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Physiol. Genomics 24: 173-180, 2006; doi:10.1152/physiolgenomics.00308.2004
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Received 23 December 2004; accepted in final form 14 November 2005.
Physiological Genomics 24:173-180 (2006)
1094-8341/05 $8.00 © 2006 American Physiological Society

Noninvasive indirect imaging of vascular endothelial growth factor gene expression using bioluminescence imaging in living transgenic mice

Yanling Wang1,2, Meera Iyer1,2, Alexander Annala1,2, Lily Wu3, Michael Carey4 and Sanjiv S. Gambhir1,2,5

1 The Crump Institute for Molecular Imaging
2 Department of Molecular and Medical Pharmacology
3 Division of Urology
4 Department of Biological Chemistry, David Geffen School of Medicine at University of California, Los Angeles
5 Departments of Radiology and Bioengineering, Bio-X Program, Molecular Imaging Program at Stanford, Stanford University School of Medicine, Stanford, California


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Vascular endothelial growth factor (VEGF) plays a critical role in the early activation of stromal tissues during wound healing and tumor growth. We report the use of a two-step transcriptional amplification (TSTA) approach to augment the transcriptional activity of the relatively weak VEGF promoter (pVEGF) using firefly luciferase (fl) reporter gene and bioluminescence imaging (BLI). In cell culture, we demonstrate that TSTA-based fl gene expression can be significantly enhanced over the direct one-step system. Using a transgenic mouse model (pVEGF-TSTA-fl), we demonstrate the induction of VEGF gene expression using a wound-healing model and a subcutaneous mammary tumor model. In skin-wounding experiments, pVEGF-induced fl expression in the wound lesion is detected on days 4 and 5 and peaks on days 15–22. Furthermore, the bioluminescence signal shows good correlation with the endogenous VEGF protein levels in the wound tissue (r2 = 0.70). In the mammary tumor model, fl expression is detected on day 3, peaks at day 17, and declines thereafter. These results support the use of noninvasive BLI for the longitudinal monitoring of VEGF induction during wound healing and tumor progression, and this mouse model should find use in various applications in which it is important to noninvasively study VEGF gene expression.

vascular endothelial growth factor induction; firefly luciferase; transgenic model; molecular imaging; two-step transcriptional amplification


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) has been recognized as one of the main regulators of angiogenesis and plays an important role in neovascularization occurring under both physiological and pathological conditions (9, 32). Upregulation of VEGF production has also been demonstrated during wound repair in keratinocytes in skin wounds in rat and mice (3). Early stages of wound repair are characterized by increased vascular permeability, and the role of VEGF in wound healing became apparent with increased VEGF production in skin wounds (3). The role of VEGF in angiogenesis during wound healing has been well documented (3, 6, 24).

Growth and metastasis of solid tumors is highly dependent on the formation of new blood vessels. Tumor-derived angiogenic cytokines have been known to play a crucial role in neovascularization. Several tumor types are known to express potent angiogenic cytokines such as basic fibroblast growth factor (FGF-2), VEGF, and interleukin-8 (1, 19, 26). Because the progressive growth of tumors beyond a minimum size is dependent on angiogenesis, the early activation of adjacent stromal tissues with VEGF is a significant event in tumor cell proliferation and survival. VEGF is known to be upregulated in many cancers, inflammatory responses, and ischemic conditions (9, 30).

VEGF gene expression is also strongly controlled by environmental stress, such as hypoxia and hypoglycemia. VEGF levels are upregulated under hypoxic conditions and stimulate angiogenesis to support wound healing or tumor growth (21). VEGF expression has also been known to be upregulated in the presence of heavy metal ions (8). VEGF's role as an angiogenesis initiator has led to its recognition as a potential target for antiangiogenic therapies. The ability to monitor upregulated VEGF levels in vivo in a noninvasive and quantitative manner will allow monitoring of early events during angiogenesis. The potential of noninvasive molecular imaging as a modality to monitor reporter gene expression in living animals has been realized in several applications (11, 12, 13). These include, among others, the use of positron emission tomography (PET) and bioluminescence imaging (BLI) (4, 14, 22, 33). Presently, there are not many direct approaches to monitor endogenous VEGF levels during wound healing, although the indirect approaches described next have been attempted. Transgenic mice carrying VEGF promoter (pVEGF) have been used to study the expression of green fluorescent protein (gfp) reporter gene during wound healing and tumor growth (10). Transgenic mice carrying the human VEGF promoter were also used to detect VEGF promoter activity in the skin (18). These approaches have been somewhat limited due to poor depth penetration of light, autofluorescence leading to background signal, and lack of quantitation. The advent of recent techniques such as multichannel imaging technology and fluorescence-mediated optical tomography may help to overcome these issues and lead to their increased use in monitoring early VEGF activation (20, 25). We have been exploring ways to indirectly measure VEGF gene expression in living mice using in vivo BLI. In the recent past, BLI has been increasingly used for the real-time analysis of gene expression in small animal models (4, 5). Technological advancements have led to the development of highly sensitive detection cameras capable of imaging small levels of light emitted from rodent tissues. A major advantage of using BLI is the very low level of background signal compared with fluorescence-based imaging strategies.

