Research advances in epigenetic modifications and post-translational modifications in endothelial-mesenchymal transition.

EndMT, a critical cellular differentiation process in embryonic development, vascular diseases (such as atherosclerosis and cardiac fibrosis), and tumor metastasis, involves complex signaling pathway interactions for its initiation and regulation. Various post-translational modifications (PTMs), including phosphorylation, acetylation, ubiquitination, SUMOylation, and O-GlcNAcylation, play central roles [ 123 – 125 ]. These modifications precisely regulate protein activity, stability, conformation, and subcellular localization, collectively forming a multi-layered and sophisticated regulatory network that determines the progression and reversibility of EndMT. Post-translational modifications of proteins, particularly phosphorylation, play a crucial role in regulating cellular functions and protein properties [ 126 – 130 ]. Phosphorylation, one of the most common and extensively studied PTMs, profoundly influences EndMT regulation by altering protein structure, activity, interactions, and subcellular localization. Phosphorylation introduces a negatively charged phosphate group into a protein, causing significant changes in protein conformation and energy landscape [ 131 ]. This modification can alter protein backbone dynamics and conformational preferences. In many cases, phosphorylation directly alters receptor function, often leading to negative regulation, such as reducing the ability to interact with biochemical effectors or decreasing ligand binding affinity [ 132 ]. Phosphorylation occurs particularly frequently in intrinsically disordered proteins (IDPs), which are highly flexible and dynamic [ 133 ]. The addition of a single phosphate group can significantly impact the function of IDPs. Furthermore, the presence of multiple phosphorylation sites increases the complexity of protein states, enabling more refined regulation. Multiple studies indicate that phosphorylation plays a key role in both the activation and inhibition of EndMT: Phosphorylation Regulation in the TGF-β Signaling Pathway: TGF-β activates the phosphorylation of downstream SMAD proteins, particularly SMAD2 and SMAD3, through its receptor kinases, thereby driving EndMT [ 134 , 135 ]. TGF-β signaling plays a central role in venous graft remodeling by mediating EndMT. Some studies also mention that TRPV4 channels mediate TGF-β2-induced EndMT through the Rho/Snail mechanotransduction pathway [ 40 ]. The ETS-Related Gene (ERG) transcription factor is important in regulating EndMT. The endothelial transcription factor ERG promotes liver homeostasis by controlling canonical TGF-β-SMAD signaling, driving the SMAD1 pathway and inhibiting SMAD3 activity. ERG binds to SMAD3, limiting its DNA contact, thereby protecting the liver during fibrosis [ 136 ]. Loss of ERG leads to EndMT and severe liver fibrosis. Phosphorylation Regulation in the NF-κB Signaling Pathway: In ischemic diseases, GTF2H4 (General Transcription Factor IIH Subunit 4) activates the NF-κB pathway through NCOA3 phosphorylation, thereby regulating partial EndMT [ 137 ]. Decursin inhibits EndMT progression by suppressing the PI3K/AKT/NF-κB and Smad signaling pathways, suggesting that phosphorylation events within these pathways regulate EndMT [ 30 ]. Phosphorylation Regulation of the CHK1-SENP2 Axis: Laminar shear stress can inhibit EndMT through phosphorylation. Research has found that Checkpoint Kinase 1 (CHK1)-associated phosphorylation of SENP2 at serine 344 (S344 in humans, S343 in corresponding mouse models) plays a role in laminar flow-regulated inhibition of EndMT and atherogenesis [ 138 ]. Using SENP2 S343A knock-in mice and endothelial cell-specific SENP2 knockout mice, researchers demonstrated that phosphorylation of SENP2 S344 is key to inhibiting EndMT [ 138 ]. MT2 Receptor and EndMT: Melatonin promotes ectopic ossification in injured Achilles tendons via the MT2 receptor, a process involving the regulation of EndMT [ 139 ]. This suggests that phosphorylation events likely occur downstream of the MT2 receptor signaling pathway, influencing EndMT. Metabolism-Associated Phosphorylation: Complex interconnections exist between phosphorylation and cellular metabolism. Metabolites can serve as donors for PTMs; for example, acetyl-CoA is the donor for acetylation reactions [ 140 ]. EndMT is closely linked to cellular metabolic state, although the metabolic status in digestive system diseases involving EndMT is not fully understood [ 141 ]. Metabolism is a key regulatory mechanism of EndMT in pulmonary arterial hypertension; for instance, METTL3 promotes EndMT in pulmonary arterial endothelial cells by regulating the TRPC6/calcineurin/NFAT signaling pathway [ 142 ]. Although specific phosphorylation details are not fully elucidated, kinase and phosphatase activities are typically involved in these pathways, implying a potential role for phosphorylation in metabolism-regulated EndMT. Diabetic complications, such as retinopathy, nephropathy, and atherosclerosis, share the common feature of myofibroblast-driven excessive deposition of extracellular matrix proteins, which is associated with EndMT. Oxidative stress is a