Epithelial-to-mesenchymal transition in cancer progression: unraveling the immunosuppressive module driving therapy resistance.

Cancer cells undergo phenotypic switching (cancer cell plasticity) in response to microenvironmental cues, including exposure to therapy/treatment. Phenotypic plasticity enables the cancer cells to acquire more mesenchymal traits promoting cancer cells' growth, survival, therapy resistance, and disease recurrence. A significant program in cancer cell plasticity is epithelial-to-mesenchymal transition (EMT), wherein a comprehensive reprogramming of gene expression occurs to facilitate the translational shift from epithelial-to-mesenchymal phenotypes resulting in increased invasiveness and metastasis. In addition, EMT plays a pivotal role in facilitating cancer cells' escape from the body's immune system using several mechanisms, such as the downregulation of major histocompatibility complex-mediated antigen presentation, upregulation of immune checkpoint molecules, and recruitment of immune-suppressive cells. Cancer cells' ability to undergo phenotypic switching and EMT-driven immune escape presents a formidable obstacle in cancer management, highlighting the need to unravel the intricate mechanisms underlying these processes and develop novel therapeutic strategies. This article discusses the role of EMT in promoting immune evasion and therapy resistance. We also discuss the ongoing research on developing therapeutic approaches targeting intrinsic and induced cell plasticity within the immune suppressive microenvironment. We believe this review article will update the current research status and equip researchers, clinicians, and other healthcare professionals with valuable insights enhancing their existing knowledge and shedding light on promising directions for future cancer research. This will facilitate the development of innovative strategies for managing therapy-resistant cancers and improving patient outcomes.

CircTLK1: A novel regulator involved in VSMC phenotypic switching and the developmental process of atherosclerosis.

Phenotypic switching of vascular smooth muscle cells (VSMCs) is essential for atherosclerosis development. Circular RNA (circRNA) is a specific non-coding RNA that is produced as a closed-loop structure in mammals, and its specific expression pattern is closely related to its cell type and tissue. To clarify the roles of circTLK1 in VSMC phenotypic switching, we performed qRT-PCR, immunoblotting, and immunostaining. qRT-PCR revealed that circTLK1 was upregulated in both mouse models of atherosclerosis in vivo and PDGF (platelet-derived growth factor)-BB-induced VSMCs in vitro. Furthermore, the overexpression of circTLK1 promoted PDGF-BB-induced VSMC phenotypic switching. Conversely, experiments performed in vivo demonstrate that the knockdown of SMC-specific circTLK1 led to a reduction in the development of atherosclerosis. The relationship between circTLK1 and miR-513a-3p and Krüppel-like factor 4 (KLF4) was detected by RNA immunoprecipitation (RIP), luciferase reporter assay, RNA pull-down, and RNA fluorescence in situ hybridization (RNA FISH). Mechanistically, circTLK1 acted as a sponge for miR-513a-3p, leading to the upregulation of KLF4, a key transcription factor for phenotypic switching. Targeting the circTLK1/miR-513a-3p/KLF4 axis may provide a potential therapeutic strategy for atherosclerosis.

Single-cell RNA sequencing reveals that NRF2 regulates vascular smooth muscle cell phenotypic switching in abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is a life-threatening disease characterized by extensive membrane destruction in the vascular wall that is closely associated with vascular smooth muscle cell (VSMC) phenotypic switching. A thorough understanding of the changes in regulatory factors during VSMC phenotypic switching is essential for managing AAA therapy. In this study, we revealed the impact of NRF2 on the modulation of VSMC phenotype and the development of AAA based on single-cell RNA sequencing analysis. By utilizing a murine model of VSMC-specific knockout of nuclear factor E2-related factor 2 (NRF2), we observed that the absence of NRF2 in VSMCs exacerbated AAA formation in an angiotensin II-induced AAA model. The downregulation of NRF2 promoted VSMC phenotypic switching, leading to an enhanced inflammatory response. Through genome-wide transcriptome analysis and loss- or gain-of-function experiments, we discovered that NRF2 upregulated the expression of VSMC contractile phenotype-specific genes by facilitating microRNA-145 (miR-145) expression. Our data identified NRF2 as a novel regulator involved in maintaining the VSMC contractile phenotype while also influencing AAA formation through an miR-145-dependent regulatory mechanism.

