Ultrasound contrast agents are widely used for diagnostic ultrasound imaging procedures, but questions have been raised about their safety based on the results of in vitro and animal studies. Notably, the combination of US and US contrast agents has been shown to produce vascular damage. A number of these studies report damage to endothelial layer of blood vessels exposed to US and US contrast agents. In this paper, we have explored the physiological consequences of endothelial damage following combined exposure to US and Optison. When combined, this treatment can have marked vascular effects in an ex vivo vascular preparation.
As shown in Figures 2 and 3, a marked reduction in contractile response was noted when Optison was combined with US treatment. Phenylephrine is an-adrenergic agonist that induces smooth muscle contraction. The inability of the aortic segment to contract implies that there is a profound injury to smooth muscle. This observation is in agreement with an earlier study which showed dose-dependent smooth muscle damage in ex vivo porcine carotid arteries exposed to both US and Optison . Similarly, cardiomyocyte viability has also been compromised by the combination of US and Optison following in vivo exposure .
Relaxation of arteries by acetylcholine is due to the release of soluble factors from endothelium. As shown in Figures 4 and 5, impaired relaxation was noted when US and Optison were combined. Brayman et al. noted that endothelial monolayers were readily damaged by US and Optison . In rabbit ears, adherence of platelets was observed following US and Optison treatment. Platelet aggregation and injury to endothelial cells were more severe when the contrast agent and US were combined . Importantly, US and Optison can induce marked microvascular damage in rat mesentery [7, 15].
Endothelial cells synthesize and release multiple forms of VEGF, which is required to maintain vascular integrity as well to promote angiogenesis . The most common forms of VEGF interact with tyrosine coupled receptors, FLT-1 and FLK-1, on the surface of the endothelium to modulate function in an auto-regulatory fashion. Staining for these three entities was markedly reduced in the US plus Optison group (Figure 6). A slightly decreased DAB signal was seen in either US or Optison treatment.
In this study, endothelial apoptosis was noted during the combined treatment of US and Optison (Figure 7). Importantly, endothelial apoptosis has been found in rabbit corneal endothelial cells treated with both US and Optison in vivo. Using confocal microscopy, US and Optison was observed to induce apoptosis at low energy. As emphasized in their study, damage was limited to the endothelial layers, and the internal elastic lamina may protect the smooth muscles cells from inertial cavitation . It should be noted that agents such as Optison have the propensity to bind to activated endothelial cells. If the binding of Optison takes place in vivo as well, atherosclerotic lesions may be at risk during US imaging.
Optison, in conjunction with US, has been used for gene therapy, drug delivery, angiogenesis studies, imaging vascular injury and evaluating cardiac function. As with any therapy, there are often untoward effects that need to be balanced the potential benefits. Given that there is the potential for widespread collateral effects, preclinical evaluation of ultrasound contrast agents is warranted which is consistent with the results presented in Figures 3, 5, 6 and 7.
The goal of US directed gene therapy is to identify and transfect selected anatomical structures with the gene(s) of interest. Vascular beds are ideal targets given the relative ease in identification and the delivery of the DNA of interest to the relevant tissues. The transfer of genetic material such as plasmid into a cell requires the brief disruption of the membrane. Additionally, signaling pathways such as ERK are activated and may play a role in the eventual expression of the transfected DNA, probably through mechanical sensing via integrins. US activates ERK ½ signaling via rock in skin fibroblasts .
Both endothelial cells and smooth muscle cells can be transfected with plasmids by Optison and US . C-myc expression was decreased following transfection with anti-sense morpholino oligomers in porcine arteries treated ex vivo. Neointimal proliferation was inhibited following balloon injury when anti-sense p53 plasmids or decoy E2F decoy oligo nucleotides, Optison and ultrasound were used to transfect rat carotid arteries [21, 22]. The contractile response to prostaglandin was reduced in porcine carotid arteries that were transfected with eNOS ex vivo. Plasmid DNA and viruses can be transduced into skeletal muscle through an intra vascular route using US and US contrast agents . In sum, the use of both Optison and US enhances the nonviral gene transfer is an alternative to using viral vectors . While these studies focused upon transfer of genetic material, there was no attempt to evaluate untoward thrombus formation.
Inflamed endothelial cells express cell adhesion molecules such as Intracellular adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM). Both ICAM and VCAM can be used to image inflammation and the inflamed tissue can be targeted for in vivo drug delivery as well. A gas-filled microbubble with anti-ICAM-1 antibody on its shell specifically binds to activated endothelial cells over expressing ICAM-1 . Significantly, the endothelial BBB can be altered by ultrasound and contrast agents and may be a means of delivering drugs to the CNS due to altered permeability [27, 28]. Other vascular effects include micro vessel rupture, and cell death in the rat spinotrapezius muscle using Optison . Further, petechiae, and capillary leakage was observed in the mouse abdomen following use of Optison . Clearly, the coupling of integrin specific MAB to US contrast reagents may generate tissue specific contrast reagents but, there are nonspecific and collateral damage that results from the combined use of Optison and Ultrasound.
