Notch in Fibrosis: EMT, Angiogenesis & Tissue Repair
Notch signaling controls EMT, angiogenesis, and macrophage polarization in fibrosis. Preclinical data on γ-secretase inhibitors and anti-Notch antibodies.
Introduction: Why Is Notch Involved in Fibrosis?
The Notch signaling pathway is one of the most evolutionarily conserved pathways, controlling cell fate decisions and tissue homeostasis from development through adulthood. Recent studies have revealed that this pathway also plays a central role in fibrosis.
In particular, Notch is deeply involved in three key fibrotic processes: epithelial-mesenchymal transition (EMT), angiogenesis, and macrophage polarization. Like Wnt/β-catenin and Hedgehog, Notch is a prime example of "developmental pathways reactivated in pathological contexts."
This article covers the molecular mechanism of Notch signaling, its role in pulmonary, hepatic, and renal fibrosis, and therapeutic strategies including γ-secretase inhibitors.
1. Molecular Mechanism of Notch Signaling
Notch Family and Ligands
Mammals have four Notch receptors and five ligands:
Notch receptors (single-pass transmembrane):
- Notch1, Notch2, Notch3, Notch4
Ligands (membrane-bound):
- Delta-like (DLL1, DLL3, DLL4): Dll4 is especially important in vascular endothelium
- Jagged (JAG1, JAG2): Jagged1 is expressed in many tissues
Because both receptors and ligands are membrane proteins, Notch signaling requires direct cell-cell contact (juxtacrine).
Activation Steps: S2/S3 Cleavage and Nuclear Translocation
The defining feature of Notch signaling is signal transduction via two-step proteolytic cleavage.
- Ligand-receptor binding: Ligand (Jagged/Dll) on an adjacent cell binds the Notch receptor extracellular domain
- S2 cleavage: ADAM10/17 (TACE) metalloprotease cleaves the extracellular domain
- S3 cleavage: γ-secretase complex (PSEN1/2, Nicastrin, APH-1, PEN-2) performs intramembrane cleavage, releasing the Notch intracellular domain (NICD)
- Nuclear translocation: NICD translocates to the nucleus and binds transcription factor CSL (RBP-Jκ)
- Transcriptional activation: Mastermind (MAML) and other coactivators are recruited to induce target genes including HES1, HES5, HEY1, HEY2
- Signal termination: NICD is degraded via ubiquitination (self-limiting)
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2. Notch and Epithelial-Mesenchymal Transition (EMT)
EMT Promotion Mechanisms
Notch signaling promotes EMT through multiple pathways:
- Induction of Snail and Slug: Notch directly upregulates Snail1/2 (SNAI1/2), transcription factors that suppress epithelial markers
- E-cadherin suppression: The Notch-HES/HEY pathway suppresses E-cadherin, disrupting epithelial polarity
- Synergy with TGF-β: The TGF-β pathway and Notch cooperate on EMT gene promoters
EMT in Pulmonary Fibrosis (IPF)
In IPF alveolar epithelial cells, Jagged1-Notch1 signaling is activated, driving Snail induction → EMT → myofibroblast accumulation. γ-secretase inhibitor administration alleviates pulmonary fibrosis in the bleomycin model.
3. Notch and Angiogenesis: Dll4/Notch1 Tip/Stalk Regulation
Aberrant Angiogenesis in Tumor and Fibrotic Tissue
Fibrotic tissues exhibit an "angiogenic paradox" where abnormal angiogenesis fails to relieve hypoxia and actually promotes fibrosis. Notch precisely regulates this angiogenesis.
- Tip vs. Stalk cells: Vascular endothelial tip cells (leading cells) strongly express Dll4 → activate Notch1 in adjacent stalk cells → stalk cells downregulate VEGFR2 and focus on division/elongation
- Disruption of Dll4-Notch1 balance: In tumors and fibrosis, disruption of this balance forms dysfunctional immature vessels
Notch3 and Vascular Pericytes
Notch3 is highly expressed in vascular pericytes and smooth muscle cells. In chronic kidney injury, Notch3 activation drives pericyte-to-myofibroblast transition (important mechanism in renal fibrosis).
4. Notch and Macrophage Polarization
The M1 vs M2 Seesaw
Notch signaling regulates macrophage polarization:
- M1 promotion: Notch1 activation induces inflammatory M1 macrophages (acute inflammation)
- M2 control: In some contexts, Notch2 regulates M2-like macrophages (pro-fibrotic)
This regulation depends on tissue/disease context, so simple "Notch inhibition = antifibrotic" is not sufficient; organ- and time-specific strategies are needed. See Fundamentals of Macrophage Polarization for details.
