Adenovirus-mediated gene transfer to glomerular cells in newborn mice

The systemic delivery of recombinant adenoviral (rAd) vectors to renal glomeruli has been problematic due to the rapid clearance of the circulating virus by the liver. We have previously shown that prolonged retention of rAd vectors in the circulation by liver bypass improves the transduction of renal glomerular cells in adult mice and rats. This study was done to determine whether newborn mice have a delayed clearance of rAd vectors from the circulation and a more efficient transduction of glomerular cells after a systemic injection of rAd vectors. Newborn (1 day old) and adult (6 months old) C57Bl6/J mice ( n =20 in each group) were injected with rAd vectors carrying the lacZ gene (rAd.lacZ) through the retro-orbital venous plexus (2×10⁹ particles/g body weight). The renal expression of Coxsackie and Adenoviral Receptors (CAR) and lacZ gene were evaluated at different time points by Western blots, immunohistochemistry, -galactosidase staining, enzyme assay activity, and RT-PCR studies in newborn and adult mice. The clearance rate of rAd.lacZ was significantly delayed in newborn mice, and the concentration of circulating virus in these mice was almost ten times higher than that in adult mice. Newborn kidneys showed increased expression of CAR, predominately localized in glomerular cells. These findings were associated with an efficient gene transfer of the lacZ gene into glomeruli and tubules of newborn mice. This study demonstrates for the first time the feasibility of using systemic intravenous injections of rAd vectors to express foreign genes in developing glomeruli of young mice.     

Adenovirus-mediated Bcl-2 gene transfer inhibits apoptosis and promotes survival of allogeneic transplanted hepatocytes

BACKGROUND: Donor hepatocyte apoptosis that is induced by host cytotoxic T lymphocytes (CTLs) limits the application of hepatocyte transplantation. Hepatocytes from Bcl-2 transgenic mice can resist the lethal effect of anti-Fas antibody. However, the anti-apoptotic effect of Bcl-2 expression on allogeneic transplanted hepatocytes remains elusive. This study tested the feasibility of Bcl-2 gene transfer as an approach to inhibit CTL-mediated apoptosis in allogeneic transplanted hepatocytes. METHODS: An adenovirus vector that encoded human Bcl-2 gene (AdCMVhBcl-2) was used to transfect cultured rat hepatocytes, which were then transplanted into allogeneic spleens. DNA fragmentation and caspase-3 activation were examined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling assay and immunohistochemistry for active caspase-3, respectively. Cocultivation of hepatocytes and allogeneic CD8(+) T lymphocytes was performed, and cytotoxicity on hepatocytes was examined by alanine transaminase release. RESULTS: Bcl-2 gene transfer inhibited apoptosis and increased liver-associated enzyme activities in allogeneic transplanted hepatocytes, which were associated with inhibition of caspase-3 activation. Alanine transaminase release in hBcl-2 modified hepatocytes was lower compared with controls, which could not be further decreased by inhibition of Fas ligand and granzyme B. CONCLUSIONS: Adenovirus-mediated Bcl-2 gene transfer blocks CTL-mediated apoptosis in allogeneic hepatocytes by inhibition of caspase-3 activation. Bcl-2 gene transfer could be used to promote survival of transplanted hepatocytes.     

A dominant negative cadherin inhibits osteoblast differentiation.

We have previously indicated that human osteoblasts express a repertoire of cadherins and that perturbation of cadherin-mediated cell-cell interaction reduces bone morphogenetic protein 2 (BMP-2) stimulation of alkaline phosphatase activity. To test whether inhibition of cadherin function interferes with osteoblast function, we expressed a truncated N-cadherin mutant (NCaddeltaC) with dominant negative action in MC3T3-E1 osteoblastic cells. In stably transfected clones, calcium-dependent cell-cell adhesion was decreased by 50%. Analysis of matrix protein expression during a 4-week culture period revealed that bone sialoprotein, osteocalcin, and type I collagen were substantially inhibited with time in culture, whereas osteopontin transiently increased. Basal alkaline phosphatase activity declined in cells expressing NCaddeltaC, relative to control cells, after 3 weeks in culture, and their cell proliferation rate was reduced moderately (17%). Finally, 45Ca uptake, an index of matrix mineralization, was decreased by 35% in NCaddeltaC-expressing cells compared with control cultures after 4 weeks in medium containing ascorbic acid and beta-glycerophosphate. Similarly, BMP-2 stimulation of alkaline phosphatase activity and bone sialoprotein and osteopontin expression also were curtailed in NCaddeltaC cells. Therefore, expression of dominant negative cadherin results in decreased cell-cell adhesion associated with altered bone matrix protein expression and decreased matrix mineralization. Cadherin-mediated cell-cell adhesion is involved in regulating the function of bone-forming cells.     

