Phenotypic Switch Research Articles

Cancer Cell Biomechanical Properties Accompany Tspan8-Dependent Cutaneous Melanoma Invasion.

The intrinsic biomechanical properties of cancer cells remain poorly understood. To decipher whether cell stiffness modulation could increase melanoma cells' invasive capacity, we performed both in vitro and in vivo experiments exploring cell stiffness by atomic force microscopy (AFM). We correlated stiffness properties with cell morphology adaptation and the molecular mechanisms underlying epithelial-to-mesenchymal (EMT)-like phenotype switching. We found that melanoma cell stiffness reduction was systematically associated with the acquisition of invasive properties in cutaneous melanoma cell lines, human skin reconstructs, and Medaka fish developing spontaneous MAP-kinase-induced melanomas. We observed a systematic correlation of stiffness modulation with cell morphological changes towards mesenchymal characteristic gains. We accordingly found that inducing melanoma EMT switching by overexpressing the ZEB1 transcription factor, a major regulator of melanoma cell plasticity, was sufficient to decrease cell stiffness and transcriptionally induce tetraspanin-8-mediated dermal invasion. Moreover, ZEB1 expression correlated with Tspan8 expression in patient melanoma lesions. Our data suggest that intrinsic cell stiffness could be a highly relevant marker for human cutaneous melanoma development.

Artesunate inhibits macrophage-like phenotype switching of vascular smooth muscle cells and attenuates vascular inflammatory injury in atherosclerosis via NLRP3

Inflammation is one of the main pathogenic factors of atherosclerosis (AS), and the phenotypic transformation of macrophages in human vascular smooth muscle cells (HVSMCs) contributes to the inflammatory injury of blood vessels and the formation of atherosclerotic plaques. Artesunate reportedly exerts anti-inflammatory activity against AS. Herein, we aimed to explore the artesunate-mediated anti-inflammatory and HVSMC phenotypic switch effects against AS and elucidate potential underlying mechanisms. In vitro, artesunate decreased expression of NLRP3, caspase-1, and interleukin (IL)− 1β. Artesunate significantly inhibited low-density lipoprotein (LDL) expression in HVSMCs and macrophages. In vivo, artesunate reduced atherosclerotic plaque formation in high-fat diet (HFD)-fed ApoE-/- mice, as well as decreased NLRP3 and CD68 expression in atherosclerotic plaques. Artesunate decreased serum levels of triglycerides and increased high-density lipoprotein levels in HFD-med mice; however, serum levels of total cholesterol and LDL were unaltered. Treatment with artesunate substantially increased α-smooth muscle actin expression in aortic tissues while inhibiting expression levels of NLRP3, IL-1β, heparinase, matrix metalloproteinase 9, and Krüppel-like factor 4 (KLF4). Collectively, our findings suggest that artesunate-mediated effects may involve inhibition of the ERK1/2/NF-κB/IL-1β pathway in HVSMCs via the downregulation of NLRP3 expression. Thus, artesunate could serve as a novel strategy to treat AS by inhibiting AS plaque formation and suppressing macrophage-like phenotype switching of HVSMCs.

Differential histone acetylation and super-enhancer regulation underlie melanoma cell dedifferentiation.

Dedifferentiation or phenotype switching refers to the transition from a proliferative to an invasive cellular state. We previously identified a 122-gene epigenetic gene signature that classifies primary melanomas as low versus high risk (denoted as Epgn1 or Epgn3). We found that the transcriptomes of the Epgn1 low-risk and Epgn3 high-risk cells are similar to the proliferative and invasive cellular states, respectively. These signatures were further validated in melanoma tumor samples. Examination of the chromatin landscape revealed differential H3K27 acetylation in the Epgn1 low-risk versus Epgn3 high-risk cell lines that corroborated with a differential super-enhancer and enhancer landscape. Melanocytic lineage genes (MITF, its targets and regulators) were associated with super-enhancers in the Epgn1 low-risk state, whereas invasiveness genes were linked with Epgn3 high-risk status. We identified the ITGA3 gene as marked by a super-enhancer element in the Epgn3 invasive cells. Silencing of ITGA3 enhanced invasiveness in both in vitro and in vivo systems, suggesting it as a negative regulator of invasion. In conclusion, we define chromatin landscape changes associated with Epgn1/Epgn3 and phenotype switching during early steps of melanoma progression that regulate transcriptional reprogramming. This super-enhancer and enhancer-driven epigenetic regulatory mechanism resulting in major changes in the transcriptome could be important in future therapeutic targeting efforts.

