1.

Introduction

Atherosclerotic cardiovascular disease remains the leading cause of death in the United States and other developed countries. Vascular gene therapy holds promise as an exciting alternative strategy to the existing therapeutic methods for dealing with the causes of atherosclerotic cardiovascular disease.¹ Many genes have been either experimentally or clinically shown to be useful to inhibit postangioplasty/in-stent restenosis, promote angiogenesis, reduce thrombogenesis, and prevent transplantation allograft vasculopathy.² A catheter-based local gene delivery approach has become a promising tool to localize a high dose of the transgene at a target site while minimizing undesirable systemic toxicity. To date, different imaging techniques, such as magnetic resonance (MR) imaging and nuclear imaging, as well as optical imaging, have been developed to monitor gene therapy.³ An in-vivo imaging modality can help answer several issues pertinent to the success of vascular gene therapy, including assessments of 1. desirable distribution and localization of transgene delivery at the target vessel wall; 2. whether there is a sufficient level of transgene expression for efficient therapeutic effect on the targets; and 3. the functional period of therapeutic genes at the targets.⁴

Optical imaging, which is based on the detection of fluorescence or emitted light from the luciferase-luciferin reaction, has shown some prominent advantages, including relative technical simplicity, portability, cost effectiveness, the ability to provide real-time imaging, and the lack of radiation. A previous in-vitro/ex-vivo study proved the principle of using optical imaging to detect vascular reporter gene expression.⁵ A subsequent study developed a surface optical imaging system, which offers the potential to detect green fluorescent protein (GFP) gene expression from superficially seated arteries.⁶

However, to detect reporter gene expression from deep-seated vessels, the surface optical imaging is limited by light signal loss due to problems of penetration depth of illumination light in tissue, and forward scattering of incident and emitted light. To circumvent these problems, an alternative strategy is to position the optical imager close to the gene-targeted vessels via a minimally invasive approach. This encouraged us to develop a percutaneous optical imaging system, which was specifically designed for in-vivo detection of imaging reporter gene expression from deep-seated arteries or organs.

2.

Materials and Methods

2.2.

Percutaneous Optical Imaging System

The percutaneous optical imaging system consisted of a dual-port fiber optic probe (Zibra Corporation, Westport, Massachusetts) [Fig. 1a ], a $250\text{-}\mathrm{W}$ broadband cold halogen light source (KL 2500 LCD, Schott, Germany), a thermally cooled charge-coupled device (CCD) camera (Sensi-Cam QE, Cooke Corporation, Michigan), and an image grabber installed on a personal computer. The fiber optic imaging probe was primarily modeled on a Y-shape milliendoscope, with one of the proximal ports connected to the cold halogen light source, which was equipped with a filter wheel containing an excitation filter [ $475\text{to}488\text{-}\mathrm{nm}$ (XF1072), Omega Opticals Incorporated, Brattleboro, Vermont) appropriate for excitation light for green fluorescence. The other proximal port was connected to the CCD camera through a bandpass emission filter [ $480\text{to}530\mathrm{nm}$ , (D505/40), Chroma Technology Corporation, Rockingham, Vermont), and a system of video relay lenses. A schematic showing the operation and layout of the optical imaging system is presented in Fig. 1b. The distal end of the probe was semi-rigid, $5\mathrm{cm}$ in length, and $1.5\mathrm{mm}$ in outer diameter. The probe consisted of an inner imaging core made of silica fiber bundles ( $10,000\text{-}\text{pixel}$ bundle) surrounded by loosely bound illumination fiber bundles made up of soft glass, and a series of objective lenses at the tip for imaging [Fig. 1c].

Fig. 1

(a) The dual-port percutaneous optical imaging probe, which can be positioned via a guiding needle. (b) The working mechanism of the percutaneous optical imaging system. (c) Schematic diagrams of the optical imaging probe, demonstrating the inner imaging core (green) surrounded by loosely bound illumination fiber bundles (yellow) (color online only).

A previous study using Monte Carlo simulation has analyzed the basic light transport of the percutaneous optical probe.⁷ Monte Carlo simulations showed that the optimal optical signal detection was at a probe tip distance of around $1.5\text{to}2\mathrm{mm}$ from the target vessel. At this distance, the incident fluorophore distribution was sufficient to produce a detectable amount of emitted fluence.⁷

2.3.

In-Vitro Experiments

2.3.1.