NEAT1 regulates VSMC differentiation and calcification in as long noncoding RNA NEAT1 enhances phenotypic and osteogenic switching of vascular smooth muscle cells in atherosclerosis via scaffolding EZH2.

Atherosclerosis (AS) is a significant contributor to cardio-cerebrovascular ischemia diseases, resulting in high mortality rates worldwide. During AS, vascular smooth muscle cells (VSMCs) play a crucial role in plaque formation by undergoing phenotypic and osteogenic switching. Long noncoding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) has previously been identified as a nuclear regulator that promotes tumorigenesis and metastasis, but its role in regulating VSMCs in AS remains unclear. Our study aimed to investigate the biological functions and specific mechanisms of NEAT1 in regulating VSMCs in AS. We found that NEAT1 was upregulated in the aortas of AS mouse models and dedifferentiated primary VSMCs. Silencing NEAT1 in vitro attenuated the proliferation, migration, and osteogenic differentiation of VSMCs, while NEAT1 overexpression had the opposite effect. Furthermore, NEAT1 promoted VSMC osteogenic differentiation and vascular calcification in both in vivo and in vitro vascular calcification models. We also discovered that NEAT1 directly activates enhancer of zeste homolog 2 (EZH2), an epigenetic enzyme that suppresses the expression of senescence- and antimigration-related genes, by translocating it into the nucleus. CUT&Tag assay revealed that NEAT1 guides EZH2 to the promoters of senescence-related genes (P16, P21, and TIMP3), methylating local histones to reduce their transcription. Our findings suggest that NEAT1 functions in AS by modulating the epigenetic function of EZH2, which enhances the proliferation, migration, and osteogenic differentiation of VSMCs. This study provides new insights into the molecular mechanisms underlying the pathogenesis of AS and highlights the potential of NEAT1 as a therapeutic target of AS.NEW & NOTEWORTHY Our study demonstrates that the upregulation of long noncoding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) promotes proliferation and migration during phenotypic switching of vascular smooth muscle cells in atherosclerosis. We also provide in vivo and in vitro evidence that NEAT1 accelerates vascular calcification. Our findings identified the direct interaction between enhancer of zeste homolog 2 (EZH2) and NEAT1 during atherosclerosis. NEAT1 is necessary for EZH2 to translocate from the cytoplasm to the nucleus, where EZH2 epigenetically inhibits the expression of genes related to senescence and antimigration.

Microvascular smooth muscle cells exhibit divergent phenotypic switching responses to platelet-derived growth factor and insulin-like growth factor 1.

OBJECTIVE: Vascular smooth muscle cell (VSMC) phenotypic switching is critical for normal vessel formation, vascular stability, and healthy brain aging. Phenotypic switching is regulated by mediators including platelet derived growth factor (PDGF)-BB, insulin-like growth factor (IGF-1), as well as transforming growth factor-ß (TGF-ß) and endothelin-1 (ET-1), but much about the role of these factors in microvascular VSMCs remains unclear. METHODS: We used primary rat microvascular VSMCs to explore PDGF-BB- and IGF-1-induced phenotypic switching. RESULTS: PDGF-BB induced an early proliferative response, followed by formation of polarized leader cells and rapid, directionally coordinated migration. In contrast, IGF-1 induced cell hypertrophy, and only a small degree of migration by unpolarized cells. TGF-ß and ET-1 selectively inhibit PDGF-BB-induced VSMC migration primarily by repressing migratory polarization and formation of leader cells. Contractile genes were downregulated by both growth factors, while other genes were differentially regulated by PDGF-BB and IGF-1. CONCLUSIONS: These studies indicate that PDGF-BB and IGF-1 stimulate different types of microvascular VSMC phenotypic switching characterized by different modes of cell migration. Our studies are consistent with a chronic vasoprotective role for IGF-1 in VSMCs in the microvasculature while PDGF is more involved in VSMC proliferation and migration in response to acute activities such as neovascularization. Better understanding of the nuances of the phenotypic switching induced by these growth factors is important for our understanding of a variety of microvascular diseases.