静脉桥基因转录及表达的实验研究

目的：研究静脉桥基因的转移及表达。方法：将转染Ａｄｖ ５－ＣＭＶ／ＬａｃＺ质粒的颈静脉桥移植于同一大鼠的颈总动脉，行β－半乳糖苷酶活性测定和组织化学染色。结果腺病毒载体能使外源性基因有效地转入静脉桥并获得高效的基因表达。结果：用腺病毒载体将治疗性基因导入静脉桥，通过基因高效表达来防止血栓形成、内膜增生和粥样硬化，可能是提高静脉桥近期和远期通畅率的一个有效方法。     

Adenovirus-mediated gene transfer into tendon and tendon sheath

In this study, we successfully transferred the Escherichia coli β-galactosidase gene, LacZ, into the chicken tendon and tendon sheath by a recombinant adenovirus. The recombinant adenovirus Adv-βgal that carried the E. coli LacZ gene was constructed by homologous recombination in 293 cells (human transformed embryonic kidney) between the expressing vector and the ClaI large fragment of adenovirus 5 genome. Each chicken received a 10 μl injection, containing 10⁵ plaque-forming units of recombinant virus Adv-βgal, into the tendon sheath of the long toe. Samples of tendon and tendon sheath were harvested at 3, 30, and 75 days after the injection. The LacZ gene transfer was detected for its coding product β-galactosidase by staining with X-gal solution. The results showed that all tendon and tendon sheath samples from the three harvest times stained positive (blue). The tendon sheath samples were more extensively stained; staining of the tendon was limited to the epitenon layer. These data suggest that a functional exogenous gene can potentially be transferred into the tendon and tendon sheath by similar techniques; such techniques may be used to improve healing and reduce adhesion formation.     

Adenovirus-mediated gene transfer of human inducible nitric oxide synthase in porcine vein grafts inhibits intimal hyperplasia.

OBJECTIVE: The aim of this study is to determine whether adenoviral inducible nitric oxide synthase (iNOS) gene transfer could inhibit intimal hyperplasia (IH) in porcine internal jugular veins interposed into the carotid artery circulation. METHODS: Porcine internal jugular veins were transduced passively with 1 x 10(11) particles of an adenoviral vector carrying either the human iNOS (AdiNOS) or beta-galactosidase (AdlacZ) cDNA for 30 minutes and then interposed into the carotid artery circulation. Segments of each vein graft were maintained in an ex vivo organ culture to measure nitrite accumulation, a marker of nitric oxide synthesis. The grafts were analyzed immunohistochemically for the presence of neutrophils, macrophages, and leukocytes by staining for myeloperoxidase, ED1, and CD45, respectively, at 3 (n = 4) and 7 (n = 4) days. Morphometric analyses and cellular proliferation (Ki67 staining) were assessed at 3 (n = 4), 7 (n = 4), and 21 days (n = 8). RESULTS: AdlacZ-treated vein grafts demonstrated high levels of beta-galactosidase expression at 3 days with a gradual decline thereafter. Nitrite production from AdiNOS-treated vein grafts was approximately fivefold greater than AdlacZ-treated grafts (P =.00001). AdiNOS or AdlacZ treatment was associated with minimal graft inflammation. Cellular proliferation rates were significantly reduced in AdiNOS-treated grafts as compared with controls at both 3 (41%, P =.000004) and 7 days (32%, P =.0001) after bypass. This early antiproliferative effect was most pronounced at the distal anastomosis (65%, P =.0005). The iNOS gene transfer reduced the intimal/medial area ratio in vein grafts at 7 (36%, P =.009) and 21 days (30%, P =.007) versus controls. This inhibition of IH was again more prominent in the distal segments of the grafts (P =.01). CONCLUSION: Adenovirus-mediated iNOS gene transfer to porcine internal jugular vein grafts effectively reduced cellular proliferation and IH. Although iNOS gene transfer reduced IH throughout the entire vein graft, the most pronounced effect was measured at the distal anastomosis. These results suggest potential for iNOS-based genetic modification of vein grafts to prolong graft patency.     

