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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.

Department of Molecular Biology, Dankook University, Yongin 168 90, KoreaReceived August 18, 2015/Revised August 24, 2015/Accepted August 25, 2015Optogenetics is the combination of optical and molecular strategies to control designated molecular and cellular activities in living tissues and cells using genetically encoded light-sensitive proteins. It involves the use of light to rapidly gate the membrane channels that allows for ion movement. Optogenetics began with the placing of light-sensitive proteins from green algae inside specific types of brain cells. The cells can then be turned on or off with pulses of blue and yellow light. Using the naturally occurring algal protein Channelrhodopsin-2 (ChR2), a rapidly gated light-sensitive cation channel, the number and frequency of action potentials can be controlled. The ChR2 provides a way to manipulate a single type of neuron while affecting no others, an unprecedented specificity. This technology allows the use of light to alter neural processing at the level of single spikes and synaptic events, yielding a widely applicable tool for neuroscientists and biomedical engineers. An improbable combination of green algae, lasers, gene therapy and fiber optics made it possible to map neural cir-cuits deep inside the brain with a precision that has never been possible before. This will help identify the causes of disorders like depression, anxiety, schizophrenia, addiction, sleep disorder, and autism. Optogenetics could improve upon existing implanted devices that are used to treat Parkinson’s dis-ease, obsessive-compulsive disorder and other ailments with pulses of electricity. An optogenetics de-vice could hit more specific subsets of brain cells than those devices can. Applications of optogenetic tools in nonneuronal cells are on the rise. Keywords

Prostatic acid phosphatase (PAP) expression increases proportionally with prostate cancer progression, making it useful in prognosticating intermediate to high-risk prostate cancers. A novel ligand that can specifically bind to PAP would be very helpful for guiding prostate cancer therapy. RNA aptamers bind to target molecules with high specificity and have key advantages such as low immunogenicity and easy synthesis. Here, human PAP-specific aptamers were screened from a 2'-fluoropyrimidine (FY)-modified RNA library by SELEX. The candidate aptamer families were identified within six rounds followed by analysis of their sequences and PAP-specific binding. A gel shift assay was used to identify PAP binding aptamers and the 6N aptamer specifically bound to PAP with a Kd value of 118 nM. RT-PCR and fluorescence labeling analyses revealed that the 6N aptamer bound to PAP-positive mammalian cells, such as PC-3 and LNCaP. IMR-90 negative control cells did not bind the 6N aptamer. Systematic minimization analyses revealed that 50 nucleotide sequences and their two hairpin structures in the 6N 2'-FY RNA aptamer were equally important for PAP binding. Renewed interest in PAP combined with the versatility of RNA aptamers, including conjugation of anti-cancer drugs and nano-imaging probes, could open up a new route for early theragnosis of prostate cancer.

Alanine. The ZnS:Mn-Gly and ZnS:Mn-Alananocrystal powders were characterized by XRD, HR-TEM, EDXS, ICP-AES, and FT-IR spectroscopy. Theoptical properties were measured by UV-Visible and photoluminescence (PL) spectroscopy. The PL spectrafor the ZnS:Mn-Gly and ZnS:Mn-Ala showed broad emission peaks at 599 nm and 607 nm with PL efficienciesof 6.5% and 7.8%, respectively. The measured averag e particle size from the HR-TEM images were 6.4 ± 0.8nm (ZnS:Mn-Gly) and 4.1 ± 0.5 nm (ZnS:Mn-Ala), which were also supported by Debye-Scherrer calculations.In addition, the degree of aggregation of the nanocrystals in aqueous solutions were measured by a hydro-dynamic light scattering method, which showed formation of sub-micrometer size aggregates for bothZnS:Mn-Gly (273 ± 94 nm) and ZnS:Mn-Ala (233 ± 34 nm) in water due to the intermolecular attractionbetween the capping amino acids molecules. Finally, the cytotoxic effects of ZnS:Mn-Gly and ZnS:Mn-Alananocrsystals over the growth of wild type E. coli were investigated. As a result, no toxicity was shown for theZnS:Mn-Gly nanocrystal in the colloidal concentration region from 1 µg/mL to 1000 µg/mL, while ZnS:Mn-Ala showed significant toxicity at 100 µg/mL.Key Words : ZnS:Mn nanocrystal, Glycine capping, Alanine capping, Cytotoxicity, AggregationIntroductionThe synthesis of nano-sized low-dimensional semicon-ductor nanocrystals and their applications have been one ofthe most attractive research subjects for the last fewdecades.

Mw = 10,000 g/mol) and O-(2-Carboxyethyl)polyethylene glycol(PEG-COOH, Mw = 10,000 g/mol) molecules. The modified PEG capped ZnS:Mn nanocrystal powders werethoroughly characterized by XRD, HR-TEM, EDXS, ICP-AES and FT-IR spectroscopy. The optical propertieswere also measured by UV/Vis and photoluminescence (PL) spectroscopies. The PL spectra showed broademission peaks at 600 nm with similar PL efficiencies of 7.68% (ZnS:Mn-PEG-NH2) and 9.18% (ZnS:Mn-PEG-COOH) respectively. The measured average particle sizes for the modified PEG capped ZnS:Mnnanocrystals by HR-TEM images were 5.6 nm (ZnS:Mn-PEG-NH2) and 6.4 nm (ZnS:Mn-PEG-COOH),which were also supported by Debye-Scherrer calculations. In addition, biological toxicity effects of thenanocrystals over the growth of wild type E. coli were investigated. They showed no biological toxicity toE. coli until very high concentration dosage of 1 mg/mL of the both nanocrystal samples.Key Words : ZnS:Mn nanocrystal, Polyethylene glycol derivatives capping, Water-dispersible nanocrystal,Biological toxicity IntroductionNanosized semiconductor materials have gained signi-ficant attention over the past decade.