Epithelial and Mesenchymal-like Pancreatic Cancer Cells Exhibit Different Stem Cell Phenotypes Associated with Different Metastatic Propensities.

Pancreatic ductal adenocarcinoma (PDAC) is mostly diagnosed at advanced or even metastasized stages, limiting the prognoses of patients. Metastasis requires high tumor cell plasticity, implying phenotypic switching in response to changing environments. Here, epithelial-mesenchymal transition (EMT), being associated with an increase in cancer stem cell (CSC) properties, and its reversion are important. Since it is poorly understood whether different CSC phenotypes exist along the EMT axis and how these impact malignancy-associated properties, we aimed to characterize CSC populations of epithelial and mesenchymal-like PDAC cells. Single-cell cloning revealed CSC (Holoclone) and non-CSC (Paraclone) clones from the PDAC cell lines Panc1 and Panc89. The Panc1 Holoclone cells showed a mesenchymal-like phenotype, dominated by a high expression of the stemness marker Nestin, while the Panc89 Holoclone cells exhibited a SOX2-dominated epithelial phenotype. The Panc89 Holoclone cells showed enhanced cell growth and a self-renewal capacity but slow cluster-like invasion. Contrarily, the Panc1 Holoclone cells showed slower cell growth and self-renewal ability but were highly invasive. Moreover, cell variants differentially responded to chemotherapy. In vivo, the Panc1 and Panc89 cell variants significantly differed regarding the number and size of metastases, as well as organ manifestation, leading to different survival outcomes. Overall, these data support the existence of different CSC phenotypes along the EMT axis in PDAC, manifesting different metastatic propensities.

Low-Dose LPS Modulates Microglia/Macrophages Phenotypic Transformation to Amplify Rehabilitation Effects in Chronic Spinal Cord Injured (CSCI) Mice.

Spinal cord injury (SCI) results in stalled motor function recovery under the chronic phase. One of the reasons due to the presence of ongoing inflammation. Therefore, regulating the status of immune cells may help reopen the window for neural repair, which represents a potential therapeutic target. In this study, we aimed to investigate whether this could be achieved in mice with cervical 5 crush CSCI (4W) by utilizing a concentration of 0.5mg/kg of lipopolysaccharide (LPS) to stimulate microglia/macrophages. Additionally, the mice underwent rehabilitation training for another 6 weeks. Our results showed that systemic injection of LPS enhanced the effects of forelimb rehabilitation training, as evaluated through single pellet grasping (SPG). Electrophysiological studies revealed the restoration of cortical drive to the injured side's forelimb muscles in the training combined with LPS group. Tract tracing studies demonstrated the reconstruction of cortical innervation to the cervical spinal cord. Furthermore, the levels of pro-inflammatory phenotype markers, such as inducible nitric oxide synthase (INOS) and CD68, decreased, while the expression of anti-inflammatory phenotype markers, including arginase 1 (ARG-1) and CD206, increased. Importantly, this phenotypic switch in microglia/macrophages was accompanied by an increase in phagocytic activity markers as indicated by BODIPY + IBA1 + staining. Collectively, our data suggests that low-dose LPS improves the effects of rehabilitation training by regulating the phenotypic transformation of microglia/macrophages in CSCI.This study provides a fresh perspective and intervention direction for the clinical treatment of chronic spinal cord injuries.

Allostery through DNA drives phenotype switching

Allostery through DNA drives phenotype switching

Cellular, Molecular and Clinical Aspects of Aortic Aneurysm-Vascular Physiology and Pathophysiology.

A disturbance of the structure of the aortic wall results in the formation of aortic aneurysm, which is characterized by a significant bulge on the vessel surface that may have consequences, such as distention and finally rupture. Abdominal aortic aneurysm (AAA) is a major pathological condition because it affects approximately 8% of elderly men and 1.5% of elderly women. The pathogenesis of AAA involves multiple interlocking mechanisms, including inflammation, immune cell activation, protein degradation and cellular malalignments. The expression of inflammatory factors, such as cytokines and chemokines, induce the infiltration of inflammatory cells into the wall of the aorta, including macrophages, natural killer cells (NK cells) and T and B lymphocytes. Protein degradation occurs with a high expression not only of matrix metalloproteinases (MMPs) but also of neutrophil gelatinase-associated lipocalin (NGAL), interferon gamma (IFN-γ) and chymases. The loss of extracellular matrix (ECM) due to cell apoptosis and phenotype switching reduces tissue density and may contribute to AAA. It is important to consider the key mechanisms of initiating and promoting AAA to achieve better preventative and therapeutic outcomes.