We have been studying the transcriptional activity of pVEGF using firefly luciferase (fl) in living mice. Our initial work in cell culture and in vivo using the vector representing the one-step system (pVEGF-fl) resulted in relatively low levels of fl expression (unpublished data). To enable sensitive monitoring of VEGF induction using in vivo imaging assays, the level of gene expression must be considerably higher. Therefore, strategies to augment the transcriptional activity of pVEGF were warranted. We have previously demonstrated that the transcriptional activity of the weak, prostate-specific promoter can be significantly enhanced using the yeast transcriptional activator GAL4-VP16 (17). This strategy, known as the two-step transcriptional amplification (TSTA) system, resulted in a 50-fold increase in prostate-specific fl expression over the one-step system (prostate-specific promoter directly driving fl expression). In the present study, we extended the utility of the TSTA system to augment pVEGF-driven fl expression levels.

The objectives of the present study were 1) to enhance the transcriptional activity of pVEGF and evaluate its induction efficacy in cell culture using hypoxia and cobalt chloride and 2) to create a transgenic mouse model to monitor induction of pVEGF-mediated fl expression during wound healing and tumor growth.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Construction of plasmids.
pVEGF-fl was made by cloning a 3,019-bp KpnI to HindIII genomic fragment (–2,274 to +745) containing the human pVEGF (31) upstream of fl cDNA in a pGL2-basic vector (Promega) (23). pVEGFVP2G5-fl was made by ligating the KpnI/HindIII fragment of human pVEGF from pVEGF-fl with the HindIII/NotI fragment of GAL4VP2 and the NotI/KpnI fragment of G5E4T-fl/pGL3 from the single TSTA vector pBCVP2G5-fl (35). pVEGFVP1G5-fl was made by ligating three DNA fragments, the KpnI/HindIII fragment of human pVEGF, the HindIII/NotI fragment of GAL4VP1 from plasmid pPSEBCGAL4VP1 (35), and the NotI/KpnI fragment of G5E4T-fl/pGL3. pVEGFVP2G2-fl was made by cloning the G2-fl fragment into KpnI and SalI sites of pVEGFVP2G5-fl. Note that "p" refers to the plasmid, and "p" denotes the promoter. The number of VP16 activation domains in the constructs was one and two (VP1 and VP2) (7). In the reporter template, the number of Gal4-binding sites upstream of fl varied from one to five (G1-fl, G2-fl, and G5-fl). The three single vectors generated by combining the effector and reporter templates are pVEGFGAL4VP2G5-fl (pVEGFVP2G5-fl), pVEGFGAL4VP1G5-fl (pVEGFVP1G5-fl), and pVEGFGAL4VP2G2-fl (pVEGFVP2G2-fl) (Fig. 1A). Figure 1B illustrates the construct used to generate transgenic mice. Note that GAL4 refers to the transactivator, and Gal4 refers to the binding sites placed upstream of the fl gene.



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  		Fig. 1. A: vectors used in the study. B: schematic illustration of the pVEGF-TSTA gene construct used to generate transgenic mice. The pVEGF is placed upstream of the GAL4-VP16 fusion protein carrying two VP16 activation domains. The reporter template comprises two Gal4-binding sites positioned upstream of the adenovirus E4 TATA minimal promoter driving the fl reporter gene. VEGF, vascular endothelial growth factor; pVEGF, promoter of VEGF; TSTA, two-step transcriptional amplification; fl, firefly luciferase.

 
Cell lines and transfection conditions.
HeLa (human cervical carcinoma) cells were obtained from American Type Culture Collection (ATCC, Manassas, VA). NK2 (FVB/N mouse mammary tumor) cells were a kind gift from Timothy Lane's laboratory at University of California, Los Angeles (UCLA). HeLa and NK2 cells were grown in DMEM medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum, 0.2 mM glutamine, 100 units/ml penicillin-G, and 100 mg/ml streptomycin. The transfections were carried out using Superfect transfection reagent (Qiagen, Valencia, CA). Plasmids used in the transient transfection of HeLa cells are listed in Fig. 1A. Twenty-four hours after transient transfection, the cells were replated into nine plates with fresh medium and grown for 24 h. After 24 h, three of the nine plates were exposed to hypoxia for 48 h (1 or 0% O2, 5% CO2, 94% N2), three plates were incubated in the presence of cobalt chloride for 48 h (final concentration of 100 µM), and three were left under normal incubation conditions for 48 h. Stable clones (expressing pVEGF-fl and pVEGFVP2G2-fl) were selected from HeLa cells with 500 µg/ml G418 and thereafter maintained at a concentration of 300 µg/ml. The stable cells were assayed for FL activity under hypoxia and cobalt chloride conditions at 6, 24, 32, 48, and 56 h.

Assay for FL activity.
After 48-h induction, the transiently transfected cells were harvested and lysed with passive lysis buffer (Promega, Madison, WI). FL activity was measured using the luciferase assay kit from Promega in a luminometer (Turner Designs, Sunnyvale, CA). The relative light units were normalized to the micrograms of protein in each well. Triplicate observations were made at each time point.

Assay of VEGF protein by ELISA.
Cell supernatants from triplicate plates were assayed for human VEGF165 isoform using a human VEGF ELISA kit (Oncogene Research Products, Boston, MA). Color development was recorded on a 96-well plate reader (GENios TECAN, Salzburg, Austria). The results were quantified using a linear range standard curve obtained with recombinant human VEGF as control.