key factor in high glucose-induced EndMT [ 79 ]. The production of reactive oxygen species (ROS) and subsequent oxidative damage to endothelial cells may induce EndMT. Antioxidants like Decursin inhibit EndMT via the PI3K/AKT/NF-κB and Smad pathways, which involve multiple phosphorylation steps [ 30 ]. Downregulation of the ETS family transcription factors ERG and FLI1 in endothelial cells triggers EndMT [ 143 ]. ETS1 influences mitochondrial dysfunction and ATP production, and leads to Occludin degradation and alterations in ZO-1/ZO-2, all of which promote EndMT [ 144 ]. In summary, phosphorylation, as a crucial PTM, plays a central role in regulating EndMT by directly or indirectly affecting protein structure, activity, interactions, and stability. It is involved in various signaling pathways such as TGF-β, NF-κB, and PI3K/AKT, and acts in concert with factors like cellular metabolism, mechanical forces, and non-coding RNAs to collectively determine endothelial cell fate. An in-depth understanding of the precise regulatory mechanisms of phosphorylation in EndMT will provide new targets and therapeutic strategies for treating EndMT-related diseases such as fibrosis, atherosclerosis, pulmonary arterial hypertension, and cancer (Table  8 ). Table 8 Regulatory roles of Post-translational phosphorylation modifications in EndMT Regulatory Factor/Pathway Phosphorylation Site/Kinase Role in EndMT References SENP2 CHK1-mediated phosphorylation at Serine 344 (S344) Under laminar flow, phosphorylation of SENP2 at S344 attenuates EndMT and reduces the incidence of atherosclerosis. [ 145 ] eIF2S1(eIF2α) Phosphorylation Phosphorylation of eIF2S1 is indispensable for the nuclear translocation of TFEB and TFE3 during endoplasmic reticulum stress. [ 146 ] GTF2H4 Phosphorylation of NCOA3 GTF2H4 activates NF-κB via phosphorylation of NCOA3, modulating partial EndMT in ischemic diseases. [ 147 ] Akt Phosphorylation Activation of the Akt pathway via phosphorylation is a critical step in the EndMT process. [ 29 ] eEF2 Phosphorylation Phosphorylation of eEF2 coordinates and regulates erythroid differentiation. [ 148 ] Notch1 Phosphorylation Phosphorylation of Notch1 is essential for its signaling transduction. [ 149 ] eIF4G Ksp1-dependent phosphorylation Under glucose deprivation, Ksp1-dependent phosphorylation regulates the post-transcriptional control of specific mRNAs. [ 150 ] Hsp90 Phosphorylation Phosphorylation serves as an important regulatory mechanism for Hsp90. [ 151 ] Integrated Stress Response Phosphorylation of eIF2α Cellular stress response kinases (GCN2, PERK, PKR, HRI) phosphorylate the eIF2α subunit, leading to downregulation of general mRNA translation initiation, while selectively upregulating the translation of ATF4. [ 152 ] Regulatory roles of Post-translational phosphorylation modifications in EndMT Acetylation, a key post-translational modification (PTM), plays a critical regulatory role in Endothelial-Mesenchymal Transition (EndMT). It influences the conversion of endothelial cells to mesenchymal cells, thereby participating in various physiological and pathological processes, including development, wound healing, cancer metastasis, organ fibrosis, and cardiovascular diseases [ 8 , 153 – 164 ]. Acetylation typically occurs on lysine residues of proteins. It is catalyzed by histone acetyltransferases (HATs), which add acetyl groups, and histone deacetylases (HDACs), which remove them; this dynamic balance determines the acetylation level of proteins [ 47 , 165 – 168 ]. Beyond histones, many non-histone proteins are also subject to acetylation, which can affect their activity, stability, subcellular localization, and interactions with other molecules, thereby regulating various intracellular signaling pathways [ 165 , 168 – 171 ]. The regulatory role of acetylation in the EndMT process is primarily manifested in the following aspects: Firstly, acetylation directly affects key signaling pathways in EndMT. Studies have shown that acetylation modifications can influence components of the TGF-β signaling pathway, thereby regulating the initiation and progression of EndMT [ 172 ]. Acetate was found to control TGF-β-induced EndMT. One study discovered that the metabolite acetate enhances TGF-β signaling by promoting p300-mediated acetylation of Smad3 in endothelial cells, thereby driving EndMT [ 172 ]. This creates a positive feedback loop where EndMT leads to more TGF-β signaling, complicating the induction and persistence of EndMT. Secondly, acetylation influences EndMT by regulating the activity of transcription factors. The occurrence of EndMT involves changes in the expression of a series of key transcription factors that regulate the downregulation of endothelial-specific genes and the upregulation of mesenchymal-specific genes [ 173 ]. Acetylation of transcription factors can alter their DNA-binding capacity, transcriptional activity, and stability. For instance, transcription factors such as Snail, Slug, Twist, and ZEB are core regulators of EMT, and EndMT is a specific type of EMT [ 174 , 175 ]. Their activity is likely finely tuned by acetylation modifications. In the EMT process of glioblastoma (GBM), acetylation (e.g., H2BK5Ac) is listed among the key