The CoREST repressor complex mediates phenotype switching and therapy resistance in melanoma.

Virtually all patients with BRAF-mutant melanoma develop resistance to MAPK inhibitors largely through nonmutational events. Although the epigenetic landscape is shown to be altered in therapy-resistant melanomas and other cancers, a specific targetable epigenetic mechanism has not been validated. Here, we evaluated the corepressor for element 1-silencing transcription factor (CoREST) epigenetic repressor complex and the recently developed bivalent inhibitor corin within the context of melanoma phenotype plasticity and therapeutic resistance. We found that CoREST was a critical mediator of the major distinct melanoma phenotypes and that corin treatment of melanoma cells led to phenotype reprogramming. Global assessment of transcript and chromatin changes conferred by corin revealed specific effects on histone marks connected to epithelial-mesenchymal transition-associated (EMT-associated) transcription factors and the dual-specificity phosphatases (DUSPs). Remarkably, treatment of BRAF inhibitor-resistant (BRAFi-R) melanomas with corin promoted resensitization to BRAFi therapy. DUSP1 was consistently downregulated in BRAFi-R melanomas, which was reversed by corin treatment and associated with inhibition of p38 MAPK activity and resensitization to BRAFi therapies. Moreover, this activity was recapitulated by the p38 MAPK inhibitor BIRB 796. These findings identify the CoREST repressor complex as a central mediator of melanoma phenotype plasticity and resistance to targeted therapy and suggest that CoREST inhibitors may prove beneficial for patients with BRAFi-resistant melanoma.

Cell polarization in ischemic stroke: molecular mechanisms and advances.

Ischemic stroke is a cerebrovascular disease associated with high mortality and disability rates. Since the inflammation and immune response play a central role in driving ischemic damage, it becomes essential to modulate excessive inflammatory reactions to promote cell survival and facilitate tissue repair around the injury site. Various cell types are involved in the inflammatory response, including microglia, astrocytes, and neutrophils, each exhibiting distinct phenotypic profiles upon stimulation. They display either proinflammatory or anti-inflammatory states, a phenomenon known as 'cell polarization.' There are two cell polarization therapy strategies. The first involves inducing cells into a neuroprotective phenotype in vitro, then reintroducing them autologously. The second approach utilizes small molecular substances to directly affect cells in vivo. In this review, we elucidate the polarization dynamics of the three reactive cell populations (microglia, astrocytes, and neutrophils) in the context of ischemic stroke, and provide a comprehensive summary of the molecular mechanisms involved in their phenotypic switching. By unraveling the complexity of cell polarization, we hope to offer insights for future research on neuroinflammation and novel therapeutic strategies for ischemic stroke.

Calcium phosphate coating enhances osteointegration of melt electrowritten scaffold by regulating macrophage polarization

The osteoimmune microenvironment induced by implants plays a significant role in bone regeneration. It is essential to efficiently and timely switch the macrophage phenotype from M1 to M2 for optimal bone healing. This study examined the impact of a calcium phosphate (CaP) coating on the physiochemical properties of highly ordered polycaprolactone (PCL) scaffolds fabricated using melt electrowritten (MEW). Additionally, it investigated the influence of these scaffolds on macrophage polarization and their immunomodulation on osteogenesis. The results revealed that the CaP coated PCL scaffold exhibited a rougher surface topography and higher hydrophilicity in comparison to the PCL scaffold without coating. Besides, the surface morphology of the coating and the release of Ca2+ from the CaP coating were crucial in regulating the transition of macrophages from M1 to M2 phenotypes. They might activate the PI3K/AKT and cAMP-PKA pathways, respectively, to facilitate M2 polarization. In addition, the osteoimmune microenvironment induced by CaP coated PCL could not only enhance the osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) in vitro but also promote the bone regeneration in vivo. Taken together, the CaP coating can be employed to control the phenotypic switching of macrophages, thereby creating a beneficial immunomodulatory microenvironment that promotes bone regeneration.Graphical abstract

Phenotypic switching of vascular smooth muscle cells in atherosclerosis, hypertension, and aortic dissection.

