The antiandrogen enzalutamide downregulates TMPRSS2 and reduces cellular entry of SARS-CoV-2 in human lung cells.

SARS-CoV-2 attacks various organs, most destructively the lung, and cellular entry requires two host cell surface proteins: ACE2 and TMPRSS2. Downregulation of one or both of these is thus a potential therapeutic approach for COVID-19. TMPRSS2 is a known target of the androgen receptor, a ligand-activated transcription factor; androgen receptor activation increases TMPRSS2 levels in various tissues, most notably prostate. We show here that treatment with the antiandrogen enzalutamide-a well-tolerated drug widely used in advanced prostate cancer-reduces TMPRSS2 levels in human lung cells and in mouse lung. Importantly, antiandrogens significantly reduced SARS-CoV-2 entry and infection in lung cells. In support of this experimental data, analysis of existing datasets shows striking co-expression of AR and TMPRSS2, including in specific lung cell types targeted by SARS-CoV-2. Together, the data presented provides strong evidence to support clinical trials to assess the efficacy of antiandrogens as a treatment option for COVID-19.

Delivery of sTRAIL variants by MSCs in combination with cytotoxic drug treatment leads to p53-independent enhanced antitumor effects.

Mesenchymal stem cells (MSCs) are able to infiltrate tumor tissues and thereby effectively deliver gene therapeutic payloads. Here, we engineered murine MSCs (mMSCs) to express a secreted form of the TNF-related apoptosis-inducing ligand (TRAIL), which is a potent inducer of apoptosis in tumor cells, and tested these MSCs, termed MSC.sTRAIL, in combination with conventional chemotherapeutic drug treatment in colon cancer models. When we pretreated human colorectal cancer HCT116 cells with low doses of 5-fluorouracil (5-FU) and added MSC.sTRAIL, we found significantly increased apoptosis as compared with single-agent treatment. Moreover, HCT116 xenografts, which were cotreated with 5-FU and systemically delivered MSC.sTRAIL, went into remission. Noteworthy, this effect was protein 53 (p53) independent and was mediated by TRAIL-receptor 2 (TRAIL-R2) upregulation, demonstrating the applicability of this approach in p53-defective tumors. Consequently, when we generated MSCs that secreted TRAIL-R2-specific variants of soluble TRAIL (sTRAIL), we found that such engineered MSCs, labeled MSC.sTRAIL(DR5), had enhanced antitumor activity in combination with 5-FU when compared with MSC.sTRAIL. In contrast, TRAIL-resistant pancreatic carcinoma PancTu1 cells responded better to MSC.sTRAIL(DR4) when the antiapoptotic protein XIAP (X-linked inhibitor of apoptosis protein) was silenced concomitantly. Taken together, our results demonstrate that TRAIL-receptor selective variants can potentially enhance the therapeutic efficacy of MSC-delivered TRAIL as part of individualized and tumor-specific combination treatments.

MnSOD protects colorectal cancer cells from TRAIL-induced apoptosis by inhibition of Smac/DIABLO release.

The mitochondrial enzyme manganese superoxide dismutase (MnSOD) has been shown to have two faces with regard to its role in tumor development. On the one side, it is well documented that overexpression of MnSOD slows down cancer cell growth, whereas on the other side MnSOD also has a metastasis-promoting activity. We set out to examine the role of MnSOD in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, thought to be a first-line tumor surveillance mechanism and failure to undergo apoptosis might contribute to metastasis formation. We show that overexpression of MnSOD at moderate levels is able to protect cells from TRAIL-induced apoptosis. While caspase-8 activation and Bid cleavage were not affected by MnSOD, we detected a marked decrease in caspase-3 activation pointing to a mitochondrial resistance mechanism. Indeed, we found that MnSOD-overexpressing cells showed reduced cytochrome c and no Smac/DIABLO release into the cytosol. The resulting lack of X-linked inhibitor of apoptosis (XIAP) inhibition by cytosolic Smac/DIABLO most likely caused the TRAIL resistance as RNAi against XIAP-rescued caspase-3 activity and TRAIL sensitivity. Our results show that reactive oxygen species are involved in TRAIL-induced Smac/DIABLO release and in TRAIL-triggered apoptosis. Hence, high levels of MnSOD, which decompose and neutralize these reactive oxygen species, might contribute to metastasis formation by allowing disseminated tumor cells to escape from TRAIL-mediated tumor surveillance. As part of TRAIL regimens, adjuvant treatment with XIAP inhibitors in the form of Smac/DIABLO mimetics or MnSOD inhibitors might be able to break TRAIL resistance of malignant tumor cells.

