transfection of PDGFRBY562D in zebrafish embryos resulted in cerebral vascular anomalies. Ruxolitinib, the JAK inhibitor, could reversed the smooth muscle cells phenotype modulation in vitro and inhibit the vascular anomalies in zebrafish induced by PDGFRB mutation.ConclusionOur findings suggested that PDGFRB somatic variants played a role in regulating smooth muscle cells phenotype modulation in fusiform aneurysms and offered a potential therapeutic option for fusiform aneurysms.

Abstract 1054: Toll-like Receptor 4 Is A New Regulator Of Smooth Muscle Cell Phenotypic Transitions

Objectives: There is growing evidence that smooth muscle cell ( SMC ) phenotypic transitions have a critical role in cardiovascular pathologies, including atherosclerosis. However, molecular mechanisms regulating different aspects of these transitions are still not fully elucidated. We previously found that the pluripotency factor OCT4 plays a protective role in SMC in atherogenesis and neointioma formation after vascular injury. Using a bioinformatics approach, we identified Toll-like receptor 4 ( TLR4 ), an integral component of the innate immune system, pivotal in eliciting inflammatory responses, as an indirect target of OCT4 in SMC. However, it is not yet elucidated whether OCT4/TLR4 axis has a role in SMC transitions. Methods and Results: First, we used blocking siRNA against Oct4 and different downstream targets of the TLR4 signaling ( Trif , Irf3 ), as well as a TLR4 inhibitor TAK242 (Resatorvid) in cultured mouse aortic SMC treated with the oxidized phospholipid, POVPC. These experiments demonstrated that blocking TLR4 signaling in wild-type SMC resulted in suppression of SMC contractile genes and up-regulation of LGALS3 (SMC transition marker). In contrast, TAK242 treatment decreased LGALS3 levels in OCT4 deficient SMC, indicating that TLR4, at least in part, is responsible for the SMC phenotype in OCT4 deficient SMC. Next, using the bioinformatics approach and Chromatin Immunoprecipitation assays, we identified the EST Proto-oncogene 1 ( ETS1 ) transcriptional factor as a target of OCT4 that directly regulates TLR4. Interestingly, on the other hand, we found that overexpression of TLR4 led to OCT4 activation in SMC, and, oppositely, the inhibition of TLR4 signaling by TAK242 resulted in significant decreases in the POVPC-induced OCT4 activation, indicating that TLR4 signaling is critical for OCT4 activation in SMC. Using an SMC-lineage tracing mouse model combined with a TLR4-IRES-YFP reporter, we demonstrated that TLR4 is highly expressed in the healthy arteries of young mice but not in the phenotypically modulated SMC within the atherosclerotic plaques. Conclusions: These results suggest a negative feedback loop between TLR4 and OCT4 regulating SMC phenotypic transitions.

Abstract 1045: Using Multiplex Imaging For Cell Phenotyping Of Early Human Atherosclerosis

Background: In the past few years, animal models and cultured cells have shown that smooth muscle cells (SMCs) and inflammatory cytokines are important new players in atherosclerotic disease. Validating the roles of these new players in human atherogenesis is crucial for the development of therapeutics targeting them. Multiplex imaging is a powerful tool for mapping cell phenotypes and microenvironment in tissue sections, enabling proof-of-concept studies using biobanked human specimens. However, this technology has seldom been applied to human atherosclerotic lesions. This study is a trial to benchmark the application of the multiplex imaging platform PhenoCycler to biobanked human coronary lesions. We hypothesize that this technology is a powerful tool to prove theories raised by previous studies: SMCs initiate foam cell formation and inflammation is associated with phenotypic switching in SMCs. Methods: We created an 8-plex imaging panel to map lesion cells previously identified as key players in atherogenesis, including foam cells from SMC and macrophage origins, CD68 + SMCs, apoptotic cells, and cells expressing inflammatory cytokines interleukin-1β and tumor necrosis factor α. They were imaged simultaneously on each section of early human lesions (n=9) to test theories related to foam cell formation, phenotypic transition, and inflammatory cytokines. Results: Foam cells of SMC origin greatly outnumbered those of macrophage origin and were enriched in the deep intima, where lipids accumulate in early atherogenesis, distinct from macrophage foam cells in the superficial intima. A higher presence of CD68 + SMCs was observed among lesion SMCs highly expressing interleukin-1β, but not TNF-α. Highly inflamed SMCs exhibited a trend of increased apoptosis compared to macrophages with similar cytokine levels. Conclusions: Our multiplex imaging results confirmed that SMCs form the majority of foam cells in early atherogenesis, and their phenotypic transition to macrophage-like is related to inflammation. Applying multiplex imaging to biobanked human lesions unlocks opportunities to prove that theories based on animal models and cultured cells apply to human atherogenesis.