Melatonin Ameliorates Atherosclerotic Plaque Vulnerability by Regulating PPARδ-Associated Smooth Muscle Cell Phenotypic Switching.

Vulnerable atherosclerotic plaque rupture, the leading cause of fatal atherothrombotic events, is associated with an increased risk of mortality worldwide. Peroxisome proliferator-activated receptor delta (PPARδ) has been shown to modulate vascular smooth muscle cell (SMC) phenotypic switching, and, hence, atherosclerotic plaque stability. Melatonin reportedly plays a beneficial role in cardiovascular diseases; however, the mechanisms underlying improvements in atherosclerotic plaque vulnerability remain unknown. In this study, we assessed the role of melatonin in regulating SMC phenotypic switching and its consequential contribution to the amelioration of atherosclerotic plaque vulnerability and explored the mechanisms underlying this process. We analyzed features of atherosclerotic plaque vulnerability and markers of SMC phenotypic transition in high-cholesterol diet (HCD)-fed apolipoprotein E knockout (ApoE-/-) mice and human aortic SMCs (HASMCs). Melatonin reduced atherosclerotic plaque size and necrotic core area while enhancing collagen content, fibrous cap thickness, and smooth muscle alpha-actin positive cell coverage on the plaque cap, which are all known phenotypic characteristics of vulnerable plaques. In atherosclerotic lesions, melatonin significantly decreased the synthetic SMC phenotype and KLF4 expression and increased the expression of PPARδ, but not PPARα and PPARγ, in HCD-fed ApoE-/- mice. These results were subsequently confirmed in the melatonin-treated HASMCs. Further analysis using PPARδ silencing and immunoprecipitation assays revealed that PPARδ plays a role in the melatonin-induced SMC phenotype switching from synthetic to contractile. Collectively, we provided the first evidence that melatonin mediates its protective effect against plaque destabilization by enhancing PPARδ-mediated SMC phenotypic switching, thereby indicating the potential of melatonin in treating atherosclerosis.

METTL14/YTHDF1 axis-modified UCHL5 aggravates atherosclerosis by activating the NLRP3 inflammasome.

BACKGROUND: Vascular smooth muscle cell (VSMC) phenotypic switching contributes to VSMC proliferation and migration in atherosclerosis (AS). Nevertheless, the regulatory mechanism of VSMC phenotypic switching during AS progression is unclear. Here, the role and regulatory mechanism of UCHL5 in VSMC phenotypic switching during AS progression were investigated. METHODS: ApoE-/- mice were fed with high fat diet to establish AS model in vivo. VSMCs stimulated by ox-LDL were used as AS cellular model. VSMC proliferation and migration were examined by CCK8 assay and transwell assay, respectively. The levels of pro-inflammatory cytokines were assessed using ELISA. The interactions between METTL14/YTHDF1, UCHL5 and NLRP3 were analyzed using RIP and/or dual-luciferase reporter gene and/or Co-IP assays. NLRP3 ubiquitination was analyzed by ubiquitination analysis. RESULTS: UCHL5 was significantly upregulated in AS patients and ox-LDL-treated VSMCs. UCHL5 silencing ameliorated plaque formation and vascular remodeling in vivo and suppressed ox-LDL-induced VSMC proliferation, migration, inflammation and phenotypic switching in vitro. Moreover, METTL14 could increase UCHL5 mRNA m6A level and promoted UCHL5 expression by recruiting YTHDF1. Moreover, UCHL5 overexpression enhanced protein stability by deubiquitinating NLRP3. Rescue studies revealed that NLRP3 overexpression abrogated UCHL5 silencing-mediated biological effects in ox-LDL-treated VSMCs. CONCLUSION: UCHL5 modified by METTL14/YTHDF1 axis could facilitate the inflammation and vascular remodeling in atherosclerosis by activating the NLRP3 inflammasome.

Hormonal influence: unraveling the impact of sex hormones on vascular smooth muscle cells.

