Ultrasound contrast-enhanced study as an imaging biomarker for anti-cancer drug treatment: preliminary study with paclitaxel in a xenograft mouse tumor model (secondary publication)

PURPOSE: The purpose of this study was to assess tumor angiogenesis using contrast-enhanced ultrasonography (CEUS) of human prostate cancer cells (PC3) that were implanted in mice before and after paclitaxel injection. METHODS: Twelve mice were injected with human PC3. The mice were grouped into two groups; one was the paclitaxel-treated group (n=6) and the other was the control group (n=6). Before administering paclitaxel into the peritoneal cavity, baseline CEUS was performed after the administration of 500 μL (1×108 microbubbles) of contrast agent. The area under the curve (AUC) up to 50 seconds after injection was derived from the time-intensity curves. After injection of paclitaxel or saline, CEUS studies were performed at the 1-week follow-up. Changes in tumor volume and the AUC in both two groups were evaluated. After CEUS, the microvessel density (MVD) was compared between the groups. RESULTS: In the paclitaxel-treated group, the AUC from CEUS showed a significant decrease 1-week after paclitaxel administration (P=0.030), even though the tumor volume showed no significant changes (P=0.116). In the control group, there was no significant decrease of the AUC (P=0.173). Pathologically, there was a significant difference in MVD between both groups (P=0.002). CONCLUSION: The AUC from the time intensity curve derived from CEUS showed an early change in response to the anti-cancer drug treatment that preceded the change in tumor size. The findings of CEUS could serve as an imaging biomarker for assessing tumor responses to anti-cancer drug treatment.

Nucleic Acid Aptamers: New Methods for Selection, Stabilization, and Application in Biomedical Science

The adoption of oligonucleotide aptamer is well on the rise, serving an ever increasing demand for versatility in biomedical field. Through the SELEX (Systematic Evolution of Ligands by EXponential enrichment), aptamer that can bind to specific target with high affinity and specificity can be obtained. Aptamers are single-stranded nucleic acid molecules that can fold into complex threedimensional structures, forming binding pockets and clefts for the specific recognition and tight binding of any given molecular target. Recently, aptamers have attracted much attention because they not only have all of the advantages of antibodies, but also have unique merits such as thermal stability, ease of synthesis, reversibility, and little immunogenicity. The advent of novel technologies is revolutionizing aptamer applications. Aptamers can be easily modified by various chemical reactions to introduce functional groups and/or nucleotide extensions. They can also be conjugated to therapeutic molecules such as drugs, drug containing carriers, toxins, or photosensitizers. Here, we discuss new SELEX strategies and stabilization methods as well as applications in drug delivery and molecular imaging.

Emerging Roles of Human Prostatic Acid Phosphatase

Prostate cancer is one of the most prevalent non-skin related cancers. It is the second leading cause of cancer deaths among males in most Western countries. If prostate cancer is diagnosed in its early stages, there is a higher probability that it will be completely cured. Prostatic acid phosphatase (PAP) is a non-specific phosphomonoesterase synthesized in prostate epithelial cells and its level proportionally increases with prostate cancer progression. PAP was the biochemical diagnostic mainstay for prostate cancer until the introduction of prostate-specific antigen (PSA) which improved the detection of early-stage prostate cancer and largely displaced PAP. Recently, however, there is a renewed interest in PAP because of its usefulness in prognosticating intermediate to high-risk prostate cancers and its success in the immunotherapy of prostate cancer. Although PAP is believed to be a key regulator of prostate cell growth, its exact role in normal prostate as well as detailed molecular mechanism of PAP regulation is still unclear. Here, many different aspects of PAP in prostate cancer are revisited and its emerging roles in other environment are discussed.

