NOTCH3 Modulation of Extracellular Matrix, Cytoskeletal Organisation and Metabolic Functions in Human Vascular Smooth Muscle Cells

NOTCH3 is a transmembrane receptor highly expressed in vascular mural cells where it contributes to blood vessel formation and homeostasis. NOTCH3 expression declines in the vasculature with aging, and dysregulated NOTCH3 signalling is implicated in pulmonary arterial hypertension, cancer progression and CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy). RNA-based approaches targeting NOTCH3 are emerging as potential therapeutic strategies, however, the consequences of NOTCH3 suppression in mature vascular smooth muscle cells (VSMCs) remain incompletely understood. Here, we investigated the molecular and functional effects of siRNA-mediated NOTCH3 knockdown in human aortic smooth muscle cells.

Transfection with NOTCH3-targeting siRNA efficiently suppressed NOTCH3 transcript and protein levels. Quantitative proteomics revealed remodelling of extracellular matrix (ECM), cytoskeletal and metabolic pathways, with enrichment of collagen biosynthesis and inhibition of glycolytic signalling. Specifically, NOTCH3 knockdown increased ECM components, including COL3A1, elevated F-actin, and upregulated the actin regulator, CTTN. In parallel, glycolytic capacity was reduced, accompanied by decreased expression of the glycolytic enzyme ENO2. Despite reduced VEGFA and alteration in angiogenic signalling proteins, endothelial network formation in co-cultures, as well as VSMC proliferation and migration remained unaffected. Finally, NOTCH3 interactome analysis revealed key collagen and actin-regulating proteins.

These findings identify NOTCH3 as an important regulator of ECM homeostasis, cytoskeletal organisation, and glycolytic metabolism. The preservation of primary cellular functions despite molecular remodelling highlights the adaptive capacity of VSMCs. These findings demonstrate that therapeutic modulation of NOTCH3 may alter vascular cell biology which warrants consideration during development of RNA-based therapeutics for CADASIL and other NOTCH3-associated diseases.

Graphical Abstract

Effects of NOTCH3 knockdown on human aortic smooth muscle cells (HAoSMCs). HAoSMCs were treated with NOTCH3 siRNA followed by stimulation with PDGF-BB or TGF-β to model VSMC activation. NOTCH3 knockdown induced molecular remodelling characterised by increased ECM-associated pathways, including COL3A1 and collagen biosynthesis, alongside alterations in cytoskeletal proteins, CTNN and F-Actin. Metabolic alterations were observed, including a decrease in ENO2 and a trend towards decreased glycolytic capacity. NOTCH3 knockdown also increased expression of the epigenetic regulator SETD7. Despite reduction in VEGFA expression, there was no change in angiogenic endothelial cell network formation when exposed to conditioned media from the HAoSMCs or in direct contact with HAoSMCs lacking NOTCH3. Functionally, migration and proliferation were also unchanged indicating preservation of key VSMC responses. Collectively, NOTCH3 siRNA causes molecular remodelling while maintaining VSMC functional capacity.

Key Points

• NOTCH3 knockdown increases COL3A1 and total collagen deposition following stimulation with PDGF and TGF-β.

• NOTCH3 knockdown suppresses glycolytic capacity and reduces the glycolytic enzyme ENO2.

• NOTCH3 knockdown increases the abundance of key actin-associated proteins, including cortactin (CTTN) and F-actin.

• The NOTCH3 interactome encompasses structural collagens (COL3A1) and actin-associated regulatory proteins (CTTN).

• NOTCH3 knockdown does not alter angiogenesis, migration, or proliferation under the investigated conditions.