Naruya Tomita

Inhibition of Renal Fibrosis by Gene Transfer of Inducible Smad7 Using Ultrasound-Microbubble System in Rat UUO Model

TGF-beta is a key mediator in renal fibrosis. Kidney-targeted gene therapy with anti-TGF-beta strategies is expected to have therapeutic potential, but this has been hampered by concerns over the safety and practicability of viral vectors and the inefficiency of nonviral transfection techniques. The present study explored the potential role of TGF-beta/Smad signaling in renal fibrosis in vivo and developed a safe and effective gene therapy to specifically block TGF-beta signaling and renal fibrosis in a rat unilateral ureteral obstruction (UUO) model by transferring a doxycycline-regulated Smad7 gene or control empty vectors using an ultrasound-microbubble (Optison)-mediated system. The Smad7 transgene expression was tightly controlled by addition of doxycycline in the daily drinking water. Groups of six rats were sacrificed at day 7, and the transfection rate, Smad7 transgene expression, and tubulointerstitial fibrosis including alpha-smooth muscle actin and collagen matrix mRNA and protein expression were determined. Compared with the non-ultrasound treatment, the combination of ultrasound with Optison largely increased the transfection rate of FITC-ODN and Smad7 transgene expression up to a 1000-fold, and this was found in all kidney tissues. Compared with normal rats, Smad7 expression within the UUO kidney was significantly reduced, and this was associated with up to a sixfold increase in Smad2 and Smad3 activation and severe tubulointerstitial fibrosis. In contrast, treatment with inducible Smad7 resulted in a fivefold increase in Smad7 expression with complete inhibition of Smad2 and Smad3 activation and tubulointerstitial fibrosis in terms of tubulointerstitial myofibroblast accumulation (85% downward arrow ) and collagen I and III mRNA and protein expression (60 to 70% downward arrow ). In conclusion, the ultrasound-mediated inducible Smad7 gene transfer is a safe, effective, and controllable gene therapy. TGF-beta-mediated renal fibrosis is regulated positively by Smad2/3, but negatively by Smad7. Target blockade of TGF-beta/Smad signaling by expression of Smad7 may provide a new therapeutic potential for renal fibrosis.

Amelioration of progressive renal injury by genetic manipulation of Klotho gene

Klotho, an antiaging gene with restricted organ distribution, is mainly expressed in the kidney tubules; the mutant mice have shortened life span, arteriosclerosis, anemia, and osteoporesis, features common to patients with chronic renal failure. Conceivably, the reduction of the Klotho gene expression may contribute to the development of kidney failure; alternatively, its overexpression may lead to the amelioration of renal injury in an ICR-derived glomerulonephritis (ICGN) mouse model with subtle immune complex-mediated disease. To address this issue, four different strains of mice were generated by cross-breeding: ICGN mice without the Klotho transgene (ICGN), ICGN mice with the Klotho transgene (ICGN/klTG), wild-type mice with the Klotho transgene (klTG), and wild-type mice without the Klotho transgene (control). At 40 weeks old, the survival rate was ≈30% in ICGN mice, and ≈70% in the ICGN/klTG group. This improvement was associated with dramatic improvement in renal functions, morphological lesions, and cytochrome c oxidase activity but a reduction in β-galactosidase activity (a senescence-associated protein), mitochondrial DNA fragmentation, superoxide anion generation, lipid peroxidation, and Bax protein expression and apoptosis. Interestingly, improvement was seen in both the tubular and glomerular compartments of the kidney, although Klotho is exclusively confined to the tubules, suggesting that its gene product has a remarkable renoprotective effect by potentially serving as a circulating hormone while mitigating the mitochondrial oxidative stress.

Direct in vivo gene introduction into rat kidney.