Generation of pVEGF-TSTA-fl transgenic mice.
To prepare a linear transgene for microinjection, the single TSTA vector in pGL3 backbone was digested with NotI and SalI. The 1.2-kb pVEGF-VP2G2-fl DNA fragment was purified by agarose gel electrophoresis and prepared for injection. The fragment was microinjected into fertilized embryos of FVB/N mice. The presence of fl in the transgenic founders was confirmed by PCR amplification of DNA obtained from tail biopsies using oligonucleotide primers 5'-CCGACTCTAGAGGATCCCC-3' (E4T) and 5'-CAGCGGATAGAATGGCGCCGGGCC-3' (fl). About 0.5–1 cm of tail was digested in tail lysis buffer containing proteinase K (0.55 mg/ml). The DNA was precipitated with isopropanol, rinsed with ethanol, and resuspended in 50 µl of buffer. One microliter was used for PCR genotyping. The founders were crossed with wild-type FVB/N mice to generate F1 offspring. The F1 mice were crossed with wild-type FVB/N mice to generate F2 offspring. Mice from F1 and F2 progeny were used in this study.

Imaging pVEGF induction during wound healing.
All animal experiments were reviewed and approved by the UCLA Animal Research Committee. Three- to six-week-old pVEGF-TSTA-fl transgenic mice were anesthetized with ketamine-xylazine (4:1). A full-thickness wound (0.5 cm in diameter) was created by excising the dorsal skin and the underlying panniculus carnosus. The mice were serially imaged with D-luciferin (150 mg/kg body wt, ip injection) in the CCD camera on days 2 and 5 and subsequently every day until day 25. A total of six mice were studied in this experimental group. To study the correlation between the observed bioluminescence signal and endogenous VEGF induction during wound healing, the fl expression in four littermates was monitored by CCD camera (Xenogen, Alameda, CA). The four mice were killed on days 19, 20, 21, and 22, and the wound skin (1-cm diameter) and the underlying panniculus carnosus were harvested and stored frozen at –80°C. To determine endogenous VEGF gene expression in the wound region, the wound tissues were homogenized in 0.5 ml of PBS. The supernatants were used for VEGF ELISA assay using a mouse VEGF ELISA kit (R&D Systems, Minneapolis, MN). The VEGF protein level was normalized to the total protein in each sample. A total of eight mice were studied in this experimental group. To visualize the kinetics of VEGF expression at early times (days 1–4), four punch wounds each were created on days 1–4 on the back of three female transgenic mice (n = 3). The mice were imaged in the CCD camera before wounding and 4 days after wounding. The wound tissues were harvested and assayed for VEGF protein levels (ELISA).

Induction of pVEGF activity in a mouse mammary tumor model.
Adult pVEGF-TSTA-fl transgenic mice were anesthetized with ketamine-xylazine (4:1). NK2 cells (mouse mammary tumor, 5 x 106) were injected subcutaneously on the lower right flanks of the mice. The mice were serially imaged with D-luciferin (150 mg/kg body wt, ip injection) in the CCD camera from day 3 until day 29. Four mice were imaged in this experimental group. NK2 cells (5 x 106) were also injected subcutaneously on the lower right flanks of naive FVB mice. The tumor growth was closely monitored every 3 days until day 29, after which the mice were killed.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Construction of the pVEGF-TSTA-fl system.
The three single vector constructs used in this study are shown in Fig. 1A. We employed a 3-kb human pVEGF to drive the expression of a GAL4-VP2 fusion protein (containing 2 VP16 activation domains) (34). The GAL4-VP2 in turn binds to two Gal4-binding sites placed upstream of a TATA minimal promoter driving fl in a single vector (Fig. 1B). This transcriptional amplification strategy results in significant amplification of fl expression when compared with the direct one-step system (pVEGF-fl). We constructed three different plasmids and evaluated their efficacy in cell culture.

TSTA system-mediated fl expression in HeLa cells is significantly greater than the one-step system.
Transient transfection of HeLa cells using the three TSTA vectors described in Fig. 1A was carried out in the absence and presence of hypoxia and cobalt chloride. The cells were also transfected with pVEGF-fl (1-step) to compare fl expression driven by the one-step system. We observe a significantly high level of FL activity under hypoxic conditions using all three plasmids. The FL induction levels for pVEGFVP1G5-fl, pVEGFVP2G5-fl, and pVEGFVP2G2-fl were 1.9, 3.9, and 5.6-fold, respectively, under hypoxia. We observe similar induction results using cobalt chloride. Furthermore, FL activity mediated by the TSTA system is significantly greater than that driven by the one-step system, pVEGF-fl (1,294-, 333-, and 47-fold for pVEGFVP2G5-fl, pVEGFVP1G5-fl, and pVEGFVP2G2-fl, respectively) (P < 0.01) under hypoxia (Fig. 2). These results demonstrate the strong ability to titrate the levels of fl expression by varying the number of VP16 activation domains and the Gal4-binding sites. We also observed that the background FL activities in the TSTA system were also amplified under normal conditions, and the plasmid pVEGFVP2G2-fl showed the highest fold induction under hypoxia with minimum background. This plasmid was subsequently used to create stable cells and a transgenic mouse line.



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  		Fig. 2. TSTA system-mediated firefly luciferase (FL) activity in HeLa cells. The three constructs, pVEGFVP1G5-fl (V1G5), pVEGFVP2G5-fl (V2G2), and pVEGFVP2G2-fl (V2G5), and one-step pVEGF-fl were used for transient transfections in HeLa cells. FL activities under normal (Norm), hypoxic (Hyp), and cobalt chloride (Co++) conditions are represented as relative light units (RLU)·min–1·µg protein–1. Each point represents the mean of three observations. Because the values from the FL activities using pVEGF-fl were significantly lower than the two-step plasmids, these are enlarged for clarity. Error bars represent the standard error between triplicate samples.