NKX2-5 regulates vessel remodeling in scleroderma-associated pulmonary arterial hypertension.

NKX2-5 is a member of the homeobox-containing transcription factors critical in regulating tissue differentiation in development. Here, we report a role for NKX2-5 in vascular smooth muscle cell phenotypic modulation in vitro and in vascular remodeling in vivo. NKX2-5 is upregulated in scleroderma patients with pulmonary arterial hypertension. Suppression of NKX2-5 expression in smooth muscle cells halted vascular smooth muscle proliferation and migration, enhanced contractility, and blocked the expression of extracellular matrix genes. Conversely, overexpression of NKX2-5 suppressed the expression of contractile genes (ACTA2, TAGLN, CNN1) and enhanced the expression of matrix genes (COL1) in vascular smooth muscle cells. In vivo, conditional deletion of NKX2-5 attenuated blood vessel remodeling and halted the progression to hypertension in a mouse chronic hypoxia model. This study revealed that signals related to injury such as serum and low confluence, which induce NKX2-5 expression in cultured cells, is potentiated by TGF-β and further enhanced by hypoxia. The effect of TGF-β was sensitive to ERK5 and PI3K inhibition. Our data suggest a pivotal role for NKX2-5 in the phenotypic modulation of smooth muscle cells during pathological vascular remodeling and provide proof of concept for therapeutic targeting of NKX2-5 in vasculopathies.

Integrative analysis of the lncRNA-miRNA-mRNA interactions in smooth muscle cell phenotypic transitions.

Objectives: We previously found that the pluripotency factor OCT4 is reactivated in smooth muscle cells (SMC) in human and mouse atherosclerotic plaques and plays an atheroprotective role. Loss of OCT4 in SMC in vitro was associated with decreases in SMC migration. However, molecular mechanisms responsible for atheroprotective SMC-OCT4-dependent effects remain unknown. Methods: Since studies in embryonic stem cells demonstrated that OCT4 regulates long non-coding RNAs (lncRNAs) and microRNAs (miRNAs), making them candidates for OCT4 effect mediators, we applied an in vitro approach to investigate the interactions between OCT4-regulated lncRNAs, mRNAs, and miRNAs in SMC. We used OCT4 deficient mouse aortic SMC (MASMC) treated with the pro-atherogenic oxidized phospholipid POVPC, which, as we previously demonstrated, suppresses SMC contractile markers and induces SMC migration. Differential expression of lncRNAs, mRNAs, and miRNAs was obtained by lncRNA/mRNA expression array and small-RNA microarray. Long non-coding RNA to mRNA associations were predicted based on their genomic proximity and association with vascular diseases. Given a recently discovered crosstalk between miRNA and lncRNA, we also investigated the association of miRNAs with upregulated/downregulated lncRNA-mRNA pairs. Results: POVPC treatment in SMC resulted in upregulating genes related to the axon guidance and focal adhesion pathways. Knockdown of Oct4 resulted in differential regulation of pathways associated with phagocytosis. Importantly, these results were consistent with our data showing that OCT4 deficiency attenuated POVPC-induced SMC migration and led to increased phagocytosis. Next, we identified several up- or downregulated lncRNA associated with upregulation of the specific mRNA unique for the OCT4 deficient SMC, including upregulation of ENSMUST00000140952-Hoxb5/6 and ENSMUST00000155531-Zfp652 along with downregulation of ENSMUST00000173605-Parp9 and, ENSMUST00000137236-Zmym1. Finally, we found that many of the downregulated miRNAs were associated with cell migration, including miR-196a-1 and miR-10a, targets of upregulated ENSMUST00000140952, and miR-155 and miR-122, targets of upregulated ENSMUST00000155531. Oppositely, the upregulated miRNAs were anti-migratory and pro-phagocytic, such as miR-10a/b and miR-15a/b, targets of downregulated ENSMUST00000173605, and miR-146a/b and miR-15b targets of ENSMUST00000137236. Conclusion: Our integrative analyses of the lncRNA-miRNA-mRNA interactions in SMC indicated novel potential OCT4-dependent mechanisms that may play a role in SMC phenotypic transitions.

Engineering Tumor Stroma Morphogenesis Using Dynamic Cell-Matrix Spheroid Assembly.