US Contrast Enhanced Study as an Imaging Biomarker of Anti-cancer Drug Treatment: Preliminary Study with Paclitaxel and a Xenograft Mouse Tumor Model

PURPOSE: We wanted to assess tumor angiogenesis of human prostate cancer cells (PC3) implanted in mice before and after paclitaxel injection via contrast-enhanced ultrasonography (CEUS). MATERIALS AND METHODS: Twelve mice were injected with human prostate cancer cells (PC3) on the back or hind limbs. The mice were grouped into two groups; one was the paclitaxel treated group (n = 6) and the other was the control group, which was treated with normal saline (n = 6). Before injection of paclitaxel into the peritoneal cavity, baseline CEUS was performed by the administration of 500 microl (1x108 microbubbles) of contrast agent. The area under the curve (AUC) up to 50 seconds after contrast injection was derived from the time-intensity curves. After injection of paclitaxel or saline, one week follow up CEUS studies were performed. The changes of the tumor volume and the AUC in both two groups were evaluated. After CEUS, the mice were sacrificed and the microvessel density (MVD) was compared. RESULTS: In the paclitaxel treated group, the AUC from CEUS showed a significant decrease one week after paclitaxel administration (p = 0.03), even though the tumor volume showed no significant changes (p = 0.116). In the control group, there was no significant decrease of the AUC (p = 0.173). Pathologically, there was a significant difference of microvessel density in both groups (p = 0.002). CONCLUSION: The AUC from the time intensity curve derived from CEUS showed early change in response to the anti-cancer drug treatment in advance of a tumor size response. The findings of CEUS could be an imaging biomarker for assessing the tumor response to anti-cancer drug treatment.

Molecular Imaging in the Age of Genomic Medicine

The convergence of molecular and genetic disciplines with non-invasive imaging technologies has provided an opportunity for earlier detection of disease processes which begin with molecular and cellular abnormalities. This emerging field, known as molecular imaging, is a relatively new discipline that has been rapidly developed over the past decade. It endeavors to construct a visual representation, characterization, and quantification of biological processes at the molecular and cellular level within living organisms. One of the goals of molecular imaging is to translate our expanding knowledge of molecular biology and genomic sciences into good patient care. The practice of molecular imaging is still largely experimental, and only limited clinical success has been achieved. However, it is anticipated that molecular imaging will move increasingly out of the research laboratory and into the clinic over the next decade. Non-invasive in vivo molecular imaging makes use of nuclear, magnetic resonance, and in vivo optical imaging systems. Recently, an interest in Positron Emission Tomography (PET) has been revived, and along with optical imaging systems PET is assuming new, important roles in molecular genetic imaging studies. Current PET molecular imaging strategies mostly rely on the detection of probe accumulation directly related to the physiology or the level of reporter gene expression. PET imaging of both endogenous and exogenous gene expression can be achieved in animals using reporter constructs and radiolabeled probes. As increasing numbers of genetic markers become available for imaging targets, it is anticipated that a better understanding of genomics will contribute to the advancement of the molecular genetic imaging field. In this report, the principles of non-invasive molecular genetic imaging, its applications and future directions are discussed.

hVEGF121 transfection and animal model for in vivo angiogenesis / 대한내과학회지

BACKGROUND: This study was designed to prove the superiority of ACP vector containing ITR, and to establish animal model quantifying angiogenesis in vivo. METHODS: hVEGF121, therapeutic gene, was inserted to various vectors (pcDNA3.1, pcDNA 3.2, pActin, pDesm, pACP vector), and these vectors were transfected to various cells using FuGENE6. We cultured for 48hrs, and then quantified amounts of hVEGF121 of supernatants by ELISA. The long-term transfection was assessed for 14 days. Optimal condition of transfection was evaluated by change of the ratio of DNA to FuGENE6, amount of DNA, and confluence of cells. ACP-hVEGF121 was transfected to C2C12 and these transfected C2C12 cells were mixed with Matrigel, and then injected to C3H mouse subcutaneously. Seven days later, hemoglobin assay and pathology of Matrigel were reviewed for angiogenesis. RESULTS: The level of hVEGF121 gene expression using pACP vector was significantly higher than those of others. In 2 weeks culture study, pACP vector showed the highest gene expression and produced VEGF until 2 weeks. The highest gene expression was obtained when the concentration of DNA was 7 microgram, the confluence was up to 80% and the ratio of DNA to FuGENE6 was 1:3. The hemoglobin level in Matrigel of VEGF group was significantly higher than the one of the control group, and active angiogenesis was noted in the VEGF group. CONCLUSIONS: pACP vector might be an efficient vector for angiogenic gene delivery, and animal model using Matrigel and transfected C2C12 cell could be a useful tool for quantitative angiogenesis assay.