We established a simple and highly efficient method for in vivo gene transfer using HVJ (Sendai virus) and liposomes. Plasmid DNA and high mobility group 1 (HMG1) protein were co-encapsulated in liposomes by agitation and sonication and were co-introduced into cells by HVJ-mediated membrane fusion. pACT SVT DNA, as a reporter gene, was introduced into the kidney of intact rats through a cannula in the renal artery, and SV40 large T antigen was detected by enzyme immunohistochemistry in glomerular cells 4 days after its introduction. This newly developed kidney-directed gene transfer method should be useful not only in basic research but also in potential gene therapeutics of renal diseases.

Intratumoral injection of oligonucleotides to the NFκB binding site inhibits cachexia in a mouse tumor model

Cancer cachexia, characterized by anorexia, weight loss and progressive tissue wasting, has been postulated to be mediated by various cytokines. However, the precise mechanism of cachexia induction is not fully explained. We have developed synthetic double-stranded oligodeoxynucleotides (ODN) as ‘decoy’ cis-elements that block the binding of nuclear factors to promoter regions of targeted genes, resulting in the inhibition of gene transactivation in vivo as well as in vitro. This novel molecular strategy could be useful for treating a broad range of human diseases including cancer. In this study, we injected decoy ODN targeting the transcriptional factor, NF-kappaB (NFκB) binding cis-elements, which are essential for transactivation of gene expression of cytokines, directly into tumors of adenocarcinoma colon26 in mice, in order to examine whether or not cachexia is alleviated by inhibiting the action of cytokines. Tumor growth was not affected by transfection of NFκB decoy ODN as compared with scrambled decoy ODN. Nevertheless, transfection of NFκB decoy, but not scrambled decoy, ODN resulted in attenuation of the reductions in body weight, epididymal fat, gastrocnemius muscle mass and food intake, which were induced by the tumor presence. Interleukin 6 mRNA in the tumor was also markedly decreased by the transfection of NFκB decoy ODN. It is known that the transcriptional factor E2F plays a pivotal role in the coordinated transactivation of cell cycle regulatory genes. Therefore, we hypothesized that the introduction of synthetic double-stranded DNA with high affinity for E2F in vivo as ‘decoy’ cis-elements might inhibit the tumor growth of colon26, resulting in turn in inhibition of cachexia induction. However, injection of E2F decoy ODN failed to inhibit tumor growth and cachexia induction, as compared with mismatched decoy ODN. Overall, the present study demonstrated that cachexia induced by adenocarcinoma colon26 was inhibited by blocking of NFκB, using a novel molecular decoy strategy, without an effect on tumor growth, and also that tumor growth and cachexia induction in the colon26 model were not affected by E2F decoy ODN. These results suggest that cytokines regulated by NFκB may play a pivotal role in the induction of cachexia by colon26, providing a new therapeutic strategy for cancer cachexia.

An efficient gene transfer method mediated by ultrasound and microbubbles into the kidney.

Safety issues are of paramount importance in clinical human gene therapy. From this point of view, it would be better to develop a novel non‐viral efficient gene transfer method. Recently, it was reported that ultrasound exposure could induce cell membrane permeabilization and enhance gene expression.

Angiogenic property of hepatocyte growth factor is dependent on upregulation of essential transcription factor for angiogenesis, ets-1.

Background—Although hepatocyte growth factor (HGF) is an angiogenic growth factor, it is still unclear how it exerts its angiogenic effects. Thus, we focused on the role of an essential transcription factor for angiogenesis, ets-1. In this study, we addressed the following specific questions: (1) what genes responsible for angiogenesis can be regulated by HGF and (2) whether upregulation of gene expression for angiogenesis is dependent on ets-1. Methods and Results—In human endothelial cells, HGF significantly stimulated the matrix-degrading pathway, such as the production of matrix metalloprotease-1 (MMP-1) through its specific receptor, c-met. In addition, HGF also significantly increased HGF itself and its specific receptor, c-met. Moreover, HGF significantly increased the transcription activity and mRNA expression of ets-1 in a time-dependent manner. Importantly, transfection of antisense ets-1 oligodeoxynucleotides (ODN) resulted in a significant reduction in MMP-1, HGF and c-met. Interestingly, HGF also stimulated ets-1 mRNA in vascular smooth muscle cells, similar to endothelial cells. Of importance, transfection of antisense ets-1 ODN resulted in a significant decrease in vascular endothelial growth factor (VEGF) and HGF expression, whereas HGF stimulated both HGF and VEGF expression. Moreover, in vivo transfection of ets-1 antisense ODN resulted in an inhibition of angiogenesis induced by the HGF gene in a rat ischemic hindlimb model. Conclusions—Here, we demonstrated that HGF stimulated the expression of MMP-1, VEGF, HGF itself, and c-met in human endothelial cells and vascular smooth muscle cells. Upregulation of angiogenesis-related genes was largely dependent on the induction of ets, especially ets-1. These data provide new information about the mechanisms of angiogenesis.