[Gene therapy strategies for colorectal cancer]. / Gentherapeutische Strategien gegen das kolorektale Karzinom.

Current advances in CRC treatment have led to significant but slight improvements in patient survival with curative outcomes only seen in earlier stage cancers. Consequently, much effort is being put into developing completely novel therapies that fulfil a number of criteria including greater efficacy and fewer side effects. Many of these conditions are met by the wide range of gene therapy strategies currently in pre-clinical or clinical trial phases. Gene therapy approaches may be broadly broken down into three main areas: Following a few tragic events in the context of clinical gene therapy studies, safety is currently the prime concern. Further progress in the field is expected from the combination of the described approaches with conventional treatment modalities.

NF-kappaB-independent sensitization of glioblastoma cells for TRAIL-induced apoptosis by proteasome inhibition.

The transcription factor nuclear factor-kappaB (NF-kappaB) is a key regulator of stress-induced transcriptional activation and has been implicated in mediating primary or acquired apoptosis resistance in various cancers. In the present study, we therefore investigated the role of NF-kappaB in regulating apoptosis in malignant glioma, a prototypic tumor refractory to current treatment approaches. Here, we report that constitutive NF-kappaB DNA-binding activity was low or moderate in eight different glioblastoma cell lines compared to Hodgkin's lymphoma cells, known to harbor aberrant constitutive NF-kappaB activity. Specific inhibition of NF-kappaB by overexpression of inhibitor of kappaB (IkappaB)alpha superrepressor did not enhance spontaneous apoptosis of glioblastoma cells. Also, overexpression of IkappaBalpha superrepressor had no significant impact on apoptosis induced by two prototypic classes of apoptotic stimuli, that is, chemotherapeutic drugs or death-inducing ligands such as TNF-related apoptosis inducing ligand (TRAIL), which are known to trigger NF-kappaB activation as part of a cellular stress response. Similarly, inhibition of NF-kappaB by the proteasome inhibitor MG132 did not increase doxorubicin (Doxo)-induced apoptosis of glioblastoma cells, although it prevented DNA binding of NF-kappaB complexes in response to Doxo. Interestingly, proteasome inhibition significantly sensitized glioblastoma cells for TRAIL-induced apoptosis. These findings indicate that the characteristic antiapoptotic function of NF-kappaB reported for many cancers is not a primary feature of glioblastoma and thus, specific NF-kappaB inhibition may not be effective for chemosensitization of glioblastoma. Instead, proteasome inhibitors, which enhanced TRAIL-induced apoptosis in an NF-kappaB-independent manner, may open new perspectives to increase the efficacy of TRAIL-based regimens in glioblastoma, which warrants further investigation.

AAV-encoded expression of TRAIL in experimental human colorectal cancer leads to tumor regression.

Gene transfer vectors based on the adeno-associated virus (AAV) are used for various experimental and clinical therapeutic approaches. In the present study, we demonstrate the utility of rAAV as a tumoricidal agent in human colorectal cancer. We constructed an rAAV vector that expresses tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/Apo2L) and used it to transduce human colorectal cancer cells. TRAIL belongs to the TNF superfamily of cytokines that are involved in various immune responses and apoptotic processes. It has been shown to induce cell death specifically in cancer cells. Transduction with AAV.TRAIL gave rise to rapid expression of TRAIL, followed by induction of apoptosis, which could be inhibited by the caspase inhibitor z-VAD.fmk, in several human colon cancer cell lines. The apoptotic mechanism included activation of caspase-3, as well as cytochrome c release from mitochondria. The outgrowth of human colorectal tumors grown in mice was completely blocked by transduction with AAV.TRAIL in vitro, while in vivo transduction significantly inhibited the growth of established tumors. AAV vectors could provide a safe method of gene delivery and offer a novel method of using TRAIL as a therapeutic protein.

Reactive oxygen species in oncogenic transformation.