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.

Mercaptoacetic acid (MAA) and mercaptopropionic acid (MPA) capped ZnS:Mn nanocrystals were synthesized and their physical characteristics were examined by XRD, HR-TEM, EDXS, and FT-IR spectroscopy. The optical properties of the MPA capped ZnS:Mn nanocrystals dispersed in aqueous solution were also measured by UV/Vis and solution photoluminescence (PL) spectra, which showed a broad emission peak around 598 nm (orange light emissions) with calculated relative PL efficiency of 5.2%. Comparative toxicity evaluation of the uncoordinated ligands, MAA and MPA, with the corresponding ZnS:Mn nanocrystals revealed that the original ligands significantly suppressed the growth of wild type E. coli whereas the ligandcapped nanocrystals did not show significant toxic effects. The reduced cytotoxicity of the conjugated ZnS:Mn nanocrystals was also observed in NIH/3T3 mouse embryonic fibroblasts. These results imply that potential toxicities of the capping ligands can be neutralized on ZnS:Mn surface.

Prostatic acid phosphatase (PAP) is one of the widely used biomarkers in the diagnosis of prostate cancer. It was initially identified in 1935 and is the most abundant phosphatase in the human prostate. PAP is a prostate-specific enzyme that is synthesized in prostate epithelial cells. It belongs to the acid phosphatase group that shows enzymatic activity in acidic conditions. PAP is abundant in prostatic fluid and is thought to have a role in fertilization and oligospermia. It also has a potential role in reducing chronic pain. But one of the most apparent functions of PAP is the dephosphorylation of macromolecules such as HER-2 and PI3P that are involved in the ERK1/2 and MAPK pathways, which in turn leads to inhibition of cell growth and tumorigenesis. Currently, clinical trials using PAP DNA vaccine are underway and FDA-approved immunotherapy using PAP is commercially available. Despite these clinically important aspects, molecular mechanisms underlying PAP regulation are not fully understood. The promoter region of PAP was reported to be regulated by NF-, TNF-, IL-1, androgen and androgen receptors. Here, the features of PAP gene and protein structures together with the function, regulation and roles of PAP in prostate cancer are discussed.

Cysteine and histidine-capped water-dispersible ZnS:Mn nanocrystals (ZnS:Mn-Cys and ZnS:Mn-His) were synthesized and their effects on E. coli and human cells were investigated. Particle sizes of these nanocrystals were found from HR-TEM images to be 3.5 nm and 4.0 nm, respectively. Their solution photoluminescence spectra showed identical broad emission peaks at 580 nm. ZnS:Mn-His significantly suppressed the growth of E. coli at and 1 mg/mL concentrations, something not observed with ZnS:Mn-Cys. Consistent with this, greater inhibition of cell proliferation and viability were observed in HEK293 and IMR90 cells in ZnS:Mn-His at and 1 mg/mL concentrations.

Colorectal cancer (CRC) is the third most common malignancy in the world. Because CRC develops slowly from removable precancerous lesions, detection of the disease at an early stage during regular health examinations can reduce both the in-cidence and mortality of the disease. Although sigmoidoscopy offers significant improvements in the detection rate of CRC, its diagnostic value is limited by its high costs and inconvenience. Therefore, there is a compelling need for the identification of noninvasive biomarkers that can enable earlier detection of CRC. Accordingly, many validation studies have been con-ducted to evaluate genetic, epigenetic or protein markers that can be detected in the stool or in serum. Currently, the fe-cal-occult blood test is the most widely used method of screen-ing for CRC. However, advances in genomics and proteomics combined with developments in other relevant fields will lead to the discovery of novel non invasive biomarkers whose use-fulness will be tested in larger validation studies. Here, non-invasive molecular biomarkers that are currently used in clin-ical settings and have the potential for use as CRC biomarkers are discussed. [BMB reports 2008; 41(10): 685-692]

Aims The endothelium has emerged recently as a therapeutic target in the treatment of hypertension because endothelial dysfunction and subsequent vascular rarefaction cause target organ damage and further elevate blood pressure (BP). It led us to hypothesize that one of the endothelial survival factors, a potent derivative of angiopoietin-1 (cartilage oligomeric matrix protein, COMP-Ang-1), could be a novel class of antihypertensive agents that maintain endothelial integrity and function, thereby preventing the development of hypertension and target organ damage. Methods and results To study the role of COMP-Ang-1 in preventing hypertension and target organ damage, a COMP-Ang-1 plasmid was electroporated into adductor muscles of 6 weeks old, pre-hypertensive, spontaneously hypertensive rats (SHRs), and the secretion of its expressed protein into the bloodstream was confirmed by western blotting. In comparison with sham and reporter gene transfer, COMP-Ang-1 gene transfer significantly prevented increases in systolic BP and reduced microvascular rarefaction and tissue damage in the heart and kidney. However, overexpression of soluble Tie2 receptor completely abolished these beneficial effects of COMP-Ang-1 gene transfer on SHRs, indicating that expressed COMP-Ang-1 protein has antihypertensive effects in SHRs by binding Tie2 receptors on the vascular endothelium. In particular, COMP-Ang-1 gene-transferred SHRs had significantly higher plasma levels of nitrite than other controls, which was found to be due to that expressed COMP-Ang-1 protein promoted nitrite synthesis by activating endothelial nitric oxide synthase, one of the Tie2 downstream-signalling molecules. Conclusion The present study suggests a new potential of endothelial survival factor, COMP-Ang-1, as an antihypertensive agent that effectively reduces the hypertension-associated cardiovascular and renal damage, as well as prevents the further elevation of BP.

