GO:1905064 negative regulation of vascular associated smooth muscle cell differentiation: Mechanism, Genes, Functions and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:1905064 describes any process that stops, prevents or reduces the frequency, rate or extent of vascular smooth muscle cell (VSMC) differentiation.
• VSMC differentiation is controlled by a network of transcription factors, including myocardin, Runx2, and SRF, which are modulated by signaling pathways such as Wnt, BMP, and PI3K/Akt.
• Negative regulators of VSMC differentiation are critical in vascular pathology: their dysregulation contributes to atherosclerosis, vascular calcification, and aortic aneurysm.
• Key negative regulators include NONO, Cdon, GDF10, GDF11, RIP2, and TWIST1, which suppress pro-differentiation or pro-calcification programs.
• Experimental approaches to study GO:1905064 include CRISPR knockout, point mutation, knock-in, and overexpression models, combined with RNA-seq, proteomics, and imaging.
• Understanding this process offers therapeutic targets for diseases characterized by VSMC phenotype switching, such as atherosclerosis and vascular calcification.
Description
Vascular smooth muscle cells (VSMCs) are the predominant cell type in the arterial media, where they maintain vascular tone and structural integrity. Under physiological conditions, VSMCs exhibit a differentiated, contractile phenotype characterized by high expression of contractile proteins such as ACTA2, MYH11, and CNN1. However, in response to injury or pathological stimuli, VSMCs can undergo phenotype switching toward a synthetic, proliferative, or osteogenic state, a process that is normally restrained by negative regulatory mechanisms. The Gene Ontology term GO:1905064, negative regulation of vascular associated smooth muscle cell differentiation, captures the biological processes that inhibit or reduce the rate of VSMC differentiation. This term is essential for understanding how vascular cells maintain homeostasis and how their dysregulation contributes to vascular disease. Recent studies have identified multiple negative regulators of VSMC differentiation, including NONO, Cdon, GDF10, GDF11, RIP2, and TWIST1, which act through diverse signaling pathways to suppress contractile gene expression or promote alternative phenotypes. These findings highlight the importance of GO:1905064 in vascular biology and disease. This article provides a comprehensive overview of the mechanisms, key genes, and research methods associated with GO:1905064, based on authoritative QuickGO data and verified PubMed literature.
negative regulation of vascular associated smooth muscle cell differentiation At A Glance
| GO ID | GO:1905064 |
|---|---|
| GO term | negative regulation of vascular associated smooth muscle cell differentiation |
| Ontology | biological_process |
| Synonym | down regulation of VSMC differentiation; inhibition of vascular smooth muscle cell differentiation |
| Major function | Suppression of the differentiation program that drives VSMCs toward a contractile phenotype |
| Related processes | Vascular calcification, atherosclerosis, aortic aneurysm, VSMC phenotype switching |
| Key regulators | NONO, Cdon, GDF10, GDF11, RIP2, TWIST1, myocardin, Runx2 |
| Research methods | CRISPR knockout, point mutation, knock-in, overexpression, RNA-seq, proteomics, imaging |
What Is GO:1905064?
GO:1905064, negative regulation of vascular associated smooth muscle cell differentiation, is defined as any process that stops, prevents, or reduces the frequency, rate, or extent of vascular smooth muscle cell differentiation. In other words, it encompasses molecular events that inhibit the transition of vascular smooth muscle cells from a synthetic or proliferative state to a differentiated, contractile phenotype. This regulation is crucial for maintaining vascular homeostasis and preventing pathological remodeling.
Why Is negative regulation of vascular associated smooth muscle cell differentiation Important in Cell Biology?
GO:1905064 is important because the balance between differentiated and dedifferentiated VSMC states is a central determinant of vascular health. Negative regulation of VSMC differentiation prevents excessive or premature differentiation, but when this regulation is disrupted, VSMCs can undergo pathological phenotype switching, leading to diseases such as atherosclerosis, vascular calcification, and aortic aneurysm. Understanding the molecular players and mechanisms of GO:1905064 can reveal therapeutic targets for these prevalent cardiovascular conditions.
• Maintains vascular homeostasis by preventing inappropriate VSMC differentiation.
• Dysregulation contributes to vascular calcification, a common complication of chronic kidney disease and aging.
• Implicated in atherosclerosis, where VSMC phenotype switching promotes plaque instability.
• Linked to aortic aneurysm formation through GDF11-mediated regulation.
• Provides potential therapeutic targets for modulating VSMC plasticity.
• Key for understanding developmental vascular biology and adult vascular repair.