Ever since ROS (reactive oxygen species) were shown to meet the criteria of true signalling molecules, such as regulated production and a specific biological function, many efforts have been made to understand the precise role of ROS. The function of ROS in pathological mechanisms is taking a more and more central role in various fields of biomedical research, including neurobiology, cardiology and cancer. An elevated oxidative status has been found in many types of cancer cells, and the introduction of chemical and enzymological antioxidants can inhibit tumour cell proliferation, pointing to a critical role of ROS in mediating loss of growth control. The present review describes ROS-regulated mechanisms that are associated with cancer and tumour invasiveness. The cellular processes that are linked to these ROS functions are mitogenic signalling and cell motility, while ROS have also been implicated in apoptosis and cellular senescence, two mechanisms regarded as being anti-tumorigenic. This "two-faced" character of free radicals will be discussed and placed in the context of the physiological conditions of the tumour cell, the different molecular backgrounds, and the specific ROS. More detailed understanding of the signalling pathways regulated by ROS in tumour cells will open up new prospects for chemo- or gene-therapeutic interventions.

Subcellular site of superoxide dismutase expression differentially controls AP-1 activity and injury in mouse liver following ischemia/reperfusion.

Acute damage following ischemia and reperfusion (I/R) in the liver is in part caused by the generation of reactive oxygen species, such as superoxides, during the reperfusion event. Gene therapy directed at attenuating mitochondrial superoxide production following warm I/R injury in the liver has demonstrated great promise in reducing acute hepatocellular damage. In the present study, we have compared the therapeutic effects of ectopic expression of mitochondrial (MnSOD) and cytoplasmic (Cu/ZnSOD) superoxide dismutase using recombinant adenoviral vectors for reducing I/R damage in the liver. Consistent with previous observations, recombinant adenoviral delivery of MnSOD to the liver significantly attenuated both acute liver damage and AP-1 activation following I/R injury to the livers of mice. However, ectopic expression of Cu/ZnSOD diminished neither I/R-induced elevations in serum alanine transaminase (ALT) nor AP-1 activation. Interestingly, baseline activation of AP-1 before I/R-induced injury was seen in livers infected with recombinant Ad.Cu/ZnSOD, but not Ad.MnSOD or Ad.LacZ, vectors. The level of Cu/ZnSOD-induced AP-1 activation was significantly reduced by ablation of Kupffer cells or by coexpression of catalase, suggesting that increased H(2)O(2) production facilitated by Cu/ZnSOD in hepatocytes and/or Kupffer cells may be responsible for AP-1 activation. In vitro reconstitution studies using hepatocyte and macrophage cell lines demonstrated that Cu/ZnSOD overexpression induces AP-1 in both cell types, and that secretion of a Cu/ZnSOD-induced macrophage factor is capable of elevating AP-1 in hepatocytes. In summary, our findings demonstrate that subcellular sites of superoxide production in the liver can differentially affect the outcome of I/R injury in the liver and selectively influence AP-1 activation.

Superoxide anions and endothelial cell proliferation in normoglycemia and hyperglycemia.

-Oxygen free radicals are believed to play a key role in cellular proliferation, and increased concentrations of these molecules have been implicated in the pathogenesis of endothelial dysfunction in diabetes mellitus. Our aim was to study the role of superoxide anions in endothelial cell proliferation under conditions of normoglycemia and hyperglycemia. Human aortic endothelial cells (HAECs) and human umbilical vein endothelial cells (HUVECs) exposed to adenoviral vectors encoding CuZnSOD (AdCuZnSOD), ss-galactosidase (Adssgal), or diluent (control) were cultured in normal glucose (NG, 5.5 mmol/L) or high glucose (HG, 28 mmol/L) medium. Cell proliferation was compared by use of [(3)H]thymidine incorporation and cell count in transduced and control cells in the setting of NG and HG. Transgene expression was detected in transduced cells by X-gal staining and by Western analysis and SOD activity assay in AdCuZnSOD-transduced cells. Superoxide production was significantly (P:<0.05) decreased in AdCuZnSOD-transduced cells cultured in both NG and HG medium. In NG, AdCuZnSOD-transduced endothelial cells had decreased proliferation compared with control cells. After 48 hours in HG, superoxide levels were increased and DNA synthesis was decreased (P:<0.05) in control and Adssgal-transduced but were not affected in AdCuZnSOD-transduced cells. In addition, after 7 days in HG, cell counts were reduced (P:<0.05) in control (73+/-2.5%) and Adssgal-transduced (75+/-3.4%) but not in AdCuZnSOD-transduced cells (89+/-3.4%). These results suggest that either a deficiency or an excess of superoxide anions inhibits endothelial cell proliferation, and the inhibitory effect of increased superoxide due to hyperglycemia can be reversed by CuZnSOD overexpression.