 
FL activity increases under hypoxia and cobalt chloride and demonstrates good correlation with endogenous VEGF levels.
HeLa cells stably transfected with pVEGF-VP2G2-fl show a significant increase in FL activity when exposed to hypoxia and cobalt chloride. The FL activity postincubation increases with time up to 56 h and demonstrates an excellent correlation with endogenous VEGF levels under hypoxia (Fig. 3A, r2 = 0.92) and in the presence of cobalt chloride (Fig. 3B, r2 = 0.96). These results demonstrate the feasibility of indirectly measuring endogenous VEGF induction by detecting FL activity in stably transfected HeLa cells. HeLa cells stably transfected with the one-step and two-step systems were assayed for FL activity after exposure to hypoxia. FL activity in cells carrying the TSTA system was threefold greater than that observed with the one-step system (data not shown).



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  		Fig. 3. Correlation between VEGF levels and FL activities in HeLa cells stably transfected with pVEGFVP2G2-fl under hypoxia (A) and cobalt chloride conditions (B). Cells were assayed for FL activity at different time points (6, 24, 32, 48, and 56 h, as described in MATERIALS AND METHODS).

 
pVEGF-driven fl expression is highly upregulated during wound healing and demonstrates a good correlation with endogenous VEGF protein levels.
The pVEGF-TSTA-fl transgenic mice were imaged before creation of a wound to obtain basal bioluminescence signal. Color images of visible light are superimposed on photographic images of mice with a scale in photons per second per square centimeter per steradian (p·s–1·cm–2·sr–1). After wound creation, the bioluminescence signal emitted from the wound lesion was detected on days 4 and 5 and increased over time with the peak signal observed on day 15. Peak bioluminescence signal was detected between days 15 and 20 across several mice. After day 22, the bioluminescence signal started to decline until day 29. Figure 4A illustrates serial CCD camera imaging in a pVEGF-TSTA-fl transgenic mouse. The bioluminescence signal on day 5 was measured at 2.7 x 106 p·s–1·cm–2·sr–1, which subsequently increased to 5.2 x 106 p·s–1·cm–2·sr–1 and 1.7 x 107 p·s–1·cm–2·sr–1 on days 9 and 15, respectively, followed by a decrease thereafter (1.5 x 105 p·s–1·cm–2·sr–1). The intensity of the background bioluminescent signal across mice varied from 1.0 to 7.7 x 105 p·s–1·cm–2·sr–1 over the duration of the experiment. To examine the correlation of fl expression with endogenous VEGF levels in the wound tissue, wounds were created in four pVEGF-TSTA-fl transgenic mice. The mice were killed, and the wound tissue was removed on days 19, 20, 21, and 22 after wounding, when the bioluminescence signal intensities in the wound area were 6.3 x 105, 3.7 x 106, 7.4 x 106, and 1.7 x 106 p·s–1·cm–2·sr–1 (Fig. 4B). The bioluminescence signal in a sample of normal skin used as a control was 4 x 105 p·s–1·cm–2·sr–1. Endogenous VEGF levels in the wound tissues in four mice measured using a mouse VEGF ELISA assay kit were 51.8, 83.2, 87.9, and 75.4 pg/ml. The VEGF level in the control mouse was 42.9 pg/ml. Figure 4C illustrates a plot of maximum bioluminescence signal vs. endogenous VEGF gene expression level in the wound tissue. A good correlation is observed between the two values (r2 = 0.70). VEGF expression in vivo was also measured in the early days (days 1–4) after wounding using a mouse VEGF ELISA assay. VEGF protein expression in the wound tissue was found to increase from day 1 to day 4 (34.0 ± 5.0, 54.0 ± 6.0, 51.0 ± 6.0, and 118.0 ± 21.0 pg VEGF/µg protein, averaged across 3 mice, P < 0.05; Fig. 5). The control sample (obtained using the bioluminescence signal in a sample of normal skin) had a VEGF protein level of 18.4 ± 8.4 pg VEGF/µg protein. Although there was evidence of increasing VEGF expression on days 1–4, bioluminescence signal was detected in the wound area only on day 4. The lack of bioluminescence signal during the early days is likely due to several factors. These include substrate delivery (not enough substrate reaching the target site), relatively low light output due to low levels of transcriptional activation, and low light transmitted due to absorption and scattering (22). Nevertheless, these findings suggest that in vivo bioluminescence imaging can be used to monitor VEGF expression in vivo a few days after skin wounding.



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  		Fig. 4. Induction of fl expression during wound healing in a pVEGF-TSTA-fl transgenic mouse. A: mouse was imaged before wound creation in the CCD camera (day 0) and imaged again subsequent to wound creation every 4–5 days using D-luciferin (150 mg/kg ip). B: optical CCD images of four pVEGF-TSTA-fl transgenic mice (mice 1–4) on days 19, 20, 21, and 22 after creation of the wound. Arrows indicate the position of the wounds. C: correlation plot of maximum bioluminescence signal (p·s–1·cm–2·sr–1) vs. endogenous VEGF levels in the wound tissue (r2 = 0.70). Error bars represent the standard error for triplicate samples in the ELISA assay.

