PB125® is a phytochemical composition providing potent Nrf2 activation as well as a number of direct actions that do not involve Nrf2. Nrf2 is a transcription actor that helps maintain metabolic balance by providing redox-sensitive expression of numerous genes controlling normal day-to-day metabolic pathways. When ordinary metabolism is upset by extraordinary events such as injury, pathogenic infection, air or water pollution, ingestion of toxins, or simply by the slow but incessant changes brought about by aging and genetic variations, Nrf2 may also be called into action by the redox changes resulting from these events, whether acute or chronic. A complicating factor in all of this is that Nrf2 levels decline with aging, leaving the elderly less able to maintain proper redox balance. The dysregulated gene expression that results can cause or exacerbate a wide variety of pathological conditions, including susceptibility to viral infections. This review examines the characteristics desirable in Nrf2 activators that have therapeutic potential, as well as some of the patterns of dysregulated gene expression commonly observed during pulmonary infections and the normalizing effects possible by judicious use of phytochemicals to increase the activation level of available Nrf2.

Since nontuberculous mycobacteria (NTM) are pervasive in the environment and NTM infections are relatively uncommon, underlying hereditary or acquired host susceptibility factors should be sought for in most NTM-infected patients. To facilitate identification of underlying risk factors, it is useful to classify NTM disease into skin-soft tissue infections, isolated NTM lung disease, and extrapulmonary visceral/disseminated disease because the latter two categories have unique sets of underlying host risk factors. Nakajima and coworkers (M. Nakajima, M. Matsuyama, M. Kawaguchi, T. Kiwamoto, et al., mBio 12:e01947- 20, 2021, https://doi.org/10.1128/mBio.01947-20) in a recent issue of mBio found that Nrf2 (nuclear factor erythroid 2-related factor 2), a transcription factor that is induced by oxidative stress but induces antioxidant molecules, provides protection against an NTM infection in a murine model. While they showed that Nrf2 induction of Nramp-1 enhanced phagosome-lysosome fusion, we discuss other potential mechanisms by which oxidative stress predisposes to and Nrf2 protects against NTM infections.

Since nontuberculous mycobacteria (NTM) are pervasive in the environment and NTM infections are relatively uncommon, underlying hereditary or acquired host susceptibility factors should be sought for in most NTM-infected patients. To facilitate identification of underlying risk factors, it is useful to classify NTM disease into skin-soft tissue infections, isolated NTM lung disease, and extrapulmonary viscera-disseminated disease because the latter two categories have unique sets of underlying host risk factors. ABSTRACT Since nontuberculous mycobacteria (NTM) are pervasive in the environment and NTM infections are relatively uncommon, underlying hereditary or acquired host susceptibility factors should be sought for in most NTM-infected patients. To facilitate identification of underlying risk factors, it is useful to classify NTM disease into skin-soft tissue infections, isolated NTM lung disease, and extrapulmonary visceral/disseminated disease because the latter two categories have unique sets of underlying host risk factors. Nakajima and coworkers (M. Nakajima, M. Matsuyama, M. Kawaguchi, T. Kiwamoto, et al., mBio 12:e01947-20, 2021, https://doi.org/10.1128/mBio.01947-20) in a recent issue of mBio found that Nrf2 (nuclear factor erythroid 2-related factor 2), a transcription factor that is induced by oxidative stress but induces antioxidant molecules, provides protection against an NTM infection in a murine model. While they showed that Nrf2 induction of Nramp-1 enhanced phagosome-lysosome fusion, we discuss other potential mechanisms by which oxidative stress predisposes to and Nrf2 protects against NTM infections.

The HIV-infected population is at a dramatically increased risk of developing pulmonary arterial hypertension (PAH), a devastating and fatal cardiopulmonary disease that is rare amongst the general population. It is increasingly apparent that PAH is a disease with complex and heterogeneous cellular and molecular pathologies, and options for therapeutic intervention are limited, resulting in poor clinical outcomes for affected patients. A number of soluble HIV factors have been implicated in driving the cellular pathologies associated with PAH through perturbations of various signaling and regulatory networks of uninfected bystander cells in the pulmonary vasculature. While these mechanisms are likely numerous and multifaceted, the overlapping features of PAH cellular pathologies and the effects of viral factors on related cell types provide clues as to the potential mechanisms driving HIV-PAH etiology and progression. In this review, we discuss the link between the DNA damage response (DDR) signaling network, chronic HIV infection, and potential contributions to the development of pulmonary arterial hypertension in chronically HIV-infected individuals.