The tumor microenvironment consists of resident tumor cells organized within a compositionally diverse, three-dimensional (3D) extracellular matrix (ECM) network that cannot be replicated in vitro using bottom-up synthesis. We report a new self-assembly system to engineer ECM-rich 3D MatriSpheres wherein tumor cells actively organize and concentrate microgram quantities of decellularized ECM dispersions which modulate cell phenotype. 3D colorectal cancer (CRC) MatriSpheres were created using decellularized small intestine submucosa (SIS) as an orthotopic ECM source that had greater proteomic homology to CRC tumor ECM than traditional ECM formulations such as Matrigel. SIS ECM was rapidly concentrated from its environment and assembled into ECM-rich 3D stroma-like regions by mouse and human CRC cell lines within 4-5 days via a mechanism that was rheologically distinct from bulk hydrogel formation. Both ECM organization and transcriptional regulation by 3D ECM cues affected programs of malignancy, lipid metabolism, and immunoregulation that corresponded with an in vivo MC38 tumor cell subpopulation identified via single cell RNA sequencing. This 3D modeling approach stimulates tumor specific tissue morphogenesis that incorporates the complexities of both cancer cell and ECM compartments in a scalable, spontaneous assembly process that may further facilitate precision medicine.

Atherosclerotic three-layer nanomatrix vascular sheets for high-throughput therapeutic evaluation

In vitro atherosclerosis models are essential to evaluate therapeutics before in vivo and clinical studies, but significant limitations remain, such as the lack of three-layer vascular architecture and limited atherosclerotic features. Moreover, no scalable 3D atherosclerosis model is available for making high-throughput assays for therapeutic evaluation. Herein, we report an in vitro 3D three-layer nanomatrix vascular sheet with critical atherosclerosis multi-features (VSA), including endothelial dysfunction, monocyte recruitment, macrophages, extracellular matrix remodeling, smooth muscle cell phenotype transition, inflammatory cytokine secretion, foam cells, and calcification initiation. Notably, we present the creation of high-throughput functional assays with VSAs and the use of these assays for evaluating therapeutics for atherosclerosis treatment. The therapeutics include conventional drugs (statin and sirolimus), candidates for treating atherosclerosis (curcumin and colchicine), and potential gene therapy (miR-146a-loaded liposomes). The high efficiency and flexibility of the scalable VSA functional assays should facilitate drug discovery and development for atherosclerosis.

Compliant substrates mitigate the senescence associated phenotype of stress induced mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs) are a promising cell population for musculoskeletal cell-based therapies due to their multipotent differentiation capacity and complex secretome. Cells from younger donors are mechanosensitive, evidenced by changes in cell morphology, adhesivity, and differentiation as a function of substrate stiffness in both two- and three-dimensional culture. However, MSCs from older individuals exhibit reduced differentiation potential and increased senescence, limiting their potential for autologous use. While substrate stiffness is known to modulate cell phenotype, the influence of the mechanical environment on senescent MSCs is poorly described. To address this question, we cultured irradiation induced premature senescent MSCs on polyacrylamide hydrogels and assessed expression of senescent markers, cell morphology, and secretion of inflammatory cytokines. Compared to cells on tissue culture plastic, senescent MSCs exhibited decreased markers of the senescence associated secretory phenotype (SASP) when cultured on 50 kPa gels, yet common markers of senescence (e.g., p21, CDKN2A, CDKN1A) were unaffected. These effects were muted in a physiologically relevant heterotypic mix of healthy and senescent MSCs. Conditioned media from senescent MSCs on compliant substrates increased osteoblast mineralization compared to conditioned media from cells on TCP. Mixed populations of senescent and healthy cells induced similar levels of osteoblast mineralization compared to healthy MSCs, further indicating an attenuation of the senescent phenotype in heterotypic populations. These data indicate that senescent MSCs exhibit a decrease in senescent phenotype when cultured on compliant substrates, which may be leveraged to improve autologous cell therapies for older donors.