• Involved in response to rapamycin and other mTOR inhibitors that promote VSMC differentiation.
• Relevant to hypertension and vascular remodeling through RIP2-myocardin signaling.
• Offers insights into sex-specific differences in VSMC phenotype regulation.
• Enables development of precision medicine approaches targeting VSMC differentiation pathways.
What Happens During negative regulation of vascular associated smooth muscle cell differentiation?
Initiation by extracellular cues
In simple terms: Signals from outside the cell start the process that stops VSMCs from becoming fully differentiated.
Negative regulation of VSMC differentiation is often initiated by extracellular ligands such as GDF10 and GDF11, which belong to the TGF-beta superfamily. GDF10 acts as a negative regulator of vascular calcification by inhibiting VSMC osteogenic differentiation. Similarly, GDF11 regulates VSMC phenotype switching to prevent aortic aneurysm formation, partly by suppressing differentiation. These ligands bind to cell surface receptors and trigger intracellular signaling cascades that ultimately inhibit the expression of contractile genes.
Intracellular signaling cascades
In simple terms: Inside the cell, a series of molecular switches relay the signal to the nucleus.
Upon receptor activation, pathways such as Wnt/Runx2 and PI3K/Akt are modulated. Cdon suppresses vascular smooth muscle calcification via repression of the Wnt/Runx2 axis, thereby inhibiting osteogenic differentiation. Rapamycin promotes VSMC differentiation through insulin receptor substrate-1/phosphatidylinositol 3-kinase/Akt2 feedback signaling, indicating that PI3K/Akt signaling can negatively regulate differentiation. RIP2 knockdown attenuates VSMC activation via negative regulation of myocardin expression, linking innate immune signaling to VSMC differentiation.
Transcriptional control
In simple terms: In the nucleus, transcription factors turn off genes that make VSMCs contractile.
The transcription factor myocardin is a master regulator of VSMC differentiation, and its suppression leads to reduced contractile gene expression. RIP2 negatively regulates myocardin expression, thereby inhibiting VSMC differentiation. TWIST1 domains regulate smooth muscle cell phenotype, with specific domains mediating repression of contractile genes. Additionally, NONO attenuates vascular calcification by inhibiting bone morphogenetic protein 2 (BMP2) transcription, which is a pro-osteogenic factor. Runx2 is a key transcription factor promoting osteogenic differentiation, and its repression by Cdon prevents calcification.
Epigenetic and post-transcriptional regulation
In simple terms: Chemical tags on DNA and RNA can also lock genes in the off position.
Emerging evidence suggests that epigenetic modifications and non-coding RNAs contribute to the negative regulation of VSMC differentiation. Although specific mechanisms are still being elucidated, the paraspeckle protein NONO has been shown to interact with RNA and regulate transcription. Further research is needed to fully understand how epigenetic changes stabilize the differentiated or dedifferentiated states.
Outcome: suppression of contractile phenotype
In simple terms: The end result is that VSMCs do not become fully contractile and may adopt other fates.
The integrated effect of these negative regulatory mechanisms is a reduction in the expression of VSMC contractile markers such as ACTA2, MYH11, and CNN1, and an increase in synthetic or osteogenic markers. This phenotype switching is a hallmark of vascular diseases including atherosclerosis and calcification. Understanding these outcomes is essential for developing therapies that can modulate VSMC plasticity.
Key Genes Involved in GO:1905064 negative regulation of vascular associated smooth muscle cell differentiation
The following genes and proteins have been experimentally implicated in the negative regulation of vascular associated smooth muscle cell differentiation (GO:1905064) or in related VSMC phenotype switching.