Nrf2 is a transcription factor that regulates cellular redox balance and the expression of a wide array of genes involved in immunity and inflammation, including antiviral actions. Nrf2 activity declines with age, making the elderly more susceptible to oxidative stress-mediated diseases, which include type 2 diabetes, chronic inflammation, and viral infections. Published evidence suggests that Nrf2 activity may regulate important mechanisms affecting viral susceptibility and replication. We examined gene expression levels by GeneChip microarray and by RNA-seq assays. We found that the potent Nrf2 activating composition PB125® downregulates ACE2 and TMPRSS2 mRNA expression in human liver-derived HepG2 cells. ACE2 is a surface receptor and TMPRSS2 activates the spike protein for SARS-Cov-2 entry into host cells. Furthermore, in endotoxin-stimulated primary human pulmonary artery endothelial cells we report the marked downregulation by PB125 of 36 genes encoding cytokines. These include IL1-beta, IL6, TNF-α the cell adhesion molecules ICAM1, VCAM1, and E-selectin, and a group of IFN-γ-induced genes. Many of these cytokines have been specifically identified in the "cytokine storm" observed in fatal cases of COVID-19, suggesting that Nrf2 activation may significantly decrease the intensity of the storm.

Chronic HIV infection in the era of anti-retroviral therapy is associated with dramatically increased risk of developing severe cardio pulmonary disease. Common to these diseases is increased oxidative burden and chronic inflammation despite low viremia and restoration of CD4+ T-cell levels. Soluble viral factors are heavily implicated in these disease processes, including the HIV Transactivator of Transcription (Tat). Tat is produced in high levels during infection and secreted from infected cells into circulation where it is internalized by bystander cells and is known to regulate inflammatory pathways and elicit a pro-oxidant environment. We have examined the effects of Tat on the anti-oxidant regulatory network driven by the transcription factor Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in primary human pulmonary arterial endothelial cells, which are heavily involved in pathogenesis of HIV associated lung diseases including pulmonary arterial hypertension and COPD. Co-expression of Tat and a luciferase reporter construct driven by the Nrf2 activated anti-oxidant response element (ARE) demonstrated markedly reduced Nrf2/ARE activity, even when stimulated by the potent Nrf2 activating compound PB125. Additionally, Heme-oxygenase-1 (HO-1) transcription was potently repressed by Tat in a cell line as well as primary endothelial cells, and treatment with PB125 failed to restore transcriptional activity. Other anti-oxidant Nrf2 genes examined included NADPH Dehydrogenase Quinone 1 (NQO1) and Sulfiredoxin-1 (SRXN1). NQO1 was repressed basally by Tat, while SRXN1 transcription was refractory to activation by PB125 in the presence of Tat. Lastly, we demonstrated that Tat expressing cells have increased indicators of oxidative stress including elevated production of reactive oxygen species, measured by electron paramagnetic resonance spectroscopy, and increased levels of nitrotyrosine content. These observations suggest a novel mechanism by which HIV Tat increases oxidative burden by dysregulation of the Nrf2/ARE pathway.

