Related Experiment Video
Updated: Aug 28, 2025
Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro
Published on: May 10, 2021
Integrin-Linked Kinase Expression in Human Valve Endothelial Cells Plays a Protective Role in Calcific Aortic Valve
Sandra Sánchez-Esteban1, Mercedes Castro-Pinto1,2, Alberto Cook-Calvete1
1Universidad de Alcalá, Dpto Biología de Sistemas/Fisiología, IRYCIS, 28871 Alcalá de Henares, Spain.
Insights
Integrin-linked kinase (ILK) downregulation in aortic valves promotes calcific aortic valve disease (CAVD) by causing endothelial dysfunction and osteogenic transformation. Restoring nitric oxide (NO) production prevents this process.
Area of Science:
- Cardiovascular Biology
- Vascular Endothelial Cell Biology
- Biomaterials and Tissue Engineering
Background:
- Calcific aortic valve disease (CAVD) is a prevalent aging-related condition.
- Endothelial dysfunction, lipid accumulation, and inflammation are key initiators of CAVD.
- Integrin-linked kinase (ILK) is implicated in cardiovascular diseases, but its role in CAVD is unclear.
Purpose of the Study:
- To investigate the role of ILK in the pathogenesis of CAVD.
- To determine the effect of ILK silencing on human valve endothelial cells (hVECs).
- To elucidate the underlying molecular mechanisms involving ILK, endothelial-to-mesenchymal transition (EndMT), and nitric oxide (NO) signaling.
Main Methods:
- ILK expression analysis in human CAVD and non-CAVD aortic valves.
- ILK silencing in hVECs (siILK-hVECs) to assess EndMT, osteogenic markers (RUNX2), and calcification.
- Nitric oxide (NO) and eNOS inhibitor treatments on siILK-hVECs.
- Analysis of Smad2 phosphorylation and TGF-β signaling.
- In vivo studies using eNOS knockout (KO) mice.
Main Results:
- ILK expression is downregulated in CAVD valves, particularly in the endothelium, and correlates with calcification.
- ILK silencing in hVECs induced EndMT, increased RUNX2 expression, promoted calcified nodule formation, and reduced NO production.
- NO treatment reversed ILK-silencing-induced EndMT and calcification, while eNOS inhibition mimicked ILK silencing.
- Mechanistically, ILK silencing increased Smad2 phosphorylation, and NO treatment reduced Smad2 activation and RUNX2.
- eNOS KO mice exhibited decreased ILK expression, increased RUNX2, and aortic valve calcification.
Conclusions:
- Endothelial ILK expression is crucial in preventing endothelial osteogenic transformation in human CAVD.
- ILK acts via the eNOS/NO pathway to inhibit EndMT and subsequent valve calcification.
- Targeting ILK or enhancing NO signaling may represent therapeutic strategies for CAVD.
Abstract:
Calcific aortic valve disease (CAVD) is highly prevalent during aging. CAVD initiates with endothelial dysfunction, leading to lipid accumulation, inflammation, and osteogenic transformation. Integrin-linked kinase (ILK) participates in the progression of cardiovascular diseases, such as endothelial dysfunction and atherosclerosis. However, ILK role in CAVD is unknown. First, we determined that ILK expression is downregulated in aortic valves from patients with CAVD compared to non-CAVD, especially at the valve endothelium, and negatively correlated with calcification markers. Silencing ILK expression in human valve endothelial cells (siILK-hVECs) induced endothelial-to-mesenchymal transition (EndMT) and promoted a switch to an osteoblastic phenotype; SiILK-hVECs expressed increased RUNX2 and developed calcified nodules. siILK-hVECs exhibited decreased NO production and increased nitrosative stress, suggesting valvular endothelial dysfunction. NO treatment of siILK-hVECs prevented VEC transdifferentiation, while treatment with an eNOS inhibitor mimicked ILK-silencing induction of EndMT. Accordingly, NO treatment inhibited VEC calcification. Mechanistically, siILK-hVECs showed increased Smad2 phosphorylation, suggesting a TGF-β-dependent mechanism, and NO treatment decreased Smad2 activation and RUNX2. Experiments performed in eNOS KO mice confirmed the involvement of the ILK-eNOS signaling pathway in valve calcification, since aortic valves from these animals showed decreased ILK expression, increased RUNX2, and calcification. Our study demonstrated that ILK endothelial expression participates in human CAVD development by preventing endothelial osteogenic transformation.
Related Concept Videos
01:17Intracellular Signaling Affects Focal Adhesions
Some...
01:15Activation of Integrins
In "outside-in signaling," external factors in the extracellular space bind to exposed ligand binding sites on integrins. This causes the inactive protein to undergo a conformational change to become active. Integrins are often clustered on the cell membrane. Repetitive and regularly spaced ligand binding...