A new method for monitoring phospholipase A (PLA) activity in venom extracts is under investigation to assess its suitability as a stability indicating assay. Lyophilized honey bee venom (HBV) and yellow jacket venom (YJV) samples were reconstituted to 100μg/mL and stored under various conditions; recommended storage (5oC ± 3oC), accelerated (25oC ± 2oC), and stressed (40oC ± 2oC). Testing was performed at various time-points using the alternative PLA assay. For each time-point, freshly reconstituted samples acted as controls. Both venom species demonstrated a temperature-dependent change in PLA activity. For example, when HBV samples were reconstituted in albumin buffered saline and kept under the recommended storage condition (5oC ± 3oC) there was essentially no change in PLA activity through 6 months. However, over the same time period under the accelerated condition (25oC ± 2oC) there was a 40% or greater decline in measured PLA activity. An even greater change was observed under the stressed condition (40oC ± 2oC), where at least a 60% loss of PLA activity was observed. The trend seen for PLA activity in YJV samples was similar, but even more pronounced for the accelerated and stressed conditions over the same time frame. Forced degradation studies have demonstrated the stability indicating potential of a new method for measuring PLA activity in venom extracts.

Background Pathological vascular remodeling in venous bypass grafts (VGs) results in smooth muscle cell (SMC) intimal hyperplasia and provides the substrate for progressive atherosclerosis, the principal cause of late VG failure. Nitric oxide (NO) bioactivity is reduced in VGs, in association with increased vascular superoxide production, but how these features relate to pathological VG remodeling remains unclear. We used gene transfer of the neuronal isoform of nitric oxide synthase (nNOS) to investigate how increased NO production modulates vascular remodeling in VGs and determined the effects on late VG phenotype. Methods and Results New Zealand White rabbits (n=60) underwent jugular-carotid interposition bypass graft surgery with intraoperative adenoviral gene transfer of nNOS or β-galactosidase. Vessels were analyzed after 3 days (early, to investigate acute injury/inflammation) or 28 days (late, to investigate SMC intimal hyperplasia). In early VGs, nNOS gene transfer significantly increased NOS activity and substantially reduced adhesion molecule expression and inflammatory cell infiltration. In late VGs, recombinant nNOS protein was no longer evident, but there were sustained effects on VG remodeling, resulting in a striking reduction in SMC intimal hyperplasia, a more differentiated intimal SMC phenotype, and reduced vascular superoxide production. Conclusions Intraoperative nNOS gene transfer has sustained favorable effects on VG remodeling and on the vascular phenotype of mature VGs. These findings suggest that early, transient modification of the response to vascular injury is a powerful approach to modulate VG biology and highlight the potential utility of NOS gene transfer as a therapeutic strategy in VGs.

Reduced production of nitric oxide (NO) in the cirrhotic liver results from a defect in hepatic endothelial cell nitric oxide synthase (ecNOS) and appears to contribute to the high intrahepatic resistance and portal hypertension typical of cirrhosis. Therefore, we postulated that targeting a heterologous NOS isoform to sinusoidal endothelial cells or other perisinusoidal cells, such as hepatic stellate cells, would counter the defect in NO production and reduce resistance to blood flow. Recombinant adenovirus (Ad) carrying the neuronal NOS gene (nNOS) targeted liver sinusoidal endothelial cells, stellate cells, and hepatocytes more efficiently than the corresponding cells in cirrhotic livers, but transduction rates were substantial even in cirrhotic animals. Expression of nNOS in each liver cell type, whether from normal or injured liver, caused increased NO production and inhibited endothelin-l-induced contractility of perisinusoidal stellate cells. Finally, in 2 different in vivo models of cirrhosis and portal hypertension, transduction of livers with recombinant Ad.nNOS significantly reduced intrahepatic resistance and portal pressure. The data highlight the feasibility of gene transfer to diseased liver and hepatic cells and demonstrate the potential of a novel therapy for portal hypertension caused by cirrhosis.

Abstract —Acute injury after adenoviral vascular gene transfer remains incompletely characterized. Here, we describe the early response (≤days) in 52 New Zealand White rabbits undergoing gene transfer (β-galactosidase or empty vector) or sham procedures to both carotid arteries. After gene transfer, arteries were either left in vivo for 1 hour to 3 days (in vivo arteries) or were excised immediately after gene transfer and cultured (ex vivo arteries). Within 1 hour, in vivo arteries receiving infectious titers of ≥4×10 9 plaque-forming units (pfu)/mL showed endothelial activation, with an acute inflammatory infiltrate developing by 6 hours. Ex vivo arteries showed endothelial activation but no inflammatory infiltrate. There were also significant differences in transgene expression between in vivo and ex vivo arteries. Ex vivo arteries showed titer-dependent increases in β-galactosidase expression through 2×10 10 pfu/mL, whereas in in vivo arteries, titers above 4×10 9 pfu/mL merely increased acute inflammatory response, without increasing transgene expression. In vivo arteries showed significant time- and titer-dependent impairment in endothelium-dependent relaxation, with no effect on contraction or nitroprusside-induced relaxation. Interestingly, however, if rabbits were made neutropenic with vinblastine, their arteries maintained full endothelium-dependent relaxation, even after very high titer vascular infection (up to 1×10 11 pfu/mL). These findings show that recombinant adenovirus triggers an early inflammatory response, and it is the inflammatory response that in turn causes functional endothelial injury. This occurs at much lower titers than previously appreciated (though the precise threshold will undoubtedly vary between laboratories). However, titers below the inflammatory threshold produce excellent transgene expression without inflammation or vascular injury.

