The application of DNA technology to regulate the transcription of disease-related genes in vivo has important therapeutic potentials. The transcription factor E2F plays a pivotal role in the coordinated transactivation of cell cycle-regulatory genes such as c-myc, cdc2, and the gene encoding proliferating-cell nuclear antigen (PCNA) that are involved in lesion formation after vascular injury. We hypothesized that double-stranded DNA with high affinity for E2F may be introduced in vivo as a decoy to bind E2F and block the activation of genes mediating cell cycle progression and intimal hyperplasia after vascular injury. Gel mobility-shift assays showed complete competition for E2F binding protein by the E2F decoy. Transfection with E2F decoy inhibited expression of c-myc, cdc2, and the PCNA gene as well as vascular smooth muscle cell proliferation both in vitro and in the in vivo model of rat carotid injury. Furthermore, 2 weeks after in vivo transfection, neointimal formation was significantly prevented by the E2F decoy, and this inhibition continued up to 8 weeks after a single transfection in a dosedependent manner. Transfer of an E2F decoy can therefore modulate gene expression and inhibit smooth muscle proliferation and vascular lesion formation in vivo.

We examined angiotensinogen gene expression in rat kidney by in situ hybridization histochemistry. Using a rat cRNA probe to angiotensinogen, we demonstrated angiotensinogen mRNA to be localized predominantly in the proximal renal tubule, with considerably lesser amounts in distal tubular segments and glomerular tufts. Previous studies have localized renin immunoreactivity to the juxtaglomerular cells, glomerular tufts, and proximal tubules. Such findings provide further evidence for a local tissue renin angiotensin system within the kidney which may influence regional function. Based on our data, we hypothesize that a major site of angiotensin production is the proximal tubule. We postulate that angiotensin synthesized in and/or around the proximal tubule may directly modulate tubular transport of sodium, bicarbonate, and water. In addition to the proximal tubule, the specific localization of the renin angiotensin components elsewhere in the kidney would also support the other proposed regional functions of the intrarenal system, including modulation of tubuloglomerular balance. (J. Clin. Invest. 1990. 85:417-423.) renin -angiotensinogen * kidney-in situ hybridization -RNA

To develop an effective strategy to prevent neointima formation after angioplasty ijury, we have identified cell-cycle regulatory proteins as targets for inhibition by using antisense oligonucleotides (ODNs). We utilized an intraluminal molecular delivery method that employs the protein coat of a Sendai virus complexed with liposomes that enhances markedly the efficiency of ODNs uptake. First, we examined the effect of antisense cdc2 kinase and proliferating-cell nuclear antigen (PCNA) ODNs in vitro. [3][4][5]. This failure may reflect the difficulty in identifying appropriate drug targets due to the complexity of the pathophysiological process of neointima formation and/or the inability to deliver sufficient quantities of drugs to the site of injury. Neointima formation after angioplasty involves a complex interaction between multiple growth factors that promote vascular smooth muscle cell (VSMC) proliferation and migration including: thrombin, platelet-derived growth factor, and basic fibroblast growth factor, to name a few (6). Given the multiplicity ofgrowth factors involved, it appears unlikely that selective inhibition of a particular growth factor will completely prevent lesion formation (2-5, 7, 8). Growth-factorinduced cell proliferation involves the sequential activation ofThe 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.intracellular proteins that promote cell-cycle progression (9, 10). Accordingly, we hypothesized that restenosis could be prevented by the blockade of genes regulating cell-cycle progression-the final common pathway. Previous studies have established that proliferating-cell nuclear antigen (PCNA; a nuclear protein required for DNA synthesis by DNA polymerase A) and p34cdc2 (a serine/threonine protein kinase) are principle components ofthe final common pathway regulating cell proliferation (9-12). PCNA and cdc2 kinase appear to play an essential role in the transition through both the G1/S and G2/M phases of the cell cycle. We therefore employed antisense oligonucleotides (ODNs) directed at the translation initiation sites ofPCNA and cdc2 mRNAs to inhibit neointimal hyperplasia. The effectiveness of the antisense approach in vivo has been reported by Simon et al. (13). Our results demonstrated the efficacy of combined administration of these ODNs on VSMC proliferation in vitro and in vivo.To enhance the efficiency of cellular uptake and the stability of antisense ODNs while minimizing nonspecific toxicity, we developed a viral protein-mediated ODN transfer technique. Phosphorothioate ODNs were complexed with liposomes and the protein coat of the inactivated hemagglutinating virus of Japan (HVJ) (14-17). This method resulted in a more rapid cellular uptake and a 10-fold higher transfection efficiency of ODN or plasmid DNA than lipofection or passive uptake methods (17). Using the HVJ method, we demonstrated that a single ...

