Abstract
Loss-of-function mutations of the gene encoding Krev interaction trapped protein 1 (KRIT1) are associated with the pathogenesis of Cerebral Cavernous Malformation (CCM), a major cerebrovascular disease characterized by abnormally enlarged and leaky capillaries and affecting 0.5% of the human population. However, growing evidence demonstrates that KRIT1 is implicated in the modulation of major redox-sensitive signaling pathways and mechanisms involved in adaptive responses to oxidative stress and inflammation, suggesting that its loss-of-function mutations may have pathological effects not limited to CCM disease. The aim of this study was to address whether KRIT1 loss-of-function predisposes to the development of pathological conditions associated with enhanced endothelial cell susceptibility to oxidative stress and inflammation, such as arterial endothelial dysfunction (ED) and atherosclerosis. Silencing of KRIT1 in human aortic endothelial cells (HAECs), coronary artery endothelial cells (HCAECs), and umbilical vein endothelial cells (HUVECs) resulted in increased expression of endothelial proinflammatory adhesion molecules vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) and in enhanced susceptibility to tumor necrosis factor alpha (TNF-α)-induced apoptosis. These effects were associated with a downregulation of Notch1 activation that could be rescued by antioxidant treatment, suggesting that they are consequent to altered intracellular redox homeostasis induced by KRIT1 loss-of-function. Furthermore, analysis of the aorta of heterozygous KRIT1+/− mice fed a high-fructose diet to induce systemic oxidative stress and inflammation demonstrated a 1.6-fold increased expression of VCAM-1 and an approximately 2-fold enhanced fat accumulation (7.5% vs 3.6%) in atherosclerosis-prone regions, including the aortic arch and aortic root, as compared to corresponding wild-type littermates. In conclusion, we found that KRIT1 deficiency promotes ED, suggesting that, besides CCM, KRIT1 may be implicated in genetic susceptibility to the development of atherosclerotic lesions.
Highlights
Endothelial dysfunction (ED) encompasses maladaptive changes in the functional phenotype of endothelial cells, which have important implications for the maintenance of vascular homeostasis and the modulation of acute and chronic oxidative stress and inflammatory responses in the arteries
The levels of proinflammatory adhesion molecules vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) expression were evaluated by western blot and qRT-PCR and the number of apoptotic cells was determined with the Annexin V binding assay
As compared to control cells, Krev interaction trapped protein 1 (KRIT1) silencing in Human Umbilical Vein Endothelial Cells (HUVECs), human aortic endothelial cells (HAECs), and Human Coronary Artery Endothelial Cells (HCAECs) resulted in an increased expression of VCAM-1 and ICAM-1 proteins both under basal conditions and, more significantly, upon cell treatment with Tumor Necrosis Factor α (TNF-α) (Figure 1A), suggesting that KRIT1 deficiency in endothelial cell (EC) induces the upregulation of proinflammatory cell adhesion molecules (CAMs)
Summary
Endothelial dysfunction (ED) encompasses maladaptive changes in the functional phenotype of endothelial cells, which have important implications for the maintenance of vascular homeostasis and the modulation of acute and chronic oxidative stress and inflammatory responses in the arteries. KRIT1 has been shown to regulate cadherin-mediated cell–cell junctions [8], integrin-mediated cell-matrix adhesion [9,10,11], Rho GTPase-mediated cytoskeleton dynamics [12,13], Delta-Notch signaling [14,15,16], vascular endothelial growth factor (VEGF) signaling [17], and mechanotransduction pathways mediated by blood flow-sensitive transcription factors of the Krüppel-like factor (KLF) family [18,19] Consistent with these pleiotropic regulatory functions, emerging evidence demonstrates that KRIT1 plays a major role in modulating distinct redox-sensitive signaling pathways and mechanisms involved in endothelial cell homeostasis and adaptive responses to oxidative stress and inflammation, including pro-oxidant and antioxidant pathways and autophagy [4,20,21,22,23,24,25,26,27,28]. These findings have pointed towards a novel unifying mechanistic scenario based on redox homeostasis and signaling that reconciles all the pleiotropic physiopathological functions of KRIT1 proposed so far [4,25]
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