The SNHG12/microRNA-15b-5p/MYLK axis regulates vascular smooth muscle cell phenotype to affect intracranial aneurysm formation

ObjectiveThis research was dedicated to investigating the impact of the SNHG12/microRNA (miR)-15b-5p/MYLK axis on the modulation of vascular smooth muscle cell (VSMC) phenotype and the formation of intracranial aneurysm (IA). MethodsSNHG12, miR-15b-5p and MYLK expression in IA tissue samples from IA patients were tested by RT-qPCR and western blot. Human aortic vascular smooth muscle cells (VSMCs) were cultivated with H2O2 to mimic IA-like conditions in vitro, and the cell proliferation and apoptosis were measured by MTT assay and Annexin V/PI staining. IA mouse models were established by induction with systemic hypertension combined with elastase injection. The blood pressure in the tail artery of mice in each group was assessed and the pathological changes in arterial tissues were observed by HE staining and TUNEL staining. The expression of TNF-α and IL-1β, MCP-1, iNOS, caspase-3, and caspase-9 in the arterial tissues were tested by RT-qPCR and ELISA. The relationship among SNHG12, miR-15b-5p and MYLK was verified by bioinformatics, RIP, RNA pull-down, and luciferase reporter assays. ResultsThe expression levels of MYLK and SNHG12 were down-regulated and that of miR-15b-5p was up-regulated in IA tissues and H2O2-treated human aortic VSMCs. Overexpressed MYLK or SNHG12 mitigated the decrease in proliferation and increase in apoptosis of VSMCs caused by H2O2 induction, and overexpression of miR-15b-5p exacerbated the decrease in proliferation and increase in apoptosis of VSMCs caused by H2O2 induction. Overexpression of miR-15b-5p reversed the H2O2-treated VSMC phenotypic changes caused by SNHG12 up-regulation, and overexpression of MYLK reversed the H2O2-treated VSMC phenotypic changes caused by up-regulation of miR-15b-5p. Overexpression of SNHG12 reduced blood pressure and ameliorated arterial histopathological damage and VSMC apoptosis in IA mice. The mechanical analysis uncovered that SNHG12 acted as an endogenous RNA that competed with miR-15b-5p, thus modulating the suppression of its endogenous target, MYLK. ConclusionDecreased expression of SNHG12 in IA may contribute to the increasing VSMC apoptosis via increasing miR-15b-5p expression and subsequently decreasing MYLK expression. These findings provide potential new strategies for the clinical treatment of IA.

RhoGDI3 at the trans-Golgi network participates in NLRP3 inflammasome activation, VSMC phenotypic modulation, and neointima formation

Background and aimsThe pathological roles and mechanisms of Rho-specific guanine nucleotide dissociation inhibitor 3 (RhoGDI3) in vascular smooth muscle cell (VSMC) phenotypic modulation and neointima formation are currently unknown. This study aimed to investigate how RhoGDI3 regulates the Nod-like receptor protein 3 (NLRP3) inflammasome in platelet-derived growth factor-BB (PDGF-BB)-induced neointima formation. MethodsFor in vitro assays, human aortic VSMCs (HA-VSMCs) were transfected with pcDNA3.1-GDI3 and RhoGDI3 siRNA to overexpress and knockdown RhoGDI3, respectively. HA-VSMCs were also treated with an NLRP3 inhibitor (CY-09) or agonist (NSS). Protein transcription and expression, cell proliferation and migration, Golgi morphology, and protein binding and colocalization were measured. For the in vivo assays, balloon injury (BI) rats were injected with recombinant adenovirus carrying RhoGDI3 shRNA. Carotid arterial morphology, protein expression and colocalization, and activation of the NLRP3 inflammasome were measured. ResultsPDGF-BB treatment induced transcription and expression of RhoGDI3 through PDGF receptor αβ (PDGFRαβ) rather than PDGFRαα or PDGFRββ in HA-VSMCs. RhoGDI3 suppression blocked PDGF-BB-induced VSMC phenotypic transformation. In contrast, RhoGDI3 overexpression further promoted PDGF-BB-induced VSMC dedifferentiation. The in vivo results also confirmed that RhoGDI3 expressed in VSMCs participated in neointima formation and muscle fiber and collagen deposition caused by balloon injury. In addition, PDGF-BB increased binding of RhoGDI3 to NLRP3 and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) at the trans-Golgi membrane, which depended on the normal Golgi network. However, recruitment of NLRP3 and ASC to the trans-Golgi network after PDGF-BB treatment was independent of RhoGDI3. Moreover, RhoGDI3 knockdown significantly inhibited ASC expression and NLRP3 inflammasome assembly and activation and reduced NLRP3 protein stability in PDGF-BB-treated HA-VSMCs. Inhibiting NLRP3 effectively prevented PDGF-BB-induced VSMC phenotypic modulation, and an NLRP3 agonist reversed the decline in VSMC phenotypic transformation caused by RhoGDI3 knockdown. Furthermore, RhoGDI3 suppression reduced the protein levels and assembly of NLRP3 and ASC, and the activation of the NLRP3 inflammasome in VSMCs in a rat balloon injury model. ConclusionsThe results of this study reveal a novel mechanism through which RhoGDI3 regulates VSMC phenotypic modulation and neointima formation by activating the NLRP3 inflammasome.