The cell cycle regulatory enzyme, cdk (cyclin-dependent kinase) 2 kinase, is activated in the rat carotid artery after balloon angioplasty injury, and may mediate smooth muscle proliferation. To

Renal angiotensinogen (ang-n) mRNA concentration in the male WKY rat increases significantly during puberty. Furthermore, renal angiotensinogen mRNA level in the adult female WKY rat is considerably lower than in the male. The present study investigates the role of androgen in differential renal ang-n mRNA expression. Northern and slot blot analyses with a-32P labeled ang-n cDNA (pRang 3) demonstrated that castration lowered ang-n mRNA levels in the male kidney by 2 60% compared with control, suggesting that androgen may be involved with renal ang-n gene regulation. Moreover, male WKY rats castrated as weanlings and normal adult female WKY rats each implanted with testosterone displayed significant (P < 0.05) increases in renal ang-n mRNA levels. Our observations, taken together with previous reports that androgen influences proximal tubule morphology and the tubular expression of transport proteins (e.g., Na+/H+ antiporter), may have important physiological implications for understanding the relationship between androgen and angiotensin in the regulation of tubular function.

Rat liver angiotensinogen cDNA (pRang 3) and mouse renin cDNA (pDD-lD2) were used to identify angiotensinogen and renin mRNA sequences in rat kidney cortex and medulla in rats on high and low salt diet. Angiotensinogen mRNA sequences were present in renal cortex and medulla in apparently equal proportions, whereas renin mRNA sequences were found primarily in renal cortex. Average relative signal of rat liver to whole kidney angiotensinogen mRNA was 100:3. Densitometric analysis of Northern blots demonstrated that renal cortical angiotensinogen mRNA concentrations increased 3.5-fold (P < 0.001) and medulla, 1.5-fold (P < 0.005) on low sodium compared with high sodium diet, whereas renal cortex renin mRNA levels increased 6.8-fold (P < 0.0005). Dietary sodium did not significantly influence liver angiotensinogen mRNA levels. These findings provide evidence for sodium regulation of renal renin and angiotensinogen mRNA expressions, which supports potential existence of an intrarenally regulated RAS and suggest that different factors regulate renal and hepatic angiotensinogen.

Previous studies have reported the presence of renin mRNAs in several mouse tissues and angiotensinogen mRNAs in various rat tissues. Clarification as to whether renin and angiotensinogen mRNAs are coexpressed in the same tissues of the same animal species is important for understanding the biology of the tissue renin-angiotensin system. We employed mouse renin cDNA and rat angiotensinogen cDNA to compare tissue distributions of renin and angiotensinogen in RNAs of the rat and mouse. Both cDNA probes readily cross-hybridize with the corresponding mRNA of the other species. Our results demonstrate several patterns of distribution. Renin and angiotensinogen mRNAs are readily detected in kidney and adrenals of both species. In brain and heart, angiotensinogen mRNAs are present in concentrations that far exceed renin mRNA levels in these organs in both species. In mouse and rat livers, angiotensinogen, but not renin, mRNA is demonstrated. In rat testis, only renin mRNA can be detected, whereas in mouse testes both renin and angiotensinogen mRNA are present. In CD-1 male mouse submandibular gland, renin mRNA exists in high concentrations, whereas angiotensinogen mRNA is present in low levels. In contrast, neither renin nor angiotensinogen mRNA could be detected in rat salivary gland. In summary, our study demonstrates the widespread codistribution of renin and angiotensinogen mRNAs in many tissues of both species, allowing for the possibility of local angiotensin production. However, tissue and species differences in these gene expressions also exist. Understanding differential tissue expressions of these genes will provide additional important insight into the biology of the renin-angiotensin system.

IntroductionIn vitro studies have demonstrated that angiotensin (Ang) II directly stimulates vascular smooth muscle cell (VSMC) The pathogenesis of vascular diseases such as hypertension, atherosclerosis, and restenosis involves a process of vascular remodeling associated with increased local expression of biologically active substances that are postulated to play pathophysiological roles. In hypertension, the arteries undergo a process of vascular hypertrophy that is associated with the activation of a local angiotensin system (1-3). The potential role of autocrine/paracrine mediators such as angiotensin in vascular pathobiology independent from systemic factors has been suggested from indirect evidence, that is, cell culture studies, morphologic analysis, and/or by systemic administration of antagonists and agonists (1-16). To elucidate the role of a specific autocrine/paracrine factor, we have developed an efficient in vivo gene transfer technique to examine the consequences of overexpression of the factor in a segment of the carotid artery in the intact rat. This approach is particularly powerful because the locally transfected vessel segment can be compared with adjacent untransfected segments as well as the contralateral vessel. Furthermore, the transfected segment is exposed to the same blood pressure and circulating factors as the control vessel. In this study, we examined the role of autocrine/paracrine angiotensin as a mediator of vascular hypertrophy in vivo. Previous data have demonstrated that angiotensin II (Ang II)' can stimulate smooth muscle cell growth and modulate extracellular matrix (10-13). Ang II is generated via an enzymatic cascade in which tissue angiotensin converting enzyme (ACE) plays a key role (17)(18)(19)