Phantoms

To investigate the performance of the percutaneous optical imaging probe, it was necessary to closely simulate the actual tissue properties, such as absorption and scattering, which modulate the incident illumination on the embedded fluorophores as well as the emitted fluorescence. The optical imaging setup for the in-vitro phantom studies is as shown in Fig. 2a . The inhomogeneous phantom was based on the model proposed by Durkin, Jaikumar, and Richards-Kortum.⁸ To model the fluorescence spectra of inhomogeneous tissue, solid phantoms were prepared with unflavored gelatin (Knox) as the matrix, because it served as a substrate with minimal absorption, scattering, and fluorescence [Fig. 2b]. Human hemoglobin containing 2% red blood cells (RBC) by volume (Sigma Chemical Company, Saint Louis, Missouri) was used as the absorber to mimic the double absorption windows of blood in this wavelength range. Polystyrene microspheres (Polysciences Incorporated, Warrington, Pennsylvania) with high diffuse reflectance and minimal fluorescence and absorption were used as the scattering media (0.625% by volume in sample solution). Initially, gelatin was dissolved in heated phosphate buffered saline (PBS), and then the hemoglobin and microspheres were stirred in. No other fluorophores were added to the phantom to prevent interference with fluorescence emitted from GFP. The phantom was allowed to cool and solidify overnight. A polyethylene terephthalate capillary tube ( $0.7\text{-}\mathrm{mm}$ inner diameter and $0.3\text{-}\mathrm{mm}$ wall thickness), embedded $6\mathrm{mm}$ below the surface, was used to mimic the blood vessel geometry. The optical images of the control, saline, and the GFP-positive cells are shown in Figs. 2c and 2d, respectively.

Fig. 2

(a) In-vitro experimental setup. The optical imaging probe vertically contacts the pink-colored phantom. (b) The human tissue-like phantom, through which a capillary tube is placed to mimic the vessel. (c) In-vitro optical images of the capillary tube filled with saline compared to (d) GFP-positive cells. Brighter light in the center of the capillary tube (arrows) is visualized with GFP/cells than with saline.

2.3.2.

Cell lines

A third-generation GFP-carrying lentivirus was used to carry and express GFP genes.⁹ It was used to transduce Hu-293 T cells. The Hu-293 T cells were cultured in high glucose Dulbecco’s modified Eagle’s medium (Invitrogen Corporation, California) containing 10% by volume of fetal bovine serum (FBS) (Sigma-Aldrich Corporation, Saint Louis, Missouri). On the day of the experiment, the adherent cells were washed with Trypsin and PBS, counted, and then washed and resuspended in PBS. Initially, the capillary tube was filled with PBS, and baseline data were obtained using the optical imaging system. The image parameters were an incident excitation $\text{power}=8\mathrm{mW}∕{\mathrm{cm}}^2$ , an exposure $\mathrm{time}=0.3\mathrm{s}$ , and a probe distance of $1\mathrm{mm}$ above the sample. Then, the PBS solution was replaced with a GFP-positive cell suspension $(7.5\text{million}\text{cells}∕\mathrm{ml})$ and was imaged using the same image capture parameters.

3.

Results

Of the in-vitro experiments, optical imaging demonstrated brighter light detected in the GFP-positive cell-containing tube than in the saline-containing tube [Figs. 2c and 2d]. Of the ex-vivo experiments, color Doppler allowed us to confirm vessel location, and the probe could be precisely positioned at a distance of $1\text{to}2\mathrm{mm}$ from the vessel under ultrasound imaging guidance (Fig. 3 ).

Fig. 3

Ultrasound-guided positioning of the optical imaging probe ex vivo. (a) The vessel [arrow on (a)] is localized by real-time ultrasound imaging and confirmed (b) by color Doppler. (c) Subsequently, the optical imaging probe (arrow) was positioned with its tip $2\mathrm{mm}$ from the vessel (the distance between the stars).

Of the in-vivo experiments using the surgery-based approach, the optical imaging of the exposed arteries showed higher average signal intensity from GFP-treated arteries than from saline-treated arteries for each of five different exposure times (Fig. 4 ). At exposure times of 10, 45, and $60\mathrm{s}$ , the average signal intensities were significantly higher in GFP-treated arteries than in saline-treated arteries $(p<0.05)$ .

Fig. 4

Data from the surgery-based optical imaging approach, presenting higher average signal intensities for GFP-treated arteries than for saline-treated arteries for all exposure times. $\ast p<0.05$ ; $\ast \ast p<0.01$ .

Of the in-vivo experiments using the percutaneous-based approach, the optical probe was successfully positioned with its tip $1\text{to}2\mathrm{mm}$ from the targeted arteries under ultrasound guidance (Fig. 5 ). The difference in emission light signals between GFP-treated arteries and saline-treated arteries could be differentiated under optical imaging (Fig. 5). Fluorescent microscopy examination confirmed that higher fluorescence in the media layer was detected in GFP-treated arteries than in saline-treated arteries.

Fig. 5

In-vivo percutaneous optical imaging of pig arteries. (a) Ultrasound imaging, demonstrating that the target femoral artery (open arrow) is imaged by the optical imaging probe (arrowheads). (b) and (c) Direct view of in-vivo optical images obtained from a percutaneous-based approach. The emission light is brighter in the (c) GFP-treated artery than in (b) the saline-treated artery. (d) and (e) Fluorescent microscopy examination of percutaneously imaged artery tissues, showing higher green fluorescence from both intima [arrows on (e)] and media (M) of (e) the GFP-treated artery compared to autofluorescence from internal elastic lamina [arrows on (d)] of (d) the saline-treated artery. (Magnification $20\times$ .)