Vascular smooth muscle cells (VSMCs) play a critical role in regulating vasotone, and their phenotypic plasticity is a key contributor to the pathogenesis of various vascular diseases. Two main VSMC phenotypes have been well described: contractile and synthetic. Contractile VSMCs are typically found in the tunica media of the vessel wall, and are responsible for regulating vascular tone and diameter. Synthetic VSMCs, on the other hand, are typically found in the tunica intima and adventitia, and are involved in vascular repair and remodeling. Switching between contractile and synthetic phenotypes occurs in response to various insults and stimuli, such as injury or inflammation, and this allows VSMCs to adapt to changing environmental cues and regulate vascular tone, growth, and repair. Furthermore, VSMCs can also switch to osteoblast-like and chondrocyte-like cell phenotypes, which may contribute to vascular calcification and other pathological processes like the formation of atherosclerotic plaques. This provides discusses the mechanisms that regulate VSMC phenotypic switching and its role in the development of vascular diseases. A better understanding of these processes is essential for the development of effective diagnostic and therapeutic strategies.

Modeling stress-induced responses: plasticity in continuous state space and gradual clonal evolution.

Mathematical models of cancer and bacterial evolution have generally stemmed from a gene-centric framework, assuming clonal evolution via acquisition of resistance-conferring mutations and selection of their corresponding subpopulations. More recently, the role of phenotypic plasticity has been recognized and models accounting for phenotypic switching between discrete cell states (e.g., epithelial and mesenchymal) have been developed. However, seldom do models incorporate both plasticity and mutationally driven resistance, particularly when the state space is continuous and resistance evolves in a continuous fashion. In this paper, we develop a framework to model plastic and mutational mechanisms of acquiring resistance in a continuous gradual fashion. We use this framework to examine ways in which cancer and bacterial populations can respond to stress and consider implications for therapeutic strategies. Although we primarily discuss our framework in the context of cancer and bacteria, it applies broadly to any system capable of evolving via plasticity and genetic evolution.

Vascular injury activates the ELK1/SND1/SRF pathway to promote vascular smooth muscle cell proliferative phenotype and neointimal hyperplasia

BackgroundVascular smooth muscle cell (VSMC) proliferation is the leading cause of vascular stenosis or restenosis. Therefore, investigating the molecular mechanisms and pivotal regulators of the proliferative VSMC phenotype is imperative for precisely preventing neointimal hyperplasia in vascular disease.MethodsWire-induced vascular injury and aortic culture models were used to detect the expression of staphylococcal nuclease domain-containing protein 1 (SND1). SMC-specific Snd1 knockout mice were used to assess the potential roles of SND1 after vascular injury. Primary VSMCs were cultured to evaluate SND1 function on VSMC phenotype switching, as well as to investigate the mechanism by which SND1 regulates the VSMC proliferative phenotype.ResultsPhenotype-switched proliferative VSMCs exhibited higher SND1 protein expression compared to the differentiated VSMCs. This result was replicated in primary VSMCs treated with platelet-derived growth factor (PDGF). In the injury model, specific knockout of Snd1 in mouse VSMCs reduced neointimal hyperplasia. We then revealed that ETS transcription factor ELK1 (ELK1) exhibited upregulation and activation in proliferative VSMCs, and acted as a novel transcription factor to induce the gene transcriptional activation of Snd1. Subsequently, the upregulated SND1 is associated with serum response factor (SRF) by competing with myocardin (MYOCD). As a co-activator of SRF, SND1 recruited the lysine acetyltransferase 2B (KAT2B) to the promoter regions leading to the histone acetylation, consequently promoted SRF to recognize the specific CArG motif, and enhanced the proliferation- and migration-related gene transcriptional activation.ConclusionsThe present study identifies ELK1/SND1/SRF as a novel pathway in promoting the proliferative VSMC phenotype and neointimal hyperplasia in vascular injury, predisposing the vessels to pathological remodeling. This provides a potential therapeutic target for vascular stenosis.Graphical abstract

Novel in vitro evidence on the beneficial effect of quercetin treatment in vascular calcification.