 


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  		Fig. 5. Kinetics of VEGF expression at early times after skin wounding. Four punch wounds each were created on days 1–4 on the back of three female transgenic mice (n = 3). The mice were imaged in the CCD camera before wounding and 4 days after wounding. On the 5th day, the wound tissues were harvested and assayed for VEGF protein levels (ELISA).

 
VEGF induction in transgenic mice can be monitored using a subcutaneous mammary tumor model.
The pVEGF-TSTA-fl mice were implanted with NK2 cells in the right bottom flank and subsequently imaged in the CCD camera from day 3 until day 29. Figure 6A shows the longitudinal monitoring of pVEGF-induced fl expression in the area around the tumor on days 3, 8, 10, 15, 17, 22, 24, and 29. Bioluminescence signal is detected on day 3 (1.1 x 106 p·s–1·cm–2·sr–1) and continues to increase until day 17 (5.8 x 106 p·s–1·cm–2·sr–1) followed by a considerable decline on days 22 (1.4 x 106 p·s–1·cm–2·sr–1) and 29 (2.8 x 105 p·s–1·cm–2·sr–1). The tumors started to regress after 2 wk, and the bioluminescence signal also showed a corresponding decline. The initial increase in signal from day 4 to day 17 is 5-fold, and the subsequent decrease on days 22 and 29 is 4-fold and 20-fold, respectively. Figure 6B shows a bar graph of the maximum photon counts in the area around the tumor (averaged across 4 mice) on different days during the imaging period. To study whether the tumor regression was specific to pVEGF-TSTA-fl transgenic mice, we injected wild-type FVB mice with NK2 cells and followed the tumor growth until day 29. We observed that the kinetics of tumor growth were similar to that observed in the transgenic mice. The tumor continued to grow until day 14 and then started to regress. By day 29, there was no visible tumor. Tumor regression with NK2 cells in FVB mice has also been observed by others (personal communication with Dr. Tim Lane at UCLA).



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  		Fig. 6. Longitudinal monitoring of VEGF promoter activity in pVEGF-TSTA-fl transgenic mice using a mouse mammary tumor model. A: pVEGF-TSTA-fl transgenic mice were injected subcutaneously with 5 x 106 NK2 cells in the lower right flank. Mice were imaged on day 4 and subsequently every few days until day 29 using D-luciferin as the reporter substrate (150 mg/kg, injected ip). CCD camera imaging revealed a detectable bioluminescence signal in the area around the tumor on day 4. Signal intensity continued to increase until day 17, after which a considerable decline in signal was observed (days 22–29). B: bar graph of maximum photons in the area around the tumor (p·s–1·cm–2·sr–1) during different days of imaging. Data were averaged across four mice.

 
pVEGF-TSTA-fl transgenic mice display normal physical characteristics with no observed deleterious effects from the transactivator.
The GAL4-VP16 transactivation system is a powerful tool for amplifying transgene expression from a weak promoter. However, it has been reported that high levels of the transactivator can be detrimental to cells (29). In the present study, comparison of litter weights, growth rates, and weaning weights indicated no abnormal differences between the transgenic and wild-type mice. Recently, we have reported the development and imaging of a prostate-specific TSTA transgenic mouse model (15). We did not observe any deleterious effects of the transactivator on the progeny of the TSTA or pVEGF-TSTA-fl transgenic mice.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Noninvasive imaging of VEGF activation during wound healing and tumor growth will provide a better understanding of the role of VEGF under these conditions. In the current work, the full-length human pVEGF was used to drive fl reporter gene expression using a TSTA strategy. Endogenous VEGF activation was accomplished in cell culture using cobalt chloride and hypoxia and in vivo using a wound-healing model and a mouse mammary tumor model in pVEGF-TSTA-fl transgenic mice. VEGF activation as visualized by fl expression was clearly demonstrated in both the wound lesion and growing tumor for up to several days. The bioluminescence signal in the tumor continued to increase until day 17, followed by a decrease that was maintained up to the end of the imaging period. A similar trend was observed in all mice studied. It is likely that the bioluminescence signal around the tumor site is the result of VEGF induction in the surrounding stroma. We studied VEGF expression in the NK2 cells and found that the cells express VEGF at high levels. However, because these cells do not carry the fl reporter gene, the bioluminescence signal observed in the area around the tumor in the days after cell implantation can be attributed to VEGF induction in the stroma. The VEGF that is made by the tumor cells does not lead to bioluminescence in the current model. The VEGF induction can also in part be from inflammatory cells. Future studies can include models in which the tumor expression of VEGF is monitored by a separate reporter gene while the current model measures stromal activation of VEGF. The spatial resolution in the current imaging does not allow us to distinguish stromal cells that may have infiltrated the tumor from the tumor itself. Evidence from a previous study suggests that the most prominent cells showing VEGF promoter activation are spindle-shaped cells that display structural features of fibroblasts (10). Future studies with this transgenic model will need to look at the cell types involved in signal production. We observed a decrease in tumor size after 14 days with a corresponding decrease in the bioluminescence signal. The regression in tumor size was observed not only with the pVEGF-TSTA-fl transgenic mice but also in naive FVB mice injected with NK2 cells. In the wound-healing experiments, pVEGF-induced fl expression in the wound area was detected on day 5, and peak bioluminescence signal was observed on days ranging from 15 to 22. The bioluminescence signal showed a good correlation with the endogenous VEGF protein levels in the wound tissue on days 5–21 (r2 = 0.70). VEGF protein levels measured in the early days after skin wounding continued to increase from day 1 to day 4. However, bioluminescence signal was detected in the wound tissue only on day 4. The absence of a signal in the early days may be related to the amount of substrate (D-luciferin) reaching the target site and absorption and scattering of light leading to a relatively low light output. In the current study, we used a tumor cell line that regressed with time. Future studies using nonregressing cell lines will be helpful in further characterizing this transgenic model. This study demonstrates that the imaging strategies employed allow noninvasive and repetitive monitoring of endogenous VEGF gene expression during wound healing and tumor progression through the use of a noninvasive reporter gene assay with excellent correlations in cell culture and good correlations in living transgenic mice.