| Gene | Major Role | Research Relevance |
|---|---|---|
| NONO | Paraspeckle protein; inhibits BMP2 transcription | Attenuates vascular calcification by suppressing osteogenic differentiation |
| Cdon | Cell surface receptor; represses Wnt/Runx2 axis | Suppresses VSMC calcification and osteogenic differentiation |
| TWIST1 | Transcription factor; regulates SMC phenotype | Domains regulate smooth muscle cell differentiation and phenotype |
| GDF11 | TGF-beta superfamily ligand | Regulates VSMC phenotype switching to prevent aortic aneurysm |
| GDF10 | TGF-beta superfamily ligand | Negative regulator of vascular calcification |
| RIP2 | Kinase; negatively regulates myocardin | Knockdown attenuates VSMC activation |
| Myocardin | Transcriptional coactivator; master regulator of VSMC differentiation | Target of negative regulation; suppression inhibits differentiation |
| Runx2 | Transcription factor; promotes osteogenic differentiation | Repressed by Cdon to inhibit calcification |
| BMP2 | Bone morphogenetic protein; pro-osteogenic | Transcription inhibited by NONO |
| ACTA2 | Smooth muscle alpha-actin; contractile marker | Downregulated during negative regulation of differentiation |
| MYH11 | Smooth muscle myosin heavy chain; contractile marker | Downregulated during phenotype switching |
| CNN1 | Calponin; contractile marker | Downregulated during dedifferentiation |
| SRF | Serum response factor; cooperates with myocardin | Involved in contractile gene expression |
| Akt2 | Kinase; PI3K/Akt pathway | Rapamycin promotes differentiation via IRS-1/PI3K/Akt2 feedback |
| IRS-1 | Insulin receptor substrate-1 | Mediates rapamycin-induced VSMC differentiation |
| mTOR | Kinase; central regulator of growth | Rapamycin inhibits mTOR to promote VSMC differentiation |
| Wnt | Secreted signaling proteins | Wnt/Runx2 axis repressed by Cdon |
How Is negative regulation of vascular associated smooth muscle cell differentiation Regulated?
The negative regulation of VSMC differentiation is itself tightly regulated by multiple signaling pathways. The mTOR pathway plays a key role: rapamycin, an mTOR inhibitor, promotes VSMC differentiation through insulin receptor substrate-1/phosphatidylinositol 3-kinase/Akt2 feedback signaling. This indicates that mTOR activity normally suppresses differentiation, and its inhibition relieves this block. Additionally, the Wnt/Runx2 axis is a target of negative regulation by Cdon; when Cdon is present, it represses Wnt signaling, leading to decreased Runx2 activity and inhibition of osteogenic differentiation. GDF11 and GDF10, members of the TGF-beta superfamily, also modulate VSMC phenotype through SMAD-dependent and independent pathways. RIP2 negatively regulates myocardin expression, thereby linking innate immune signaling to VSMC differentiation. These regulatory layers ensure that VSMC differentiation is precisely controlled in response to environmental cues.
negative regulation of vascular associated smooth muscle cell differentiation and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| NONO | Vascular calcification | KO and overexpression in VSMCs; calcification assays |
| Cdon | Vascular calcification | KO and overexpression; Wnt/Runx2 reporter assays |
| GDF11 | Aortic aneurysm | KO and knock-in; aneurysm mouse models |
| GDF10 | Vascular calcification | KO and overexpression; calcification assays |
| RIP2 | Hypertension, atherosclerosis | KO and knockdown; VSMC activation assays |
Vascular calcification
Vascular calcification is characterized by the deposition of calcium phosphate crystals in the arterial wall, often accompanied by osteogenic differentiation of VSMCs. Negative regulators of VSMC differentiation, such as NONO and Cdon, protect against calcification by inhibiting BMP2 transcription and repressing the Wnt/Runx2 axis, respectively. GDF10 also acts as a negative regulator of vascular calcification. Loss of these protective mechanisms contributes to calcification, a common complication in chronic kidney disease, diabetes, and aging.
Aortic aneurysm
Aortic aneurysm formation is associated with VSMC phenotype switching and loss of contractile function. GDF11 regulates VSMC phenotype switching to prevent aortic aneurysm formation, suggesting that negative regulation of differentiation may be protective in this context. Dysregulation of GDF11 or its downstream effectors could lead to weakened vascular walls and aneurysm progression.
Atherosclerosis
Atherosclerosis involves the accumulation of plaques in arteries, where VSMCs undergo phenotype switching from contractile to synthetic states. This switching is influenced by negative regulators of differentiation. For example, TWIST1 domains regulate smooth muscle cell phenotype, and their dysregulation may contribute to plaque instability. RIP2 knockdown attenuates VSMC activation via negative regulation of myocardin, linking inflammation to VSMC phenotype in atherosclerosis.
Hypertension and vascular remodeling
Hypertension is associated with vascular remodeling, in which VSMCs dedifferentiate and proliferate. RIP2 negatively regulates myocardin expression, and its knockdown attenuates VSMC activation, suggesting that RIP2 may contribute to pathological remodeling in hypertension. Understanding these mechanisms could lead to new antihypertensive therapies targeting VSMC differentiation.
