Single Mutation in the NFU1 Gene Metabolically Reprograms Pulmonary Artery Smooth Muscle Cells.

OBJECTIVE: NFU1 is a mitochondrial iron-sulfur scaffold protein, involved in iron-sulfur assembly and transfer to complex II and LAS (lipoic acid synthase). Patients with the point mutation NFU1G208C and CRISPR/CAS9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9)-generated rats develop mitochondrial dysfunction leading to pulmonary arterial hypertension. However, the mechanistic understanding of pulmonary vascular proliferation due to a single mutation in NFU1 remains unresolved. Approach and Results: Quantitative proteomics of isolated mitochondria showed the entire phenotypic transformation of NFU1G206C rats with a disturbed mitochondrial proteomic landscape, involving significant changes in the expression of 208 mitochondrial proteins. The NFU1 mutation deranged the expression pattern of electron transport proteins, resulting in a significant decrease in mitochondrial respiration. Reduced reliance on mitochondrial respiration amplified glycolysis in pulmonary artery smooth muscle cell (PASMC) and activated GPD (glycerol-3-phosphate dehydrogenase), linking glycolysis to oxidative phosphorylation and lipid metabolism. Decreased PDH (pyruvate dehydrogenase) activity due to the lipoic acid shortage is compensated by increased fatty acid metabolism and oxidation. PASMC became dependent on extracellular fatty acid sources due to upregulated transporters such as CD36 (cluster of differentiation 36) and CPT (carnitine palmitoyltransferase)-1. Finally, the NFU1 mutation produced a dysregulated antioxidant system in the mitochondria, leading to increased reactive oxygen species levels. PASMC from NFU1 rats showed apoptosis resistance, increased anaplerosis, and attained a highly proliferative phenotype. Attenuation of mitochondrial reactive oxygen species by mitochondrial-targeted antioxidant significantly decreased PASMC proliferation. CONCLUSIONS: The alteration in iron-sulfur metabolism completely transforms the proteomic landscape of the mitochondria, leading toward metabolic plasticity and redistribution of energy sources to the acquisition of a proliferative phenotype by the PASMC.

Rats with a Human Mutation of NFU1 Develop Pulmonary Hypertension.

NFU1 is a mitochondrial protein that is involved in the biosynthesis of iron-sulfur clusters, and its genetic modification is associated with disorders of mitochondrial energy metabolism. Patients with autosomal-recessive inheritance of the NFU1 mutation G208C have reduced activity of the respiratory chain Complex II and decreased levels of lipoic-acid-dependent enzymes, and develop pulmonary arterial hypertension (PAH) in ∼70% of cases. We investigated whether rats with a human mutation in NFU1 are also predisposed to PAH development. A point mutation in rat NFU1G206C (human G208C) was introduced through CRISPR/Cas9 genome editing. Hemodynamic data, tissue samples, and fresh mitochondria were collected and analyzed. NFU1G206C rats showed increased right ventricular pressure, right ventricular hypertrophy, and high levels of pulmonary artery remodeling. Computed tomography and angiography of the pulmonary vasculature indicated severe angioobliterative changes in NFU1G206C rats. Importantly, the penetrance of the PAH phenotype was found to be more prevalent in females than in males, replicating the established sex difference among patients with PAH. Male and female homozygote rats exhibited decreased expression and activity of mitochondrial Complex II, and markedly decreased pyruvate dehydrogenase activity and lipoate binding. The limited development of PAH in males correlated with the preserved levels of oligomeric NFU1, increased expression of ISCU (an alternative branch of the iron-sulfur assembly system), and increased complex IV activity. Thus, the male sex has additional plasticity to overcome the iron-sulfur cluster deficiency. Our work describes a novel, humanized rat model of NFU1 deficiency that showed mitochondrial dysfunction similar to that observed in patients and developed PAH with the same sex dimorphism.