post-translational modifications [ 176 ]. Furthermore, an imbalance between HDACs and HATs is a significant factor in the occurrence of EndMT in various diseases [ 165 , 168 , 177 – 179 ]. HDAC inhibitors (HDACis), as potential therapeutic agents, can intervene in EndMT-related diseases by modulating protein acetylation levels. For example, in chronic cardiovascular diseases, EndMT is a major contributor to cardiac fibrosis, and there are currently no specific therapies to halt or reverse cardiac fibrosis [ 8 , 158 ]. Understanding the molecular mechanisms of EndMT, including acetylation regulation, is crucial for developing new therapeutic strategies. Beyond the direct effects on transcription factors, acetylation can also influence EndMT by regulating cytoskeletal reorganization. For instance, acetylation of α-tubulin stabilizes microtubule structures, thereby affecting cell morphology, motility, and cytoskeletal remodeling, which are key aspects of the phenotypic changes during EndMT. In the nucleus, acetylation of histones (H2A, H2B, H3, H4) and non-histone proteins (such as p53, NFκB, USF-1), catalyzed by HATs, can activate gene transcription, leading to the expression of genes like BDNF, GDNF, Hsp70, Akt, and ERK, which are associated with cell survival, growth, and stress responses [ 47 ]. Conversely, HDACs remove acetyl groups, repressing gene transcription. In the cytoplasm, acetylation of α-tubulin is dynamically regulated by HATs and HDACs. Stable acetylated microtubules are crucial for the intracellular transport and release of BDNF, impacting intercellular communication and neural function. EndMT not only plays important roles in physiological developmental processes but is also implicated in various pathological conditions, such as cancer progression, vascular fibrosis, cardiac fibrosis, pulmonary arterial hypertension, atherosclerosis, and ectopic ossification [ 29 , 54 , 155 , 174 , 180 – 188 ]. Therefore, a deeper understanding of the regulatory mechanisms of acetylation in EndMT holds promise for providing new targets and strategies for treating these diseases. In chronic cardiovascular diseases, EndMT is a significant pathogenic process. Although EndMT plays a key role in development and disease, its specific molecular mechanisms, particularly the molecular basis for the induction and persistence of TGF-β-driven EndMT, require further exploration. Targeted intervention in metabolic reprogramming is considered a strategy to control EMT in aggressive tumors, which also applies to EndMT, as changes in metabolites can affect the activity of acetylating and deacetylating enzymes. In summary, the post-translational modification acetylation plays a complex and precise regulatory role in the initiation and progression of EndMT by modulating components of key signaling pathways, the activity of transcription factors, and cytoskeletal reorganization. Further research into the specific roles of acetyltransferases (HATs), deacetylases (HDACs), and their substrates in EndMT will contribute to the development of effective interventions for EndMT-related diseases (Table  9 ). Table 9 Regulatory roles of Post-translational acetylation modifications in EndMT Regulatory Aspect Specific Role Participating Molecules/Pathways References Gene Transcription Regulation Acetylation of transcription factors regulates downstream gene expression; Acetyltransferases and deacetylases modulate transcription factor activity. Transcription factors (e.g., Snail, Twist, Slug), Acetyltransferases (KATs), Deacetylases (KDACs) [ 153 , 189 , 190 ] Protein Stability Acetylation influences protein degradation pathways, thereby regulating protein half-life; Acetylation alters protein conformation, affecting its interactions with other molecules. p53, β-catenin, E-cadherin [ 153 , 189 ] Signaling Pathway Regulation Acetyltransferases/deacetylases affect the activity of key signaling molecules, modulating EndMT-related pathways; Acetylation alters the localization and interactions of signaling components; Acetylation is involved in regulating Receptor Tyrosine Kinase (RTK) signaling. TGF-β, Rho/Snail, Wnt/β-catenin, STAT3, TRPC6/Calcineurin/NFAT [ 172 , 54 , 191 ] Metabolic Regulation Acetylation regulates metabolic enzyme activity, influencing cellular metabolic status and thereby affecting EndMT; Acetylation participates in regulating mitochondrial function and energy metabolism; Short-chain fatty acids (e.g., acetate) influence EndMT via acetylation. Metabolic enzymes (e.g., TCA cycle enzymes), Acetyl-CoA, Acetate [ 172 ] Endothelial Cell Function ANGPTL4 suppresses EndMT by protecting KLF2; Acetylation regulates endothelial cell migration, adhesion, and permeability; CD45 modulates EndMT in endothelial cells. KLF2, ANGPTL4, CD45 [ 182 ] Inflammatory Response Suppressing inflammatory activity in vascular endothelial cells reduces EndMT; Acetylation modulates the expression and activity of inflammation-related molecules; 27-Hydroxycholesterol (27HC)-induced EndMT promotes breast cancer cell migration via STAT3 signaling. Inflammatory factors (e.g., IL-6, TNF-α), STAT3, 27HC [ 54 ] Cytoskeletal Remodeling Tubulin acetylation affects cell morphology and motility; Acetylation regulates the assembly