Hypoxia inhibits expression and function of mitochondrial thioredoxin 2 to promote pulmonary hypertension.

Pulmonary hypertension (PH) is characterized by increased pulmonary vascular resistance, pulmonary vascular remodeling, and increased pulmonary vascular pressures that often result in right ventricular dysfunction, leading to right heart failure. Evidence suggests that reactive oxygen species (ROS) contribute to PH pathogenesis by altering pulmonary vascular cell proliferation and intracellular signaling pathways. However, the role of mitochondrial antioxidants and oxidant-derived stress signaling in the development of hypoxia-induced PH is largely unknown. Therefore, we examined the role of the major mitochondrial redox regulator thioredoxin 2 (Trx2). Levels of Trx2 mRNA and protein were examined in human pulmonary arterial endothelial cells (HPAECs) and smooth muscle cells (HPASMCs) exposed to hypoxia, a common stimulus for PH, for 72 h. Hypoxia decreased Trx2 mRNA and protein levels. In vitro overexpression of Trx2 reduced hypoxia-induced H2O2 production. The effects of increased Trx2 protein level were examined in transgenic mice expressing human Trx2 (TghTrx2) that were exposed to hypoxia (10% O2) for 3 wk. TghTrx2 mice exposed to hypoxia had exacerbated increases in right ventricular systolic pressures, right ventricular hypertrophy, and increased ROS in the lung tissue. Trx2 overexpression did not attenuate hypoxia-induced increases in Trx2 oxidation or Nox4 expression. Expression of a dominant negative C93S Trx2 mutant that mimics Trx2 oxidation exacerbated hypoxia-induced increases in HPASMC H2O2 levels and cell proliferation. In conclusion, Trx2 overexpression failed to attenuate hypoxia-induced HPASMC proliferation in vitro or hypoxia-induced PH in vivo. These findings indicate that strategies to enhance Trx2 expression are unlikely to exert therapeutic effects in PH pathogenesis.

Ubiquitin-activating enzyme activity contributes to differential accumulation of mutant huntingtin in brain and peripheral tissues.

Huntington's disease (HD) belongs to a family of neurodegenerative diseases caused by misfolded proteins and shares the pathological hallmark of selective accumulation of misfolded proteins in neuronal cells. Polyglutamine expansion in the HD protein, huntingtin (Htt), causes selective neurodegeneration that is more severe in the striatum and cortex than in other brain regions, but the mechanism behind this selectivity is unknown. Here we report that in HD knock-in mice, the expression levels of mutant Htt (mHtt) are higher in brain tissues than in peripheral tissues. However, the expression of N-terminal mHtt via stereotaxic injection of viral vectors in mice also results in greater accumulation of mHtt in the striatum than in muscle. We developed an in vitro assay that revealed that extracts from the striatum and cortex promote the formation of high-molecular weight (HMW) mHtt compared with the relatively unaffected cerebellar and peripheral tissue extracts. Inhibition of ubiquitin-activating enzyme E1 (Ube1) increased the levels of HMW mHtt in the relatively unaffected tissues. Importantly, the expression levels of Ube1 are lower in brain tissues than peripheral tissues and decline in the nuclear fraction with age, which is correlated with the increased accumulation of mHtt in the brain and neuronal nuclei during aging. Our findings suggest that decreased targeting of misfolded Htt to the proteasome for degradation via Ube1 may underlie the preferential accumulation of toxic forms of mHtt in the brain and its selective neurodegeneration.

Preferential accumulation of N-terminal mutant huntingtin in the nuclei of striatal neurons is regulated by phosphorylation.

An expanded polyglutamine tract (>37 glutamines) in the N-terminal region of huntingtin (htt) causes htt to accumulate in the nucleus, leading to transcriptional dysregulation in Huntington disease (HD). In HD knock-in mice that express full-length mutant htt at the endogenous level, mutant htt preferentially accumulates in the nuclei of striatal neurons, which are affected most profoundly in HD. The mechanism underlying this preferential nuclear accumulation of mutant htt in striatal neurons remains unknown. Here, we report that serine 16 (S16) in htt is important for the generation of small N-terminal fragments that are able to accumulate in the nucleus and form aggregates. Phosphorylation of N-terminal S16 in htt promotes the nuclear accumulation of small N-terminal fragments and reduces the interaction of N-terminal htt with the nuclear pore complex protein Tpr. Mouse brain striatal tissues show increased S16 phosphorylation and a decreased association between mutant N-terminal htt and Tpr. These findings provide mechanistic insight into the nuclear accumulation of mutant htt and the selective neuropathology of HD, revealing potential therapeutic targets for treating this disease.