5. Involvement in Organ-Specific Fibrosis
Liver Fibrosis
- Ductular reaction: Notch is essential (Jagged1/Notch2) for ductular reaction seen in chronic liver injury
- MASH: Correlations between ductular reaction and Notch have been reported in MASH progression
- Hepatic stellate cell activation: Notch3 promotes myofibroblast differentiation
Renal Fibrosis
- Tubulointerstitial fibrosis: Notch1/Notch2 promote EMT-like changes in tubular epithelium
- Podocyte injury: Notch activation induces podocyte dysfunction in diabetic nephropathy
- Pericyte-to-myofibroblast transition: Notch3-dependent
Cardiac Fibrosis
- Post-infarction remodeling: Notch3 activation drives cardiac fibroblast-to-myofibroblast transition
- Atrial fibrillation: Aberrant Notch signaling in atrial tissue contributes to fibrosis
Skin Fibrosis (Scleroderma)
- Dermal fibroblasts: Jagged1/Notch1 activation → enhanced α-SMA, Col1a1 expression
- Also involved in keloid formation
6. Crosstalk with Other Pathways
Interaction with TGF-β
The TGF-β/Smad pathway directly induces Notch ligand (Jag1) expression. Conversely, Notch signaling enhances Smad transcriptional activity, forming a TGF-β-Notch positive feedback loop.
Coordination with Wnt
The Wnt/β-catenin pathway and Notch cooperate during development and in fibrotic tissue through common target genes.
Relationship with Hedgehog
In ductular reaction driven by the Hedgehog pathway, Notch and Hh cooperate to promote cholangiocyte differentiation.
7. Therapeutic Strategies
γ-Secretase Inhibitors (GSI)
Inhibitors of the enzyme responsible for Notch S3 cleavage. Originally developed for Alzheimer's disease (Aβ production suppression), but Notch-dependent applications in tumors and fibrosis are under study.
| Compound | Status | Indication Research |
|---|---|---|
| DAPT | Research use | Standard tool in preclinical fibrosis models |
| MK-0752 | Phase I/II | T-ALL, breast cancer |
| Crenigacestat (LY3039478) | Clinical development | Solid tumors, desmoid tumors |
| Nirogacestat | FDA approved (2023) | Desmoid tumors |
Challenges: Systemic GSI administration has serious gastrointestinal toxicity (goblet cell hyperplasia). Optimization of route and dose is required for antifibrotic use.
Anti-Notch Ligand Antibodies
- Demcizumab (anti-DLL4): Developed for tumor angiogenesis inhibition. Potential application in fibrotic angiogenesis
- Anti-Jagged1 antibody: Alleviates liver and renal fibrosis in preclinical studies
Selective Notch Receptor Inhibition
- Notch1 selective inhibitor: Next-generation approach avoiding pan-Notch inhibition toxicity
- Notch3 selective inhibitor: Promising for pericyte-mediated fibrosis
8. Practical Preclinical Evaluation
Markers to Measure
- NICD (intracellular domain): Direct marker of activated Notch. IHC/WB with anti-cleaved-Notch1 antibodies
- Hes1, Hey1 mRNA: Target genes. Quantify by qPCR
- Jag1, Dll4: Ligand expression. Evaluate tissue-specifically
- EMT markers: E-cadherin↓, N-cadherin↑, Snail↑
- Fibrosis markers: hydroxyproline, α-SMA, Sirius Red
Recommended Models
- Bleomycin pulmonary fibrosis: EMT/Notch1 axis
- UUO: Notch3-pericyte transition
- CCl4 liver fibrosis: Ductular reaction, Notch2
- Bleomycin skin fibrosis: Scleroderma model
Conclusion
The Notch signaling pathway integratively controls the multi-faceted fibrotic processes of EMT, angiogenesis, and macrophage polarization. γ-Secretase inhibitors and anti-ligand antibodies show promise in preclinical studies, but overcoming gastrointestinal toxicity and achieving tissue specificity are key for clinical application.
Combination inhibition with other "developmental pathways" like TGF-β, Wnt, and Hedgehog, organ-specific delivery, and time-specific intervention will define the direction of next-generation antifibrotic therapy.
Related Articles
- TGF-β/Smad Pathway: The Master Switch of Fibrosis
- Wnt/β-catenin: Why a Developmental Pathway Drives Fibrosis
- Hedgehog/GLI Pathway: Hepatic Stellate Cell Activation
- JAK/STAT Pathway and Fibrosis
- Fundamentals of Macrophage Polarization
- Myofibroblast Origins and Activation
References
- Kopan R, Ilagan MX. The canonical Notch signaling pathway: unfolding the activation mechanism. Cell. 2009;137(2):216-233.
- Hu B, Phan SH. Mechanisms of disease: the role of Notch signaling in pulmonary fibrosis. Int J Clin Exp Pathol. 2014;7(11):7346-7356. PMID: 25550767
- Morell CM, et al. Notch signalling beyond liver development: emerging concepts in liver repair and oncogenesis. Clin Res Hepatol Gastroenterol. 2013.
- Bielesz B, et al. Epithelial Notch signaling regulates interstitial fibrosis development in the kidneys of mice and humans. J Clin Invest. 2010;120(11):4040-4054.
- Noseda M, et al. Notch and minimizing the need for pathway redundancy. Trends Cell Biol. 2005.