Graphene is a next-generation biomaterial with increasing biomedical applicability. As a new class of one-atom-thick nanosheets, it is a true two-dimensional honeycomb network nanomaterial that attracts interest in various scientific fields and is rapidly becoming the most widely studied carbon-based material. Since its discovery in 2004, its unique optical, mechanical, electronic, thermal, and magnetic properties are the basis of exploration of the potential applicability of graphene. Graphene materials, such as graphene oxide and its reduced form, are studied extensively in the biotechnology arena owing to their multivalent functionalization and efficient surface loading with various biomolecules. This review provides a brief summary of the recent progress in graphene and graphene oxide biological research together with current findings to spark novel applications in biomedicine. Graphene-based applications are progressively developing; hence, the opportunities and challenges of this rapidly growing field are discussed together with the versatility of these multifaceted materials.

Intramyocardial administration of gene therapy vectors expressing angiogenic factors have been attempted as an alternative to conventional surgical methods for the management of myocardial ischemia. In this study, we havedeveloped the pGT 2 -VEGF, a plasmid DNA vector expressing human VEGF165, for the management of ischemic cardiovascular disease and investigated in vivo pharmacokinetics and tissue distribution of pGT 2 -VEGF after intramyocardial and intravenous administration in rats. A high concentration of pGT 2 -VEGF was observed in the heart after intramyocardial injection of 300 μg, which is in line with the assumption that direct intramyocardial delivery enables extended localization at the administration site. Leakage of the pGT 2 -VEGF to the blood circulation was observed after intramyocardial injection, with an area under the curve (AUC) of 3.8 μg min/mL, as compared with 37.3 μg min/mL after intravenous injection of the same dose. The pGT 2 -VEGF concentration in blood peaked at 5 minutes after intramyocardial administration and declined rapidly to undetectable levels by 2 hours post-administration. In tissue distribution studies, pGT 2 -VEGF peaked at 5 minutes post-administration in various organs but was undetectable at 2 hours in all organs except heart, lung, and liver. Taken together, the results suggest that intramyocardial-delivered pGT 2 -VEGF was degraded rapidly in vivo and mainly persisted in target tissues, the heart. In addition, intramyocardial-administered pGT 2 -VEGF was expressed for longer periods than the persistence of the pGT 2 -VEGF plasmid DNA in a target tissue. Therefore, a direct myocardial injection of pGT 2 -VEGF might be useful for local therapeutic angiogenesis.

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 microg/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.

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 mug 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.

Previously we demonstrated that a group I ribozyme can perform trans-splicing to repair sickle β-globin transcripts upon transfection of in vitro transcribed ribozyme into mammalian cells. Here, we sought to develop expression cassettes that would yield high levels of active ribozyme after gene transfer. Our initial expression constructs were designed to generate trans-slicing ribozymes identical to those used in our previous RNA transfection studies with ribozymes containing 6-nucleotide long internal guide sequences. The ribozymes expressed from these cassettes, however, were found to be unable to repair sickle β-globin RNAs. Further experiments revealed that two additional structural elements are important for ribozyme-mediate RNA repair: the P10 interaction formed between the 5′ end of the ribozyme and the beginning of the 3′ exon and an additional base-pairing interaction formed between an extended guide sequence and the substrate RNA. These optimized expression cassettes yield ribozymes that are able to amend 10%–50% of the sickle β-globin RNAs in transfected mammalian cells. Finally, a ribozyme with a 5-bp extended guide sequence preferentially reacts with sickle β-globin RNAs over wild-type β-globin RNAs, although the wild-type β-globin transcript forms only a single mismatch with the ribozyme. These results demonstrate that trans-splicing ribozyme expression cassettes can be generated to yield ribozymes that can repair a clinically relevant fraction of sickle β-globin RNAs in mammalian cells with greatly improved specificity.

In an effort to develop a guiding and monitoring tool for transmyocardial gene transfer, we have evaluated the feasibility of intracardiac echocardiography (ICE) to guide percutaneous endomyocardial gene transfer (PEGT), and monitor complications, in a pig model. ICE (5.5‐ 10 MHz), complemented by fluoroscopy, was utilized to guide a needle injection into the heart in 19 normal pigs. Using this system, we injected Evans blue dye into eight pigs (group I), a mixture of pCK-CAT plasmid and India ink into seven pigs (group II), and pCK-LacZ plasmid into four pigs (group III). In all pigs, ICE contributed to the injection procedure by guiding the catheter to anatomically distinct sites, and by assisting stabilization of the catheter‐ endocardial contact. ICE predicted the injection sites correctly in 56 of 64 sites (87.5%) in group I, and in 42 of 42 sites (100%) in group II. Leakage of injectate into the left ventricular cavity could be detected by the microbubbles generated. The sites of injections appeared as foci of bright myocardial echodensity, which persisted until the end of the procedure. The procedures were not associated with significant morbidity or mortality. The expression of the chloramphenicol acetyltransferase (CAT) gene was identified in 40 sites from 42 injections (95.2%) in group II. In group III, histology showed positive b -galactosidase staining of myocytes limited around the needle track with low transfection efficiency ( ,1%). These results suggest that real-time ICE monitoring proves safe and useful during PEGT for guiding needle injection, monitoring leakage, ensuring delivery of injectate into the myocardium, and instantly diagnosing cardiac complications, resulting in successful gene transfer.