and disassembly of cytoskeletal proteins. Tubulin, α-tubulin-N-acetyltransferase 1, Histone deacetylase 6, Sirtuin-2 [ 192 ] Regulatory roles of Post-translational acetylation modifications in EndMT Ubiquitination and deubiquitination, as key forms of post-translational modifications (PTMs), play a crucial regulatory role in EndMT [ 176 , 193 , 194 ]. The processes of ubiquitination and deubiquitination during EndMT represent a critical pathogenic mechanism in cardiovascular diseases (such as atherosclerosis, pulmonary arterial hypertension, valvular diseases, and fibroelastosis), embryonic development, as well as renal fibrosis and cancer [ 5 , 156 , 161 , 163 , 164 , 183 , 195 ]. Ubiquitination involves the covalent attachment of ubiquitin (Ub) molecules to substrate proteins through a cascade of enzymes (E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes, and E3 ubiquitin ligases) [ 193 , 194 , 196 ]. This process typically requires energy from ATP hydrolysis. Ubiquitin molecules can be attached singly (monoubiquitination) or in chains (polyubiquitination), forming different types of polyubiquitin chains, each potentially leading to distinct functional consequences such as protein degradation, signal transduction, or altered subcellular localization [ 197 ]. Deubiquitination is catalyzed by deubiquitinating enzymes (DUBs), which remove ubiquitin from substrate proteins by cleaving the isopeptide or thioester bonds between ubiquitin and the substrate or within ubiquitin chains. With approximately 100 DUBs in cells, they are essential for maintaining the balance of intracellular ubiquitination levels and can reverse the effects of ubiquitination, thereby regulating protein stability, activity, and localization. Ubiquitination and deubiquitination, as key forms of post-translational modifications (PTMs), play a crucial regulatory role in EndMT [ 176 , 193 , 194 ]. The processes of ubiquitination and deubiquitination during EndMT represent a critical pathogenic mechanism in cardiovascular diseases (such as atherosclerosis, pulmonary arterial hypertension, valvular diseases, and fibroelastosis), embryonic development, as well as renal fibrosis and cancer [ 5 , 156 , 161 , 163 , 164 , 183 , 195 ]. Ubiquitination involves the covalent attachment of ubiquitin (Ub) molecules to substrate proteins through a cascade of enzymes (E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes, and E3 ubiquitin ligases) [ 193 , 194 , 196 ]. This process typically requires energy from ATP hydrolysis. Ubiquitin molecules can be attached singly (monoubiquitination) or in chains (polyubiquitination), forming different types of polyubiquitin chains, each potentially leading to distinct functional consequences such as protein degradation, signal transduction, or altered subcellular localization [ 197 ]. Deubiquitination is catalyzed by deubiquitinating enzymes (DUBs), which remove ubiquitin from substrate proteins by cleaving the isopeptide or thioester bonds between ubiquitin and the substrate or within ubiquitin chains. With approximately 100 DUBs in cells, they are essential for maintaining the balance of intracellular ubiquitination levels and can reverse the effects of ubiquitination, thereby regulating protein stability, activity, and localization. Snail is a pivotal transcription factor that acts as a primary driver of EndMT by repressing endothelial genes (e.g., VE-cadherin) and activating mesenchymal programs. Its activity is directly controlled by ubiquitination. Glycogen synthase kinase-3β (GSK-3β) promotes the phosphorylation-dependent ubiquitination of Snail, targeting it for proteasomal degradation and thereby terminating the EndMT program. Conversely, deubiquitinating enzymes such as USP20 counteract this by stabilizing Snail, ensuring its nuclear retention and sustained transcriptional repression of endothelial identity, thus initiating and maintaining EndMT [ 197 – 200 ]. The Hippo pathway effector YAP (Yes-associated protein) is a potent regulator of cell growth and plasticity whose stability is regulated by ubiquitination. When the Hippo pathway is inhibited, deubiquitinating enzymes like JOSD1 stabilize YAP, promoting its nuclear translocation [ 201 ]. In the nucleus, YAP forms a complex with TEAD transcription factors, driving the expression of pro-mesenchymal and pro-fibrotic genes (e.g., CTGF, CYR61). In endothelial cells, sustained YAP activation directly induces EndMT-like phenotypes, characterized by loss of endothelial markers and gain of mesenchymal markers such as α-SMA. YAP/TEAD signaling exhibits extensive crosstalk with other EndMT pathways; for instance, TGF-β signaling can inhibit Hippo pathway components to stabilize YAP, creating a synergistic loop that amplifies the mesenchymal transition and contributes to tissue fibrosis and tumor microenvironment remodeling [ 202 , 203 ]. The PD-1/PD-L1 axis is a key immune checkpoint, and its expression is regulated by ubiquitination. E3 ubiquitin ligases such as STUB1 promote PD-L1 degradation, while deubiquitinases like USP22 enhance its stability [ 198 ]. Although PD-L1 is not a direct inducer of EndMT, its stabilization in endothelial or stromal cells fosters an immunosuppressive tumor microenvironment. This