Redox imbalance results in damage to cellular macromolecules and interferes with signaling pathways, leading to an inflammatory cellular and tissue environment. As such, the cellular oxidative environment is tightly regulated by several redox-modulating pathways. Many viruses have evolved intricate mechanisms to manipulate these pathways for their benefit, including HIV-1, which requires a pro-oxidant cellular environment for optimal replication. One such virulence factor responsible for modulating the redox environment is the HIV Transactivator of transcription (Tat). Tat is of particular interest as it is actively secreted by infected cells and internalized by uninfected bystander cells where it can elicit pro-oxidant effects resulting in inflammation and damage. Previously, we demonstrated that Tat regulates basal expression of Superoxide Dismutase 2 (sod2) by altering the binding of the Sp-transcription factors at regions relatively near (approx. -210 nucleotides) upstream of the transcriptional start site. Now, using in silico analysis and a series of sod2 promoter reporter constructs, we have identified putative clusters of Sp-binding sites located further upstream of the proximal sod2 promoter, between nucleotides -3400 to -210, and tested their effect on basal transcription and for their sensitivity to HIV-1 Tat. In this report, we demonstrate that under basal conditions, maximal transcription requires a cluster of Sp-binding sites in the -584 nucleotide region, which is extremely sensitive to Tat. Using chromatin immunoprecipitation (ChIP) we demonstrate that Tat results in altered occupancy of Sp1 and Sp3 at this distal Tat-sensitive regulatory element and strongly stimulated endogenous expression of SOD2 in human pulmonary artery endothelial cells (HPAEC). We also report altered expression of Sp1 and Sp3 in Tat-expressing HPAEC as well as in the lungs of HIV-1 infected humanized mice. Lastly, Tat co-immunoprecipitated with endogenous Sp3 but not Sp1 and did not alter the acetylation state of Sp3. Thus, here, we have defined a novel and important cis-acting factor in HIV-1 Tat-mediated regulation of SOD2, demonstrated that modulation of Sp1 and Sp3 activity by Tat promotes SOD2 expression in primary human pulmonary artery endothelial cells and determined that pulmonary levels of Sp3 as well as SOD2 are increased in the lungs of a mouse model of HIV infection.

Despite the longevity afforded by modern anti‐retroviral therapy, long‐term HIV infection increases the risk for pulmonary co‐morbidities associated with cell cycle dysregulation including lung cancer and pulmonary arterial hypertension. As such, we postulate that select viral factors, in particular the HIV transactivator of transcription (Tat), interfere with cellular processes involving tumor suppressors and other cell cycle regulators during chronic infection. Tat is well known to inhibit the activity of the tumor suppressor Tat Interacting Protein 60 kDa (Tip60), an acetyltransferase critical in the progression to cell cycle arrest and/or apoptosis. In response to stressors such as DNA damage and oxidative stress, Tip60 activates the cell cycle master regulator kinase Ataxia Telangiectasia Mutated (ATM) via acetylation, initiating a signaling cascade that culminates in the activation of a DNA damage response resulting in both short and long term cellular arrest. Thus, we hypothesize that inhibition of Tip60 by Tat interferes with cellular arrest in responses to these stressors and results in abnormal proliferation. We addressed the hypothesis using HeLa cells stably transfected to express Tat (HeLa‐Tat III ). Utilizing an ATP based luminescent cell viability assay, we observed accelerated cell proliferation in HeLa‐Tat III compared to its HeLa wild‐type counterpart. Furthermore, immunoprecipitation experiments in HeLa‐WT cells confirmed a physical interaction between Tat and Tip60. Acetylation by Tip60 results in autophosphorylation of ATM at ser1981, and this phosphorylation event is a canonical marker for ATM activation. As such, we determined the phosphorylation state of ATM ser1981 after treatment with doxorubicin in HeLa‐WT and HeLa‐Tat III cells. We found that, following treatment with doxorubicin, ATM phosphorylation at ser1981 was severely attenuated in HeLa‐Tat III cells compared to HeLa‐WTs, suggesting a role for Tat in the inhibition of ATM activation. Taken together, these data support the role of the HIV Tat protein in dysregulation of cell cycle arrest via ATM signaling pathways. Whether HIV promotes cellular proliferation in co‐morbidities such as lung cancer and pulmonary arterial hypertension via these pathways remains to be addressed. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