MUC1 contributes to goblet cell metaplasia and MUC5AC expression in response to cigarette smoke in vivo.

Goblet cell metaplasia (GCM) and mucin overproduction are a hallmark of chronic rhinosinusitis (CRS) and chronic obstructive pulmonary disease (COPD). In the airways, cigarette smoke (CS) induces activation of the epidermal growth factor receptor (EGFR) leading to GCM and overexpression of the gel-forming mucin MUC5AC. Although previous studies have demonstrated that a membrane-bound mucin, MUC1, modulates the activation of CS-induced EGFR, the role of MUC1 in CS-induced GCM and mucin overproduction has not been explored. In response to CS exposure, wild-type (WT) rats displayed Muc1 translocation from the apical surface of airway epithelium to the intracellular compartment of hyperplastic intermediate cells, EGFR phosphorylation, GCM, and Muc5ac overproduction. Similarly, human CRS sinonasal tissues demonstrated hyperplasia of intermediate cells enriched with MUC1 in the intracellular compartment, which was accompanied by GCM and increased MUC5AC expression. To further evaluate the role of Muc1 in vivo, a Muc1 knockout (KO) rat (MUC in humans and Muc in animals) was developed. In contrast to WT littermates, Muc1-KO rats exhibited no activation of EGFR, and were protected from GCM and Muc5ac overproduction. Genetic knockdown of MUC1 in human lung or Muc1 knockout in primary rat airway epithelial cells led to significantly diminished EGF-induced MUC5AC production. Together, these findings suggest that MUC1-dependent EGFR activation mediates CS-induced GCM and mucin overproduction. Strategies designed to suppress MUC1-dependent EGFR activation may provide a novel therapeutic approach for treating mucin hypersecretion in CRS and COPD.

Inositol monophosphatase 1 as a novel interacting partner of RAGE in pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a lethal disease characterized by progressive pulmonary vascular remodeling. The receptor for advanced glycation end products (RAGE) plays an important role in PAH by promoting proliferation of pulmonary vascular cells. RAGE is also known to mediate activation of Akt signaling, although the particular molecular mechanism remains unknown. This study aimed to identify the interacting partner of RAGE that could facilitate RAGE-mediated Akt activation and vascular remodeling in PAH. The progressive angioproliferative PAH was induced in 24 female Sprague-Dawley rats ( n = 8/group) that were randomly assigned to develop PAH for 1, 2, or 5 wk [right ventricle systolic pressure (RVSP) 56.5 ± 3.2, 63.6 ± 1.6, and 111.1 ± 4.5 mmHg, respectively, vs. 22.9 ± 1.1 mmHg in controls]. PAH triggered early and late episodes of apoptosis in rat lungs accompanied by RAGE activation. Mass spectrometry analysis has identified IMPA1 as a novel PAH-specific interacting partner of RAGE. The proximity ligation assay (PLA) confirmed the formation of RAGE/IMPA1 complex in the pulmonary artery wall. Activation of IMPA1 in response to increased glucose 6-phosphate (G6P) is known to play a critical role in inositol synthesis and recycling. Indeed, we confirmed a threefold increase in G6P ( P = 0.0005) levels in lungs of PAH rats starting from week 1 that correlated with accumulation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3), membrane translocation of PI3K, and a threefold increase in membrane Akt levels ( P = 0.02) and Akt phosphorylation. We conclude that the formation of the newly discovered RAGE-IMPA1 complex could be responsible for the stimulation of inositol pathways and activation of Akt signaling in PAH.

Novel Populations of Lung Capillary Endothelial Cells and Their Functional Significance.