From negative regulation of vascular associated smooth muscle cell differentiation-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does loss of NONO increase VSMC calcification? | NONO knockout VSMCs or mice, followed by calcification assays |
| Does Cdon overexpression suppress Wnt/Runx2 signaling? | Cdon overexpression in VSMCs, Wnt reporter assays |
| What domains of TWIST1 regulate SMC phenotype? | TWIST1 domain deletion mutants in SMCs |
| Does GDF11 gain-of-function prevent aneurysm? | GDF11 knock-in or overexpression in mouse models |
| Is RIP2 required for myocardin suppression? | RIP2 knockout or knockdown VSMCs, myocardin expression analysis |
| Does rapamycin promote differentiation via Akt2? | Akt2 knockout VSMCs treated with rapamycin |
How to Study the negative regulation of vascular associated smooth muscle cell differentiation Process
| Method | What It Measures | Typical Application |
|---|---|---|
| RNA-seq | Global gene expression changes | Identify contractile vs. synthetic markers after gene manipulation |
| Proteomics | Protein abundance and modifications | Study signaling pathways (e.g., Akt, RIP2) |
| Immunofluorescence | Protein localization and expression | Visualize VSMC phenotype in tissues |
| Alizarin red staining | Calcium deposition | Quantify vascular calcification in vitro |
| Western blot | Protein levels and phosphorylation | Validate knockout or overexpression effects |
| Luciferase reporter assay | Transcriptional activity | Measure Runx2 or myocardin promoter activity |
| EdU incorporation | Cell proliferation | Assess synthetic VSMC phenotype |
| Chromatin immunoprecipitation (ChIP) | Protein-DNA interactions | Determine transcription factor binding (e.g., NONO, TWIST1) |
Transcriptomic analysis (RNA-seq)
RNA sequencing can quantify changes in contractile gene expression (e.g., ACTA2, MYH11, CNN1) and identify global shifts in VSMC phenotype upon manipulation of negative regulators. For example, RNA-seq of NONO knockout VSMCs revealed upregulation of osteogenic markers. This method is essential for unbiased discovery of pathways affected by GO:1905064 regulators.
Proteomics and phosphoproteomics
Mass spectrometry-based proteomics can measure protein abundance and post-translational modifications, such as phosphorylation, in response to signaling changes. This is particularly useful for studying pathways like PI3K/Akt, where rapamycin-induced differentiation involves Akt2 phosphorylation. Phosphoproteomics can identify downstream targets of kinases like RIP2.
Imaging and immunohistochemistry
Immunofluorescence and immunohistochemistry can visualize the expression and localization of contractile proteins (e.g., ACTA2) and osteogenic markers (e.g., Runx2) in VSMCs and tissue sections. This approach has been used to confirm VSMC phenotype switching in aortic aneurysm models and calcification studies.
Functional assays for calcification and proliferation
Alizarin red staining and calcium quantification are standard for assessing vascular calcification in vitro and in vivo. Proliferation and migration assays (e.g., EdU incorporation, scratch wound) can evaluate the synthetic phenotype. These functional readouts directly link molecular changes to disease-relevant outcomes.
How CRISPR Can Be Used to Study GO:1905064 negative regulation of vascular associated smooth muscle cell differentiation
Knockout
CRISPR knockout of negative regulators such as NONO, Cdon, or RIP2 can reveal their role in suppressing VSMC differentiation. For example, NONO knockout increases BMP2 transcription and promotes calcification. Cdon knockout enhances Wnt/Runx2 signaling and osteogenic differentiation. RIP2 knockout attenuates VSMC activation by increasing myocardin expression. These models are invaluable for establishing causality.
Point Mutation
Point mutations can dissect specific domains or phosphorylation sites. For instance, mutating TWIST1 domains can identify which regions are required for regulating smooth muscle cell phenotype. Similarly, point mutations in Akt2 can test its role in rapamycin-induced differentiation. Such models provide mechanistic insights beyond simple knockout.
Knock-in
Knock-in of tagged or reporter alleles allows real-time monitoring of gene expression and protein localization. For example, a GFP knock-in at the ACTA2 locus can track VSMC differentiation in live cells. Knock-in of disease-associated mutations in GDF11 or GDF10 can model their effects on VSMC phenotype. These models are powerful for studying dynamic processes.
Overexpression
Overexpression of negative regulators can suppress VSMC differentiation and prevent calcification. For example, Cdon overexpression represses Wnt/Runx2 and inhibits calcification. GDF10 overexpression reduces vascular calcification. Overexpression models are useful for gain-of-function studies and for validating therapeutic targets.