Our initial attempts to drive the reporter gene directly with the pVEGF were not robust enough for imaging in living mice. Although gene induction using the one-step system was possible, the absolute level of reporter activity was found to be very low. For this reason, we employed the TSTA system that we have previously validated (17, 35) to help overcome the relatively weak transcriptional activity of pVEGF. This led to significant gene induction and a high absolute reporter signal after induction, but at the expense of a higher background signal before induction. The absolute levels of reporter gene expression varied based on the choice of the V2G2, V1G5, or V2G5 TSTA strategies (Fig. 2). We chose the V2G2 TSTA system for the current transgenic model because we wanted to maximize the induced fl bioluminescent signal while not increasing the background preinduction signal too much. The background signal, due to basal levels of reporter gene expression before induction of pVEGF, did not pose a significant problem because it is still relatively low for in vivo imaging studies compared with the signal after gene induction. The background signal for V1G5 and V2G5 is considerably higher based on the results in Fig. 2. Future studies may be able to further optimize maximizing the induced reporter signal while minimizing the preinduction background signal.

A key goal in developing the transgenic model for this work was to allow for noninvasive monitoring of endogenous VEGF gene expression. A single previous study has used gfp as a reporter (10) to monitor VEGF levels but failed to demonstrate whether the qualitative signal correlates with endogenous VEGF gene expression. In fact, no attempts to relate the signal to endogenous VEGF gene expression were undertaken in that work. Furthermore, gfp has some specific limitations for applications in living subjects because of poor depth penetration and background autofluorescence (2). The development of newer and more advanced techniques such as fluorescence-mediated optical tomography and multichannel imaging may lead to considerable improvements in relation to depth penetration and quantitation. Improvements in BLI, including BLI tomography, may also help quantitation and depth information when using the transgenic developed in this study. The current mouse model incorporating amplified promoter activity and bioluminescence imaging should be very useful to the research community. Recently, Zhang et al. (36) have reported the kinetics of VEGF receptor type 2 (VEGFR2) gene expression in different pathophysiological processes using BLI (36). The authors reported the generation of a transgenic mouse model wherein fl reporter gene expression is driven by a murine VEGFR2 promoter. The authors observed induction of VEGFR2-fl expression in endothelial cells during wound healing. The VEGFR2 transgenic model represents a one-step system where a 0.5-kb fragment from the first VEGFR2 intron was placed downstream of the fl gene. This led to increased VEGFR2 expression in endothelial cells. The transgenic developed in this work allows the study of VEGF gene expression, whereas the above study allows the study of VEGFR2 expression, and both models should complement each other. In the present study, the use of the fl reporter coupled with the TSTA strategy allows very low background signal and highly sensitive detection at all depths throughout the mouse. Although in the current work we have focused on superficial wounds, the strategies developed should also facilitate signal detection at any depth within rodents. In the current work, we successfully demonstrated that the reporter signal correlates both in cell culture (r2 > 0.90) and in living transgenic mice (r2 = 0.70) with endogenous VEGF gene expression. The correlation was not as high in vivo as it was in cell culture, likely reflecting that it is harder to measure absolute regional VEGF levels in the wound and limitations due to heterogeneity within a given region. Future studies will have to further explore this correlation with more animals under more experimental conditions in addition to wound healing. Also, additional studies to look at serum VEGF in addition to regional tissue VEGF should add further utility.

The TSTA strategy has been used by us previously to create a transgenic model for prostate-specific expression (16). In that model and in the current model, we did not observe any gross toxicity with expression of GAL4 and VP16. Although there may be some subtle effects related to expression of these foreign genes, we did not observe anything at a more gross level in the current study. Future studies will need to continue to explore any potential toxicity of this system in transgenic mice models. An ideal system would enable monitoring of VEGF activation in any tissue at any depth with high sensitivity in a living intact subject. In this study, we have developed a transgenic mouse that allows noninvasive assessment of VEGF in various tissues and has a slightly poorer level of detection compared with direct tissue biopsy. The key advantage is the ability to serially monitor the same mouse over time to study changes in VEGF activation. This transgenic mouse model can potentially be useful in evaluating in vivo any new drugs that directly or indirectly modulate VEGF transcriptional activation. Molecular imaging of living subjects is allowing many applications in which therapies can be tested in vivo. This allows optimization of in vivo pharmacokinetics and can accelerate drug testing in preclinical models. In addition, this mouse can be mated with mice carrying other reporters to study activation of multiple genes. For example, we are now studying the interaction of expression of the Cox-2 gene with the VEGF gene by mating the two appropriate mice carrying different reporters. As many different reporter mice are developed with different pathways that can be monitored, this will likely lead to insights into cell biology and testing of novel therapies. We therefore feel this mouse model will be useful for many different purposes.