Atrial natriuretic peptide (ANP) is a cardiac hormone that elicits a profound diuresis, natriuresis, and hypotension. As a preliminary study toward ANP gene therapy of cardiovascular disorders, we have cloned a cDNA for mouse preproANP and carried out expression studies in muscle cells. The expression cassette, which was flanked by ITRs from AAV-2, consisted of HCMV IE enhancer/promoter, preproANP gene, and polyadenylation signal from bovine growth hormone. We transfected this expression vector into primary skeletal myoblasts and examined the following points: (1) secretion of immunoreactive ANP, (2) biological activity, and (3) nature of secreted ANP(s). The conditioned media from cells transfected with ANP vector had significantly higher levels of irANP in comparison to mock control. The secreted irANP had biological activity as confirmed by the elevated level of intracellular cGMP in human umbilical vein endothelial cells. Reverse-phase HPLC analysis showed that the processed form of ANP was the predominant form. These results demonstrate that preproANP gene could be ectopically expressed and correctly processed in skeletal myoblasts, which has implications for development of muscle-based ANP gene therapy.

Background Efficient and stable transfer of therapeutic DNA into injured myocardium would be an initial step towards a genetic treatment aimed at myocardial repair after myocardial infarction. Proliferating cardiac fibroblasts in the healing myocardium could be a compelling target for retroviral infection. We evaluated the feasibility of direct in vivo gene transfer into injured myocardium using a high-titer, stable retroviral vector. Methods Using the TE-FLY-A-based MFG retroviral vector harboring nlsLacZ reporter, the gene transfer efficiency was assessed first in vitro in rat cardiac fibroblasts, followed by in vivo evaluation in healing rat myocardium after local freeze–thaw injury. A total of 2.5×107 infectious units of retrovirus were injected into the injured region of a beating rat heart. The transduced cells were identified by X-gal staining and immunohistochemistry. Results Highly efficient transduction of cardiac fibroblasts was observed in vitro with 98% of the cells transduced with single infection. The cell proliferation index in the cardiac granulation tissue appeared maximal 3 days after cryoinjury. Retroviral injection into the injured beating heart induced gene expression localized to the wound repair region. One week after retrovirus injection, 14% of the cells in the reparative tissue were β-gal-positive, while 4% were β-gal-positive after 4 weeks. The transduced cells were mostly myofibroblasts. Conclusions Local gene transfer to the healing rat heart is feasible by retrovirus in vivo. This observation may serve as a useful guide for the development of gene therapy aimed at myocardial repair after myocardial infarction. Copyright © 2000 John Wiley & Sons, Ltd.

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.

Few studies have examined in detail the combined effects of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) gene delivery on collateral development. Here, we evaluated the potential synergism of naked DNA vectors encoding VEGF and bFGF using a skeletal-muscle based ex vivo angiogenesis assay and compared tissue perfusion and limb loss in a murine model of hindlimb ischemia. In the ex vivo angiogenesis assay, the VEGF+bFGF combination group had a larger capillary sprouting area than those of the LacZ, VEGF, and bFGF groups. Consistent with these results, regional blood flow recovery on day 14 was also highest in the VEGF+bFGF combination group, followed by the bFGF, VEGF, and LacZ groups. The limb loss frequency was 0% in the combination group, whereas the limb loss frequencies of the other groups were 7-29%. The ischemic muscles of the combination group revealed evidence of increased angiogenesis and arteriogenesis and the upregulated expression of genes that may be associated with arteriogenesis, such as those for cardiac ankyrin repeat protein, early growth response factor-1, and transforming growth factor-beta1. Our study has implications for the development of a combined gene therapy for the vascular occlusive diseases.

Objective: Ischemia-dependent upregulation of angiopoietin2 (Ang2) led us to hypothesize the potentially proangiogenic Ang2-Tie2 signaling in endothelial progenitor cells (EPCs). Given the well-known vascular destabilizing action of Ang2 in mature endothelium, we investigated the yet unidentified mechanism behind cell-dependent differential activity of Ang2. Methods and results: Both in vitro and in vivo experiments showed that Ang2 promoted angiogenicity of human cord blood-derived EPCs, where Ang2 directly activated Tie2 and its related downstream signaling molecules. However, Ang2 had no such effect in fully differentiated human umbilical vein endothelial cells (HUVECs) under the same condition. Such a cell-dependent Tie2 activation by Ang2 was explained by comparing EPCs and HUVECs, where most Tie2 receptors in EPCs were found to be present unbound to Tie1, whereas those in HUVECs existed as heterocomplexes with Tie1. When Tie2 in HUVECs was prevented from forming heterocomplexes by silencing Tie1 expression, they underwent rapid phosphorylation upon Ang2 treatment, as shown in EPCs. Conclusions: In contrast with its roles in mature endothelial cells, Ang2 has proangiogenic activities in EPC directly through Tie2 signaling pathway. Such a cell-dependent differential reactivity of Ang2 was for the first time found to be modulated by physical association between Tie1 and Tie2, which inhibited Ang2-mediated Tie2 activation.

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 beta-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 microg and 25 microg 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.

Endothelial progenitor cells (EPCs) act as endothelial precursors that promote new blood vessel formation and increase angiogenesis by secreting growth factors and cytokines in ischemic tissues. These facts prompt the hypothesis that EPC transplantation should accelerate the wound-repair process by facilitating neovascularization and the production of various molecules related to wound healing. In a murine dermal excisional wound model, EPC transplantation accelerated wound re-epithelialization compared with the transplantation of mature endothelial cells (ECs) in control mice. When the wounds were analyzed immunohistochemically, the EPC-transplanted group exhibited significantly more monocytes/macrophages in the wound at day 5 after injury than did the EC-transplanted group. This observation is consistent with enzyme-linked immunosorbent assay results showing that EPCs produced in abundance several chemoattractants of monocytes and macrophages that are known to play a pivotal role in the early phase of wound healing. At day 14 after injury, the EPC-transplanted group showed a statistically significant increase in vascular density in the granulation tissue relative to that of the EC-transplanted group. Fluorescence microscopy revealed that EPCs preferentially moved into the wound and were directly incorporated into newly formed capillaries in the granulation tissue. These results suggest that EPC transplantation will be useful in dermal wound repair and skin regeneration, because EPCs both promote the recruitment of monocytes/macrophages into the wound and increase neovascularization.

