Caveolin-1 is a primary determinant of endothelial stiffening associated with dyslipidemia, disturbed flow, and ageing

Figures

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  Figure 1. Caveolin-1 is required for oxLDL uptake and oxLDL-mediated stiffening in MAECs. (A) Representative confocal images showing the endothelial markers PECAM-1 and vWF in mouse aortic endothelial cells (MAECs). (B) Representative amplification profile of Cav-1 mRNA expression in mouse aortic endothelial cells (n = 4). (C) Representative confocal images of Cav-1 IHC staining in WT and Cav-1 KO ECs and Cav-1 rescue using Cav-1 adenovirus. (D) Representative confocal images of DiI-oxLDL uptake into WT, Cav-1 KO and Cav-1 adenovirus treated Cav-1 KO ECs (left) and summarized data of DiI-oxLDL uptake (right; mean ± SD; n = 5–8, 20–40 cells per experiment). (E) Histograms (left) and mean ± SD elastic modulus values (stiffness) of WT, Cav-1 KO and Cav-1 KO MAECs reconstituted with Cav-1 adenovirus after exposure to oxLDL or vehicle for 48 h (15–25 cells per condition/experiment, n = 4–10 independent experiments). Scale bars represent 20 µm. *P < 0.05 by ANOVA.

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  Figure 2. Cav1 regulates the expression of oxLDL scavenger receptor CD36. (A) Real-time PCR plot showing decreased CD36 mRNA expression in Cav-1 KO MAECs. (B) Representative 3D rendered confocal images showing CD36 expression in WT, CD36 KO and Cav-1 KO MAECs and (C) average integrated 3D fluorescence intensity of CD36 (n = 4, 5–8 cells per condition/experiment). Scale bars are 10 µm. *P < 0.05 by ANOVA.

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  Figure 3. Disturbed flow-induced increase in oxLDL uptake and endothelial stiffening is mediated by Cav-1. (A) Representative confocal images of Cav-1 specific fluorescence (green, top) in WT ECs and DiI-oxLDL uptake (red) in WT (middle) and Cav-1 KO (bottom) in mouse microvascular ECs (MMVECs) exposed to laminar flow (LF) or disturbed flow (DF). Scale bars are 20 µm. (B) DiI-oxLDL uptake in Cav-1 KO and WT MMVECs (20–40 cells per condition/experiment; Mean ± SD, n = 6). (C) CD36 expression in WT versus Cav-1 KO MMVECs (n = 4). (D) Histograms of elastic modulus values from Cav-1 KO and WT ECs exposed to oxLDL (1 µg/ml, 48 h) during laminar and disturbed flow (15–25 cells per condition/experiment, n = 5) and (E) summarized (mean ± SD; n = 6) elastic modulus values for WT and Cav-1 KO MMVECs following 48 h exposure to laminar or disturbed flow in the presence of oxLDL. *P < 0.05 by ANOVA.

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  Figure 4. Absence of caveolin-1 abrogates endothelial stiffening in the pro-atherogenic aortic arch. (A) Histograms of elastic modulus values recorded from the descending aorta (left) and the aortic arch (right) of WT and (B) Cav-1 KO male 4 month old mice with representative confocal images to confirm lack of Cav-1 expression in Cav-1 KO mice (scale bars are 15 µm) and (C) data summary from AFM recording in (A,B) (Each circle is data from one mouse; error bars are SD from 10 to 15 measurements per aortic segment; horizontal line depicts mean of the total mice, n = 5–6 mice with 10–15 measurements per sample). *P < 0.05 by ANOVA.

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  Figure 5. Absence of caveolin-1 abrogates endothelial stiffening induced by high fat diet. (A,B) Increase in body weight and plasma concentration of oxLDL in Cav-1 KO mice fed a HFD for 1 month. (C) Histograms of elastic modulus of DA and Arch in Cav-1 KO male mice fed regular chow (RC) or HFD for 1 month. (D) Summary of aortic elastic modulus recordings in Cav-1 KO mice. *P < 0.05 by ANOVA.

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  Figure 6. Age-associated increase in mouse aortic endothelial stiffening is mitigated by Cav-1 deletion. (A) Representative 3D confocal images (scale bars are 15 µm) of Cav-1 expression in the descending aorta and aortic arch in young (3–4 month old) and older (18–20 month old) WT mice (n = 3–4 mice per condition per age, 2–4 3D images per mouse) and (B) its quantification. (C) Summary of aortic elastic modulus values for WT and Cav-1 KO mice at each age. (D) Exponential curves representing mouse age versus elastic modulus. *P < 0.05 using ANOVA.

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  Figure 7. Cav-1 deletion in advanced aged mice reduces endothelial stiffening and no elastic modulus differences between the sexes. (A) Histograms of elastic modulus values from the aortic arch of advanced age (20–25 months) WT and Cav-1 KO males as compared to females with (B) Data summary of descending aorta and aortic arch from AFM recording in male and female WT and Cav-1 KO mice (n = 4–7 mice, 10–20 measurements per sample). *P < 0.05 compared to same vessel type and sex in WT mice as tested by ANOVA and a Student’s t-test.

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  Figure 8. Age-associated increase in mouse aortic sub-endothelial layer stiffening is mitigated by Cav-1 deletion. Summary of sub-endothelial aortic elastic modulus recordings (average of DA and Arch) from WT and Cav-1 KO mice at three age groups. *P < 0.05 using ANOVA.