Sex hormones play a pivotal role as endocrine hormones that exert profound effects on the biological characteristics and vascular function of vascular smooth muscle cells (VSMCs). By modulating intracellular signaling pathways, activating nuclear receptors, and regulating gene expression, sex hormones intricately influence the morphology, function, and physiological state of VSMCs, thereby impacting the biological properties of vascular contraction, relaxation, and growth. Increasing evidence suggests that abnormal phenotypic changes in VSMCs contribute to the initiation of vascular diseases, including atherosclerosis. Therefore, understanding the factors governing phenotypic alterations in VSMCs and elucidating the underlying mechanisms can provide crucial insights for refining interventions targeted at vascular diseases. Additionally, the varying levels of different types of sex hormones in the human body, influenced by sex and age, may also affect the phenotypic conversion of VSMCs. This review aims to explore the influence of sex hormones on the phenotypic switching of VSMCs and the development of associated vascular diseases in the human body.

The rod cell, a small form of Candida albicans, possesses superior fitness to the host gut and adaptation to commensalism.

Candida albicans deploys various morphological forms through complex switching mechanisms, ensuring its survival and thriving as a commensal or pathogen in vastly different human niches. In this study, we demonstrate that a novel ''rod'' morphological form of C. albicans coexists and is interchangeable with previously reported white, gray, and opaque forms, constituting a tetra-stable phenotypic switching system. Rod cells arise from the efg1 mutant of SC5314 cells or from the clinical BJ1097 strain cultured under glucose-free conditions. They are characterized by a distinct gene expression profile and can be stably maintained through in vitro passaging or in vivo inhabitation of the gastrointestinal (GI) tract of mice. Remarkably, the majority of the efg1 mutant cells become rod cells in N-acetylglucosamine (GlcNAc)-containing medium, and the GlcNAc sensor Ngs1 is instrumental in converting the white or gray cells to the rod cells. Conversely, glucose inhibits rod cells through Cph1; consequently, the loss of Cph1 in the efg1 mutantcells permits their conversion to rod cells in glucose-replete media. Notably, rod cells of the efg1/ cph1 mutant display superior adaptation and longer persistence in the murine GI environment than wild-type white cells. Taken together, these findings establish rod cells as a previously unappreciated form that is not only morphologically and transcriptionally distinguishable but also defined by specific genetic and environmental determinants, shedding light on complex fungus-host interactions.

Smooth muscle α-actin missense variant promotes atherosclerosis through modulation of intracellular cholesterol in smooth muscle cells.

AIMS: The variant p.Arg149Cys in ACTA2, which encodes smooth muscle cell (SMC)-specific α-actin, predisposes to thoracic aortic disease and early onset coronary artery disease in individuals without cardiovascular risk factors. This study investigated how this variant drives increased atherosclerosis. METHODS AND RESULTS: Apoe-/- mice with and without the variant were fed a high-fat diet for 12 weeks, followed by evaluation of atherosclerotic plaque formation and single-cell transcriptomics analysis. SMCs explanted from Acta2R149C/+ and wildtype (WT) ascending aortas were used to investigate atherosclerosis-associated SMC phenotypic modulation. Hyperlipidemic Acta2R149C/+Apoe-/- mice have a 2.5-fold increase in atherosclerotic plaque burden compared to Apoe-/- mice with no differences in serum lipid levels. At the cellular level, misfolding of the R149C α-actin activates heat shock factor 1, which increases endogenous cholesterol biosynthesis and intracellular cholesterol levels through increased HMG-CoA reductase (HMG-CoAR) expression and activity. The increased cellular cholesterol in Acta2R149C/+ SMCs induces endoplasmic reticulum stress and activates PERK-ATF4-KLF4 signaling to drive atherosclerosis-associated phenotypic modulation in the absence of exogenous cholesterol, while WT cells require higher levels of exogenous cholesterol to drive phenotypic modulation. Treatment with the HMG-CoAR inhibitor pravastatin successfully reverses the increased atherosclerotic plaque burden in Acta2R149C/+Apoe-/- mice. CONCLUSION: These data establish a novel mechanism by which a pathogenic missense variant in a smooth muscle-specific contractile protein predisposes to atherosclerosis in individuals without hypercholesterolemia or other risk factors. The results emphasize the role of increased intracellular cholesterol levels in driving SMC phenotypic modulation and atherosclerotic plaque burden.

Smooth-Muscle-Cell-Specific Deletion of CD38 Protects Mice from AngII-Induced Abdominal Aortic Aneurysm through Inhibiting Vascular Remodeling.