Vascular enlargement is a characteristic feature of angiopoietin-1 (Ang1)-induced changes in adult blood vessels. However, it is unknown whether tissues having Ang1-mediated vascular enlargement have more blood flow or whether the enlargement is reversible. We have recently created a soluble, stable and potent Ang1 variant, COMP-Ang1. In the present study, we investigated the effects of varied dose and duration of COMP-Ang1 on vascular enlargement and blood flow in the tracheal microvasculature of adult mice and explored a possible mechanism of long-lasting vascular enlargement. We found that COMP-Ang1 administered by adenoviral vector induced long-lasting vascular enlargement and increased tracheal blood flow. In contrast, short-term administration of COMP-Ang1 recombinant protein induced transient vascular enlargement that spontaneously reversed within a month. In both cases, the vascular enlargement resulted from endothelial proliferation. The COMP-Ang1–induced vascular remodeling is mediated mainly through Tie2 activation. Sustained overexpression of Tie2 could participate in the maintenance of vascular changes. Together, our findings indicate that sustained treatment with COMP-Ang1 can produce long-lasting vascular enlargement and increased blood flow.

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 pattern in p38 MAPK. Blocking studies using pre-treatment of the inhibitor of each pathway 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.

C-reactive protein (CRP), a predictor of future cardiovascular diseases, has been reported to damage the vascular wall by inducing endothelial dysfunction and inflammation. This proatherogenic CRP was speculated to have a role in attenuating angiogenic functions of human endothelial progenitor cells (EPCs), possibly impairing vascular regeneration and increasing cardiovascular vulnerability to ischemic injury. Herein, we investigated the direct effect of CRP on angiogenic activity and gene expression in human EPCs. Incubation of EPCs with human recombinant CRP significantly inhibited EPC migration in response to vascular endothelial growth factor, possibly by decreasing the expression of endothelial nitric oxide synthase and subsequent nitric oxide production. In addition, CRP-treated EPCs showed the reduced adhesiveness onto an endothelial cell monolayer. When assayed for the gene expression of arteriogenic chemo-cytokines, CRP substantially decreased their expression levels in EPC, in part due to the upregulation of suppressors of cytokine signaling proteins. These results suggest that CRP directly attenuates the angiogenic and possibly arteriogenic functions of EPCs. This CRP-induced EPC dysfunction may impair the vascular regenerative capacity of EPCs, thereby leading to increased risk of cardiovascular diseases.

Objective— Increased risk of cardiovascular disease in patients with chronic renal failure (CRF) has been explained by accelerated atherosclerosis and impaired angiogenesis, in which endothelial progenitor cells (EPCs) may play key roles. We hypothesized that altered EPC biology may contribute to the pathophysiology of CRF. Methods and Results— EPCs were isolated from CRF patients on maintenance hemodialysis (n=44) and from a normal control group (n=30). CRF patients showed markedly decreased numbers of EPC (44.6%) and colonies (75.3%) when compared with the controls ( P P =0.040) and 48.8% decrease in EPC incorporation into human umbilical vein endothelial cells (HUVEC) ( P r =−0.461, P =0.010) and normal group ( r =−0.367, P =0.016) significantly correlated with the numbers of EPC. Indeed, the number of circulating EPC was significantly lower in CRF patients than in normal group under the same burden of risk factors ( P r =0.427, P =0.004). Conclusions— EPC biology, which is critical for neovascularization and the maintenance of vascular function, is 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.

To test the hypothesis that overexpression of early growth response factor-1 (Egr-1) contributes to the revascularization of ischemic limbs, a constitutively active form of Egr-1 (Egr-1*) was made and evaluated in vitro and in vivo. Analyses of the transduced myocytes revealed significant upregulation of bFGF, PDGF-A, PDGF-B, IGF-II, and TGF-beta1. A coculture assay of the paracrine effects indicated that Ad-Egr-1* promoted proliferation and migration of endothelial cells. When Ad-Egr-1* was injected into the tibialis anterior muscle of mice, followed by explant culture in growth factor-reduced Matrigel, many capillary-like structures were observed in the Egr-1* group compared with minimal sprouting from the LacZ group, suggesting an angiogenic potential of Egr-1*. Next we evaluated Ad-Egr-1* in a murine model of hindlimb ischemia. Compared with slow revascularization in the control PBS or LacZ group, a rapid increase in tissue perfusion was observed in the Egr-1* group and the difference in flux ratio was statistically significant at day 7. In the injected muscle, expression of Egr-1*, upregulation of its target genes, and increased number of vessels staining positive for smooth muscle alpha-actin were observed. These results suggest that Egr-1 plays an important role in vascular recovery after occlusion and could be a potential target for therapeutic angiogenesis.