The introduction of antiretroviral therapies (ART) is largely responsible for the development of chronic HIV infection, but long‐term exposure to viral proteins can contribute to oxidative stress and chronic inflammation which could promote the development of HIV‐Associated co‐morbidities such as pulmonary arterial hypertension (HA‐PAH). Previously, we have reported that the HIV transcriptional regulator, Tat, can induce oxidative responses by depleting cellular glutathione and repressing the mitochondrial manganese‐superoxide dismutase (MnSOD, sod2); there are Tat‐regulated elements within the proximal region of the human sod2 promoter, designated Tat‐sensitive Sp‐responsive elements (TSS). Tat alters the pattern of binding by the Sp1 and Sp3 transcription factors on the sod2 TSS, resulting in transcriptional repression. Recently, we observed evidence of Tat‐regulated regions containing predicted Sp‐binding sequences within more distal sections of the sod2 promoter, which we have termed the DTSS (Distal Tat‐sensitive Sp‐responsive elements). The previous discoveries that Tat regulates the proximal promoter by changing the pattern of Sp binding prompted us to investigate if Tat influences transcription from the distal elements via modulation of Sp1 and Sp3 ratios. In this study, we transiently transfected luciferase‐reporter plasmids containing varying regions of the sod2 promoter in the presence or absence of a Tat‐expressing plasmid into human pulmonary artery endothelial cells (HPAEC). To further investigate the DTSS and the occupancy of Sp1 and Sp3, we collected Sp1 and Sp3‐bound DNA via Chromatin Immunoprecipitation (ChIP). Our data shows that under basal conditions certain regions of this promoter are highly expressed while others are repressed by an already existing biological ratio of Sp1 and Sp3. However, in the presence of Tat, all of these regions are repressed. ChIP analysis shows that Tat changes the amounts of Sp1 and Sp3 bound at the predicted Sp‐binding clusters. With this data we conclude that Tat represses sod2 by altering the ratio of Sp1 and Sp3 at both proximal and distal regions thereby extending the known regulatory sequences of the sod2 gene that are responsive to HIV Tat.Support or Funding InformationNational Institutes of Health (NIH) 1RO1HL125050‐01This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Human immunodeficiency virus (HIV) is an incurable and life‐threatening disease impacting over 36 million people world‐wide. Antiretroviral treatment (ART) suppresses the virus and allows many patients to live longer lives. However, even patients undergoing ART are susceptible to complications in many organ systems including the lung. HIV has been successfully linked to pulmonary complications including: tuberculosis, cytomegalovirus, Hodgkin's lymphoma, chronic obstructive pulmonary disease (COPD), and pulmonary arterial hypertension (PAH). Some of these diseases have a higher prevalence amongst patients infected with HIV. For example, in HIV patients, the incidence of PAH is 1:200, which is approximately 300‐fold higher than idiopathic PAH in the general population. Due to the limited host specificity of HIV, there are not many well‐developed animal models to study these complications. However, previous research in NOD‐Scid IL‐2 receptor γ chain knockout mice engrafted with human hematopoietic stem cells (hCD34+) and surgically implanted with human fetal thymic and liver tissue (NSG‐BLT mice) has shown disseminated HIV infection, making NSG‐BLT mice a promising in vivo model. In this study, we aimed to confirm HIV dissemination to the lungs in these mice and to examine pulmonary pathologies after an acute infection period. We infected NSG‐BLT mice with HIV for 5, 10, or 15 weeks and looked for viral RNA and pro‐viral DNA in the lungs using qPCR and nested PCR, respectively. To begin investigating possible lung pathology, we measured cytokines from lung tissue and cell‐free bronchoalveolar (BAL) fluid using an ELISA‐based protein array. Of the many proteins that were dysregulated in HIV infected mice, RAGE and IL‐25 (also known as IL‐17) are of particular interest because they have been implicated in endothelial dysfunction or vascular remodeling. This suggests a role of these proteins in HIV‐directed lung pathology in these mice. We are currently confirming the dysregulation of these genes by qPCR. In conclusion, NSG‐BLT mice constitute a useful model for studying pulmonary dissemination and pathologies during HIV infection.Support or Funding InformationNational Institutes of Health (NIH) 1R01HL125050‐01This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Occlusive arterial remodeling driven by hyperproliferation of endothelial and smooth muscle cells is characteristic of pulmonary arterial hypertension (PAH) and known to be driven by pulmonary vascular inflammation and oxidative stress. Human immunodeficiency virus (HIV) is well-documented to increase the risk for PAH up to 300-fold, leading to HIV-Associated Pulmonary Arterial Hypertension (HA-PAH) in 1:200 HIV infected individuals. Previous research has shown that the viral protein Tat (trans-activator of transcription), is a major effector of oxidative stress in HIV patients. Two Tat isoforms are commonly detected during HIV infection, full-length Tat101 and Tat86. Tat101 is encoded by two exons, whereas Tat86 is encoded by the first exon plus half of the second exon. It has been shown that deletion of the second exon has no substantial effect on HIV-1 LTR transactivation, and therefore the Tat86 isoform has been sufficient for most HIV research to date. However little is known about the direct role of the second exon on subsequent oxidative stress and other cellular responses such as inflammation, proliferation or apoptosis. We measured nitrotyrosine content, carbonyl proteins, lipid peroxidation and total antioxidant capacity in Human Pulmonary Artery Endothelial cells (HPAEC) expressing either Tat86 or Tat101 to determine if both of the Tat isoforms induce oxidative stress in these pulmonary vascular cells that could contribute to HA-PAH.