NF-kappaB kinetics predetermine TNF-alpha sensitivity of colorectal cancer cells.

BACKGROUND: Tumour necrosis factor (TNF)-alpha has considerable anti-tumour activity and may have potential as a treatment for metastatic colorectal cancer. However, TNF-alpha responses in patients and cell lines are variable and TNF-alpha treatment is associated with dose limiting clinical toxicity. Activation of NF-kappaB is protective against TNF-alpha induced cell death, and this may explain tumour resistance. METHODS: In order to provide further understanding of determinants of TNF-alpha responses, we studied TNF-alpha induced NF-kappaB activation and variable tumour responses. We analysed the kinetics of TNF-alpha induced NF-kappaB activation in colorectal cancer cells and determined whether it is possible to sensitize colorectal tumour cells to TNF-alpha by modulation of NF-kappaB signalling. RESULTS: We demonstrated that sustained NF-kappaB activation exceeding 16 h was observed in HRT18 and SW480 cells and was associated with TNF-alpha resistance. In contrast, transient NF-kappaB activation in HCT116 cells was associated with sensitivity to cytotoxic TNF-alpha effects, suggesting that NF-kappaB kinetics may have utility as clinical marker of TNF-alpha tumour resistance. Despite variable TNF-alpha responses and NF-kappaB kinetics, all three colorectal cancer cell lines were highly sensitive to treatment with the TNF-related apoptosis-inducing ligand (TRAIL) which induced only transient NF-kappaB activation. This further supports the notion of a pre-determined NF-kappaB response influencing receptor-mediated cell death. We also show that stable transfection and adenoviral-mediated expression of IkappaB(A32/36) can be used to confer TNF-alpha sensitivity to colorectal tumour cells previously resistant. CONCLUSIONS: These findings indicate that a combined approach using gene therapy and recombinant TNF-alpha merits further appraisal. Furthermore, the kinetics of the TNF-alpha response could be determined using a 'test-dose' to indicate whether individual patients might benefit from this gene therapy approach.

The yeast TEL1 gene partially substitutes for human ATM in suppressing hyperrecombination, radiation-induced apoptosis and telomere shortening in A-T cells.

Homozygous mutations in the human ATM gene lead to a pleiotropic clinical phenotype of ataxia-telangiectasia (A-T) patients and correlating cellular deficiencies in cells derived from A-T donors. Saccharomyces cerevisiae tel1 mutants lacking Tel1p, which is the closest sequence homologue to the ATM protein, share some of the cellular defects with A-T. Through genetic complementation of A-T cells with the yeast TEL1 gene, we provide evidence that Tel1p can partially compensate for ATM in suppressing hyperrecombination, radiation-induced apoptosis, and telomere shortening. Complementation appears to be independent of p53 activation. The data provided suggest that TEL1 is a functional homologue of human ATM in yeast, and they help to elucidate different cellular and biochemical pathways in human cells regulated by the ATM protein.

Immunolocalization and adenoviral vector-mediated manganese superoxide dismutase gene transfer to experimental oral tumors.

The anti-oxidant enzyme system protects cellular macromolecules against damage from reactive oxygen species. One component of this system, manganese superoxide dismutase (MnSOD), has also been shown to display tumor suppressor gene-like activity. The purpose of this study was to examine changes in MnSOD expression during hamster cheek pouch carcinogenesis, and the effects of MnSOD overexpression using an adenoviral vector. Tumor induction was carried out using 7,12-dimethylbenz[alpha]anthracene. Animals were killed at periodic intervals, and cheek pouch tissues were excised and examined for MnSOD expression by immunohistochemistry and digital image analysis. We observed a reduction in MnSOD expression as early as 2 weeks after the start of carcinogen application. Low MnSOD expression persisted until the end of the 23-week experimental period. Solid hamster cheek pouch carcinoma xenografts were then established in nude mice. An adenoviral vector encoding the human MnSOD gene was delivered to the xenografts by direct injection. We observed high, immediate expression of MnSOD in the xenografts that persisted for 10 days following cessation of viral construct delivery. Delivery of the MnSOD construct resulted in a maximal 50% reduction in tumor growth compared with untreated controls. Our results suggest that MnSOD may be a tumor suppressor gene in the hamster cheek pouch model system.