Angiogenesis is required for tumor growth and metastasis, and inhibition of angiogenesis is a promising approach for anticancer therapy. Tie2 (a.k.a Tek) is an endothelium-specific receptor tyrosine kinase known to play a role in tumor angiogenesis. To explore the therapeutic potential of blocking the Tie2 pathway, an adenoviral vector was constructed to deliver a recombinant, soluble Tie2 receptor (AdExTek) capable of blocking Tie2 activation. Two days after i.v. injection of AdExTek, the plasma concentration of ExTek exceeded 1 mgyml and was maintained for about 8 days. Administration of AdExTek to mice with two different well established primary tumors, a murine mammary carcinoma (4T1) or a murine melanoma (B16F10.9), significantly inhibited the growth rate of both tumors (64% and 47%, respectively). To study the effect of ExTek on tumor metastasis, both tumor cell lines were coinjected i.v. with either AdExTek or a control virus. Mice coinjected with control virus developed numerous large, well vascularized lung metastases. In contrast, mice coinjected with AdExTek virus developed few, if any, grossly apparent metastases, and histologic examination revealed only small avascular clusters of tumor cells. Administration of AdExTek also inhibited tumor metastasis when delivered at the time of surgical excision of primary tumors in a clinically relevant model of tumor metastasis. This study demonstrates the potential utility of gene therapy for systemic delivery of an antiangiogenic agent targeting an endotheliumspecific receptor, Tie2. One of the most remarkable advances in our understanding of cancer pathogenesis is the notion that the progression of solid tumors depends on tumor angiogenesis. This fundamental principle states that tumor growth beyond a few mm3 in size strictly depends on tumor angiogenesis (1). By extrapolation, the same is true for tumor metastasis (2, 3). Consistent with this notion, recent studies indicate that tumors with a luxuriant vasculature have a higher fraction of dividing cells and lower necrosis rates than tumors with a poorly developed vasculature (4, 5). Moreover, clinical studies have shown a direct correlation between the density of tumor vessels and an adverse prognosis in patients with a variety of solid tumors, including breast, colon, lung, kidney, bladder, and head and neck tumors (6–14). Taken together, these studies suggest that the ability of a tumor to induce neovascularization determines its rate of growth and its likelihood of metastasis. Considering the importance of vascular growth in tumor progression, therapeutic approaches targeting the tumor endothelium may provide long-term, effective control of the disease. Angiogenic polypeptide growth factors, such as fibroblast growth factor and vascular endothelial growth factor (VEGF), are produced by tumor cell lines in vitro and by tumors in vivo and are likely to be key regulators of tumor angiogenesis (15). VEGF is currently the leading candidate for an endogenous mediator of tumor angiogenesis, because blocking the VEGF pathway inhibits the growth of several murine tumors and human tumor xenografts (16–19). However, a recent study has demonstrated that although many tumors are inhibited by blockade of the VEGFyVEGF receptor pathway, others are unaffected, which suggests that alternative pathways for vascular growth can drive tumor angiogenesis (17). Tie2 (a.k.a. Tek) is a newly cloned endothelium-specific receptor tyrosine kinase that has crucial roles during the development of the embryonic vasculature (20–23). Disruption of Tie2 function in transgenic mice results in embryonic lethality because of defects in vascular development characterized by a reduction in endothelial cell number and a defect in the morphogenesis of microvessels (24, 25). Disrupting the function of the Tie2 ligand, angiopoietin (Ang) 1 and overproduction of Ang2, an inhibitory ligand, yielded a phenotype similar to the Tie2 knockout, confirming the importance of the Tie2yAng1 pathway during embryonic vascular development (26, 27). To determine whether Tie2 played a role in pathologic angiogenesis in adult tissues, we have demonstrated that blocking Tie2 activation by local administration of a recombinant, soluble Tie2 receptor (ExTek) could inhibit tumor angiogenesis and tumor growth (28). These findings demonstrated a role for the Tie2 pathway in the formation of the tumor vasculature and suggested that targeting the Tie2 pathway might yield useful anticancer therapy. Although our previous findings demonstrated a role for the Tie2 pathway in tumor angiogenesis, local application is not likely to be a clinically useful means of delivering a Tie2 inhibitor because many primary tumors will be inaccessible and metastases may be too numerous. In addition, using a recombinant protein is likely to be problematic because of the expense and inconvenience involved with frequent dosing, especially if delivered systemically. Gene therapy using viral vectors offers promise as an approach to the long-term delivery of therapeutic proteins (29). Adenoviruses are common and relatively benign human pathogens that have not been associated with persistent infections or neoplasias in humans (30). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1998 by The National Academy of Sciences 0027-8424y98y958829-6$2.00y0 PNAS is available online at http:yywww.pnas.org. This paper was submitted directly (Track II) to the Proceedings office. Abbreviations: VEGF, vascular endothelial growth factor; Ang, angiopoietin; pfu, plaque-forming units; TBST, 10 mM TriszCl, pH 8.0y150 mM NaCly0.1% Tween20. **To whom reprint requests should be addressed at: Duke University Medical Center, Box 3623, Durham, NC 27710. e-mail: kgp@ hodgkin.mc.duke.edu.

