Contrast Ultrasound Imaging Does Not Affect Heat Shock Protein 70 Expression in Cholesterol-Fed Rabbit Aorta

Abstract

Objectives: Diagnostic ultrasound imaging is enhanced by the use of circulating microbubble contrast agents (UCAs), but the interactions between ultrasound, UCAs, and vascular tissue are not fully understood. We hypothesized that ultrasound with a UCA would stress the vascular tissue and increase levels of heat shock protein 70 (Hsp70), a cellular stress protein.

Methods: Male New Zealand White rabbits (n = 32) were fed a standard chow diet (n = 4) or a 1% cholesterol, 10% fat, and 0.11% magnesium diet (n = 28). At 21 days, 24 rabbits on the cholesterol diet were either exposed to ultrasound (3.2-MHz f/3 transducer; 2.1 MPa; mechanical index, 1.17; 10 Hz pulse repetition frequency; 1.6 microseconds pulse duration; 2 minutes exposure duration at 4 sites along the aorta) with the UCA Definity (1× concentration, 1 mL/min; Lantheus Medical Imaging, North Billerica, MA) or sham exposed with a saline vehicle injection (n = 12 per group). Four rabbits on the cholesterol diet and 4 on the chow diet served as cage controls and were not exposed to ultrasound or restrained for blood sample collection. Animals were euthanized 24 hours after exposure, and aortas were quickly isolated and frozen in liquid nitrogen. Aorta lysates from the area of ultrasound exposure were analyzed for Hsp70 level by Western blot. Blood plasma was analyzed for cholesterol, Hsp70, and von Willebrand factor, a marker of endothelial function.

Results: Plasma total cholesterol levels increased to an average of 705 mg/dL. Ultrasound did not affect plasma von Willebrand factor, plasma Hsp70, or aorta Hsp70. Restraint increased Hsp70 (P < .001, analysis of variance).

Conclusions: Restraint, but not ultrasound with the UCA or cholesterol feeding, significantly increased Hsp70.

Keywords: atherosclerosis; cardiovascular disease; contrast agents; endothelium; heat shock protein 70; microbubbles; ultrasound.

Figure 1

Study design with 32 New Zealand White (NZW) rabbits. After 3 weeks on either the chow diet or the 1% cholesterol, 10% fat, and 0.11% magnesium diet, rabbits (n = 24) underwent ultrasound (US) imaging at 2.1 MPa with intravenous administration of the Definity UCA or a sham procedure. Serial saphenous vein blood samples were collected from these animals under restraint. An additional 4 rabbits on the cholesterol diet and the 4 on chow served as cage controls. Rabbits were euthanized 24 hours after the ultrasound procedure (day 22), and aorta tissue was collected for analysis.

Figure 2

Plasma cholesterol and vWF. A, Plasma total cholesterol (TC) was measured by an enzymatic colorimetric kit. B, Plasma vWF was measured by ELISA. TG indicates triglycerides.

Figure 3

Aorta tissue Hsp70 analysis on day 22. Aorta tissue from the site of exposure was carefully dissected from the surrounding tissue and frozen in liquid nitrogen. Tissue lysates were analyzed for Hsp70 by Western blot, with β-actin as a loading control. Western blot bands shown are representative of the mean of each group. Bars indicate SEM; + and − indicate presence and absence of the treatment.

Figure 4

Plasma Hsp70. A, Plasma Hsp70 was measured by ELISA. B, There was no correlation between plasma and tissue Hsp70 levels. Bars indicate SEM.