immune-privileged niche indirectly supports EndMT by enabling the survival and persistence of mesenchymal-like cells derived from EndMT, thereby linking immune evasion to fibrosis and tumor progression [ 204 – 206 ]. Furthermore, connections between the PD-1/PD-L1 axis and pro-fibrotic pathways exist; for example, YAP activation can upregulate PD-L1 expression, suggesting a coordinated mechanism whereby ubiquitination controls both immune evasion and endothelial plasticity [ 207 , 208 ]. The ubiquitination-regulated pathways of Snail, YAP, and PD-L1 do not operate in isolation but engage in extensive crosstalk, often mediated by master regulators like TGF-β. TGF-β signaling can simultaneously promote Snail stabilization, YAP nuclear translocation, and PD-L1 expression, creating a robust, self-reinforcing network that drives EndMT and fibrotic progression [ 203 , 205 , 209 ]. Understanding these interconnected ubiquitination-dependent mechanisms reveals promising therapeutic avenues. Strategies may include inhibiting the YAP/TEAD complex, targeting specific deubiquitinases (e.g., JOSD1 for YAP, USP20 for Snail), or employing dual-targeting approaches against both PD-L1 and TGF-β signaling to disrupt the immunosuppressive and pro-fibrotic microenvironment simultaneously [ 201 , 202 , 209 – 211 ]. In summary, ubiquitination and deubiquitination, as important PTMs, finely regulate the EndMT process by modulating the stability, activity, and subcellular localization of key proteins. These modifications play decisive roles in various physiological and pathological conditions, including tumor immune escape, cancer progression, cardiovascular diseases, and organ fibrosis. An in-depth understanding of the regulatory networks of ubiquitination and deubiquitination in EndMT holds promise for providing new therapeutic targets and strategies (Table  10 ). Table 10 Regulatory roles of Post-translational ubiquitination and deubiquitination in EndMT Regulatory Protein Core Function and Mechanism Potential Impact on EndMT References Snail Ubiquitination (e.g., by GSK-3β): Promotes degradation and terminates EMT. Deubiquitination (e.g., by USP20): Enhances stability and initiates EMT. Direct Driver Its stability directly determines the initiation of EndMT, acting as a core molecular “switch”. [ 197 – 200 ] PD-L1 Ubiquitination (e.g., by STUB1): Promotes degradation and enhances immunity. Deubiquitination (e.g., by USP22): Enhances stability and suppresses immunity. Indirect Influence Modulates the immune microenvironment, creating conditions of “immune escape” that facilitate EndMT. [ 198 ] PBX1 Ubiquitination: Promotes degradation and inhibits tumor progression. Deubiquitination: Enhances stability and promotes tumor progression. Indirect Driver As a transcription factor, it indirectly influences EndMT by regulating EMT-related genes. [ 212 ] Cyclin B1 Ubiquitination (e.g., by APC/C): Promotes degradation and arrests the cell cycle. Deubiquitination (e.g., by USP39): Enhances stability and drives cell cycle progression. Provides Momentum By driving cell proliferation, it supplies the foundation for cellular behavioral changes during EndMT. [ 213 , 214 ] YAP Ubiquitination (e.g., by LATS1/2): Promotes degradation and inhibits growth. Deubiquitination (e.g., by JOSD1): Enhances stability and promotes growth. Synergistic Driver As a key effector of the Hippo pathway, its activity directly promotes EndMT phenotypes such as cell growth and migration. [ 201 ] Regulatory roles of Post-translational ubiquitination and deubiquitination in EndMT Ubiquitination (e.g., by GSK-3β): Promotes degradation and terminates EMT. Deubiquitination (e.g., by USP20): Enhances stability and initiates EMT. Direct Driver Its stability directly determines the initiation of EndMT, acting as a core molecular “switch”. Ubiquitination (e.g., by STUB1): Promotes degradation and enhances immunity. Deubiquitination (e.g., by USP22): Enhances stability and suppresses immunity. Indirect Influence Modulates the immune microenvironment, creating conditions of “immune escape” that facilitate EndMT. Ubiquitination: Promotes degradation and inhibits tumor progression. Deubiquitination: Enhances stability and promotes tumor progression. Indirect Driver As a transcription factor, it indirectly influences EndMT by regulating EMT-related genes. Ubiquitination (e.g., by APC/C): Promotes degradation and arrests the cell cycle. Deubiquitination (e.g., by USP39): Enhances stability and drives cell cycle progression. Provides Momentum By driving cell proliferation, it supplies the foundation for cellular behavioral changes during EndMT. Ubiquitination (e.g., by LATS1/2): Promotes degradation and inhibits growth. Deubiquitination (e.g., by JOSD1): Enhances stability and promotes growth. Synergistic Driver As a key effector of the Hippo pathway, its activity directly promotes EndMT phenotypes such as cell growth and migration. Beyond the major modifications discussed, other PTMs provide fine-tuned regulation in EndMT [ 215 ]. SUMOylation, analogous to ubiquitination but functionally distinct, modifies target proteins by covalently attaching SUMO (Small Ubiquitin-like Modifier) proteins. Research indicates that SUMOylation of Snail