The introduction of antiretroviral therapies is largely responsible for the development of chronic HIV infection, but long-term exposure to viral proteins can contribute to pulmonary and cardiac complications such as HIV-Associated pulmonary arterial hypertension (HA-PAH). Oxidative stress and inflammation are two major drivers of pulmonary pathology in idiopathic pulmonary arterial hypertension (iPAH) and could also be involved in the pathogenesis of HA-PAH. We have reported previously that the HIV transcriptional regulator, Tat (trans-activator of transcription), can induce oxidative responses by depleting cellular glutathione and contributing to the inhibition of mitochondrial manganese-superoxide dismutase (MnSOD, sod2). We also previously defined Tat-regulated elements within the proximal regions of the human sod2 promoter, designated Tat-sensitive Sp-responsive elements (TSS). We’ve seen that Tat alters the pattern of binding by the Sp1 and Sp3 transcription factors on the sod2 TSS, causing transcriptional repression. Recently we observed evidence of Sp-binding sites containing Tat-regulated regions within more distal sections of the sod2 promoter which we have termed the DTSS (Distal Tat-sensitive Sp elements). The previous discoveries that Tat regulates the proximal promoter by changing the patterns of Sp binding prompted us to investigate the mechanisms of Tat regulation on the distal regions of the promoter. In this study, we transiently transfected pGL3B luciferase-reporter plasmids containing varying regions of the sod2 promoter in the presence or absence of Tat to determine if Tat influences the transcription of this promoter through distal elements. Our data suggests that Tat-mediated regulation of the sod2 promoter is different in Human Pulmonary Artery Endothelial cells (HPAEC) than in HeLa cells. We also present evidence that the different isoforms of Tat, full-length Tat101 and Tat86, regulate the sod2 promoter differently. We therefore hypothesize that the interactions of Tat with Sp, and possibly other, transcription factors are contributing to the observed differences in sod2 regulation in the different cells types.

RATIONALE:Lung infections caused by opportunistic or virulent pathogens are a principal cause of morbidity and mortality in HIV infection. It is unknown whether HIV infection leads to changes in basal lung microflora, which may contribute to chronic pulmonary complications that increasingly are being recognized in individuals infected with HIV.OBJECTIVES:To determine whether the immunodeficiency associated with HIV infection resulted in alteration of the lung microbiota.METHODS:We used 16S ribosomal RNA targeted pyrosequencing and shotgun metagenomic sequencing to analyze bacterial gene sequences in bronchoalveolar lavage (BAL) and mouths of 82 HIV-positive and 77 HIV-negative subjects.MEASUREMENTS AND MAIN RESULTS:Sequences representing Tropheryma whipplei, the etiologic agent of Whipple's disease, were significantly more frequent in BAL of HIV-positive compared with HIV-negative individuals. T. whipplei dominated the community (>50% of sequence reads) in 11 HIV-positive subjects, but only 1 HIV-negative individual (13.4 versus 1.3%; P = 0.0018). In 30 HIV-positive individuals sampled longitudinally, antiretroviral therapy resulted in a significantly reduced relative abundance of T. whipplei in the lung. Shotgun metagenomic sequencing was performed on eight BAL samples dominated by T. whipplei 16S ribosomal RNA. Whole genome assembly of pooled reads showed that uncultured lung-derived T. whipplei had similar gene content to two isolates obtained from subjects with Whipple's disease.CONCLUSIONS:Asymptomatic subjects with HIV infection have unexpected colonization of the lung by T. whipplei, which is reduced by effective antiretroviral therapy and merits further study for a potential pathogenic role in chronic pulmonary complications of HIV infection.

