Piezo-type mechanosensitive ion channel component 1 (PIEZO1) is upregulated in peripheral arterial disease (PAD) and a novel murine PAD model

Figures

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  Fig 1. Murine arteries stiffen with aging, femoral arteries (FAs) are stiffer than carotid arteries (CAs), and exercise modifies FA stiffening with aging. (A) Overview of grouping and experimental procedures for biomechanical comparison between the FA and CA. We use the term sedentary to contrast cage activity to that of running wheels. We have added data from the literature to characterize the exercise difference between cage and running wheel conditions in Supplementary Fig 1 (online only). (B) Younger FAs have divergent mechanics compared with CAs, with increased stiffness under physiologic pressures (40-140 mmHg). N = 6/group. (C) Arterial compliance in older FAs is lower than in older CAs. N = 6 for CA; N = 7 for FA. (D) Older FAs (N = 7) have less compliance over lower pressure testing than younger FAs (N = 6). (E) Exercise favorably modifies compliance in older FAs. N = 6 for exercise; N = 7 for nonexercise. (F) Physiologic stretch is a measure of longitudinally stiffness. Regardless of exercise status and artery type, the physiologic stretch of older arteries is significantly lower than that of younger arteries. Additionally, whether with or without exercise, FAs exhibit…

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  Fig 2. Partial femoral ligation (PFL) induces low wall shear stress (WSS) and exacerbates femoral artery (FA) stiffness. (A) Overview of experimental procedures for nonatherogenic PFL mice. (B) The schematic diagram of the PFL model in mouse. Briefly, the saphenous artery, proximal caudal FA, superficial caudal epigastric artery, and medial proximal genicular artery are ligated (red). (C) Following PFL, the left FA experiences sustained low WSS, as shown by temporal WSS (acquired by ultrasound measurement preoperatively, and on day 7 and day 28 after PFL). Meanwhile, the right FA remains unmanipulated and maintains normal WSS. (D) Similar to aging, the physiologic stretch of FAs under low WSS is stiffer than that under normal WSS. N = 11 for males; N = 10 for females. (E) Represented graph of Cauchy circumferential stress in FAs at the in vivo axial stretch. (F) The rate of stress increment as strain increases in D is quantified using Elastic Modulus. N = 11/group. (G) k1∗k2 represents the vessel stiffness at large deformations (higher strains). Similar to aging, low WSS increases FA stiffness, as demonstrated by increased stiffness factor k1∗k2. N = 11/group. Data are represented as…

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  Fig 3. Under atherogenic conditions, low wall shear stress (WSS) accelerates femoral artery (FA) stiffness. (A) Overview of experimental procedures for atherogenic partial femoral ligation (PFL) mice. (B) Representative ultrasound images of femoral hemodynamics 4 weeks post PFL demonstrate sustained low flow velocities and increased arterial stiffness, characterized by decreased peak systolic velocity (PSV) (C), and reduced arterial Green strain Eθθ(D). N = 4 for males; N = 3 for females. Light blue line, intima-media thickness (IMT); blue arrow, PSV; yellow arrow, end-diastolic velocity (EDV). (E) Velocity time integral (VTI) represents the distance the blood column travels in one heartbeat cycle, which is suppressed by PFL. N = 4 for males; N = 3 for females. (F) The physiologic stretch is stiffer after PFL. N = 7 for males; N = 9 for females. Data are represented as mean ± standard error of the mean. Blue indicates male; pink indicates female. Statistical significance was assessed by a two-sample paired Student t-test for normality or a nonparametric Wilcoxon matched-pairs signed rank test for non-normality. Twelve- to 20-week-old mice were used for this experiment. HFD, High-fat diet.

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  Fig 4. Under atherogenic conditions, partial femoral ligation (PFL) induces focal atherogenic plaque in femoral arteries (FAs). (A) Representative gross view of dissected FAs in atherogenic mice post 4 weeks PFL ligation (yellow arrow indicates plaque). (B) Histological staining of atherogenic femoral arteries demonstrates increased intima-media thickness (IMT) (C), robust femoral plaque (D), significant increases in lipid lesion area (E), and CD68+ cell accumulation (F) in PFL FAs. N = 5/group. Data were represented as mean ± standard error of the mean. Blue indicates male; pink indicates female. Statistical significance was assessed by a two-sample paired Student t-test for normality or a nonparametric Wilcoxon matched-pairs signed rank test for non-normality. H&E, Hematoxylin and eosin; ORO, oil red O.

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  Fig 5. Increased Piezo-type mechanosensitive ion channel component 1 (PIEZO1) expression after partial femoral ligation (PFL) in murine femoral arteries (FAs) and in human peripheral arterial disease (PAD) arteries with severe stenosis. (A) To better understand gene regulation of PAD arteries in patients, we compared gene array data from arteries with higher-grade stenosis (disturbed flow) vs low-grade stenosis. The volcano plot of microarray data shows increased PIEZO1 expression in human FAs with PAD grade V (N = 7) vs grade I (N = 10). (B) Low wall shear stress (WSS) increases PIEZO1 expression in both male and female FAs, demonstrated by representative immunofluorescence staining of PIEZO1 (red), (C) N = 4 for males; N = 5 for females. (D) PIEZO1 expression in the FA (muscular artery) is much higher than in the carotid artery (CA) (elastic artery), as demonstrated by representative immunofluorescence staining of PIEZO1 (red) and corresponding statistical results (E). N = 5 for CA; N = 9 for FA. Data are represented as mean ± standard error of the mean. Blue indicates male; pink indicates female. Statistical significance was assessed by a two-sample paired or unpaired Student t-test…

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  Fig 6. Piezo-type mechanosensitive ion channel component 1 (PIEZO1) is increased in multiple vascular cells in femoral artery (FA) atherogenic plaque. (A) Upregulated expression of PIEZO1 (red) in CDH5+ (green) cells in FAs after partial femoral ligation (PFL), as demonstrated by representative immunofluorescence images. (B) Increased expression of PIEZO1 (red) in alpha smooth muscle actin-positive (α-SMA+) (green) cells in FAs after PFL, illustrated by representative immunofluorescence images. (C) Increased expression of PIEZO1 (green) in CD68+ (red) cells in FAs after PFL, as demonstrated by representative immunofluorescence images. N = 4 for males; N = 5 for females. Data are represented as mean ± standard error of the mean. Blue indicates male; pink indicates female. Statistical significance was assessed by a two-sample paired Student t-test for normality or a nonparametric Wilcoxon matched-pairs signed rank test for non-normality. MFI, Mean fluorescence intensity.