Vascular calcification is a pathological chronic condition characterized by calcium crystal deposition in the vessel wall and is a recurring event in atherosclerosis, chronic kidney disease, and diabetes. The lack of effective therapeutic treatments opened the research to natural products, which have shown promising potential in inhibiting the pathological process in different experimental models. This study investigated the anti-calcifying effects of Quercetin and Berberine extracts on vascular smooth muscle cells (VSMCs) treated with an inorganic phosphate solution for 7days. Quercetin has shown the highest anti-calcifying activity, as revealed by the intracellular quantitative assay and morphological analysis. Confocal microscopy revealed downregulation of RUNX2, a key marker for calcified phenotype, which was otherwise upregulated in calcified VSMCs. To investigate the anti-inflammatory activity of Quercetin, culture media were subjected to immunometric assays to quantify the levels of IL-6 and TNF-α, and the caspase-1 activity. As expected, calcified VSMCs released a large quantity of inflammatory mediators, significantly decreasing in the presence of Quercetin. In summary, our findings suggest that Quercetin counteracted calcification by attenuating the VSMC pathological phenotypic switch and reducing the inflammatory response. In our opinion, these preliminary in vitro findings could be the starting point for further investigations into the beneficial effects of Quercetin dietary supplementation against vascular calcification.

CircNRIP1 promotes proliferation, migration and phenotypic switch of Ang II-induced HA-VSMCs by increasing CXCL5 mRNA stability via recruiting IGF2BP1

Circular RNA (circRNA) has been found to be differentially expressed and involved in regulating the processes of human diseases, including thoracic aortic dissection (TAD). However, the role and mechanism of circNRIP1 in the TAD process are still unclear. GEO database was used to screen the differentially expressed circRNA and mRNA in type A TAD patients and age-matched normal donors. Angiotensin II (Ang II)-induced human aortic vascular smooth muscle cells (HA-VSMCs) were used to construct TAD cell models. The expression levels of circNRIP1, NRIP1, CXC-motif chemokine 5 (CXCL5) and IGF2BP1 were detected by quantitative real-time PCR. Cell proliferation and migration were determined by EdU assay, transwell assay and wound healing assay. The protein levels of synthetic phenotype markers, contractile phenotype markers, CXCL5 and IGF2BP1 were tested by western blot analysis. The interaction between IGF2BP1 and circNRIP1/CXCL5 was confirmed by RIP assay, and CXCL5 mRNA stability was assessed by actinomycin D assay. CircNRIP1 was upregulated in TAD patients and Ang II-induced HA-VSMCs. Knockdown of circNRIP1 suppressed Ang II-induced proliferation, migration and phenotypic switch of HA-VSMCs. Also, high expression of CXCL5 was observed in TAD patients, and its knockdown could inhibit Ang II-induced HA-VSMCs proliferation, migration and phenotypic switch. Moreover, CXCL5 overexpression reversed the regulation of circNRIP1 knockdown on Ang II-induced HA-VSMCs functions. Mechanistically, circNRIP1 could competitively bind to IGF2BP1 and subsequently enhance CXCL5 mRNA stability. CircNRIP1 might contribute to TAD progression by promoting CXCL5 mRNA stability via recruiting IGF2BP1.

S-propargyl-cysteine promotes the stability of atherosclerotic plaque via maintaining vascular muscle contractile phenotype.

Introduction: Plaque rupture in atherosclerosis contributes to various acute cardiovascular events. As a new sulfide-containing donor, S-propargyl-cysteine (SPRC) has been reported to play a beneficial role in cardioprotection, potentially through its anti-inflammatory, anti-oxidative and anti-atherogenic activities. Our previous study observed an increase in eNOS phosphorylation in endothelial cells. However, it remains unclear whether SPRC influences vascular smooth muscle cells (VSMCs) within the plaque and if this effect contributes to plaque stabilization. Methods: An atherosclerotic unstable plaque mouse model was established by subjecting ApoE-/- mice to tandem stenosis of the right carotid artery along with a Western diet. Daily SPRC administration was conducted for 13weeks. Plaque morphology and stability were assessed using MRI scanning and histopathological staining. In our in vitro studies, we stimulated human artery vascular smooth muscle cells (HAVSMCs) with platelet-derived growth factor-BB (PDGF-BB), both with and without 100μM SPRC treatment. Cell phenotype was assessed using both Western blot and Real-time PCR. Cell proliferation was assessed using the BrdU cell proliferation kit and immunofluorescence of Ki-67, while cell migration was measured using scratch wound healing and transwell assay. MiR-143-3p overexpression and knockdown experiments were used to investigate whether it mediates the effect of SPRC on VSMC phenotype. Results and Discussion: SPRC treatment reduced plasma lipid levels, increased collagen content and decreased cell apoptosis in atherosclerotic plaques, indicating improved plaque stability. Both in vivo and in vitro studies elucidated the role of SPRC in preserving the contractile phenotype of VSMCs through up-regulation of miR-143-3p expression. Furthermore, SPRC suppressed the pro-proliferation and pro-migration effects of PDGF-BB on HAVSMCs. Overall, these findings suggest that the inhibitory effect of SPRC on phenotype switch from contractile to synthetic VSMCs may contribute to its beneficial role in enhancing plaque stability.