Therapeutic approaches for ischemia/reperfusion injury in the liver.

Organ injury caused by transient ischemia followed by reperfusion is associated with a number of clinically and environmentally induced conditions. Ischemia/reperfusion (I/R) conditions arise during surgical interventions such as organ transplantation and coronary bypass surgery, and in diseases such as stroke and cardiac infarct. The destructive effects of I/R arise from the acute generation of reactive oxygen species subsequent to reoxygenation, which inflict direct tissue damage and initiate a cascade of deleterious cellular responses leading to inflammation, cell death, and organ failure. This review summarizes existing and potential approaches for treatment that have been developed from research using model systems of I/R injury. Although I/R injury in the liver is emphasized, other organ systems share similar pathophysiological mechanisms and therapeutic approaches. We also review current knowledge of the molecular events controlling cellular responses to I/R injury, such as activation of AP-1 and NF-kappaB pathways. Therapeutic strategies aimed at ameliorating I/R damage are focused both on controlling ROS generated at the time of oxygen reperfusion and on intervening in the activated signal transduction cascades. Potential therapies include pharmacological treatment with small molecules, antibodies to cytokines, or free-radical scavenging enzymes, such as superoxide dismutase or catalase. Additionally, the use of gene therapy approaches may significantly contribute to the development of strategies aimed at inhibiting of I/R injury.

Effects of antioxidant enzyme overexpression on the invasive phenotype of hamster cheek pouch carcinoma cells.

To examine the role of reactive oxygen species on the invasive phenotype of cancer cells, we overexpressed manganese- and copper-zinc-containing superoxide dismutases (MnSOD, CuZnSOD) and catalase (Cat) in hamster cheek pouch carcinoma (HCPC-1) cells in vitro using adenoviral vector-mediated gene transfer. Hamster cheek pouch carcinoma cells were transduced with these adenoviral vector constructs alone, or in combination, at concentrations [i.e., multiplicity of infectivity (MOI)] of 100 MOI each. The Escherichia coli beta-galactosidase reporter construct was used as a control virus. Protein expression was examined by Western blot analysis and enzymatic activities were measured using spectrophotometry. To observe the effects of transgene overexpression on in vitro tumor cell invasion, we used the membrane invasion culture system, an accurate and reliable method for examining tumor cell invasion, in vitro. This assay measures the ability of tumor cells to invade a basement membrane matrix consisting of type IV collagen, laminin, and gelatin. MnSOD overexpression resulted in a 50% increase in HCPC-1 cell invasiveness (p < .001); co-overexpression of MnSOD with Cat partially inhibited this effect (p < .05). Moreover, co-overexpression of both SODs resulted in a significant increase in invasiveness compared with the parental HCPC-1 cells (p < .05). These changes could not be correlated with the 72 kDa collagenase IV or stromolysin activities using zymography, or the downregulation of the adhesion molecules E-cadherin or the alpha4 subunit of the alpha4beta1 integrin. These results suggest that hydrogen peroxide may play a role in the process of tumor cell invasion, but that the process does not rely on changes in matrix metalloproteinase activity in the cells, or the expression of cell adhesion molecules.

Overexpression of human catalase inhibits proliferation and promotes apoptosis in vascular smooth muscle cells.