Tumors are dependent upon the formation of neo-vessels for continued growth. Noninvasive, in vivo, imaging of vasculature is extremely important for identifying tumors. Most importantly, this permits a means of screening anti-tumor regimens in preclinical models and clinical applications. These imaging techniques also permit high-resolution, volumetric assessments of tumor vascularity. In a preclinical model of breast cancer is shown that correlates with other ultrasonographic measures of blood flow, which may provide greater sensitivity to the microvasculature in real time . The endothelium of tumor neo-vessels express vascular cell endothelial growth factor receptor 2 (VEGFR2). UCA MicroMarker has been with conjugated to anti VEGFR2 have been used to follow angiogenesis in a preclinical murine model for breast tumors . In a human xenograft melanoma model, immuohistochemical COX-2 staining of excised tumors correlated with the contrast-enhanced ultrasound image . The imaging of angiogenesis is dependent upon the expression of tumor or endothelial markers such as VEGFR2. The expression of VEGFR2 may be variable, depending upon the growth stage/size and in humans, clonality. These imaging techniques also permit high-resolution, volumetric assessments of tumor vascularity. The utility of following VEGF receptor and signaling kinases as a marker of endothelial integrity is amply demonstrated in this paper.
Inappropriate thrombus formation in the heart, brain or in a peripheral site is the hallmark of vascular disease. Imaging thrombus is an important application of Optison and US. Abciximab, which recognizes glycoprotein IIb/IIIa receptor was conjugated to Optison and the immuno bubbles enhance the image of arterial thrombus in vivo . In contrast, US and USCA can be used in combination to break apart moderate sized clots . In the rabbit ear, US and contrast agent were directed against the auricular vein. When fibrinogen was administered, the vein was occluded by an a thrombus . These examples illustrate the usefulness in identifying thrombus as well as inducing thrombus to inhibit blood flow to a target lesion. The loss of vascular function that we observed in this study illustrates the collateral damage that can be induced.
IVUS imaging of coronary and peripheral arteries is extremely useful technique to image plaque formation and vessel patency. Vulnerable plaque is an arterial lesion that has a propensity for rupture and thrombus formation. IVUS and contrast agent permits the visualization of vasa vasorum density and a combination of lipid core, cap thickness and calcification may help identify the plaques most likely to rupture .
Valvular stenosis can easily be visualized by ultrasound examination of the heart. The micro bubbles enhance the image of the ventricle making it easier to identify thrombus, calculate the volume of ejected blood and visualize wall motion. These functional studies are crucial for the clinical assessments of patients .
There are contradictory reports regarding the effect Optison on human cardiac function . Perventricular contractions (PVCs) were noted in the human heart . Troponin T was elevation was seen with Optison however, there were no negative histologic findings were seen . In an other study, no changes were seen in PVCs, Troponin I, CK, CK-MB in the human heart .
In a preclinical model using the rat heart, PVCs, and microvascular leakage was noted with Optison [43, 44]. Importantly, micro lesions were seen histologically with inflammatory infiltrates 24 hours post exposure in the rat heart [44, 45]. In glass catfish model, US and USCA revealed focal damage in the tail of fish. Importantly, this was a real time assessment of the damage, which was significant . Our results from this study are consistent with vascular damage that may contribute to the arrhythmias seen in vivo.
Rat hearts were subjected to both US and Optison, and cardiac arrhythmias were induced. Cessation of US treatment reversed the effect. However, no histological effect was seen . Rat hearts were treated with ultrasound and Optison, and the RNA was prepared for microarray analysis. The only gene up-regulated was carbonic anhydrase, so there was not dominant gene induction .
Use of ultrasound contrast agents, newly-developed microbubble-based products which are administered intravenously to enhance the ultrasound image quality, present new challenges with regard to clinical safety because of the locally destructive forces of inertial cavitation caused by the interaction of ultrasound with the micro bubbles. These destructive forces can damage the endothelium and smooth muscle of the vascular wall. The target patient population that may be exposed to microbubble/ultrasound is large and continues to grow as new applications and new products in this class are developed.
For example, increased permeability due to contrast agent-induced vascular damage can capitalized upon therapeutically to deliver genes and other large molecules across endothelium. However, an adverse event that may occur from microbubble-induced vascular lesions may be the initiation or acceleration of atherosclerotic progression. Since this modality is being considered for delivery of drugs and genes, an even larger patient population (who originally had no cardiovascular disease) will be exposed to long-term risks. Therefore, it is critical to identify the ultrasound and microbubble exposure conditions which cause damage to the vascular endothelium and determine whether microbubble-induced vascular damage increases the risk of atherosclerosis in selected populations.
There are several limitations in the present study: First, it was performed ex vivo under static conditions with no blood flow. Second, only one concentration of Optison was employed. In earlier studies (Miller, Dou, and Song 601–07; [6, 12, 39, 49], the microbubble concentration ranged from 0.01% to 2% and maximal RBC hemolysis was seen at 1%. In our experiment, we used 1% Optison, so we are using concentrations that are consistent with previous work. In clinical use, the recommended doses range from 0.5 to 5 ml of Optison infused in a 10 min period.
For these studies, we used a 2 MHz US wave employing a four-cycle tone burst simulating a pulsed Doppler mode and having an MI of 1.9, the maximum setting available on a clinical imaging unit. It is not clear whether similar effects could occur at MI values less than 1.9.
In conclusion, 2 MHz US with an MI of 1.9 and 1% Optison altered contraction and relaxation in rat dorsal aortas exposed ex vivo. The changes in arterial function may be due to damage of the endothelium and smooth muscle. This study provides insight into functional parameters of vascular function that may be compromised by US and Optison treatment.