Although currently studied with a bioluminescent reporter, future transgenics should be able to be developed with multimodality tri-fusion reporters (27, 28). This would allow imaging of the same animal using fluorescence, bioluminescence, and PET. This will likely provide greater tomographic and quantitative detail than the current model. With continued refinement and exploration of other models, the VEGF transgenic mouse model described in the present study should be useful to investigators for many different applications in which repetitive monitoring of endogenous VEGF gene expression is desired.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grants P50-CA-86306 (to S. S. Gambhir) and SAIRP-R24-CA92865 (to S. S. Gambhir) and Department of Energy Contract DE-FC03-87ER60615 (to S. S. Gambhir).


   ACKNOWLEDGMENTS
 
We extend our sincere thanks to Tom Fielder (Univ. of California, Irvine) for the generation of the transgenic mice.


   FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: S. S. Gambhir, Stanford Univ. School of Medicine, James H. Clark Center, 318 Campus Dr., 1E, Stanford, CA 94305-5427 (e-mail: sgambhir{at}stanford.edu)

10.1152/physiolgenomics.00308.2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Abdulrauf SI, Edvardsen K, Ho KL, Yang XY, Rock JP, and Rosenblum ML. Vascular endothelial growth factor expression and vascular density as prognostic markers of survival in patients with low-grade astrocytoma. J Neurosurg 88: 513–520, 1998.[Medline]
  2. Billinton N and Knight AW. Seeing the wood through the trees: A review of techniques for distinguishing green fluorescent protein from endogenous autofluorescence. Anal Biochem 291: 175–197, 2001.[CrossRef][ISI][Medline]
  3. Brown LF, Yeo KT, Berse B, Yeo TK, Senger DR, Dvorak AM, and Livingston WVD. Expression of vascular permeability factor (vascular endothelial growth factor) by epidermal keratinocytes during wound healing. J Exp Med 176: 1375–1379, 1992.[Abstract/Free Full Text]
  4. Contag CH and Bachmann MH. Advances in in vivo bioluminescence imaging of gene expression. Annu Rev Biomed Eng 4: 235–260, 2002.[CrossRef][ISI][Medline]
  5. Contag PR, Olomu IN, Stevenson DK, and Contag CH. Bioluminescent indicators in living mammals. Nat Med 4: 245–247, 1998.[CrossRef][ISI][Medline]
  6. Detmar M, Brown LF, Claffey KP, Yeo KT, Kocher O, Jackman RW, Berse B, and Dvorak AM. Overexpression of vascular permeability factor/vascular endothelial growth factor and its receptors in psoriasis. J Exp Med 180: 1141–1146, 1994.[Abstract/Free Full Text]
  7. Emami KH and Carey M. A synergistic increase in potency of a multimerized VP16 transcriptional activation domain. EMBO J 11: 5005–5012, 1992.[ISI][Medline]
  8. Eyssen-Hernandez R, Ladoux A, and Frelin C. Differential regulation of cardiac heme oxygenase-1 and vascular endothelial growth factor mRNA expressions by hemin, heavy metals, heat shock and anoxia. FEBS Lett 382: 229–233, 1994.
  9. Ferrara N. Molecular and biological properties of vascular endothelial growth factor. Semin Cancer Biol 77: 527–543, 1999.
  10. Fukumura D, Xavier R, Sugiura T, Chen Y, Park EC, Lu NF, Selig M, Nielsen G, Taksir T, Jain RK, and Seed B. Tumor induction of VEGF promoter activity in stromal cells. Cell 94: 715–725, 1998.[CrossRef][ISI][Medline]
  11. Gambhir SS, Barrio JR, Herschman HR, and Phelps ME. Assays for noninvasive imaging of reporter gene expression. Nucl Med Biol 26: 481–490, 1999.[CrossRef][Medline]
  12. Gambhir SS, Barrio JR, Phelps ME, Iyer M, Namavari M, Satyamurthy N, Wu L, Green LA, Bauer E, MacLaren DC, Nguyen K, Berk AJ, Cherry SR, and Herschman HR. Imaging adenoviral-directed reporter gene expression in living animals with positron emission tomography. Proc Natl Acad Sci USA 96: 2333–2338, 1999.[Abstract/Free Full Text]
  13. Gambhir SS, Barrio JR, Wu L, Iyer M, Namavari M, Satyamurthy N, Bauer E, Parrish C, MacLaren DC, Borghei AR, Green LA, Sharfstein S, Berk AJ, Cherry SR, Phelps ME, and Herschman HR. Imaging of adenoviral-directed herpes simplex virus type 1 thymidine kinase reporter gene expression in mice with radiolabeled ganciclovir. J Nucl Med 39: 2003–2011, 1998.[Abstract/Free Full Text]
  14. Gambhir SS, Bauer E, Black ME, Liang Q, Kokoris MS, Barrio JR, Iyer M, Namavari M, Phelps ME, and Herschman HR. A mutant herpes simplex virus type 1 thymidine kinase reporter gene shows improved sensitivity for imaging reporter gene expression with positron emission tomography. Proc Natl Acad Sci USA 97: 2785–2790, 2000.[Abstract/Free Full Text]
  15. Iyer M, Salazar F, Lewis X, Zhang L, Carey M, Wu L, and Gambhir SS. Noninvasive imaging of enhanced prostate-specific gene expression using a two-step transcriptional amplification-based lentivirus vector. Mol Ther 10: 545–552, 2004.[CrossRef][Medline]
