Fig 1.
Representative sequence of co-registered MR images and histological data along the length of the instrumented carotid artery.
(A) Diagram of the flow environment in the instrumented versus contralateral control carotid arteries. The cuff creates three distinct hemodynamic conditions, low WSS upstream of the cuff, high WSS within the cuff, and multidirectional (MD) WSS downstream of the cuff [11]. (B-D) Longitudinal sequence of DCE-MR images and Ktrans maps co-registered to histology sections stained with oil red O to identify lipid from the (B) low WSS, (C) cuff, and (D) multidirectional WSS regions in the instrumented artery of a representative mouse at 9 weeks after cuff placement. (E) Data from the contralateral control artery. Note, the cuff prevents reliable acquisition of Ktrans maps, so they are not shown in this region.
Fig 2.
(A) HRTEM images of FA-Gd NPs, white circles indicate average diameter (4.5 nm) of the NPs. Blue arrow indicates lattice fringe of NPs. (B) FT-IR spectra of folic acid and FA-Gd NPs. The black spectrum corresponds to free FA, while the red spectrum corresponds to FA-Gd NPs. The COOH peak was shifted from 1680 to 1527 cm-1, which confirmed the FA coating on to the Gd NPs. (C) R1 map of FA-Gd NP phantoms for determination of R1 relaxivity (3.14 mM-1s-1). (D) Zeta potential distribution of FA-Gd NPs, mean value of -40.5 mV.
Fig 3.
Plaque burden and NP accumulation over the length of the instrumented artery.
(A) Plaque burden in mice injected with NPs at 9 weeks after cuff placement, (B) NP accumulation assessed via Ktrans at 5 weeks after cuff placement, and (C) Ktrans at 9 weeks after cuff placement as a function of position (in mm) from the center of the cuff (no plaque developed in the cuff region and there are no Ktrans values due to the cuff creating an unreliable signal; thus, no data are presented in this region). Data points represent the mean plaque burden and Ktrans values for each mouse injected with NPs (n = 5) at each MRI slice location. Bars are mean±SD. These data were not evaluated for statistical differences between vessel regions.
Table 1.
Pharmacokinetics of FA-Gd in ApoE-/- mice determined by MRI.
Fig 4.
NP accumulation assessed via Ktrans in disturbed and normal WSS conditions.
Ktrans in the low and multidirectional (MD) WSS regions of the instrumented carotid arteries and contralateral control carotid arteries at 5 and 9 weeks after cuff placement. Each data point represents the mean Ktrans from the three DCE-MRI slices closest to the cuff within each WSS region of the instrumented artery and the mean Ktrans from the central portion of the control artery for each mouse that was injected with NPs (n = 5). Bars are mean±SD. *P<0.05 is considered statistically significant.
Fig 5.
Constituents and size of plaques induced by low WSS at 5 versus 9 weeks.
(A) Diagram of the instrumented carotid artery highlighting the focus of this figure on the low WSS (upstream) region. (B) Representative histology sections stained with oil red O to detect lipid and (C) plot of mean lipid area normalized by intima area across all mice at 5 (n = 7 mice) and 9 (n = 10) weeks after cuff placement. (D) Representative histology sections stained with picrosirius red to detect collagen, (E) plot of mean collagen area normalized by intima area at 5 (n = 7) and 9 (n = 8) weeks, and (F) plot of mean collagen distribution from the IEL to the lumen normalized by the value at the IEL at 5 and 9 weeks. (G) Representative histology sections stained for α-SMA, (H) plot of mean α-SMA intensity normalized by intima area at 5 (n = 8) and 9 (n = 10) weeks, and (I) plot of mean α-SMA intensity distribution from the IEL to the lumen normalized by the value at the IEL at 5 and 9 weeks. (J) Plot of mean plaque burden at 5 and 9 weeks. In the scatter plots, each data point represents the average of all viable histological sections for the given stain in the plaque region of the upstream vessel segment of one mouse. Bars are mean±SD. *P<0.05 is considered statistically significant.
Fig 6.
Constituents and size of plaques induced by multidirectional WSS at 5 versus 9 weeks.
(A) Diagram of the instrumented carotid artery highlighting the focus of this figure on the multidirectional WSS (downstream) region. (B) Representative histology sections stained with oil red O to detect lipid and (C) plot of mean lipid area normalized by intima area across all mice at 5 (n = 7 mice) and 9 (n = 10) weeks after cuff placement. (D) Representative histology sections stained with picrosirius red to detect collagen, (E) plot of mean collagen area normalized by intima area at 5 (n = 7) and 9 (n = 8) weeks, and (F) plot of mean collagen distribution from the IEL to the lumen normalized by the value at the IEL at 5 and 9 weeks. (G) Representative histology sections stained for α-SMA, (H) plot of mean α-SMA intensity normalized by intima area at 5 (n = 8) and 9 (n = 10) weeks, and (I) plot of mean α-SMA intensity distribution from the IEL to the lumen normalized by the value at the IEL at 5 and 9 weeks. (J) Plot of mean plaque burden at 5 and 9 weeks. In the scatter plots, each data point represents the average of all viable histological sections for the given stain in the plaque region of the downstream vessel segment of one mouse. Bars are mean±SD. *P<0.05 is considered statistically significant.