The role of the lung's microcirculation and capillary endothelial cells in normal physiology and the pathobiology of pulmonary diseases is unequivocally vital. The recent discovery of molecularly distinct aerocytes and general capillary (gCaps) endothelial cells by single-cell transcriptomics (scRNAseq) advanced the field in understanding microcirculatory milieu and cellular communications. However, increasing evidence from different groups indicated the possibility of more heterogenic structures of lung capillaries. Therefore, we investigated enriched lung endothelial cells by scRNAseq and identified five novel populations of gCaps with distinct molecular signatures and roles. Our analysis suggests that two populations of gCaps that express Scn7a(Na+) and Clic4(Cl-) ion transporters form the arterial-to-vein zonation and establish the capillary barrier. We also discovered and named mitotically-active "root" cells (Flot1+) on the interface between arterial, Scn7a+, and Clic4 + endothelium, responsible for the regeneration and repair of the adjacent endothelial populations. Furthermore, the transition of gCaps to a vein requires a venous-capillary endothelium expressing Lingo2. Finally, gCaps detached from the zonation represent a high level of Fabp4, other metabolically active genes, and tip-cell markers showing angiogenesis-regulating capacity. The discovery of these populations will translate into a better understanding of the involvement of capillary phenotypes and their communications in lung disease pathogenesis.

Acquirement of the autonomic nervous system modulation evaluated by heart rate variability in medaka (Oryzias latipes).

Small teleosts have recently been established as models of human diseases. However, measuring heart rate by electrocardiography is highly invasive for small fish and not widely used. The physiological nature and function of vertebrate autonomic nervous system (ANS) modulation of the heart has traditionally been investigated in larvae, transparent but with an immature ANS, or in anesthetized adults, whose ANS activity may possibly be disturbed under anesthesia. Here, we defined the frequency characteristics of heart rate variability (HRV) modulated by the ANS from observations of heart movement in high-speed movie images and changes in ANS regulation under environmental stimulation in unanesthetized adult medaka (Oryzias latipes). The HRV was significantly reduced by atropine (1 mM) in the 0.25-0.65 Hz and by propranolol (100 µM) at 0.65-1.25 Hz range, suggesting that HRV in adult medaka is modulated by both the parasympathetic and sympathetic nervous systems within these frequency ranges. Such modulations of HRV by the ANS in adult medaka were remarkably suppressed under anesthesia and continuous exposure to light suppressed HRV only in the 0.25-0.65 Hz range, indicating parasympathetic withdrawal. Furthermore, pre-hatching embryos did not show HRV and the power of HRV developed as fish grew. These results strongly suggest that ANS modulation of the heart in adult medaka is frequency-dependent phenomenon, and that the impact of long-term environmental stimuli on ANS activities, in addition to development of ANS activities, can be precisely evaluated in medaka using the presented method.

Gravity-dependent changes in bioconvection of Tetrahymena and Chlamydomonas during parabolic flight: increases in wave number induced by pre- and post-parabola hypergravity.

Bioconvection emerges in a dense suspension of swimming protists as a consequence of their negative-gravitactic upward migration and later settling as a blob of density greater than that of water. Thus, gravity is an important parameter governing bioconvective pattern formation. However, inconsistencies are found in previous studies dealing with the response of bioconvection patterns to increased gravity acceleration (hypergravity); the wave number of the patterns has been reported to decrease during the hypergravity phases of parabolic aircraft flight, while it increases in centrifugal hypergravity. In this paper, we reassess the responses of bioconvection to altered gravity during parabolic flight on the basis of vertical and horizontal observations of the patterns formed by Tetrahymena thermophila and Chlamydomonas reinhardtii. Spatiotemporal analyses of the horizontal patterns revealed an increase in the pattern wave number in both pre- and post-parabola hypergravity. Vertical pattern analysis was generally in line with the horizontal pattern analysis, and further revealed that hypergravity-induced changes preceded at the top layer of the suspensions while microgravity-induced changes appeared to occur from the bottom part of the settling blobs. The responses to altered gravity were rather different between the two sample species: T. thermophila tended to drastically modify its bioconvection patterns in response to changes in gravity level, while the patterns of C. reinhardtii responded to a much lesser extent. This difference can be attributed to the distinct physical and physiological properties of the individual organisms, suggesting a significant contribution of the gyrotactic property to the swimming behavior of some protists.