Abdominal aortic aneurysm (AAA) is a serious vascular disease which is associated with vascular remodeling. CD38 is a main NAD+-consuming enzyme in mammals, and our previous results showed that CD38 plays the important roles in many cardiovascular diseases. However, the role of CD38 in AAA has not been explored. Here, we report that smooth-muscle-cell-specific deletion of CD38 (CD38SKO) significantly reduced the morbidity of AngII-induced AAA in CD38SKOApoe-/- mice, which was accompanied with a increases in the aortic diameter, medial thickness, collagen deposition, and elastin degradation of aortas. In addition, CD38SKO significantly suppressed the AngII-induced decreases in α-SMA, SM22α, and MYH11 expression; the increase in Vimentin expression in VSMCs; and the increase in VCAM-1 expression in smooth muscle cells and macrophage infiltration. Furthermore, we demonstrated that the role of CD38SKO in attenuating AAA was associated with the activation of sirtuin signaling pathways. Therefore, we concluded that CD38 plays a pivotal role in AngII-induced AAA through promoting vascular remodeling, suggesting that CD38 may serve as a potential therapeutic target for the prevention of AAA.

[Mechanism of salidroside in inhibiting proliferation, migration and promoting phenotypic switching of arterial smooth muscle cells].

This study aims to examine the effect of salidroside(SAL) on the phenotypic switching of human aortic smooth muscle cells(HASMC) induced by the platelet-derived growth factor-BB(PDGF-BB) and investigate the pharmacological mechanism. Firstly, the safe concentration of SAL was screened by the lactate dehydrogenase release assay. HASMC were divided into control, model, and SAL groups, and the cells in other groups except the control group were treated with PDGF-BB for the modeling of phenotypic switching. Cell proliferation and migration were detected by the cell-counting kit(CCK-8) assay and Transwell assay, respectively. The cytoskeletal structure was observed by F-actin staining with fluorescently labeled phalloidine. The protein levels of proliferating cell nuclear antigen(PCNA), migration-related protein matrix metalloprotein 9(MMP-9), fibronectin, α-smooth muscle actin(α-SMA), and osteopontin(OPN) were determined by Western blot. To further investigate the pharmacological mechanism of SAL, this study determined the expression of protein kinase B(Akt) and mammalian target of rapamycin(mTOR), as well as the upstream proteins phosphatase and tensin homologue(PTEN) and platelet-derived growth factor receptor ß(PDGFR-ß) and the downstream protein hypoxia-inducible factor-1α(HIF-1α) of the Akt/mTOR signaling pathway. The results showed that the HASMCs in the model group presented significantly increased proliferation and migration, the switching from a contractile phenotype to a secretory phenotype, and cytoskeletal disarrangement. Compared with the model group, SAL weakened the proliferation and migration of HASMC, promoted the expression of α-SMA(a contractile phenotype marker), inhibited the expression of OPN(a secretory phenotype marker), and repaired the cytoskeletal disarrangement. Furthermore, compared with the control group, the modeling up-regulated the levels of phosphorylated Akt and mTOR and the relative expression of PTEN, HIF-1α, and PDGFR-ß. Compared with the model group, SAL down-regulated the protein levels of phosphorylated Akt and mTOR, PTEN, PDGFR-ß, and HIF-1α. In conclusion, SAL exerts a protective effect on the HASMCs exposed to PDGF-BB by regulating the PDGFR-ß/Akt/mTOR/HIF-1α signaling pathway.

Reactive oxygen species-induced long intergenic noncoding RNA p21 accelerates abdominal aortic aneurysm formation by promoting secretary smooth muscle cell phenotypes.