Olmez, Geetha A. Shetty, Scot A. Youngblood, John Pawloski, Timothy McMahon, Jonathan S. Keith M. Channon, HuSheng Qian, Valentina Neplioueva, Michael A. Blazing, Ercument Arteries From Normal and Cholesterol-Fed Rabbits In Vivo Gene Transfer of Nitric Oxide Synthase Enhances Vasomotor Function in Carotid Print ISSN: 0009-7322. Online ISSN: 1524-4539 Copyright © 1998 American Heart Association, Inc. All rights reserved. is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Circulation doi: 10.1161/01.CIR.98.18.1905 1998;98:1905-1911 Circulation. http://circ.ahajournals.org/content/98/18/1905 World Wide Web at: The online version of this article, along with updated information and services, is located on the

Gene transfer technologies offer great potential both to investigate and alter the course of vessel wall pathophysiology. Replication-deficient adenovirus vectors appear particularly useful for the transfection of blood vessels, because of their ability to accommodate large cDNA inserts and to rapidly and efficiently infect mammalian endothelial and smooth muscle cells. The potential effects of transfection with a replication-deficient adenovirus on vasomotor function have not, however, been described. This study reports gene transfer experiments with a replication-deficient adenovirus containing a cytomegalovirus promotor and a nuclear localizing variant of the bacterial beta-galactosidase gene. Excised carotid artery segments from 6 rabbits were divided into four segments and infected in pairs for thirty minutes with four viral titers (control [0], 2.5x10(9), 5x10(9), and 1x10(10) pfu/ml) at 37 degrees C in serum-free culture media (M199). Expression of beta-galactosidase and in vitro vasomotor function were determined after seventy-two hours' incubation. Isometric tension studies were performed to examine the response of the vessel segments to the contractile agonist norepinephrine. beta-galactosidase was expressed in all vessel segments exposed to the adenovirus. There was a significant difference in the sensitivity to norepinephrine (P < 0.04) of the segments treated with the highest and lowest viral titers (mean +/- sem, -log(10) [EC(50)] of 5.98 +/- 0.09, 5.77 +/- 0.11, 5.68 +/- 0.14, and 5.52 +/- 0.13 for the control, 2.5x10(9), 5x10(9), and 1x10(10) plaque-forming units (pfu)/mL groups respectively). Replication-deficient adenovirus vectors can rapidly and efficiently infect rabbit carotid vessels, but there is, however, a dose-dependent alteration of in vitro vessel vasomotor function that may be mediated by a cytotoxic effect of the viral vector. Although further in vivo work is required to verify these results, this effect needs to be taken into account in studies involving adenoviral gene transfer.

Gene therapy involves transferring foreign genes into unable to multiply and cause progressive infection). Specifically, current vectors for gene therapy have a host cells, where expression of the gene either restores an inherited genetic defect, or modulates an deletion in the E1 region, a part of the viral genome with key roles in regulating expression of other viral acquired disease process. The key to successful gene therapy is an efficient method of gene delivery into genes and in promoting viral replication (Figure 2a). Recombinant adenoviruses are generated in the 293 the target cells: genetically-engineered viruses have received much attention as vectors for gene therapy, cell line, which complements the missing E1 function and allows replication-deficient vectors to be propagas viruses are exquisitely adapted to entering cells, transferring their genetic material, and taking over ated. A new recombinant adenovirus encoding a particular gene of interest is generated by first cloning the host cell machinery to synthesize viral proteins. This review focuses on the use of adenoviral vectors the transgene into a plasmid incorporating an expression cassette and portions of the adenoviral genome. for gene transfer. Particular attention is given to recent developments in recombinant adenovirus Recombination between the plasmid and the ‘backbone’ of the adenoviral genome results in a virus technology which may signal a renewed optimism for their use in human gene therapy. encoding the desired transgene.