The role of reactive oxygen species, such as superoxide anions (O(2). (-)) and hydrogen peroxide (H(2)O(2)), in modulating vascular smooth muscle cell proliferation and viability is controversial. To investigate the role of endogenously produced H(2)O(2), rat aortic smooth muscle cells were infected with adenoviral vectors containing cDNA for human catalase (AdCat) or a control gene, beta-galactosidase (AdLacZ). Infection with AdCat resulted in dose-dependent increases in intracellular catalase protein, which was predominantly localized to peroxisomes. After infection with 100 multiplicity of infection (MOI) of AdCat, cellular catalase activity was increased by 50- to 100-fold, and intracellular H(2)O(2) concentration was reduced, as compared with control. Infection with AdCat reduced [(3)H]thymidine uptake, an index of DNA synthesis, in cells maintained in medium supplemented with 2% serum (0.37+/-0.09 disintegrations per minute per cell [AdLacZ] versus 0.22+/-0.08 disintegrations per minute per cell [AdCat], P<0.05). Five days after infection with 100 MOI of AdCat, cell numbers were reduced as compared with noninfected or AdLacZ-infected cells (157 780+/-8413 [AdCat], P<0.05 versus 233 700+/-3032 [noninfected] or 222 410+/-5332 [AdLacZ]). Furthermore, the number of apoptotic cells was increased 5-fold after infection with 100 MOI of AdCat as compared with control. Infection with AdCat resulted in induction of cyclooxygenase (COX)-2, and treatment with a COX-2 inhibitor overcame the AdCat-induced reduction in cell numbers. These findings indicate that overexpression of catalase inhibited smooth muscle proliferation while increasing the rate of apoptosis, possibly through a COX-2-dependent mechanism. Our results suggest that endogenously produced H(2)O(2) importantly modulates survival and proliferation of vascular smooth muscle cells.

Intratracheal injection of adenovirus containing the human MnSOD transgene protects athymic nude mice from irradiation-induced organizing alveolitis.

PURPOSE: A dose and volume limiting factor in radiation treatment of thoracic cancer is the development of fibrosis in normal lung. The goal of the present study was to determine whether expression prior to irradiation of a transgene for human manganese superoxide dismutase (MnSOD) or human copper/zinc superoxide dismutase (Cu/ZnSOD) protects against irradiation-induced lung damage in mice. METHODS AND MATERIALS: Athymic Nude (Nu/J) mice were intratracheally injected with 10(9) plaque-forming units (PFU) of a replication-incompetent mutant adenovirus construct containing the gene for either human MnSOD, human copper/zinc superoxide dismutase (Cu/ZnSOD) or LacZ. Four days later the mice were irradiated to the pulmonary cavity to doses of 850, 900, or 950 cGy. To demonstrate adenoviral infection, nested reverse transcriptase-polymerase chain reaction (RT-PCR) was carried out with primers specific for either human MnSOD or Cu/ZnSOD transgene on freshly explanted lung, trachea, or alveolar type II cells, and immunohistochemistry was used to measure LacZ expression. RNA was extracted on day 0, 1, 4, or 7 after 850 cGy of irradiation from lungs of mice that had previously received adenovirus or had no treatment. Slot blot analysis was performed to quantitate RNA expression for IL-1, tumor necrosis factor (TNF)-alpha, TGF-beta, MnSOD, or Cu/ZnSOD. Lung tissue was explanted and tested for biochemical activity of MnSOD or Cu/ZnSOD after adenovirus injection. Other mice were sacrificed 132 days after irradiation, lungs excised, frozen in OCT, (polyvinyl alcohol, polyethylene glycol mixture) sectioned, H&E stained, and evaluated for percent of the lung demonstrating organizing alveolitis. RESULTS: Mice injected intratracheally with adenovirus containing the gene for human MnSOD had significantly reduced chronic lung irradiation damage following 950 cGy, compared to control mice or mice injected with adenovirus containing the gene for human Cu/ZnSOD or LacZ. Immunohistochemistry for LacZ protein in adenovirus LacZ (Ad-LacZ)-injected mice demonstrated expression of LacZ in both the upper and lower airway. Nested RT-PCR showed lung expression of MnSOD and Cu/ZnSOD for at least 11 days following infection with each respective adenovirus construct. Nested RT-PCR using primers specific for human MnSOD demonstrated increased expression of the human MnSOD transgene in the trachea and alveolar type II cells 4 days after virus injection on the day of irradiation. At this time point, increased biochemical activity of MnSOD and Cu/ZnSOD respectively, was detected in lungs from these two adenovirus groups, compared to each other or to control or adenovirus LacZ mice. Slot blot analysis of RNA from lungs of mice in each group following 850 cGy irradiation demonstrated decreased expression of mRNA for interleukin-I (IL-1), TNF-alpha, and transforming growth factor-beta (TGF-beta) in the MnSOD adenovirus-injected mice, compared to irradiated control, LacZ, or Cu/ZnSOD adenovirus-injected, irradiated mice. Mice receiving adenovirus MnSOD showed decreased organizing alveolitis at 132 days in all three dose groups, compared to irradiated control or Ad-LacZ, or Ad-Cu/ZnSOD mice. CONCLUSIONS: Overexpression of MnSOD in the lungs of mice prior to irradiation prevents irradiation-induced acute and chronic damage quantitated as decreased levels of mRNA for IL-1, TNF-alpha, and TGF-beta in the days immediately following irradiation, and decrease in the percent of lung demonstrating fibrosis or organizing alveolitis at 132 days. These data provide a rational basis for development of gene therapy as a method of protection of the normal lung from acute and chronic sequelae of ionizing irradiation.