Adenovirus-mediated gene transfer in the midgestation fetal mouse.

BACKGROUND: The development of strategies for gene transfer in utero will make possible the amelioration, and eventually the cure, of genetic diseases associated with pre- and postnatal morbidity and mortality. We have developed a murine model for in utero, intrahepatic, adenovirus-mediated gene transfer in Day 15 fetuses and compared the level and distribution of luciferase reporter gene expression in newborns with those observed in adult animals injected intravenously. MATERIAL AND METHODS: CD-1 fetuses underwent intrahepatic injection on Day 15 of gestation with 1 x 10(7) particle-forming units (PFU) of an E1- and E3-deleted recombinant adenovirus containing the luciferase reporter gene or with normal saline. At birth, pups were euthanized, and the brain, heart, intestine, liver, lungs, and spleen harvested and analyzed for luciferase activity. RESULTS: Two adenovirus-injected litters proceeded to term and one female aborted. Tissues from 10 newborn mice in the experimental group and 5 newborns in the control group were analyzed; tissues from the remaining newborns were reserved for other studies. High-level luciferase expression was detected in all adenovirus-injected newborn livers. Lower levels of luciferase activity were detected in distant organs. Hepatic toxicity as determined by serum transaminase elevations was observed in adult, but not in newborn mice previously injected with the adeno-luciferase virus. CONCLUSIONS: In utero intrahepatic gene delivery with adenoviral vectors in the developing murine fetus is feasible and produces high-level gene expression. These studies suggest that viral and nonviral gene delivery vectors may be useful in the development of future approaches to prenatal treatment of genetic disorders.     

Vitamin E inhibits proliferation of primary cultured human mesangial and endothelial cells

BACKGROUND: Vitamin E (VE) has been used as an antioxidant and has been suggested to inhibit the proliferation of mesangial cells in rat and vascular endothelial cells. The direct effect of VE on primary cultures of mesangial cells (MC) and endothelial cells (EC) from the human glomerulus was studied. METHODS: (1) MC (in 17 or 2.5% FCS DMEM) or EC (in 10 or 5% FCS CSC) at 5,000 cells/well was incubated with serial concentrations of VE from 0.05 to 50 microg/ml (0.06 to 60 IU/l). (2) MC was cocultured with 160, 80, 40 or 20 microg/ml of low-density lipoprotein (LDL) or oxidized LDL (ox-LDL) in 17 or 2.5% FCS DMEM with or without VE. After 3 days of incubation at 37 degrees C in 5% CO(2), cell proliferation was measured by the Premix WST-1 Assay System. RESULTS: The concentration of VE that significantly inhibited the proliferation of MC cultured in 17 or 2.5% FCS DMEM was 50 or 2.5 microg/ml (60 or 3.0 IU/l), respectively, and that of EC in 10 or 5% FCS medium was 50 or 25 microg/ml (60 or 30 IU/l). VE at 25 microg/ml (30 IU/l) inhibited the LDL proliferative effect on MC cultured in 2.5 FCS DMEM by 21.79-93.21% in a LDL concentration-dependent manner. There was little difference between the effects of LDL and ox-LDL on the VE inhibitory effect on MC under our experimental conditions. CONCLUSION: VE at low concentrations had no effect on the proliferation of both MC and EC, but at high concentrations, it showed an inhibitory effect on both cells.     

BC047440基因RNA干扰抑制肝癌HepG2细胞增殖

目的 构建shRNA并干扰肝癌HepG2细胞中BC047440基因,观察其对肝癌HepG2细胞增殖的影响.方法 根据BC047440基因序列,设计和合成两条特异表达该基因shRNA序列并克隆载体pGenesil-1,转染肝癌HepG2细胞并用定量PCR观察抑制BC047440基因表达的效果,并通过MTT法和流式细胞仪检测细胞增殖及细胞周期的变化.结果 酶切和序列测定提示成功构建了特异表达BC047440基因的shRNA1和shRNA2两个质粒.shRNA1和shRNA2对BC047440 mRNA的抑制率分别为80.22%和58.63%.干扰BC047440基因后,肝癌HepG2细胞增殖受到明显抑制,主要停滞在S期.结论 构建的shRNA能有效干扰肝癌HepG2细胞BC047440基因的表达,并抑制肝癌HepG2细胞的增殖,提示BC047440基因和肝癌HepG2细胞增殖有关.     