Whether long noncoding RNAs participate in the formation of abdominal aortic aneurysms (AAAs) through the regulation of SMC phenotypic switching is unknown. lincRNA-p21 induced by reactive oxygen species (ROS) is likely functionally associated with SMC phenotypic switching. We thus investigated the role of lincRNA-p21 in SMC phenotypic switching-associated AAA formation and its underlying mechanisms. An analysis of human and mouse abdominal aortic samples revealed that the lincRNA-p21 levels were significantly higher in AAA tissue. Stimulation with hydrogen peroxide upregulated the expression of lincRNA-p21 in a dose-dependent manner and converted SMCs from a contractile phenotype to a synthetic, proteolytic, and proinflammatory phenotype in vitro. Moreover, lincRNA-p21 promoted fracture of elastic fibres, reconstruction of the vascular wall, and AAA formation in vivo by modulating SMC phenotypic switching in two mouse models of AAA induced by angiotensin II or porcine pancreatic elastase (PPE) perfusion. Using a bioinformatics prediction method and luciferase reporter gene assays, we further proved that lincRNA-p21 sponged miR-204-5p to release the transcriptional activity of Mekk3 and promoted the NF-κB pathway and thereby played a role in the SMC phenotypic switch and AAA formation. The ROS levels were positively correlated with the lincRNA-p21 levels in human and mouse AAA tissues. The knockdown of lincRNA-p21 in a PPE-induced mouse AAA model increased the miR-204-5p levels and reduced the expression of Mekk3, whereas lincRNA-p21 overexpression had the opposite effect. Collectively, the results indicated that ROS-induced lincRNA-p21 sponges miR-204-5p to accelerate synthetic and proinflammatory SMC phenotypes through the Mekk3/NF-κB pathway in AAA formation. Thus, lincRNA-p21 may have therapeutic potential for AAA formation.

Calcium promotes vascular smooth muscle cell phenotypic switching in Marfan syndrome.

Fibrillin 1 (Fbn1) mutations cause Marfan syndrome (MFS), with aortic root dilatation, dissection, and rupture. Few studies reported the blood calcium and lipid profile of MFS, and the effect of vascular smooth muscle cell (VSMC) phenotypic switching on MFS aortic aneurysm is unclear. Here, we aimed to investigate the role of calcium-related VSMC phenotypic switching in MFS. We retrospectively collected MFS patients' clinical data, performed bioinformatics analysis to screen the enriched biological process in MFS patients and mice, and detected markers of VSMC phenotypic switching on Fbn1C1039G/+ mice and primary aortic vascular smooth muscle cells. We found that patients with MFS have elevated blood calcium levels and dyslipidemia. Furthermore, the calcium concentration levels were increased with age in MFS mice, accompanied by the promoted VSMC phenotypic switching, and SERCA2 contributed to maintaining the contractile phenotype of VSMCs. This study provides the first evidence that the increased calcium is associated with the promoted VSMC phenotype switching in MFS. SERCA may become a novel therapeutic target for suppressing aneurysm progression in MFS.

Altered phagocytosis and morphogenesis of phenotypic switching-derived strains of the pathogenic Candida tropicalis co-cultured with phagocytic cells.

BACKGROUND AND OBJECTIVE: Candida tropicalis is among the most prevalent human pathogenic yeast species. Switch states of C. tropicalis differ in virulence traits. Here, we evaluate the effect of phenotypic switching on phagocytosis and yeast-hyphae transition in C. tropicalis. METHODS: C. tropicalis morphotypes included a clinical strain and two switch strains (rough variant and rough revertant). In vitro, phagocytosis assay was performed using peritoneal macrophages and hemocytes. The proportion of hyphal cells was ascertained by scoring morphology using optical microscopy. Expression of the WOR1 (White-opaque regulator 1) and EFG1 (Enhanced filamentous growth protein 1) was determined by quantitative PCR. RESULTS: The rough variant was more resistant to in vitro phagocytosis by peritoneal macrophages than that observed for the clinical strain, while hemocytes phagocytosed clinical and rough variant to the same extent. The rough revertant was more phagocytosed than the clinical strain by both phagocytes. During co-incubation with phagocytic cells, the clinical strain of C. tropicalis exists mainly as blastoconidia. The co-culture of the rough variant with macrophages resulted in a higher percentage of hyphae than blastoconidia cells, while in co-culture with hemocytes, no differences were observed between the percentage of hyphae and blastoconidia. The expression levels of WOR1 in the rough variant co-cultured with phagocytes were significantly higher than they were in the clinical strain. CONCLUSIONS: Differences on phagocytosis and hyphal growth between switch states cells of C. tropicalis co-cultured with phagocytic cells were observed. The pronounced hyphal growth may affect the complex host-pathogen relationship and favor the pathogen to escape phagocytosis. The pleiotropic effects of phenotypic switching suggest that this event may contribute to the success of infection associated with C. tropicalis.