Ribozyme Oligonucleotides Against Transforming Growth Factor-β Inhibited Neointimal Formation After Vascular Injury in Rat Model Potential Application of Ribozyme Strategy to Treat Cardiovascular Disease

BackgroundBecause the mechanisms of atherosclerosis or restenosis after angioplasty have been postulated to involve an increase in transforming growth factor (TGF)-&bgr;, a selective decrease in TGF-&bgr; may have therapeutic value. Thus, we used the ribozyme strategy to actively cleave the targeted gene to selectively inhibit TGF-&bgr; expression. Methods and ResultsWe constructed ribozyme oligonucleotides (ONs) targeted to the sequence of the TGF-&bgr; gene that shows 100% homology among the human, rat, and mouse species. The specificity of ribozyme against TGF-&bgr; gene was confirmed by selective inhibition of TGF-&bgr; mRNA in cultured vascular smooth muscle cells as well as balloon-injured blood vessels in vivo. Importantly, the marked decrease in TGF-&bgr; resulted in significant inhibition of neointimal formation after vascular injury in a rat carotid artery model (P <0.01), whereas DNA-based control ONs and mismatched ribozyme ONs did not have any inhibitory effect on neointimal formation. Inhibition of neointimal formation was accompanied by (1) a reduction in collagen synthesis and mRNA expression of collagen I and III and (2) a significant decrease in DNA synthesis as assessed by proliferating cell nuclear antigen staining. Moreover, we modified ribozyme ONs containing phosphorothioate DNA and RNA targeted to the TGF-&bgr; gene. Of importance, modified ribozyme ONs showed a further reduction in TGF-&bgr; expression. ConclusionsOverall, this study provides the first evidence that selective blockade of TGF-&bgr; resulted in inhibition of neointimal formation, accompanied by a reduction in collagen synthesis and DNA synthesis in a rat model. We anticipate that modification of ribozyme ON pharmacokinetics will facilitate the potential clinical utility of the ribozyme strategy.

Transient Decrease in High Blood Pressure by In Vivo Transfer of Antisense Oligodeoxynucleotides Against Rat Angiotensinogen

The renin-angiotensin system plays an important role in blood pressure regulation. Angiotensinogen, which is mainly produced in the liver, is a unique component of the renin-angiotensin system, because angiotensinogen is only known as a substrate for angiotensin I generation. It is unclear whether circulating angiotensinogen is a rate-limiting step in blood pressure regulation. Recent findings of genetic studies and analyses suggest that the angiotensinogen gene may be a candidate as a determinant of hypertension. To test the hypothesis that angiotensinogen may modulate blood pressure, we transfected antisense oligonucleotides against rat angiotensinogen into the rat liver via the portal vein using liposomes that contain viral agglutinins to promote fusion with target cells, a technique that has been reported to be highly efficient. Transfection of antisense oligonucleotides resulted in a transient decrease in plasma angiotensinogen levels in spontaneously hypertensive rats from day 1 to day 7 after the injection, consistent with the reduction of hepatic angiotensinogen mRNA. Plasma angiotensin II concentration was also decreased in rats transfected with antisense oligonucleotides. Moreover, a transient decrease in blood pressure from day 1 to day 4 was observed, whereas transfection of sense and scrambled oligonucleotides did not result in any changes in plasma angiotensinogen level, blood pressure, or angiotensinogen mRNA level. Overall, our results demonstrate that transfection of antisense oligonucleotides against rat angiotensinogen resulted in a transient decrease in the high blood pressure of spontaneously hypertensive rats, accompanied by a decrease in angiotensinogen and angiotensin II levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Transcription factor decoy for NFκB inhibits TNF-α-induced cytokine and adhesion molecule expression in vivo