Cardiac expression profiles of the naked DNA vectors encoding vascular endothelial growth factor and basic fibroblast growth factor

We investigated expression profiles and biological effects of the naked DNA vectors in the heart. To this end, naked DNA vector was injected into the apex of the beating rat heart after thorocotomy. When the expression of LacZ reporter was examined by reverse transcription-PCR and histochemical staining for b-galactosidase, LacZ expression was detected only in the heart, suggesting limited dissemination of the injected vector in vivo. Even within the heart, LacZ expression was limited to the injection area (apex). Similar observations were made with other transgenes such as VEGF and basic fibroblast growth factor (bFGF), where 77% and 69% of the total transgene exprssion were detected in the heart segments containing the apex. Although VEGF and bFGF expressions were detected until 2 weeks after DNA injection, the highest levels of VEGF and bFGF were observed on day 5 and day 1, respectively. The optimal doses of the vectors were 10 mg and 25 mg for the VEGF and bFGF vectors, respectively. Interestingly, injection of bFGF vector led to 50% increase in the level of endogenous murine VEGF expression. Consistent with this finding, the number of vessels that stained positive for alpha-smooth muscle actin was increased in the bFGF vector-injected heart. These results suggest that simple injection of naked DNA vector may be sufficient to induce significant angiogenesis in the myocardium and that naked DNA gene therapy may be a feasible approach for the treatment of ischemic heart disease.

Monocrotaline-induced pulmonary hypertension correlates with upregulation of connective tissue growth factor expression in the lung

Pulmonary hypertension (PH) is characterized by structural and functional changes in the lung including proliferation of vascular smooth muscle cells (VSMCs) and excessive collagen synthesis. Although connective tissue growth factor (CTGF) is known to promote cell proliferation, migration, adhesion, and extracellular matrix production in various tissues, studies on the role of CTGF in pulmonary hypertension have been limited. Here, we examined CTGF expression in the lung tissues of male Sprague Dawley rats treated with monocrotaline (MCT, 60 microgram/kg), a pneumotoxic agent known to induce PH in animals. Establishment of PH was verified by the significantly increased right ventricular systolic pressure and right ventricle/left ventricle weight ratio in the MCT-treated rats. Histological examination of the lung revealed profound muscular hypertrophy in the media of pulmonary artery and arterioles in MCT-treated group. Lung parenchyma, vein, and bronchiole did not appear to be affected. RT-PCR analysis of the lung tissue at 5 weeks indicated significantly increased expression of CTGF in the MCT-treated group. In situ hybridization studies also confirmed abundant CTGF mRNA expression in VSMCs of the arteries and arterioles, clustered pneumocytes, and infiltrated macrophages. Interestingly, CTGF mRNA was not detected in VSMCs of vein or bronchiole. In saline-injected control, basal expression of CTGF was seen in bronchial epithelial cells, alveolar lining cells, and endothelial cells. Taken together, our results suggest that CTGF upregulation in arterial VSMC of the lung might be important in the pathogenesis of pulmonary hypertension. Antagonizing the role of CTGF could thus be one of the potential approaches for the treatment of PH.