Statistical inference of the rates of cell proliferation and phenotypic switching in cancer.

Recent evidence suggests that nongenetic (epigenetic) mechanisms play an important role at all stages of cancer evolution. In many cancers, these mechanisms have been observed to induce dynamic switching between two or more cell states, which commonly show differential responses to drug treatments. To understand how these cancers evolve over time, and how they respond to treatment, we need to understand the state-dependent rates of cell proliferation and phenotypic switching. In this work, we propose a rigorous statistical framework for estimating these parameters, using data from commonly performed cell line experiments, where phenotypes are sorted and expanded in culture. The framework explicitly models the stochastic dynamics of cell division, cell death and phenotypic switching, and it provides likelihood-based confidence intervals for the model parameters. The input data can be either the fraction of cells or the number of cells in each state at one or more time points. Through a combination of theoretical analysis and numerical simulations, we show that when cell fraction data is used, the rates of switching may be the only parameters that can be estimated accurately. On the other hand, using cell number data enables accurate estimation of the net division rate for each phenotype, and it can even enable estimation of the state-dependent rates of cell division and cell death. We conclude by applying our framework to a publicly available dataset.

Metabolic and phenotypic plasticity may contribute for the higher virulence of Trichosporon asahii over other Trichosporonaceae members.

BACKGROUND: The Trichosporonaceae family comprises a large number of basidiomycetes widely distributed in nature. Some of its members, especially Trichosporon asahii, have the ability to cause human infections. This ability is related to a series of virulence factors, which include lytic enzymes production, biofilm formation, resistance to oxidising agents, melanin and glucuronoxylomannan in the cell wall, metabolic plasticity and phenotypic switching. The last two are poorly addressed within human pathogenic Trichosporonaceae. OBJECTIVE: These factors were herein studied to contribute with the knowledge of these emerging pathogens and to uncover mechanisms that would explain the higher frequency of T. asahii in human infections. METHODS: We included 79 clinical isolates phenotypically identified as Trichosporon spp. and performed their molecular identification. Lactate and N-acetyl glucosamine were the carbon sources of metabolic plasticity studies. Morphologically altered colonies after subcultures and incubation at 37°C indicated phenotypic switching. RESULTS AND CONCLUSION: The predominant species was T. asahii (n = 65), followed by Trichosporon inkin (n = 4), Apiotrichum montevideense (n = 3), Trichosporon japonicum (n = 2), Trichosporon faecale (n = 2), Cutaneotrichosporon debeurmannianum (n = 1), Trichosporon ovoides (n = 1) and Cutaneotrichosporon arboriforme (n = 1). T. asahii isolates had statistically higher growth on lactate and N-acetylglucosamine and on glucose during the first 72 h of culture. T. asahii, T. inkin and T. japonicum isolates were able to perform phenotypic switching. These results expand the virulence knowledge of Trichosporonaceae members and point for a role for metabolic plasticity and phenotypic switching on the trichosporonosis pathogenesis.

Smooth Muscle Heterogeneity and Plasticity in Health and Aortic Aneurysmal Disease.

Vascular smooth muscle cells (VSMCs) are the predominant cell type in the medial layer of the aorta, which plays a critical role in the maintenance of aortic wall integrity. VSMCs have been suggested to have contractile and synthetic phenotypes and undergo phenotypic switching to contribute to the deteriorating aortic wall structure. Recently, the unprecedented heterogeneity and diversity of VSMCs and their complex relationship to aortic aneurysms (AAs) have been revealed by high-resolution research methods, such as lineage tracing and single-cell RNA sequencing. The aortic wall consists of VSMCs from different embryonic origins that respond unevenly to genetic defects that directly or indirectly regulate VSMC contractile phenotype. This difference predisposes to hereditary AAs in the aortic root and ascending aorta. Several VSMC phenotypes with different functions, for example, secreting VSMCs, proliferative VSMCs, mesenchymal stem cell-like VSMCs, immune-related VSMCs, proinflammatory VSMCs, senescent VSMCs, and stressed VSMCs are identified in non-hereditary AAs. The transformation of VSMCs into different phenotypes is an adaptive response to deleterious stimuli but can also trigger pathological remodeling that exacerbates the pathogenesis and development of AAs. This review is intended to contribute to the understanding of VSMC diversity in health and aneurysmal diseases. Papers that give an update on VSMC phenotype diversity in health and aneurysmal disease are summarized and recent insights on the role of VSMCs in AAs are discussed.

