Original contribution
Effects of ultrasound-targeted microbubble destruction on cardiac gene expression

https://doi.org/10.1016/j.ultrasmedbio.2003.12.006Get rights and content

Abstract

Ultrasound (US) contrast agents are increasingly used in diagnostic echocardiography. Recent studies have suggested unanticipated effects of microbubble destruction. This study was designed to evaluate gene regulation caused by US-mediated destruction of microbubbles in the heart. During IV infusion of Optison™, triggered US was applied to rat hearts to destroy microbubbles. A control group received only saline and US. RNA was isolated from hearts 24 and 72 h after treatment. Analysis with a deeply representative murine cardiac-specific microarray was used to identify regulated genes. Real-time polymerase chain reaction (PCR) was then applied to verify regulated genes. Microarray analysis revealed only 5 regulated genes in the 24-h group and 4 in the 72-h group. Of these genes, only carbonic anhydrase was significantly upregulated in the 24-h Optison™ group (4.3 fold; p = 0.0005) when examined in individual animals by real-time PCR. By this very sensitive technique, the bioeffects of microbubble destruction are negligible. (E-mail: [email protected])

Introduction

Ultrasonic contrast agents have become an important tool in diagnostic echocardiography due to their physical properties, which allow both visualization and controlled destruction by ultrasound (US). During myocardial contrast echocardiography (MCE), microbubbles are destroyed at defined time points in the myocardial vasculature. During some applications, the destruction is facilitated by intermittent use of high mechanical index US, either in a triggered mode with increasing time intervals (Wei et al., 2001, Villanueva et al., 2001) or manually after complete opacification of the myocardium in a real-time mode (Bekeredjian et al., 2002, Masugata et al., 2001).

Recently, US targeted microbubble destruction (UTMD) has been used to deliver bioactive substances to the heart or other US accessible organs (Shohet et al., 2000, Bekeredjian et al., 2003). This technique is based upon incorporating or attaching DNA or viral vectors to microbubbles. These “loaded” microbubbles can be visualized in the target organ after IV infusion and, then, be destroyed by using high mechanical index US; thus, releasing the transported substance into the surrounding tissue. Several studies have examined the biophysical effects of UTMD. Although some studies show that microbubble destruction has no adverse effects (Borges et al. 2002), others have demonstrated the potential to increase vascular permeability (Price et al. 1998), to cause hemorrhage (Miller and Quddus 2000), rupture lipid membranes (Marmottant and Hilgenfeldt 2003) and to produce free radicals (Kondo et al. 1998). It has been suggested that UTMD alone can cause an arteriogenic stimulus in skeletal muscle (Song et al. 2002). These findings raise the question of potential in vivo effects on the heart. Despite the previous demonstration of no detectable effect on left ventricular function or histologic findings, a slight release of troponin (0.012 to 0.278 ng/mL) after UTMD was observed in our laboratory (Chen et al. 2002). Any potential safety concerns for this technique, which is widely and increasingly applied, often to seriously ill patients, needs to be evaluated with the most sensitive techniques available. If the bioeffects seen in other studies are due to disruption of cellular physiology by microbubble destruction, one would expect that these effects should be reflected in gene regulation. It is also important to establish any effect of UTMD on gene expression as a prerequisite for interpretation of research efforts to deliver gene therapy vectors with this technique. This study was designed to determine if UTMD itself causes gene regulation, either as a secondary effect of cellular damage or as an unanticipated primary effect of sonographic bubble destruction.

Section snippets

Animal preparation and sonication

Animal studies were approved by the University of Texas Southwestern Medical Center Animal Research Committee and were performed in accordance with their guidelines. A total of 14 Sprague Dawley rats (240 to 260 g, Charles River, Wilmington, MA) were anesthetized with 100 mg/kg ketamine (IP) and 5 mg/kg xylazine (IP) before the experiment. A polyethylene tube (PE 50, Becton Dickinson, Sparks, MD) was inserted into the right internal jugular vein by cutdown. A total of seven rats were randomly

Microarray analysis

Signal intensity and reproducibility were high and background was low for the microarray hybridizations, despite the use of a heterologous (rat on mouse) probe (Fig. 2). In the hybridizations of 24-h samples, a total of five genes were more than 2.5-fold up- or down-regulated on at least three slides: three known genes and two that were similar only to otherwise uncharacterized ESTs. Four were up-regulated and one was down-regulated in the Optison™ vs. saline sample (Table 1). In the

Discussion

Various in vivo and ex vivo studies have shown that US targeted microbubble destruction (UTMD) can cause adverse effects, such as capillary rupture and erythrocyte extravasation. We had previously demonstrated that UTMD has no effects on histology and left ventricular function, but can cause a mild, transient, troponin leak (Chen et al. 2002). This concern regarding potential bioeffects of UTMD on the heart has recently become more important with the broadening of US contrast applications. In

Acknowledgements

This work was supported by an AHA National Grant-in-Aid 0150586N, the NIH/NHLBI Program in Genomic Applications and by the NIH RO-1 HL64041.

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