Adenovirus-mediated gene transfer using in-situ perfusion of the liver graft

To establish an efficient technique for adenovirus-mediated gene transfer in liver transplantation, we evaluated the in situ perfusion of liver grafts. The grafts were perfused in situ with 1 × 10¹⁰ of E1-deleted, replication-defective adenoviral vectors encoding the LacZ gene driven by the human CMV promoter, either through the hepatic artery (group 1) or the portal vein (group 2). Group 3 animals served as negative controls; their liver grafts were perfused with lactated Ringer's solution through the portal vein. PCR confirmed the presence of viral DNA in every graft perfused with viral vectors. In X-gal staining, positive staining was observed almost exclusively at the portal triad in group 1, whereas in group 2 minimal staining was observed, predominantly in the parenchymal area. Protein production from the transfected gene was confirmed by a functional protein assay; the values were 0.16 % ± 0.07 % liver protein in group 1, 0.13 % ± 0.02 % in group 2, and 0.007 % ± 0.0003 % in group 3 on postoperative day 2. In conclusion, in situ perfusion of the viral vectors through the hepatic artery resulted in an effective expression of the transfected gene, predominantly at the portal triad. Received: 15 July 1996 Received after revision: 1 November 1996 Accepted: 12 November 1996     

Inhibition of NF2-negative and NF2-positive primary human meningioma cell proliferation by overexpression of merlin due to vector-mediated gene transfer.

OBJECT: The absence of in vitro models of neurofibromatosis Type 2 (NF2)-defective meningiomas has limited investigative efforts to study the biological effects of this gene in the pathogenesis of these tumors. The goals of this report are to show that gene transfer vectors can efficiently express the wild-type NF2 transgene into primary meningioma cells and to determine effects on cellular proliferation. METHODS: In this study, the authors have compared the transducing capacities of a retrovirus, an adenovirus, and a herpes simplex virus amplicon vector for use in primary human meningioma cells harvested from human tumors excised from patients with and without NF2. Transduction efficiencies with the latter vector approached 100% and it was selected to transfer the wild-type NF2 transgene into these cells. Western blot analysis confirmed that vector-mediated gene transfer mediated the expression of the NF2-encoded polypeptide merlin. Overexpression of merlin significantly inhibited the proliferation of both NF2-negative and NF2-positive human meningioma cells when compared to the proliferation of cells transduced with a control vector. CONCLUSIONS: This study demonstrates the feasibility of using vector-mediated gene transfer to study wild-type NF2 gene function in short-term cultures of primary human meningioma cells.     

Adenovirus-mediated Bak gene transfer induces apoptosis in mesothelioma cell lines

OBJECTIVE: Conventional treatment for mesothelioma is largely ineffective. We therefore evaluated the novel approach of adenoviral gene transfer of the proapoptotic Bcl-2 family member Bak in mesothelioma cancer cell lines, which are sensitive and resistant to adenoviral p53. METHODS: Binary adenoviral Bak (Ad/GT-Bak and Ad/GV16) and LacZ (Ad/GT-LacZ and Ad/GV16) vectors were used for transduction of the mesothelioma cell lines I-45 (p53 resistant) and REN (p53 sensitive). Protein levels were determined by Western blotting. Apoptosis was assessed by morphologic changes, caspase-3 cleavage, and fluorescence-activated cell sorter analysis of subdiploid populations. Cell viability was determined with the XTT assay. Statistical analysis was performed with analysis of variance and the Student t test. RESULTS: High levels of Bak gene transfer were seen after coadministration of Ad/GT-Bak and Ad/GV16 in both mesothelioma cell lines. Apoptosis was induced 24 hours after Bak but not LacZ gene transfer ([Bak: I-45, 36%; REN, 25%] vs [LacZ: I-45, 1%; REN, 3%], P <.05]) in p53-sensitive (REN) and p53-resistant (I-45) cell lines. Cellular viability was significantly decreased 48 to 72 hours after Bak gene transfer compared with control vector in both cell lines (72 hours: Bak I-45, 1.4% +/- 1.0%, and Bak REN, 4.7% +/- 1%, vs Lac-Z I-45, 83% +/- 3%, and Lac-Z REN, 100% +/- 1%; P <.05). CONCLUSIONS: Adenovirus-mediated overexpression of the Bak gene induces apoptosis and decreased cellular viability in p53-sensitive and p53-resistant mesothelioma cells. These data suggest that the gene transfer of proapoptotic Bcl-2 family members may represent a novel gene therapy strategy to treat mesothelioma.     