Induction of ferroptosis promotes vascular smooth muscle cell phenotypic switching and aggravates neointimal hyperplasia in mice.

BACKGROUND: Stent implantation-induced neointima formation is a dominant culprit in coronary artery disease treatment failure after percutaneous coronary intervention. Ferroptosis, an iron-dependent regulated cell death, has been associated with various cardiovascular diseases. However, the effect of ferroptosis on neointima formation remains unclear. METHODS: The mouse common right carotid arteries were ligated for 16 or 30 days, and ligated tissues were collected for further analyses. Primary rat vascular smooth muscle cells (VSMCs) were isolated from the media of aortas of Sprague-Dawley (SD) rats and used for in vitro cell culture experiments. RESULTS: Ferroptosis was positively associated with neointima formation. In vivo, RAS-selective lethal 3 (RSL3), a ferroptosis activator, aggravated carotid artery ligation-induced neointima formation and promoted VSMC phenotypic conversion. In contrast, a ferroptosis inhibitor, ferrostatin-1 (Fer-1), showed the opposite effects in mice. In vitro, RSL3 promoted rat VSMC phenotypic switching from a contractile to a synthetic phenotype, evidenced by increased contractile markers (smooth muscle myosin heavy chain and calponin 1), and decreased synthetic marker osteopontin. The induction of ferroptosis by RSL3 was confirmed by the increased expression level of ferroptosis-associated gene prostaglandin-endoperoxide synthase 2 (Ptgs2). The effect of RSL3 on rat VSMC phenotypic switching was abolished by Fer-1. Moreover, N-acetyl-L-cysteine (NAC), the reactive oxygen species inhibitor, counteracted the effect of RSL3 on the phenotypic conversion of rat VSMCs. CONCLUSIONS: Ferroptosis induces VSMC phenotypic switching and accelerates ligation-induced neointimal hyperplasia in mice. Our findings suggest inhibition of ferroptosis as an attractive strategy for limiting vascular restenosis.

Endoplasmic reticulum stress promotes nuclear translocation of calmodulin, which activates phenotypic switching of vascular smooth muscle cells.

BACKGROUND AND AIMS: Increased endoplasmic reticulum (ER) stress is strongly associated with the phenotypic switching of vascular smooth muscle cells (VSMCs) in atherosclerosis. Depletion of the ER Ca2+ content is one of the leading causes of increased ER stress in VSMCs. The ryanodine receptor (RyR) is a major Ca2+ release channel in the sarcoplasmic reticulum membrane. Calmodulin (CaM), which binds to RyR (CaM-RyR), stabilizes the closed state of RyR in the resting state in normal cells. Defective CaM-RyR interactions can cause abnormal Ca2+ leakage through RyR, resulting in decreased Ca2+ content, indicating that defective CaM-RyR interactions may be a cause of increased ER stress. Herein, we used a mouse VSMCs to assess whether CaM-RyR plays a pivotal role in VSMCs phenotypic switching, which is caused by ER stress, and whether dantrolene, which enhances the binding affinity of CaM to RyR, affects VSMCs phenotypic switching. METHODS AND RESULTS: Tunicamycin was used to mimic ER stress in vitro. Tunicamycin-induced ER stress caused CaM to dissociate from the RyR and translocate to the nucleus, which stimulated phenotypic switching through the activation of MEF2 and KLF5. Dantrolene suppressed tunicamycin-induced apoptosis, ER stress (restoring ER Ca2+ content), and phenotypic switching of VSMCs. Suramin, which directly unbinds CaM from RyR, promoted nuclear CaM accumulation with parallel VSMCs phenotypic switching, and dantrolene prevented these effects. CONCLUSIONS: We observed that ER stress causes CaM translocation to the nucleus and drives the phenotypic switching of VSMCs. Thus, restoration of the binding affinity of CaM to RyR may be a therapeutic target for atherosclerosis.