Abstract 18578: BMP9 Regulates Endothelial Expression of Mediators of Pulmonary Vascular Remodeling

Background: Loss-of-function mutations in GDF2 /BMP9 are found in heritable pulmonary arterial hypertension (PAH), suggesting a protective role, while protective or pathogenic effects are found in experimental PAH studies depending on context. The net contribution of BMP9 in PAH thus remains controversial. Hypothesis: BMP9 regulates the secretome of pulmonary microvascular endothelium (PMVEC) to contribute to pulmonary artery smooth muscle cell (PASMC) phenotype modulation. Methods: We tested recombinant BMP9, BMP9/BMP10 trap ALK1-Fc, and BMP9 neutralizing antibody before and after development of pulmonary hypertension (PH) in distinct experimental models. We analyzed the impact of BMP9 on the PMVEC transcriptome, intersection with human and experimental PAH lung transcriptomes, and tested the impact of EC-derived genes on PASMC phenotype and growth. Results: ALK1-Fc and anti-BMP9 prevented or reversed experimental PH administered before or after onset of experimental PH. Recombinant disulfide-linked BMP9 was not protective in SUGEN/Hypoxia (SU-Hx)- or monocrotaline (MCT)-induced experimental PH, in contrast to previous studies using incompletely disulfide-linked BMP9, suggesting dissociated monomers might function as competitive BMP9 antagonists. Consistent with this interpretation, BMP9 upregulated PMVEC genes associated with pulmonary vascular remodeling in experimental and human PAH including CXCL12, IGFBP4, ET-1, VEGF-A, and PDGF-BB. Stimulation of PMVEC with BMP9 induced a contractile phenotype ( CNN1;TAGLN ) in co-cultured PASMC. Administration of ALK1-Fc or anti-BMP9 attenuated expression of several of these growth factors in SU-Hx PH. In experimental PH ALK1-Fc and ACTRIIA-Fc elicited peripheral edema, a phenotype associated with use of ALK1-Fc in human trials. Conclusions: BMP9 contributes to experimental PH by regulating expression of EC-derived molecules modulating PASMC growth and phenotype. In contrast to broad strategies that inhibit BMP9 and BMP10 associated with HHT mimicry in clinical trials and rodent PH models, treatment with anti-BMP9 was well-tolerated. Selective antagonism of BMP9 represents a potential therapeutic strategy for human PAH.

Smooth muscle cells-specific loss of OCT4 accelerates neointima formation after acute vascular injury.

There is growing evidence that smooth muscle cell (SMC) phenotypic transitions play critical roles during normal developmental and tissue recovery processes and in pathological conditions such as atherosclerosis. However, the molecular mechanisms responsible for these transitions are not well understood. Recently, we found that the embryonic stem cell/induced pluripotent stem cell (iPSC) factor OCT4, which was believed to be silenced in somatic cells, plays an atheroprotective role in SMC, and regulates angiogenesis after corneal alkali burn and hindlimb ischemia by mediating microvascular SMC and pericyte migration. However, the kinetics of OCT4 activation in arterial SMC and its role in acute pathological conditions are still unknown. Here, using an Oct4-IRES-GFP reporter mouse model, we found that OCT4 is reactivated in the carotid artery 18 hours post-acute ligation-induced injury, a common in vivo model of the SMC phenotypic transitions. Next, using a tamoxifen-inducible Myh11-CreERT2 Oct4 knockout mouse model, we found that the loss of OCT4, specifically in SMC, led to accelerated neointima formation and increased tunica media following carotid artery ligation, at least in part by increasing SMC proliferation within the media. Bulk RNA sequencing analysis on the cultured SMC revealed significant down-regulation of the SMC contractile markers and dysregulation of the genes belonging to the regulation of cell proliferation and, positive and negative regulation for cell migration ontological groups following genetic inactivation of Oct4. We also found that loss of Oct4 resulted in suppression of contractile SMC markers after the injury and in cultured aortic SMC. Further mechanistic studies revealed that OCT4 regulates SMC contractile genes, ACTA2 and TAGLN, at least in part by direct binding to the promoters of these genes. These results demonstrate that the pluripotency factor OCT4 is quickly activated in SMC after the acute vascular injury and inhibits SMC hyperproliferation, which may be protective in preventing excessive neointima formation.