Vascular endothelial growth factor-induced angiogenic gene therapy in patients with peripheral artery disease

This phase 1 clinical trial tested the safety of intramuscular gene transfer by using naked plasmid DNA encoding the gene for VEGF, and analyzed the potential therapeutic benefits in patients with severe peripheral arterial disease (PAD). This study was an open-labeled, dose- escalating, single-center trial on nine male patients with severe debilitating PAD who had not responded to conventional therapy. Seven had Buerger's disease and two had arteriosclerosis obliterans. Plasmid DNA (pCK) containing human VEGF165 was given by eight intramuscular injections in and around the area in need of new blood vessels. The study evaluated three escalating total doses (2, 4, and 8 mg of pCK- VEGF165), with half of each total dose given four weeks apart. The follow-up duration was nine months. The gene injections were well tolerated without significant side effects or laboratory abnormalities related to gene transfer. Three patients showed transient edema in their extremities. Ischemic pain of the affected limb was relieved or improved markedly in six of seven patients. Ischemic ulcers healed or improved in four of six patients. The mean ankle-brachial index (ABI) improved significantly. Six of nine patients showed an increase in collateral vessels around the injection sites demonstrated by digital subtraction angiography. However, there was no relationship between the degree of ABI improvement and the dose given. Mean plasma levels of VEGF did not increase significantly. In conclusion, intramuscular injections of pCK- VEGF165 can be performed safely to induce therapeutic angiogenesis in patients with severe PAD.

Aldosterone Upregulates Connective Tissue Growth Factor Gene Expression via p38 MAPK Pathway and Mineralocorticoid Receptor in Ventricular Myocytes

The effect of aldosterone on connective tissue growth factor (CTGF) was examined in rat embryonic ventricular myocytes. Upon aldosterone treatment, CTGF expression was significantly increased in a dose and time-dependent manner. To explore the molecular mechanism for this upregulation, we examined the role of mineralocorticoid receptor. Pre-treatment of an antagonist (spironolactone) at 5-fold excess of aldosterone blocked the CTGF induction by aldosterone, suggesting that the upregulation was mediated by mineralocorticoid receptor. Aldosterone treatment resulted in activation of ERK1/2, p38 MAPK, and JNK pathways with a more transient pat-tern in p38 MAPK. Blocking studies using pre-treatment of the inhibitor of each path-way revealed that p38 MAPK cascade may be important for aldosterone-mediated CTGF upregulation as evidenced by the blocking of CTGF induction by SB203580 (p38 MAPK inhibitor), but not by PD098059 (ERK1/2 inhibitor) and JNK inhibitor I. Interestingly, JNK inhibitor I and PD098059 decreased the basal level of CTGF expression. On the other hand, pre-treatment of spironolactone abrogated the p38 MAPK activation, indicating that mineralocorticoid receptor mechanism is linked to p38 MAPK pathway. Taken together, our findings suggest that aldosterone induces CTGF expression via both p38 MAPK cascade and mineralocorticoid receptor and that cross-talk exists between the two pathways.

Decreased Number and Impaired Angiogenic Function of Endothelial Progenitor Cells in Patients with Chronic Renal Failure

BACKGROUND AND OBJECTIVES: The increased risk of cardiovascular disease in patients with chronic renal failure (CRF) has been explained by accelerated atherosclerosis and impaired angiogenesis, where endothelial progenitor cells (EPC) may play key roles. It was hypothesized that : "an altered EPC biology may contribute to the pathophysiology of CRF". SUBJECTS AND METHODS: EPC were isolated from CRF patients on maintenance hemodialysis (n=44) and from a normal control group (n=30). After morphological and immunological characterization, the number and in vitro angiogenic function of the EPC were evaluated. RESULTS: CRF patients showed markedly decreased numbers of EPC (44.6%) and colonies (75.3%) compared to the controls (p<0.001). These findings were corroborated by a 30.5% decrease in the migratory function in response to vascular endothelial growth factor (VEGF)(p=0.040) and by a 48.8% decrease in EPC incorporation into human umbilical vein endothelial cells (HUVEC)(p<0.001). In addition, The Framingham's risk factor scores of both the CRF (r=-0.461, p=0.010) and normal groups (r=-0.367, p=0.016) were significantly correlated with the numbers of EPC. Indeed, under the same burden of risk factors the number of circulating EPC was significantly lower in CRF patients than in the normal group (p<0.001). A significant correlation was also observed between the dialysis dose (Kt/V) and EPC incorporation into the HUVEC (r=0.427, p=0.004). CONCLUSION: The EPC biology, which is critical for neovascularization and the maintenance of vascular function, was altered in CRF. Our data strongly suggest that dysfunction of circulating EPC has a role in the progression of cardiovascular disease in patients with CRF.