An angiogenesis assay based on gene transfer would be extremely useful for angiogenic gene therapy. A simple, reproducible, and quantitative assay to test angiogenic genes would provide more accurate predictions than conventional peptide-based assays. Here, we have developed a semiquantitative angiogenesis assay utilizing gene transfer into skeletal muscle, which is a target tissue for ischemic limb diseases. To facilitate quick and clean analysis, a naked plasmid DNA vector combined with an electroporation procedure was used for gene transfer. When the plasmid vector encoding vascular endothelial growth factor cDNA (pJDK-VEGF165) was injected into the tibialis anterior muscle of BALB/c mice, followed by in vivo electroporation and explant culture in growth factor-reduced Matrigel, the outward migration of sprouting cells was observed as early as day 2. The cells soon formed capillary networks, which peaked at day 7 and persisted until day 14. The capillary-like structures were positive for von Willebrand factor, platelet endothelial cell adhesion molecule, and vimentin, suggesting they were endothelial cells. There was little, if any, sprouting or formation of capillaries from the control vector (pJDK)-injected group. Consistent with the region of sprouting and network formation, the amount of secreted VEGF increased in the conditioned medium of explant cultures. The angiogenic potential of connective tissue growth factor (CTGF) was examined using the new assay. Whereas the CTGF gene alone induced weak sprouting activity, it appeared to inhibit the angiogenic activity of the VEGF165 gene during cotreatment. This attenuating activity of CTGF on VEGF was reproduced in vivo in a murine model of hindlimb ischemia. In a group of mice treated with both pJDK-CTGF and pJDK-VEGF165, the blood flow measured by laser Doppler imaging was significantly lower than that of the pJDK-VEGF165-treated group 10 days after femoral artery excision. These results are consistent with recent reports that suggest that CTGF inhibits VEGF. This confirms the usefulness of this novel ex vivo assay in assessing the angiogenic capacity of genes of interest. In summary, this new gene-based angiogenesis assay should be widely applicable in the study of angiogenic or antiangiogenic genes because it can readily predict the angiogenic potential of specific genes and their combinations.

Direct injection of the vascular endothelial growth factor (VEGF) gene plasmid DNA into the myocardium was shown to induce development of new blood vessels to increase the circulation in the heart of patients with coronary artery diseases. However, such angiogenic gene therapy (via naked DNA) was limited by low level of gene expression. Furthermore, the temporal and spatial characteristics of VEGF gene transfer in the heart are not known. In this study, we demonstrated that a plasmid vector, containing the human cytomegalovirus immediate early (HCMV IE) promoter and enhancer, induces greater expression of gene in the rat heart monitored by gene fused to the chloramphenicol acetyl transferase (CAT) reporter, than four different viral and cellular promoters. Interestingly, expression of VEGF121 protein showed an earlier peak, a shorter duration, and a wider distribution than that of CAT only. Therefore, a plasmid vector with an HCMV IE promoter/enhancer provides clear advantages over other previously developed plasmids. Furthermore, expression profile of VEGF121 gene may provide useful information in the design of angiogenic gene therapy in the heart.

Vascular endothelial growth factor (VEGF) has proven to be one of the most effective growth factors for therapeutic angiogenesis. The biological efficacy of the adeno-associated virus (AAV) vector has recently been demonstrated in muscle tissues, including the heart. Apart from these promising insights into VEGF and the AAV vector, studies on VEGF gene transfer using the AAV vector have been limited. Here, we evaluate AAV-mediated VEGF gene transfer, both in vitro and in vivo, using the AAV-mVEGF vector that contains cDNA for murine VEGF(120) within an HCMV-driven expression cassette. Transient transfection of AAV-mVEGF plasmid significantly increased mVEGF expression in 293T cells. The secreted VEGF in the conditioned medium had strong biological activity, as confirmed by the Miles' vascular permeability assay. Transduction of 293T and HeLa cells with AAV-mVEGF stock of high titer, that is essentially adenovirus-free, showed significantly increased mVEGF expression above that of AAV-eGFP-transduced cells. When human umbilical vein endothelial cells were transduced, a higher level of mVEGF expression, together with higher cell counts, was observed compared to AAV-eGFP-transduced cells. In vivo transduction of mouse tibialis anterior muscle resulted in an increased level of mVEGF expression, and higher capillary-to-myofibre ratio, 8 weeks post-transduction. In a rat hindlimb ischemia model, regional blood flow, as well as the capillary-to-myofibre ratio, was significantly increased at 4 weeks post-transduction. These findings demonstrate the efficient delivery of the VEGF gene using an AAV vector, which has implications for angiogenic gene therapy in ischemic diseases.

Dihydroneopterin triphosphate (H2NTP) 2′-epimerase from Escherichia coli catalyzes the epimerization of H2NTP to dihydromonapterin triphosphate (H2MTP). The enzyme was purified 954-fold to apparent homogeneity by a combination of ammonium sulfate fractionation and column chromatography of Cibacron blue 3GA dye ligand, phenyl-Sepharose CL-4B, methotrexate-agarose, and Superdex 200 HR 10/30 FPLC column. The molecular mass of the epimerase determined on a Superdex column was 82.6 kDa, while the subunit molecular mass determined on SDS-polyacrylamide gel electrophoresis was 13.7 kDa. This implies that the epimerase most probably exists as homohexamer. The 20-amino acid sequence from the N terminus was determined (AQPAAIIRIKNLRLRTFIGI). Based on this sequence, the gene encoding the epimerase was cloned using a simple polymerase chain reaction approach. Translation of the nucleotide sequence of the cloned gene revealed the presence of an open reading frame containing 120 amino acids with a predicted molecular mass of 13,993 Da. The epimerase gene located in a 2.3-kilobase BamHI-EcoRI fragment from Kohara's clone 406 was overexpressed 300-fold, which was confirmed by the prominent increase in the 14-kDa protein band on SDS-polyacrylamide electrophoresis gels. It showed no homology with the sequences of isomerases or other enzymes in GenBank/EMBL data bases.