Role of ZIP kinase in development of myofibroblast differentiation from HPMCs.

During the development of pleural fibrosis, pleural mesothelial cells (PMCs) undergo phenotypic switching from differentiated mesothelial cells to mesenchymal cells (MesoMT). Here, we investigated how external stimuli such as TGF-β induce HPMC-derived myofibroblast differentiation to facilitate the development of pleural fibrosis. TGF-β significantly increased di-phosphorylation but not mono-phosphorylation of myosin II regulatory light chain (RLC) in HPMCs. An increase in RLC di-phosphorylation was also found at the pleural layer of our carbon black bleomycin (CBB) pleural fibrosis mouse model, where it showed filamentous localization that coincided with alpha smooth muscle actin (αSMA) in the cells in the pleura. Among the protein kinases that can phosphorylate myosin II RLC, ZIPK (zipper-interacting kinase) protein expression was significantly augmented after TGF-β stimulation. Furthermore, ZIPK gene silencing attenuated RLC di-phosphorylation, suggesting that ZIPK is responsible for di-phosphorylation of myosin II in HPMCs. Although TGF-β significantly increased the expression of ZIP kinase protein, the change in ZIP kinase mRNA was marginal, suggesting a posttranscriptional mechanism for the regulation of ZIP kinase expression by TGF-β. ZIPK gene knockdown (KD) also significantly reduced TGF-β-induced upregulation of αSMA expression. This finding suggests that siZIPK attenuates myofibroblast differentiation of HPMCs. siZIPK diminished TGF-β-induced contractility of HPMCs consistent with siZIPK-induced decrease in the di-phosphorylation of myosin II RLC. The present results implicate ZIPK in the regulation of the contractility of HPMC-derived myofibroblasts, phenotype switching, and myofibroblast differentiation of HPMCs.NEW & NOTEWORTHY Here, we highlight that ZIP kinase is responsible for di-phosphorylation of myosin light chain, which facilitates stress fiber formation and actomyosin-based cell contraction during mesothelial to mesenchymal transition in human pleural mesothelial cells. This transition has a significant impact on tissue remodeling and subsequent stiffness of the pleura. This study provides insight into a new therapeutic strategy for the treatment of pleural fibrosis.

The functional role of cellular senescence during vascular calcification in chronic kidney disease.

Vascular calcification (VC) has emerged as a key predictor of cardiovascular events in patients with chronic kidney disease (CKD). In recent years, an expanding body of research has put forth the concept of accelerated vascular aging among CKD patients, highlighting the significance of vascular cells senescence in the process of VC. Within the milieu of uremia, senescent vascular endothelial cells (VECs) release extracellular microvesicles (MV) that promote vascular smooth muscle cells (VSMCs) senescence, thereby triggering the subsequent osteogenic phenotypic switch and ultimately contributing to the VC process. In addition, senescent vascular progenitor or stem cells with diminished ability to differentiate into VECs and VSMCS, compromise the repair of vascular integrity, on the other hand, release a cascade of molecules associated with senescence, collectively known as the senescence-associated secretory phenotype (SASP), perpetuating the senescence phenomenon. Furthermore, SASP triggers the recruitment of monocytes and macrophages, as well as adjacent VECs and VSMCs into a pro-adhesive and pro-inflammatory senescent state. This pro-inflammatory microenvironment niche not only impacts the functionality of immune cells but also influences the differentiation of myeloid immune cells, thereby amplifying the reduced ability to effectively clear senescent cells of senescent macrophages, promoted calcification of VSMCs. The objective of this paper is to provide a comprehensive review of the contribution of vascular cell senescence to the emergence and advancement of VC. Gaining a comprehensive understanding of the involvement of cellular senescence within the vessel wall is pivotal, especially when it comes to its intersection with VC. This knowledge is essential for advancing groundbreaking anti-aging therapies, aiming to effectively mitigate cardiovascular diseases.