4.

Discussion

Clinical imaging of vascular gene therapy includes two components: 1. assessing the success of the primary gene delivery procedure; and 2. tracking the functional period of vascular gene expression after the primary gene delivery.⁴ The current study outlines a potential method of exploring the second component, tracking vascular gene expression. In the scientific field of gene therapy, colored fluorescent protein genes, such as GFP genes, have been widely used as imaging reporters for in-vivo optical imaging.^{9, 11} GFP does not require the injection of any secondary substrates and does not appear to interfere with cell growth and function.¹²

Optical imaging is a promising molecular imaging tool to characterize and measure biologic processes at the cellular and molecular level in vivo.³ However, the primary disadvantage of in-vivo optical imaging is the limited ability to either send the excitation light into or detect the emission light from deep-seated tissues and organs in the body. Thus, the penetration depth of light in tissue prevents it from being an ideal imaging tool for whole body imaging of large animals and humans. This disadvantage is more obvious when using surface optical imaging to noninvasively detect fluorescent signals from deep-seated arteries, such as the iliac and renal arteries, as well as the aorta, which are the primary targets of gene therapy of atherosclerosis. To deal with this problem, we attempted to develop a percutaneous optical imaging system, expecting to detect vascular reporter gene expression using a minimally invasive approach. Our study presents encouraging evidence that a percutaneous optical imaging approach may help overcome the limited light penetration of optical imaging, which presents the potential to provide a useful, minimally invasive molecular imaging method to monitor reporter gene expression in vessels or deep-seated organs.

For percutaneous imaging, accuracy in positioning the probe with respect to the blood vessel is of paramount importance, as it affects the optimal probe distance from the region of maximum gene expression and the best focus point. Our ex-vivo study demonstrated that under ultrasound imaging guidance, the optical imaging probe could be precisely positioned within a distance of $1\text{to}2\mathrm{mm}$ of the target vessel. Subsequent surgery-based experiments further confirmed the performance of the percutaneous optical imaging probe in vivo. Finally, we successfully performed the entire procedure of percutaneously imaging fluorescent gene expression from vasculatures using an ultrasound-guided, minimally invasive approach in a preclinical setting, the living pig.

In current practice, the confirmation of successful gene transfection/expression depends primarily on different laboratory tests of harvested tissues obtained from either autopsy or biopsy. In its present state, our percutaneous optical imaging system may provide a useful minimally invasive imaging tool for basic science on cardiovascular gene therapy. The ultimate goal of developing in-vivo imaging techniques for gene therapy is to track the expression of therapeutic genes. Recent studies have presented the possibility of simultaneously encoding two different genes from the same vector.¹³ This kind of dual gene vector can be used to carry and express two genes simultaneously, e.g., an optical imaging reporter gene (such as the GFP gene) and a therapeutic gene [such as the vascular endothelial growth factor (VEGF) gene]. Thus, direct detection of the GFP gene by optical imaging should indirectly assess the functionality of VEGF, because the two genes are simultaneously expressed with the same vector.

The current report focused only on establishing the “proof of principle” of a new concept or a new technical development, using a minimally invasive and cost-effective percutaneous optical imaging method to detect fluorescent reporter gene expression in vivo. Further work is required to optimize the probe design with higher-grade optical fibers to reduce transmission losses, improve numerical aperture for higher signal capture efficiency, refine the cross section construction of the probe for simultaneous optimization of excitation and emission fluence, and apply a laser light source that would greatly enhance the incident illumination compared to a halogen light source because of higher coupling efficiency. Limitation of this study is the lack of sham transduced controls. It is essential to optimize the parameters for both local gene delivery and optical imaging, which requires the comparison between different gene/vector groups with controls.

Fluorescence microscopy identifies a more pronounced difference among the images, as it detects the fluorescence emitted from processed tissue sections embedded on slides, with no interfering media. The signal intensity obtained using the percutaneous probe does not reach a similar level of clarity because of interfering tissue and blood, and its own system limitations because of the sensitivity of the optical components, as mentioned before. Fluorophores with a higher range of excitation and emission wavelengths would reduce the autofluorescence from tissue within the body. Such an optical system may be extended for other methods of optical imaging, including the detection of light signals from luciferase and therapeutic genes labeled with other fluorophores.

In conclusion, currently there are no imaging techniques available for detecting gene expression from vasculatures in vivo. We have presented a technical development that uses a percutaneous optical imaging system as an in-vivo imaging tool to detect fluorescent reporter gene expression from vasculatures, thus providing a minimally invasive approach with the potential to monitor gene therapy in deep-seated vasculatures and organs in vivo.