Respiratory surfaces are exposed to billions of particulates and pathogens daily. A protective mucus barrier traps and eliminates them through mucociliary clearance (MCC). However, excessive mucus contributes to transient respiratory infections and to the pathogenesis of numerous respiratory diseases. MUC5AC and MUC5B are evolutionarily conserved genes that encode structurally related mucin glycoproteins, the principal macromolecules in airway mucus. Genetic variants are linked to diverse lung diseases, but specific roles for MUC5AC and MUC5B in MCC, and the lasting effects of their inhibition, are unknown. Here we show that mouse Muc5b (but not Muc5ac) is required for MCC, for controlling infections in the airways and middle ear, and for maintaining immune homeostasis in mouse lungs, whereas Muc5ac is dispensable. Muc5b deficiency caused materials to accumulate in upper and lower airways. This defect led to chronic infection by multiple bacterial species, including Staphylococcus aureus, and to inflammation that failed to resolve normally. Apoptotic macrophages accumulated, phagocytosis was impaired, and interleukin-23 (IL-23) production was reduced in Muc5b(-/-) mice. By contrast, in mice that transgenically overexpress Muc5b, macrophage functions improved. Existing dogma defines mucous phenotypes in asthma and chronic obstructive pulmonary disease (COPD) as driven by increased MUC5AC, with MUC5B levels either unaffected or increased in expectorated sputum. However, in many patients, MUC5B production at airway surfaces decreases by as much as 90%. By distinguishing a specific role for Muc5b in MCC, and by determining its impact on bacterial infections and inflammation in mice, our results provide a refined framework for designing targeted therapies to control mucin secretion and restore MCC.

Chronic human immunodeficiency virus infection is associated with higher incidence of pulmonary complications including hypertension, vasculopathy, lymphocytic alveolitis, and interstitial pneumonitis not attributed to either opportunistic infections or presence of the virus. The Tat (transactivator of transcription) protein, a required transactivator for expression of full-length viral genes, is pleiotropic and influences expression of cellular inflammatory genes. Tat-dependent transactivation of cellular genes requires specific mediators, including NF-κB, widely recognized as sensitive to changes in cellular oxidant burden. We hypothesized that overproduction of Tat leads to increased oxidant burden and to alterations in basal inflammatory status as measured by NF-κB activation. We engineered transgenic mouse lines that express Tat (86-amino-acid isoform) in the lung under the control of the surfactant protein C promoter. Tat-transgenic mice exhibit increased pulmonary cellular infiltration, increased nitrotyrosine and carbonyl protein modifications, and increased levels of NF-κB, MnSOD, and thioredoxin-interacting protein. These data indicate that Tat increases oxidant burden and resets the threshold for inflammation, which may increase susceptibility to secondary injuries.

RATIONALE HIV-infected patients with pulmonary arterial hypertension have histologic manifestations that are indistinguishable from those found in patients with idiopathic pulmonary arterial hypertension. In addition, the role of pleiotropic viral proteins in the development of plexiform lesions in HIV-related pulmonary hypertension (HRPH) has not been explored. Simian immunodeficiency virus (SIV) infection of macaques has been found to closely recapitulate many of the characteristic features of HIV infection, and thus hallmarks of pulmonary arterial hypertension should also be found in this nonhuman primate model of HIV. OBJECTIVES To determine whether pulmonary arterial lesions were present in archived SIV-infected macaque lung tissues from Johns Hopkins University and two National Primate Research Centers. METHODS Archived macaque and human lung sections were examined via immunohistochemistry for evidence of complex vascular lesions. RESULTS Complex plexiform-like lesions characterized by lumenal obliteration, intimal disruption, medial hypertrophy, thrombosis, and recanalized lumena were found exclusively in animals infected with SHIV-nef (a chimeric viral construct containing the HIV nef gene in an SIV backbone), but not in animals infected with SIV. The mass of cells in the lesions were factor VIII positive, and contained cells positive for muscle-specific and smooth muscle actins. Lung mononuclear cells were positive for HIV Nef, suggesting viral replication. Endothelial cells in both the SHIV-nef macaques and patients with HRPH, but not in patients with idiopathic pulmonary arterial hypertension, were also Nef positive. CONCLUSIONS The discovery of complex vascular lesions in SHIV-nef- but not SIV-infected animals, and the presence of Nef in the vascular cells of patients with HRPH, suggest that Nef plays a key role in the development of severe pulmonary arterial disease.