Fig 7.
Correlation between α-SMA and NP accumulation assessed via Ktrans at 9 weeks after cuff placement.
(A) Diagram of the instrumented carotid artery highlighting the focus of this part of the figure on the low WSS (upstream) region. (B) A representative histology section and higher magnification insert of the plaque region, where the lumen and IEL are identified. (C-E) The correlation between α-SMA and Ktrans in different regions of the plaques induced by low WSS, including the (C) entire plaque, (D) plaque cap (13.3 μm), and (E) plaque body minus the cap. (F) Diagram of the instrumented carotid artery highlighting the focus of this part of the figure on the multidirectional (MD) WSS (downstream) region. (G) A representative histology section and higher magnification insert of the plaque region. (H-J) The correlation between α-SMA and Ktrans in different regions of the plaques induced by multidirectional WSS, including the (H) entire plaque, (I) plaque cap (13.3 μm), and (J) plaque body minus the cap. Each data point of each plot represents the mean α-SMA fluorescence intensity normalized by intimal area across all histological sections associated with a DCE-MRI slice, from which Ktrans was obtained (in mice with viable histological sections associated with more than one DCE-MRI slice, more than one pairing was used; all mice injected with NPs (n = 5) are represented in all plots). The black line in each plot represents a linear regression of the data to visualize the trend. Spearman’s correlation coefficient, ρ, and associated p-value are also given. *P<0.05 is considered statistically significant.
Fig 8.
Correlation between collagen and NP accumulation assessed via Ktrans at 9 weeks after cuff placement.
(A) Diagram of the instrumented carotid artery highlighting the focus of this part of the figure on the low WSS (upstream) region. (B) A representative histology section and higher magnification insert of the plaque region, where the lumen and IEL are identified. (C-E) The correlation between collagen and Ktrans in different regions of the plaques induced by low WSS, including the (C) entire plaque, (D) plaque cap (13.3 μm), and (E) plaque body minus the cap. (F) Diagram of the instrumented carotid artery highlighting the focus of this part of the figure on the multidirectional WSS (downstream) region. (G) A representative histology section and higher magnification insert of the plaque region. (H-J) The correlation between collagen and Ktrans in different regions of the plaques induced by multidirectional (MD) WSS, including the (H) entire plaque, (I) plaque cap (13.3 μm), and (J) plaque body minus the cap. Each data point of each plot represents the mean collagen area normalized by intimal area across all histological sections associated with a DCE-MRI slice, from which Ktrans was obtained (in mice with viable histological sections associated with more than one DCE-MRI slice, more than one pairing was used; all mice injected with NPs (n = 5) are represented in all plots). The black line in each plot represents a linear regression of the data to visualize the trend. Spearman’s correlation coefficient, ρ, and associated p-value are also given. *P<0.05 is considered statistically significant.
Fig 9.
Correlation between plaque area and NP accumulation assessed via Ktrans at 9 weeks after cuff placement.
(A) Diagram of the instrumented carotid artery highlighting the focus of this part of the figure on the low WSS (upstream) region. (B-D) The correlation between plaque area and Ktrans in different regions of the plaques induced by low WSS, including the (B) entire plaque, (C) plaque cap (13.3 μm), and (D) plaque body minus the cap. (E) Diagram of the instrumented carotid artery highlighting the focus of this part of the figure on the multidirectional WSS (downstream) region. (F-H) The correlation between plaque area and Ktrans in different regions of the plaques induced by multidirectional (MD) WSS, including the (F) entire plaque, (G) plaque cap (13.3 μm), and (H) plaque body minus the cap. Each data point of each plot represents the mean plaque area across all histological sections associated with a DCE-MRI slice, from which Ktrans was obtained (in mice with viable histological sections associated with more than one DCE-MRI slice, more than one pairing was used; all mice injected with NPs (n = 5) are represented in all plots). The black line in each plot represents a linear regression of the data to visualize the trend. Spearman’s correlation coefficient, ρ, and associated p-value are also given. *P<0.05 is considered statistically significant.
Fig 10.
Characteristics of plaques induced by low WSS compared to multidirectional (MD) WSS.
Plaque size and constituents assessed over the whole plaque, including: (A) Plaque burden (n = 10 mice), (B) α-SMA intensity (n = 10), (C) lipid (n = 10), (D) collagen (n = 8), and (E) area of Ki67 in the nucleus of cells normalized by total cell nuclear (DAPI) area (n = 5). Each data point of each plot represents the average of a given readout across all viable histological sections in each vessel segment of one mouse. Bars are mean±SD. *P<0.05 is considered statistically significant.