Adenovirus-mediated p53 gene transfer to rat testis impairs spermatogenesis

The tumor suppressor protein p53 participates in normal cell differentiation as well as induction of programmed cell death. The authors investigated the effect of p53 overexpression on spermatogenesis by transferring p53 gene into the rat testes. Replication-deficient recombinant adenovirus vectors were constructed to include cytomegalovirus (CMV) promoter driving wild-type p53 (Ad-CMV-p53) or beta-galactosidase (Ad-CMV-beta-gal). Virus was delivered to cells of the tubules by slow retrograde injection through the rete testis. At 0, 4, 7, and 14 days, testes were removed, weighed, and analyzed histopathologically, including immunohistochemistry for p53, Bcl-2, Bax, and interleukin-1beta converting enzyme (ICE). Testicular weight was decreased in Ad-CMV-p53 group at 14 days after injection, while no change occurred in phosphate-buffered saline-injected controls or Ad-CMV-beta-gal-infected testes. Beyond 4 days, cell degradation in tubules interfered with immunohistochemical observation in the Ad-CMV-p53 group. At 4 days, p53 was expressed mostly in spermatocytes. Bax showed greater expression in the p53 group than in the control or Ad-CMV-beta-gal group. ICE, expressed mostly in spermatids, was more abundant in the p53 group than in controls. Overall, p53 overexpression in the testis impaired spermatogenesis.     

Ad-hBMP-2转染体外培养人退变腰椎间盘细胞的研究

[目的]研究人骨形态发牛蛋白-2腺病毒表达载体（Ad—hBMP-2）转染体外培养人退变腰椎问盘细胞，分析其对腰椎间盘细胞影响.[方法]成人退变腰椎间盘细胞体外培养，通过免疫组化和染色体分析鉴定椎间盘细胞。利用免疫荧光和蛋白印迹法检测不同转染剂量对细胞BMP-2表达的影响。[结果]体外培养成人退变腰椎间盘细胞第2代鉴定具有其结构功能。转染后通过免疫荧光和蛋白印迹法可见随转染剂量增加BMP-2表达量逐渐增加。[结论]Ad—hBMP-2可高效转染体外培养人退变椎间盘细胞，同时随转染剂量增加BMP-2表达量逐渐增加。     

Adenovirus-mediated gene transfer using ex vivo perfusion of the heart graft

A replication-deficient adenovirus was used for ex vivo gene transfer into rat heart grafts under conditions simulating clinical transplantation. The adenoviral vector, AdHCMVsp1LacZ, containing an expression cassette of Escherichiae coli lacZ, was used to perfuse heart grafts during cold ischemia before transplantation. Heart grafts were perfused with University of Wisconsin (UW) solution containing either 0 pfu, 5×10¹⁰ pfu, or 1×10¹¹ pfu of viral vector, and were preserved for either 2 or 4 h and then transplanted into syngeneic recipients. The animals were killed at 1, 7, and 14 days after transplantation. The infection rate was assessed by histochemical staining for -galactosidase. Using polymerase chain reaction (PCR), viral DNA presence was confirmed in every graft perfused with viral vectors. The protein production from the transfected gene was confirmed by a functional protein assay. An efficient gene transfer was achieved with an infection rate of 1%–1.5% for all cardiac myocytes, as assessed by 5-bromo-4-chloro-indolyl--d-galactopyranoside (X-gal) staining. All studies were negative in the control grafts. Gene expression persisted for at least 10 days after transplantation. We thus conclude that an efficient adenovirus-mediated gene transfection and expression of gene products can be achieved in ex vivo perfusion of the heart graft during cold preservation.