The expression of manganese superoxide dismutase (MnSOD) is regulated by agents associated with cancer development. It has been shown that infection with the human immunodeficiency virus type 1 (HIV-1) is associated with the development of liver cancer and that the transactivating transcriptional factor (Tat) of human HIV-1 reduces the expression of MnSOD in several cell types. However, the role of Tat in the expression of MnSOD in hepatocellular carcinoma is unknown. Furthermore, the precise mechanisms whereby Tat suppresses MnSOD expression in hepatocellular carcinoma cells remain unclear. In this report, we build on our original observations that Tat changes the distribution of Sp family members on the MnSOD promoter, which accounts for Tat-dependent changes in basal expression. In hepatic cells, Tat expression upregulates Sp1/Sp3, which play different roles in regulating MnSOD transcription. While overexpression of Sp1 stimulates, overexpression of Sp3 represses transcriptional activity. The transcription repression effect of Sp3 is not due to Sp3 competing for the binding site with Sp1 because only the full-length Sp3 but not the truncated Sp3 suppresses MnSOD promoter activity. These findings suggest a novel mechanism by which Tat modulates the repression of the MnSOD gene and establish a link between HIV infection and liver cancer.

We report herein the novel observation that alterations in oxidant/antioxidant balance are evident and cause vascular dysfunction in aortae of prediabetic nonobese-diabetic mice (NOD). We found that nitrotyrosine, a biochemical marker of oxidant stress, was higher in the NOD aortae when compared to age-matched non-autoimmune BALB/c controls or the diabetes-resistant NOD congenic strain, NOD.Lc7. The oxidant stress was localized to the intimal and medial layers, and endothelium-dependent relaxation to acetylcholine was decreased in isolated aortic rings from NOD mice. Inhibition of nitric oxide synthesis caused an endothelium-dependent contraction, and treatment with either a selective thromboxane A2/prostaglandin H2 receptor antagonist or a non-isozyme-specific cyclooxygenase inhibitor reversed this effect. Aortic rings from NOD.Lc7 did not display the paradoxical vasoconstriction. Furthermore, the vascular dysfunction was caused by oxidative stress, as treatment with a superoxide dismutase mimetic in vivo or with native antioxidant enzymes ex vivo inhibited the tissue oxidant stress and restored endothelium-dependent relaxation. Endothelial function was also restored by the inhibitors of NAD(P)H oxidase, diphenylene iodonium or apocynin. Our studies indicate that an oxidant stress that occurs prior to the onset of diabetes in this mouse model contributes to endothelial dysfunction independently of overt diabetes.

Regulation of the basal manganese superoxide dismutase (SOD2) promoter depends on the transcriptional activity of the Sp family of transcription factors. Here we report that reduced expression in the presence of Tat is independent of induction with Tumor necrosis factor α and that Tat affects the interaction of Sp1 and Sp3 with the basal promoter. Footprinting and electrophoretic mobility shift assay (EMSA) analyses with extracts from HeLa cells showed that Sp1/Sp3 complexes populate the proximal SOD2 promoter, and that Tat leads to an increase in the binding activity of Sp3. In Drosophila S2 cells, both Sp1 and Sp3 activated the basal SOD2 promoter (88.1 ± 39.4 fold vs. 10.3 ± 3.5 fold, respectively), demonstrating a positive, yet lower transcriptional regulatory function for Sp3. Additionally, the inability of Sp3 to synergistically affect promoter activity indicates an efficient competition of Sp3 with Sp1 for the multiple Sp binding sites in the SOD2 basal promoter. Tat potentiated both Sp1 and Sp3 activation of the promoter in S2 cells, though the activity of Sp3 was still lower than that of Sp1. Thus, the consequence of a shift by Tat to increased Sp3-containing complexes on the basal SOD2 promoter is decreased SOD2 expression. Together, our studies demonstrate the functional importance of the interaction of Sp1, Sp3, and Tat, revealing a possible mechanism for the attenuation of basal manganese superoxide dismutase expression.