is required for its maximal transcriptional repressor activity, potentially affecting its interactions with other corepressors or its nuclear localization. SUMOylation of CD155 and nuclear receptors also influences their functions; for instance, SUMOylation of Nurr1 reduces its transcriptional activity, while the Nurr1-CoREST complex can suppress pro-inflammatory gene expression [ 216 , 217 ]. SUMOylation of α-synuclein is associated with cytotoxicity and misfolding. O-GlcNAcylation involves the attachment of a single O-linked N-acetylglucosamine (O-GlcNAc) moiety to serine/threonine residues of proteins [ 218 – 220 ]. Functioning as a nutrient and stress sensor, O-GlcNAcylation is particularly prominent in diabetes-associated EndMT. Under hyperglycemic conditions, increased flux through the hexosamine biosynthesis pathway enhances the activity of O-GlcNAc transferase (OGT). OGT may modify transcription factors like Snail and Sp1, enhancing their stability and transcriptional activity, thereby linking metabolic dysregulation to EndMT activation. For example, deficiency of Angiotensin-Converting Enzyme (ACE) enhances OGT-mediated glycosylation of PSAP, subsequently regulating GPR37-dependent macrophage-nucleus pulposus cell crosstalk and TGFβ signaling to alleviate intervertebral disc degeneration. O-GlcNAcylation also plays a role in α-synuclein aggregation. Other PTMs include: Neddylation, involving the covalent attachment of the Nedd8 protein to target proteins, plays a role in activating EndMT drivers like ZEB and HIF1α.Myristoylation, the covalent attachment of myristic acid to proteins, has roles in this process, exemplified by the myristoylation of Protein Kinase C [ 125 ].Protein Tyrosine Nitration (PTN), a PTM that regulates signal transduction and inflammatory responses, is associated with neurodegenerative and cardiovascular diseases [ 221 ].Poly(ADP-ribosyl)ation, a PTM regulating protein function via poly(ADP-ribose) polymers, is often associated with DNA damage repair and DNA-interacting proteins [ 222 ]. Crosstalk between these diverse PTMs further adds layers of complexity and precision to the regulatory network, such as the phosphorylation-acetylation switch in STAT1 signaling. Understanding the intricate networks of these PTMs and their roles in EndMT is crucial for developing innovative therapeutic strategies against EndMT-related diseases like vascular fibrosis and tumor metastasis.

EndMT, as a core pathological process linking multiple major diseases, is regulated by mechanisms far beyond the traditional linear signaling pathway model. This review systematically elaborates on the complex regulatory network formed by epigenetic modifications and post-translational modifications in this process. At the epigenetic level, DNA methylation, histone modifications, and non-coding RNAs dynamically regulate chromatin structure and gene expression through synergistic actions, determining the expression profile of EndMT-related genes. Specifically, DNA methylation precisely controls gene promoter activity via a “writer-reader-eraser” mechanism; histone modifications alter chromatin states through various means like acetylation, methylation, and lactylation; and non-coding RNAs form intricate regulatory networks through mechanisms such as ceRNA and epigenetic regulation. These epigenetic mechanisms collectively shape the epigenetic landscape of EndMT. At the post-translational modification level, various modifications including phosphorylation, acetylation, and ubiquitination precisely regulate protein activity, stability, and subcellular localization, thereby influencing the function of key signaling pathways and transcription factors. These different regulatory layers do not exist in isolation but are interconnected through metabolic reprogramming and the dual functions of enzymes, forming tight cross-talk. Metabolites like Acetyl-CoA and SAM serve as critical bridges linking cellular metabolic status to the epigenetic and PTM networks. Enzymes with multiple substrate specificities, such as p300/CBP and EZH2, act as important molecular hubs integrating different regulatory tiers. This regulatory network, through the establishment of positive feedback loops, endows the EndMT phenotype with a stable “cellular memory,” which partially explains the irreversibility of conditions like fibrosis. Research on EndMT still faces many challenges and opportunities. At the mechanistic level, emerging technologies like single-cell multi-omics and spatial transcriptomics are needed to deeply resolve the dynamic changes of different modification types during EndMT and their complex “crosstalk” mechanisms. Special attention should be paid to how the metabolic microenvironment influences the balance of the epigenetic and PTM networks by modulating metabolite levels. Furthermore, developing specific therapeutic drugs based on identified key regulatory factors is an important direction, such as inhibitors targeting DNMTs, HDACs, EZH2, and compounds modulating the activity of specific E3 ligases. Concurrently, identifying specific epigenetic marks and PTM signatures in blood or tissues holds promise for providing novel diagnostic and prognostic biomarkers for related diseases.