  16. Iyer M, Salazar F, Lewis X, Zhang L, Wu L, Carey M, and Gambhir SS. Noninvasive imaging of a transgenic mouse model using a prostate-specific two-step transcriptional amplification strategy. Transgenic Res 14: 47–55, 2005.[CrossRef][ISI][Medline]
  17. Iyer M, Wu L, Carey M, Wang Y, Smallwood A, and Gambhir SS. Two-step transcriptional amplification as a method for imaging reporter gene expression using weak promoters. Proc Natl Acad Sci USA 98: 14595–14600, 2001.[Abstract/Free Full Text]
  18. Kishimoto J, Ehama R, Ge Y, Kobayashi T, Nishiyama T, Detmar M, and Burgeson RE. In vivo detection of human vascular endothelial growth factor promoter activity in transgenic mouse skin. Am J Pathol 157: 103–110, 2000.[Abstract/Free Full Text]
  19. Kumar R, Kuniyasu H, Bucana CD, Wilson MR, and Fidler IJ. Spatial and temporal expression of angiogenic molecules during tumor growth and progression. Oncol Res 10: 301–311, 1998.[ISI][Medline]
  20. Mahmood U, Tung CH, Tang Y, and Weissleder R. Feasibility of in vivo multichannel optical imaging of gene expression: experimental study in mice. Radiology 224: 446–451, 2002.[Abstract/Free Full Text]
  21. Marti HH and Riasu W. Systemic hypoxia changes the organ-specific distribution of vascular endothelial growth factor and its receptors. Proc Natl Acad Sci USA 95: 15809–15814, 1998.[Abstract/Free Full Text]
  22. Massoud TF and Gambhir SS. Molecular imaging in living subjects: seeing fundamental biological processes in a new light. Genes Dev 17: 545–80, 2003.[Free Full Text]
  23. Mueller DM, Vigne JL, Minchenko A, Lebovic DI, Leitman DC, and Taylor RN. Regulation of vascular endothelial growth factor (VEGF) gene transcription by estrogen receptors alpha and beta. Proc Natl Acad Sci USA 97: 10972–10977, 2000.[Abstract/Free Full Text]
  24. Nissen NN, Polverini PJ, Koch AE, Violin MV, Gamelli RL, and DiPietro LA. Vascular endothelial growth factor mediates angiogenic activity during the proliferative phase of wound healing. Am J Pathol 152: 1445–1452, 1998.[Abstract]
  25. Ntziachristos V, Bremer C, and Weissleder R. Fluorescence imaging with near-infrared light: New technological advances that enable in vivo molecular imaging. Eur Radiol 13: 195–208, 2003.[ISI][Medline]
  26. Potgens AJ, Westphal HR, de Waal RM, and Ruiter DJ. The role of vascular permeability factor and basic fibroblast growth factor in tumor angiogenesis. Biol Chem Hoppe Seyler 376: 57–70, 1995.[ISI][Medline]
  27. Ray P, De A, Min JJ, Tsien RY, and Gambhir SS. Imaging tri-fusion multimodality reporter gene expression in living subjects. Cancer Res 64: 1323–1330, 2004.[Abstract/Free Full Text]
  28. Ray P, Wu AM, and Gambhir SS. Optical bioluminescence and positron emission tomography imaging of a novel fusion reporter gene in tumor xenografts of living mice. Cancer Res 63: 1160–1165, 2003.[Abstract/Free Full Text]
  29. Sadowski I, Ma J, Triezenberg S, and Ptashne M. GAL4-VP16 is an unusually potent transcriptional activator. Nature 335: 563–564, 1988.[CrossRef][Medline]
  30. Senger DR, Van de Water L, Brown LF, Nagy JA, Yeo KT, Yeo TK, Berse B, Jackman RW, Dvorak AM, and Dvorak HF. Vascular permeability factor (VPF, VEGF) in tumor biology. Cancer Metastasis Rev 12: 303–324, 1993.[CrossRef][ISI][Medline]
  31. Tischer E, Mitchell R, Hartman T, Silva M, Gospodarowicz D, Fiddes JC, and Abraham JA. The human gene for vascular endothelial growth factor—multiple protein forms are encoded through alternative exon splicing. J Biol Chem 266: 11947–11954, 1991.[Abstract/Free Full Text]
  32. Veikkola T and Alitalo K. VEGFs, receptors and angiogenesis. J Mol Med 9: 211–220, 1999.[CrossRef]
  33. Wu JC, Sundaresan G, Iyer M, and Gambhir SS. Noninvasive optical imaging of firefly luciferase reporter gene expression in skeletal muscles of living mice. Mol Ther 4: 297–306, 2001.[CrossRef][ISI][Medline]
  34. Wu L, Matherly J, Smallwood A, Belldegrun AS, and Carey M. Chimeric PSA enhancers exhibit augmented activity in prostate cancer gene therapy vector. Gene Ther 8: 1416–1426, 2001.[CrossRef][Medline]
  35. Zhang L, Adams JY, Billick E, Ilagan R, Iyer M, Le K, Smallwood A, Gambhir SS, Carey M, and Wu L. Molecular engineering of a two-step transcription amplification (TSTA) system for transgene delivery in prostate cancer. Mol Ther 5: 223–232, 2002.[CrossRef][ISI][Medline]
  36. Zhang N, Fang Z, Contag PR, Purchio AF, and West DB. Tracking angiogenesis induced by skin wounding and contact hypersensitivity using a Vegfr2-luciferase transgenic mouse. Blood 103: 617–626, 2004.[Abstract/Free Full Text]



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