Macrophages promote the transition from myocardial ischemia reperfusion injury to cardiac fibrosis in mice through GMCSF/CCL2/CCR2 and phenotype switching.

Following acute myocardial ischemia reperfusion (MIR), macrophages infiltrate damaged cardiac tissue and alter their polarization phenotype to respond to acute inflammation and chronic fibrotic remodeling. In this study we investigated the role of macrophages in post-ischemic myocardial fibrosis and explored therapeutic targets for myocardial fibrosis. Male mice were subjected to ligation of the left coronary artery for 30 min. We first detected the levels of chemokines in heart tissue that recruited immune cells infiltrating into the heart, and found that granulocyte-macrophage colony-stimulating factor (GMCSF) released by mouse cardiac microvascular endothelial cells (MCMECs) peaked at 6 h after reperfusion, and c-c motif chemokine ligand 2 (CCL2) released by GMCSF-induced macrophages peaked at 24 h after reperfusion. In co-culture of BMDMs with MCMECs, we demonstrated that GMCSF derived from MCMECs stimulated the release of CCL2 by BMDMs and effectively promoted the migration of BMDMs. We also confirmed that GMCSF promoted M1 polarization of macrophages in vitro, while GMCSF neutralizing antibodies (NTABs) blocked CCL2/CCR2 signaling. In MIR mouse heart, we showed that GMCSF activated CCL2/CCR2 signaling to promote NLRP3/caspase-1/IL-1β-mediated and amplified inflammatory damage. Knockdown of CC chemokine receptor 2 gene (CCR2-/-), or administration of specific CCR2 inhibitor RS102895 (5 mg/kg per 12 h, i.p., one day before MIR and continuously until the end of the experiment) effectively reduced the area of myocardial infarction, and down-regulated inflammatory mediators and NLRP3/Caspase-1/IL-1β signaling. Mass cytometry confirmed that M2 macrophages played an important role during fibrosis, while macrophage-depleted mice exhibited significantly reduced transforming growth factor-β (Tgf-β) levels in heart tissue after MIR. In co-culture of macrophages with fibroblasts, treatment with recombinant mouse CCL2 stimulated macrophages to release a large amount of Tgf-β, and promoted the release of Col1α1 by fibroblasts. This effect was diminished in BMDMs from CCR2-/- mice. After knocking out or inhibiting CCR2-gene, the levels of Tgf-β were significantly reduced, as was the level of myocardial fibrosis, and cardiac function was protected. This study confirms that the acute injury to chronic fibrosis transition after MIR in mice is mediated by GMCSF/CCL2/CCR2 signaling in macrophages through NLRP3 inflammatory cascade and the phenotype switching.

OxPhos in adipose tissue macrophages regulated by BTK enhances their M2-like phenotype and confers a systemic immunometabolic benefit in obesity.

Bruton's tyrosine kinase (BTK) is a non-receptor bound kinase involved in pro-inflammatory signalling in activated macrophages, however, its role within adipose tissue macrophages remains unclear. We have demonstrated that BTK signalling regulates macrophage M2-like polarisation state by up-regulating subunits of mitochondrially encoded electron transport chain Complex I (ND4 and NDL4) and Complex IV (mt-CO1, mt-CO2 and mt-CO3) resulting in an enhanced rate of oxidative phosphorylation (OxPhos) in an NF-κB independent manner. Critically, BTK expression is elevated in adipose tissue macrophages from obese individuals with diabetes, while key mitochondrial genes (mtC01, mtC02 and mtC03) are decreased in inflammatory myeloid cells from obese individuals. Inhibition of BTK signalling either globally (Xid mice) or in myeloid cells (LysMCreBTK), or therapeutically (Acalabrutinib) protects HFD-fed mice from developing glycaemic dysregulation by improving signalling through the IRS1/Akt/GSK3β pathway. The beneficial effects of acalabrutinib treatment are lost in macrophage ablated mice. Inhibition of BTK signalling in myeloid cells but not B-cells, induced a phenotypic switch in adipose tissue macrophages from a pro-inflammatory M1-state to a pro-resolution M2-like phenotype, by shifting macrophage metabolism towards OxPhos. This reduces both local and systemic inflammation and protected mice from the immunometabolic consequences of obesity. Therefore, in BTK we have identified a macrophage specific, druggable target that can regulate adipose tissue polarisation and cellular metabolism that can confer systematic benefit in metabolic syndrome.