Probing the Utility of Vascular Smooth Muscle Cells as a Target Cell for ex vivo Cardiovascular Gene Therapy

BACKGROUND AND OBJECTIVES: Compared to other target cells examined for gene therapy, vascular smooth muscle cells (VSMCs) have the unique advantages including proximity to blood stream and relative abundance in vasculature. With an ultimate goal of developing VSMC-based therapies for cardiovascular disorders, we explored the utility of VSMC as a target cell for ex vivo gene therapy using a set of retroviral vectors. MATERIALS AND METHODS: Cultured VSMCs were transduced with replication-defective recombinant retroviruses harboring LacZ, nlsLacZ, mVEGF, mGM-CSF or bacterial CAT reporter. The VSMCs were examined for G418-selection, transduction efficiency, the level of transgene expression, and longevity of gene expression. ResultsVSMCs were readily transduced with different kinds of retroviral vectors. The bacterial neo r gene-transduced VSMCs were successfully selected with G418. The G418-selected VSMCs could express the transduced genes at a level comparable to NIH3T3. The level of transgene expression did not appear to be affected by the increasing number of passages. CONCLUSION: The results demonstrate an efficient transduction of VSMCs by retroviral vectors in vitro and an sustained expression of retrovirally transduced genes in VSMCs. VSMCs could be one of the ideal target cells for ex vivo cardiovascular gene therapy employing retroviral vector.

The Effects of Aldosterone and Cytokines IL-1beta, TNF-alpha on the Expression of Angiotensin Converting Enzyme Gene in Vascular Smooth Muscle Cells

BACKGROUND: It has been suggested that all components of the renin-angiotensin-aldosterone system (RAAS) are present in the vascular wall and that the vascular RAAS modulates vascular tone and vascular hypertrophy. One of the catalytic step in the RAAS cascade is the local conversion of angiotensin I to angiotensin II (Ang II) by angiotensin converting enzyme (ACE). One of the major sources of ACE in the vasculature is vascular smooth muscle cells (VSMC). Here, we provide insight into the intrinsic mechanisms by which the components of RAAS regulate gene expression of ACE in cultured smooth muscle cells of the rat and we also investigated the effects of cytokines on ACE mRNA. METHODS: RNA was extracted from the primary cultured VSMCs. We analyzed the expression levels of ACE by competitive reverse transcription-PCR using recombinant RNA as an internal standard. RESULTS: 1) ACE mRNA level was increased markedly by aldosterone in a dose- and time-dependent manner, indicating that there exists positive feedback mechanism within RAAS. 2) The induction of ACE mRNA by aldosterone was inhibited by spironolactone. 3) Aldosterone-stimulated expression of ACE was also inhibited by Ang II, which shows that Ang II acts as a negative regulator of the expression of ACE in RAAS cascade. 4) Interleukin-1beta or TNF-alpha did not induce ACE mRNA expression. 5) However, mixture of interleukin-1betaand TNF-alpha(CytoMix) significantly increased the expression of ACE. It was also shown that CytoMix increased aldosterone-stimulated ACE mRNA expression in an additative manner. CONCLUSION: These results indicate that the expression of ACE in smooth muscle cells is modulated by the components of RAAS and cytokines. The intrinsic positive and negative feedback controls of RAAS would play an important role in the pathogenesis of vascular diseases.