Age-dependent hypertension in Mpv17-deficient mice, a transgenic model of glomerulosclerosis and inner ear disease.

The mutant mouse strain Mpv17-/-, carries a retroviral germline integration that inactivates the Mpv17 gene. Mpv17-deficient mice develop progressive glomerulosclerosis and sensineural deafness at early age. Characteristic basement membrane alterations are found in both sites of pathology. Mpv17 is a peroxisomal protein involved in the metabolism of reactive oxygen species, yet its molecular function is unknown. Dysregulation of antioxidant enzymes and basal membrane components has been established in this model and successful therapeutic intervention with antioxidants prove the causal role of reactive oxygen species in the development of the disease phenotype. We here investigated if the Mpv17-/- mice might be hypertensive. Indeed, our study revealed that Mpv17-/- mice developed significant systemic hypertension and tachycardia between 4 weeks and 5 months of age, accompanied by polyuria and elevated natriuresis. Judging from serum and urine parameters, the hypertensive condition develops concomitantly with the renal disease. Biochemical and pharmacological studies that used the endothelin receptor antagonist bosentan and the angiotensin converting enzyme inhibitor cilazapril indicated no involvement of the endothelin and renin-angiotensin systems in this hypertension, suggesting a potential novel mechanism of blood pressure regulation in this new murine hypertension model. Thus, Mpv17-/- mice unravel an intriguing new association between a defect in reactive oxygen metabolism and the age-dependent development of hypertension.

Recombinant adenovirus-mediated cardiac gene transfer of superoxide dismutase and catalase attenuates postischemic contractile dysfunction.

BACKGROUND: Coronary revascularization entails obligatory myocardial ischemia followed by reperfusion with occasional resultant postischemic contractile dysfunction, a state associated with significant morbidity and mortality. This injury is attributed in part to oxygen free radicals and has been partially ameliorated with exogenous antioxidants, a strategy limited by agent instability, low titer, and inadequate cardiomyocyte uptake. Cardiac gene transfer with antioxidant encoding vectors may significantly enhance intracellular free radical scavenger activity. METHODS AND RESULTS: C57/BL6 neonatal mice (age, 2 days; n = 131) underwent intrapericardial delivery of recombinant adenoviruses encoding superoxide dismutase (SOD) and catalase (Cat) (n = 76) or beta-galactosidase (LacZ) as a control (n = 55). After 3 days, hearts were explanted, and SOD and Cat transgene expression was detected by Western blot analysis. Spectrophotometric enzyme assays demonstrated enhanced SOD activity 1.6-fold (P < 0.0001) and Cat 3.6-fold (P < 0.00001) in experimental versus LacZ hearts. Isolated perfused hearts were subjected to 5 minutes of warm ischemia, and at 5, 10, and 15 minutes after initiation of reperfusion, LacZ controls lost 24%, 33%, and 41% of peak systolic apicobasal force, respectively, whereas experimental hearts lost 5%, 12%, and 20% (P < 0.001, each time point). In controls, rate of force generation diminished 8%, 17%, and 35%; in experimental hearts, it increased 1% at 5 minutes and decreased 5% and 15% and 10 and 15 minutes (P < 0.01, P < 0.05, P < 0.05). LacZ hearts exhibited dysfunction similar to hearts from uninjected animals (P = NS, each time point). CONCLUSIONS: Adenovirus-mediated cardiac gene transfer and expression of SOD and Cat augment antioxidant enzyme activity and minimize contractile dysfunction after ischemic reperfusion in the isolated perfused neonatal mouse heart.