The expression of several cytokines and adhesion molecules is regulated by the transcription factor NFκB, which is activated by tumor necrosis factor alpha (TNF-α). In this study, we employed a mouse model of nephritis induced by TNF-α to examine whether inhibition of NFκB activity using transcription factor decoy oligonucleotides (ODN) blocks cytokine and adhesion molecule expression and attenuates the renal inflammatory response. First, we developed a method for delivering FITC-ODN in vivo into mouse kidney glomeruli by employing HVJ-liposome. Then, in order to study the feasibility of decoy strategy in vivo, the reporter gene chloramphenicol acetyltransferase (CAT) driven by three tandemly repeated NFκB binding sequences was transfected into the kidney. Intrapenetorial injection of TNF-α stimulated CAT expression in vivo, and the increase in CAT expression was completely abolished by NFκB decoy ODN, but not scrambled ODN. Therefore, we examined the effect of NFκB decoy ODN transfection on TNF-α-induced endogenous interleukin (IL)-1α, IL-1β, IL-6, ICAM-1 and VCAM-1 gene expression as assessed by RT-PCR and Northern blotting. Our present data showed that NFκB decoy, but not scrambled, ODN abolished TNF-α induced gene expression in vivo, as well as glomerular inflammation as assessed by CD45 staining. Taken together, our results suggest the potential utility of NFκB decoy strategy for molecular therapy to glomerular inflammatory diseases.

Transfection of NFκB-decoy oligodeoxynucleotides using efficient ultrasound-mediated gene transfer into donor kidneys prolonged survival of rat renal allografts

Nuclear factor κB (NFκB) plays a pivotal role in the coordinated transactivation of a series of genes of cytokines and adhesion molecules that are highly involved in the onset of acute rejection in organ transplantation. We previously developed decoy cis-elements oligo deoxyribonucleic acid against NFκB (NFκB-decoy) that effectively inhibited the activation of major inflammatory mediators in vitro and in vivo. Accordingly, we hypothesized that transfection of NFκB-decoy into the donor kidney would prevent acute rejection and prolong graft survival, and thus provide effective therapy for renal acute rejection. To transfect NFκB-decoy, we employed a novel approach using ultrasound exposure with an echocardiographic contrast agent, Optison, and clearly demonstrated successful transfection of NFκB-decoy into renal tissue. The therapeutic effect of NFκB-decoy on renal allografts was then evaluated in a rat renal allograft model (Wistar–Lewis). In the control group, graft function significantly deteriorated with marked destruction of renal tissue, accompanied by increased production of major inflammatory mediators, and all animals died of renal failure by 9 days. In contrast, graft function (serum creatinine on day 2, NFκB-treated: 0.97±0.16 versus control: 1.84±0.23 mg/dl, P<0.01) and histological structure were well preserved with significantly decreased expression of NFκB-regulated cytokines and adhesion molecules, including IL-1, iNOS, MCP-1, TNF-α, and ICAM-1, in allografts transfected with NFκB-decoy. As a result, animal survival was significantly prolonged in this group as compared to controls (14.2±5.2 versus 7.1±1.2 days, P<0.01). Thus, we established a novel ultrasound–Optison-mediated gene transfection approach and demonstrated the significant prolongation of graft survival by the successful transfection of NFκB-decoy into the donor kidney in a rat renal allograft model.