Prostate cancer is becoming one of the most popular non-skin cancers in Korea due to continued increase of western life styles. Since earlier detection of prostate cancer can lead to complete cure, a lot of studies have been done on prostate cancer markers such as prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), prostate acid phosphatase (PAP) and prostate stem cell antigen (PSCA). Among these biomarkers, PAP is gaining renewed interest because of its potential merits in diagnosis and therapeutics. PAP, which was initially identified in 1935, is an abundant enzyme that is synthesized in prostate epithelial cells and has enzymatic activity in acidic conditions. PAP expression is androgen-independent and increases in proportion to prostate cancer progression. Indeed, PAP expression level has high correlation with cause-specific survival and its dephosphorylation activity is believed to play an important role in the incidence and development of prostate cancer. Here, we screened the in vitro transcribed RNA libraries to get aptamer(s) that can selectively bind to PAP. Aptamers are oligonucleic acid molecules that bind to a specific target through formation of tertiary structures. These chemical antibodies can bind their target molecules with a high affinity and specificity and have the advantage of being less immunogenic in vivo. For in vitro selection, the ectracellular domain of PAP were cloned from PC3 cells and subjected to 6 rounds of SELEX procedure. This resulted in enrichment of the RNA aptamers that bound to the PAP ectodomain with 3-fold higher affinity than the library control as evidenced by real-time RT-PCR and gel shift assay. Sequence analysis of the aptamers revealed three distinct groups (G1, G2, N) having variable sequences within the region comprising nucleotides 43 to 87. The Kd values of the best clones with the highest affinity were within 120nM. These clones could also bind to the PAP-positive cells such as PC3 and LNCaP cells. Interestingly, 2'-OH-modification of the aptamers led to loss of binding, implying that 2'-F modification may be essential in binding to PAP. Characterization of these aptamers is currently underway and initial minimization results suggest that the structure coming from the 50 nucleotides is essential for binding to PAP. With further optimization, these PAP RNA aptamers could find wide application in theragonosis of prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C141.

To develop a potent hypoxia-inducible vector, we evaluated the usefulness of chimeric combinations of the early growth response factor-1 (Egr-1)-binding site (EBS) from the Egr-1 promoter, the metal-response element (MRE) from the metallothionein gene, and the hypoxia-response element (HRE) from the phosphoglycerate kinase 1 (PGK1) gene. In transient trasfection assays, combination of three copies of HRE (3xHRE) with either EBS or MRE resulted in a significant increase in hypoxia-responsiveness. With three-enhancer combination such as the EBS-MRE-3xHRE (E-M-H), a hypoxia induction ratio of 69 was achieved. The expression induced from E-M-H-pGL3 was 2.4-fold higher than that induced from H-pGL3 and even surpassed the expression from a human cytomegalovirus (HCMV) promoter-driven vector. The high inducibility of E-M-H was confirmed by validation studies in different cells and by expressing other cDNAs. The E-M-H was also tested for induction by hypoxia mimetics and other stresses such as heat, low glucose, low pH, and hydrogen peroxide. Co2+, deferoxamine, and Ni2+turned out to be potent inducers whereas hydrogen peroxide worked as a moderate inducer. Gel shift assay together with functional overexpression studies suggested that increased levels of hypoxia-inducible factor 1|[alpha]| (HIF-1|[alpha]|), metal transcription factor-1 (MTF-1), and Egr-1 may be associated with the high inducibility of E-M-H enhancer. In vivo evaluation of the E-M-H vector in ischemic calf muscle revealed that luciferase expression was significantly higher in E-M-H group than in control, H, or HCMV group. With its high induction capacity and versatile means of modulation, the novel chimeric enhancer should find wide application in the treatment of ischemic diseases and cancer.

Therapeutic angiogenesis to enhance collateral vessel growth is a promising strategy for the treatment of vascular occlusive diseases. Although local delivery of angiogenic growth factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) has shown favorable results, few studies have examined in detail the combined effects of VEGF and bFGF gene delivery on collateral development. Here, we evaluated potential synergism of the naked DNA vectors encoding VEGF and bFGF by using a skeletal muscle-based ex vivo angiogenesis assay and by comparing blood flow recovery and limb loss score in a murine model of hindlimb ischemia (C57BL/6) where femoral artery was excised. In the ex vivo angiogenesis assay, VEGF+bFGF combination group displayed larger sprouting area than LacZ, VEGF, or bFGF group. Reflecting this capillary sprouting data, the combination group had the highest amount of secreted VEGF in the conditioned medium. Consistent with these ex vivo results, regional blood flow recovery at Day 14 as measured by Laser Doppler Imaging was also highest in the VEGF+bFGF combination group followed by bFGF, VEGF, and LacZ control group. The limb loss score was lowest (no necrosis) in the combination group, whereas there were 1|[sim]|4 cases of limb or toe necrosis in other groups. Currently, histological and genetic analyses of the ischemic muscles in the combination group are underway to find the evidence of increased angiogenesis and/or arteriogenesis and upregulation of the genes that may be associated with arteriogenesis, such as cardiac ankyrin repeat protein (CARP), TGF-|[beta]|1, and early growth response factor-1 (Egr-1). Our study has an implication for the development of combined arteriogenic gene therapy that uses the two best characterized angiogenic growth factors, VEGF and bFGF.