Changes in the Microbiome of Cryptosporidium-Infected Mice Correlate to Differences in Susceptibility and Infection Levels.

Cryptosporidium spp. are opportunistic protozoan parasites that infect epithelial cells of the small intestine, causing diarrheal illness in humans. Differences in severity may be due to the immunological status of the host, malnutrition or prior exposure but may also be due to differences in the host gut flora. We examined changes in bacterial flora following antibiotic treatment to determine how cryptosporidial infections and gut integrity were affected by alterations in the microbiome. DNA was extracted from fecal and intestinal samples during peak infection. V4 region amplicons were generated and sequenced using 16sRNA on an Illumina MiSeq. Species evenness and richness were estimated using the Shannon diversity index. There was a significant decrease in anaerobes and overgrowth of Enterobacteriaceae in mice treated with cloxacillin. We also examined levels of short-chain fatty acids in fecal samples. There was a significant decrease in acetate, propionate, and butyrate in these same mice. Concurrent with the shift in bacterial infection was a significant increase in severity of cryptosporidial infection and increase in gut permeability. Treatment with other antibiotics significantly altered the microbiome but did not change the infection, suggesting that specific alterations in the host microbiome allow for more favorable growth of the parasite.

HIV X4 Variants Increase Arachidonate 5-Lipoxygenase in the Pulmonary Microenvironment and are associated with Pulmonary Arterial Hypertension.

Pulmonary Arterial Hypertension (PAH) is overrepresented in People Living with Human Immunodeficiency Virus (PLWH). HIV protein gp120 plays a key role in the pathogenesis of HIV-PAH. Genetic changes in HIV gp120 determine viral interactions with chemokine receptors; specifically, HIV-X4 viruses interact with CXCR4 while HIV-R5 interact with CCR5 co-receptors. Herein, we leveraged banked samples from patients enrolled in the NIH Lung HIV studies and used bioinformatic analyses to investigate whether signature sequences in HIV-gp120 that predict tropism also predict PAH. Further biological assays were conducted in pulmonary endothelial cells in vitro and in HIV-transgenic rats. We found that significantly more persons living with HIV-PAH harbor HIV-X4 variants. Multiple HIV models showed that recombinant gp120-X4 as well as infectious HIV-X4 remarkably increase arachidonate 5-lipoxygenase (ALOX5) expression. ALOX5 is essential for the production of leukotrienes; we confirmed that leukotriene levels are increased in bronchoalveolar lavage fluid of HIV-infected patients. This is the first report associating HIV-gp120 genotype to a pulmonary disease phenotype, as we uncovered X4 viruses as potential agents in the pathophysiology of HIV-PAH. Altogether, our results allude to the supplementation of antiretroviral therapy with ALOX5 antagonists to rescue patients with HIV-X4 variants from fatal PAH.

Hypoxia-induced alterations in the lung ubiquitin proteasome system during pulmonary hypertension pathogenesis.

Pulmonary hypertension (PH) is a clinical disorder characterized by sustained increases in pulmonary vascular resistance and pressure that can lead to right ventricular (RV) hypertrophy and ultimately RV failure and death. The molecular pathogenesis of PH remains incompletely defined, and existing treatments are associated with suboptimal outcomes and persistent morbidity and mortality. Reports have suggested a role for the ubiquitin proteasome system (UPS) in PH, but the extent of UPS-mediated non-proteolytic protein alterations during PH pathogenesis has not been previously defined. To further examine UPS alterations, the current study employed C57BL/6J mice exposed to normoxia or hypoxia for 3 weeks. Lung protein ubiquitination was evaluated by mass spectrometry to identify differentially ubiquitinated proteins relative to normoxic controls. Hypoxia stimulated differential ubiquitination of 198 peptides within 131 proteins ( p < 0.05). These proteins were screened to identify candidates within pathways involved in PH pathogenesis. Some 51.9% of the differentially ubiquitinated proteins were implicated in at least one known pathway contributing to PH pathogenesis, and 13% were involved in three or more PH pathways. Anxa2, App, Jak1, Lmna, Pdcd6ip, Prkch1, and Ywhah were identified as mediators in PH pathways that undergo differential ubiquitination during PH pathogenesis. To our knowledge, this is the first study to report global changes in protein ubiquitination in the lung during PH pathogenesis. These findings suggest signaling nodes that are dynamically regulated by the UPS during PH pathogenesis. Further exploration of these differentially ubiquitinated proteins and related pathways can provide new insights into the role of the UPS in PH pathogenesis.