The activation of EndMT is a complex and highly regulated process that relies not only on the coordinated action of transcription factors and signaling pathways but also profoundly on intricate interactions between cellular metabolism, epigenetic modifications, and PTMs [ 223 , 224 ]. These regulatory mechanisms do not operate in isolation; rather, they interact across multiple layers to collectively determine cell fate and disease progression [ 225 , 226 ]. Cellular metabolites play a central role in connecting epigenetic modifications and PTMs [ 227 ]. The cofactors for many epigenetic marks and PTMs are themselves key intermediates of cellular metabolism [ 46 , 228 ], including Acetyl-CoA, S-adenosylmethionine (SAM), and ATP levels. Acetyl-CoA is an essential substrate for the acetylation of both histones and non-histone proteins (e.g., transcription factors) [ 46 , 229 ]. Acetylation generally promotes gene transcription by loosening chromatin structure (e.g., acetylation of histones H3 and H4) and can enhance the DNA-binding capacity and transcriptional activity of transcription factors (like Smad proteins), thereby cooperatively activating the mesenchymal gene program [ 229 – 231 ]. During EndMT activation, cells often undergo metabolic reprogramming, such as a shift towards glycolysis. This alteration increases the abundance of Acetyl-CoA, consequently impacting both histone hyperacetylation and the acetylation of pro-EndMT transcription factors [ 68 ]. SAM is the universal methyl donor for DNA and histone methylation [ 232 ]. DNA methylation is typically associated with gene silencing, and histone methylation (e.g., H3K27me3) also regulates gene expression by altering chromatin structure [ 45 , 233 ]. Fluctuations in SAM levels directly influence the occurrence of these methylation events.ATP levels directly affect kinase activity, thereby regulating protein phosphorylation [ 234 , 235 ]. During EndMT activation, metabolic reprogramming, such as the Warburg effect (a preference for glycolysis over oxidative phosphorylation for energy production), often occurs [ 68 ]. This shift in metabolic state directly alters the intracellular abundance of Acetyl-CoA, SAM, and ATP, thereby simultaneously influencing the status of both epigenetic modifications and PTMs. For instance, under high glucose or hypoxic conditions, elevated Acetyl-CoA can drive both histone hyperacetylation and the acetylation of pro-EndMT transcription factors (like Smads), synergistically activating the mesenchymal gene program from two levels. Thus, metabolites act as a link connecting extracellular microenvironmental signals with the intracellular epigenetic/PTM regulatory network, enabling the cell to coordinately adjust its gene expression program and protein functions based on energy and material status, thereby deciding whether to initiate EndMT. Many regulatory enzymes themselves possess dual or multiple substrate specificities, enabling them to catalyze both epigenetic modifications and PTMs, thus serving as direct molecular hubs connecting these two regulatory layers. CBP/p300, these coactivators, possessing histone acetyltransferase (HAT) activity, are a prime example [ 46 , 229 ]. They can not only acetylate histones H3 and H4 to relax chromatin structure and facilitate transcription but also directly acetylate key EndMT transcription factors like Smad2/3 and Snail, enhancing their DNA-binding ability and transcriptional activity. This means that activation of p300/CBP can powerfully drive the expression of pro-EndMT genes from two directions simultaneously: “opening chromatin” and “activating the transcriptional machinery.” EZH2, as the core catalytic subunit of the Polycomb Repressive Complex 2 (PRC2), EZH2 traditionally silences endothelial genes by catalyzing the trimethylation of histone H3 lysine 27 (H3K27me3) [ 125 , 236 ]. However, research has found that EZH2 also possesses non-canonical functions; it can directly methylate non-histone substrates, such as the transcription factor STAT3 [ 237 ]. Methylation of STAT3 enhances its tyrosine phosphorylation and transcriptional activity, subsequently activating another set of gene programs promoting cell proliferation and survival, working in concert with the canonical H3K27me3-dependent pathway to drive EndMT progression [ 237 ]. EZH2 functions within complex signaling networks, its activity is regulated by various factors, and it interacts with other transcription factors and epigenetic modifying enzymes. This dual role of such enzymes greatly enhances the complexity and integration of the regulatory network. Epigenetic modifications and PTMs collaborate to establish and stabilize the cellular phenotype of EndMT through the formation of positive feedback loops, enabling its maintenance even in the absence of the initial inducing signal [ 238 ]. This mechanism explains the “cellular memory” of EndMT [ 239 ]. A classic loop pattern is: Transcription factors regulated by PTMs can directly regulate the expression of epigenetic modifying enzymes or recruit them to specific gene loci. For example, in the TGF-β signaling pathway, Smad complexes activated by phosphorylation and acetylation, along with Snail protein entering the nucleus, not only directly activate target genes but also recruit histone deacetylases (e.g., HDAC1) and histone methyltransferases (e.g., EZH2) to