How EDITGENE Supports negative regulation of vascular associated smooth muscle cell differentiation Research
Researchers studying negative regulation of vascular associated smooth muscle cell differentiation-related genes often need to determine whether a candidate gene is causally involved in suppressing or promoting VSMC phenotype switching. This requires precise genetic manipulation, such as knockout, point mutation, knock-in, or overexpression, followed by functional assays. EDITGENE provides a comprehensive suite of CRISPR-based services to accelerate such investigations.
Contact EDITGENE today to design your custom CRISPR model for negative regulation of vascular associated smooth muscle cell differentiation research.
Frequently Asked Questions About negative regulation of vascular associated smooth muscle cell differentiation
What is GO:1905064?
GO:1905064 is a Gene Ontology term for negative regulation of vascular associated smooth muscle cell differentiation, describing any process that stops, prevents, or reduces the rate of VSMC differentiation.
What genes are involved in negative regulation of VSMC differentiation?
Key genes include NONO, Cdon, GDF10, GDF11, RIP2, TWIST1, myocardin, and Runx2, among others.
How does NONO regulate vascular calcification?
NONO attenuates vascular calcification by inhibiting bone morphogenetic protein 2 (BMP2) transcription, thereby suppressing osteogenic differentiation of VSMCs.
What is the role of Cdon in VSMC calcification?
Cdon suppresses vascular smooth muscle calcification via repression of the Wnt/Runx2 axis, inhibiting osteogenic differentiation.
How does GDF11 affect aortic aneurysm?
GDF11 regulates VSMC phenotype switching to prevent aortic aneurysm formation, likely by promoting a contractile phenotype.
What is the function of RIP2 in VSMC activation?
RIP2 knockdown attenuates VSMC activation via negative regulation of myocardin expression, linking innate immune signaling to VSMC phenotype.
How does rapamycin affect VSMC differentiation?
Rapamycin promotes VSMC differentiation through insulin receptor substrate-1/phosphatidylinositol 3-kinase/Akt2 feedback signaling.
What experimental models are used to study GO:1905064?
Common models include CRISPR knockout, point mutation, knock-in, and overexpression in VSMCs, combined with RNA-seq, proteomics, and functional assays.
Why is negative regulation of VSMC differentiation important?
It maintains vascular homeostasis and prevents pathological phenotype switching that leads to calcification, atherosclerosis, and aneurysm.
What diseases are associated with dysregulated VSMC differentiation?
Vascular calcification, atherosclerosis, aortic aneurysm, and hypertension are associated with dysregulated VSMC differentiation.
Conclusion
GO:1905064, negative regulation of vascular associated smooth muscle cell differentiation, is a critical biological process that maintains vascular homeostasis by preventing inappropriate VSMC differentiation. Key regulators such as NONO, Cdon, GDF10, GDF11, RIP2, and TWIST1 modulate this process through diverse signaling pathways, and their dysregulation contributes to vascular calcification, atherosclerosis, and aortic aneurysm. Understanding the molecular mechanisms and employing advanced CRISPR models are essential for developing targeted therapies. EDITGENE provides comprehensive services to support research in this field, from knockout and knock-in models to library screening and bioinformatics.
References
- 2. Lu Y et al.. 2024. Paraspeckle protein NONO attenuates vascular calcification by inhibiting bone morphogenetic protein 2 transcription.. Kidney Int 105(6):1221-1238 PMID: 38417578
- 3. Ahn BY et al.. 2023. Cdon suppresses vascular smooth muscle calcification via repression of the Wnt/Runx2 Axis.. Exp Mol Med 55(1):120-131 PMID: 36609601
- 4. Dy DCM et al.. 2025. Functional analysis of TWIST1 domains regulating smooth muscle cell phenotype.. Front Cardiovasc Med 12:1659847 PMID: 41246007
- 5. Su X et al.. 2026. GDF11 Regulates Vascular Smooth Muscle Cell Phenotype Switching to Prevent Aortic Aneurysm Formation.. Cardiovasc Drugs Ther 40(3):883-895 PMID: 41240221
- 6. Platko K et al.. 2024. GDF10 is a negative regulator of vascular calcification.. J Biol Chem 300(11):107805 PMID: 39307303
- 7. Zhang L et al.. 2022. RIP2 Knockdown Attenuates Vascular Smooth Muscle Cells Activation via Negative Regulating Myocardin Expression.. Am J Hypertens 35(5):454-461 PMID: 35099539
- 8. Martin KA et al.. 2007. Rapamycin promotes vascular smooth muscle cell differentiation through insulin receptor substrate-1/phosphatidylinositol 3-kinase/Akt2 feedback signaling.. J Biol Chem 282(49):36112-20 PMID: 17908691