Top of pageAbstract We hypothesized that the overexpression of early growth response factor-1 (Egr-1), which plays a pivotal role in the activation of many genes under stress conditions, might induce multiple genes involved in vessel formation and thereby contribute to the revascularization of ischemic muscle. To test this hypothesis, adenoviral vectors carrying the genes for Egr-1 or Egr-1|[ast]| (a constitutively active form of Egr-1) were made and transduced into primary human skeletal myocytes. RNA and protein analyses revealed significant upregulation of angiogenesis-related genes, such as bFGF, PDGF-A, PDGF-B, IGF-II, and TGF-|[beta]|1. A co-culture assay of the paracrine effects of transduced myocytes indicated that the Ad-Egr-1|[ast]| group significantly promoted proliferation of endothelial cells and regrowth from the wound edge following scratch injury. To further evaluate the angiogenic potential of Egr-1|[ast]|, Ad-Egr-1|[ast]| was injected into the tibialis anterior muscle of mouse (Balb/c), followed by explant culture in growth factor-reduced Matrigel. Many sprouted capillary-like structures were observed in the Egr-1|[ast]| group compared with minimal sprouting from the LacZ group, suggesting an angiogenic activity of Egr-1|[ast]|. Next we evaluated Ad-Egr-1|[ast]| in vivo in a murine model of hindlimb ischemia (Balb/c). Compared with the slow revascularization in the control group (PBS or Ad-LacZ), a rapid increase in tissue perfusion was observed in the Ad-Egr-1|[ast]| group and the difference in the measured flux ratio was statistically significant at day 7. Expression of Egr-1|[ast]|, upregulation of its target genes, and increased number of vessels that stained positive for smooth muscle |[alpha]|-actin (|[alpha]|-SMA) were also observed in the injected muscle. These results suggest that Egr-1 plays an important role in vascular recovery after occlusion and could be a potential target for therapeutic angiogenesis.

Top of pageAbstract Previous work has shown that both group I ribozymes and SMaRT™ pre-trans-splicing molecules (PTMs) can be designed to trans-splice into mutant RNA targets and subsequently correct these transcripts. The technology's therapeutic potential hinges on specific and efficient trans-splicing with the intended transcript. We are using quantitative PCR to assess these two parameters with a β-globin target in mammalian cells. Both ribozyme and PTM RNA repair approaches are being used and the results will provide the first direct comparison between these two potential therapeutics. Preliminary efficiency experiments using co-transfection of β-globin target and ribozyme expression vectors into 293T cells followed by QC-RT-PCR resulted in 5–50% trans-splicing levels depending on the promoter used. We are now testing 3' and 5' PTM constructs against the same β-globin target and assessing trans-splicing levels in 293T cells.

Objective—Increased risk of cardiovascular disease in patients with chronic renal failure (CRF) has been explained by accelerated atherosclerosis and impaired angiogenesis, in which endothelial progenitor cells (EPCs) may play key roles. We hypothesized that altered EPC biology may contribute to the pathophysiology of CRF. Methods and Results—EPCs were isolated from CRF patients on maintenance hemodialysis (n44) and from a normal control group (n30). CRF patients showed markedly decreased numbers of EPC (44.6%) and colonies (75.3%) when compared with the controls (P0.001). These findings were corroborated by 30.5% decrease in EPC migratory function in response to vascular endothelial growth factor (VEGF) (P0.040) and 48.8% decrease in EPC incorporation into human umbilical vein endothelial cells (HUVEC) (P0.001). In addition, Framingham’s risk factor score of both CRF (r0.461, P0.010) and normal group (r0.367, P0.016) significantly correlated with the numbers of EPC. Indeed, the number of circulating EPC was significantly lower in CRF patients than in normal group under the same burden of risk factors (P0.001). A significant correlation was also observed between dialysis dose (Kt/V) and EPC incorporation into HUVEC (r0.427, P0.004). Conclusions—EPC biology, which is critical for neovascularization and the maintenance of vascular function, is 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. (Arterioscler Thromb Vasc Biol. 2004;24:1246-1252.)

Background and Objectives:For the development of an arteriogenic gene therapy in peripheral artery occlusive disease, we developed a novel angiogenesis assay, with electroporation-mediated naked DNA delivery to the skeletal muscle. Materials and Methods:The levels of the expression CAT were compared between pJDK and pcDNA3.1, in HeLa and C2C12 cell lines, and skeletal muscle. The well known angiogenic gene, pJDK-hVEGF165, was injected, intramuscularly, into the tibialis anterior muscle of Balb/C mice, which was followed by electroporation. Two days later, the anterior tibialis muscles were divided into halves, embedded, and cultured in growth factor-reduced Matrigel. The capillary network area formed by the newly sprouting tube-like structures was calculated. For validation of this ex vivo assay, the connective tissue growth factor gene (pJDK-CTGF) was tested both by this new assay, and by the mice-hind limb ischemia model, with Laser Doppler imaging. Results:The pJDK showed a significantly higher level of CAT expression than the pcDNA3.1. From the pJDK-hVEGF165 injected explants, endothelial cell migration and tube-like formation occurred on day 2, and the capillary network formation peaked on day 7. The capillary network formation in the pJDK-hVEGF165 group was markedly increased to that in the pJDK group. From the skeletal muscle assay, the pJDK-CTGF showed no angiogenic activity or attenuated VEGF-induced capillary network formation. The LDI flux ratio, on day 10 in the mice-hind limb ischemia model, for the mice treated with the pJDK-CTGF and pJDK-hVEGF165 was significantly lower than that of the mice treated with the pJDK-hVEGF165 alone. Conclusion:The skeletal muscle ex vivo assay, using an electroporationmediated naked DNA delivery, is a simple, quantitative and reproducible method for assessing angiogenic genes. CTGF could be an anti-angiogenic factor due to its inhibition of VEGF. (Korean Circulation J 2003;33 (4):321-332)