the promoter regions of endothelial-specific genes like CDH5 (encoding VE-cadherin) [ 135 , 240 , 241 ]. HDACs and EZH2 subsequently remove histone acetylation marks and deposit the repressive H3K27me3 mark at these sites, thereby silencing these genes in a more stable and long-lasting manner. The establishment of this silenced state further reduces the expression of endothelial identity genes, consolidating the mesenchymal phenotype. The stable mesenchymal phenotype, in turn, may sustain the activity of transcription factors and the expression of epigenetic modifying enzymes through continuous signal output, forming a self-reinforcing cycle. Epigenetic regulation in cancer cells involves the concerted action of “writers,” “erasers,” and “readers,” affecting processes like EMT and metastasis [ 239 ]. This positive feedback loop, initiated by PTMs and solidified by epigenetic modifications, ensures that the EndMT phenotype can be long-“remembered” and resistant to external signals attempting to reverse it. This is also a key reason why diseases like fibrosis are often progressive and difficult to reverse. In summary, the complex interactions between cellular metabolism, epigenetic modifications, and protein PTMs form the core of the EndMT regulatory network. Metabolites act as bridges connecting extracellular environmental signals to the intracellular regulatory network, while dual-function enzymes directly integrate epigenetic and protein PTM signals. Through positive feedback loops, these mechanisms collectively establish a stable EndMT cellular phenotype, enabling cells to maintain their mesenchymal characteristics in the face of a changing microenvironment, which holds significant implications for disease pathogenesis and progression.

Cardiovascular diseases, organ fibrosis, and tumor metastasis represent major global health threats. Endothelial-mesenchymal transition (EndMT) is a differentiation process in which endothelial cells lose their specific characteristics and acquire mesenchymal features. It is closely associated with the development of various pathological conditions, including atherosclerotic plaque formation, ischemic heart disease, pulmonary arterial hypertension, and diabetes [ 1 – 4 ]. EndMT, a specialized form of epithelial-mesenchymal transition (EMT), is characterized by phenotypic remodeling of endothelial cells. This involves the loss of specific markers such as VE-cadherin and the gain of mesenchymal markers including α-smooth muscle actin, fibronectin, and N-cadherin [ 5 ]. This transition leads to the dissolution of cell junctions, reorganization of the cytoskeleton, and confers enhanced migratory and invasive capabilities to the cells [ 6 , 7 ]. Traditionally, research on EndMT regulatory mechanisms has primarily focused on extracellular signaling molecules (e.g., the TGF-β superfamily), downstream signaling pathways (e.g., Smad, MAPK, PI3K/Akt), and key transcription factors (e.g., Snail, Slug, Twist, ZEB family) [ 8 , 9 ]. However, this linear regulatory network falls short in fully explaining the persistence, reversibility, and precise signal integration observed during EndMT. Epigenetic modifications (such as DNA methylation, histone modifications, and non-coding RNA-mediated regulation) and post-translational modifications (PTMs, including phosphorylation, acetylation, and ubiquitination) play crucial roles in regulating gene transcription and protein function. Together, they determine the “initiation,” “maintenance,” and “termination” of the EndMT program [ 1 ]. By systematically reviewing recent advances in the roles of epigenetic and post-translational modifications in EndMT, we can achieve a more comprehensive understanding of its underlying mechanisms. This endeavor holds promise for developing novel diagnostic biomarkers and targeted therapeutic strategies for fibrosis, cancer, and vascular diseases [ 10 ]. The transforming growth factor-beta (TGF-β)/Smad, Wnt/β-catenin, and Notch signaling pathways are among the most critical pathways that orchestrate EndMT. These pathways do not operate in isolation; their activities are profoundly influenced and fine-tuned by epigenetic and post-translational modifications, which ultimately determine the transcriptional output of EndMT [ 11 ]. Transcriptional regulatory networks are pivotal in the initiation and execution of EndMT. Snail family members SNAI1 and SNAI2 function as primary “master switches.” Zinc finger E-box-binding homeobox (ZEB) proteins ZEB1 and ZEB2 facilitate the transition to a mesenchymal state by repressing epithelial markers. Epigenetic and post-transcriptional fine-tuning also play key roles in EndMT; histone modifications and non-coding RNAs can alter the chromatin accessibility at EndMT-related gene loci, thereby promoting or inhibiting their expression. In summary, EndMT is a complex biological process meticulously regulated by multiple signaling pathways, transcription factors, as well as epigenetic and post-transcriptional networks. In-depth investigation into the molecular mechanisms and regulatory pathways of EndMT is crucial for developing novel therapeutic approaches for related diseases (Fig. 1 ). Fig. 1 Research Progress on Epigenetic Modifications and Post-translational Modifications in EndMT Research Progress on Epigenetic Modifications and Post-translational Modifications in EndMT