Inhibition of Anaplerosis Attenuated Vascular Proliferation in Pulmonary Arterial Hypertension.

Vascular remodeling is considered a key event in the pathogenesis of pulmonary arterial hypertension (PAH). However, mechanisms of gaining the proliferative phenotype by pulmonary vascular cells are still unresolved. Due to well-established pyruvate dehydrogenase (PDH) deficiency in PAH pathogenesis, we hypothesized that the activation of another branch of pyruvate metabolism, anaplerosis, via pyruvate carboxylase (PC) could be a key contributor to the metabolic reprogramming of the vasculature. In sugen/hypoxic PAH rats, vascular proliferation was found to be accompanied by increased activation of Akt signaling, which upregulated membrane Glut4 translocation and caused upregulation of hexokinase and pyruvate kinase-2, and an overall increase in the glycolytic flux. Decreased PDH activity and upregulation of PC shuttled more pyruvate to oxaloacetate. This results in the anaplerotic reprogramming of lung vascular cells and their subsequent proliferation. Treatment of sugen/hypoxia rats with the PC inhibitor, phenylacetic acid 20 mg/kg, starting after one week from disease induction, significantly attenuated right ventricular systolic pressure, Fulton index, and pulmonary vascular cell proliferation. PC inhibition reduced the glycolytic shift by attenuating Akt-signaling, glycolysis, and restored mitochondrial pyruvate oxidation. Our findings suggest that targeting PC mediated anaplerosis is a potential therapeutic intervention for the resolution of vascular remodeling in PAH.

Necrosis-Released HMGB1 (High Mobility Group Box 1) in the Progressive Pulmonary Arterial Hypertension Associated With Male Sex.

Damage-associated molecular patterns, such as HMGB1 (high mobility group box 1), play a well-recognized role in the development of pulmonary arterial hypertension (PAH), a progressive fatal disease of the pulmonary vasculature. However, the contribution of the particular type of vascular cells, type of cell death, or the form of released HMGB1 in PAH remains unclear. Moreover, although male patients with PAH show a higher level of circulating HMGB1, its involvement in the severe PAH phenotype reported in males is unknown. In this study, we aimed to investigate the sources and active forms of HMGB1 released from damaged vascular cells and their contribution to the progressive type of PAH in males. Our results showed that HMGB1 is released by either pulmonary artery human endothelial cells or human pulmonary artery smooth muscle cells that underwent necrotic cell death, although only human pulmonary artery smooth muscle cells produce HMGB1 during apoptosis. Moreover, only human pulmonary artery smooth muscle cell death induced a release of dimeric HMGB1, found to be mitochondrial reactive oxygen species dependent, and TLR4 (toll-like receptor 4) activation. The modified Sugen/Hypoxia rat model replicates the human sexual dimorphism in PAH severity (right ventricle systolic pressure in males versus females 54.7±2.3 versus 44.6±2 mm Hg). By using this model, we confirmed that necroptosis and necrosis are the primary sources of circulating HMGB1 in the male rats, although only necrosis increased circulation of HMGB1 dimers. Attenuation of necrosis but not apoptosis or necroptosis prevented TLR4 activation in males and blunted the sex differences in PAH severity. We conclude that necrosis, through the release of HMGB1 dimers, predisposes males to a progressive form of PAH.

Early progression of pulmonary hypertension in the monocrotaline model in males is associated with increased lung permeability.

BACKGROUND: The mechanisms involved in pulmonary hypertension (PH) development in patients and pre-clinical models are poorly understood. PH has a well-established sex dimorphism in patients with increased frequency of PH in females, and more severe disease with poor survival prognosis in males. Previously, we found that heme signaling plays an essential role in the development phase of the Sugen/Hypoxia (SU/Hx) model. This study is focused on the elucidation of sex differences in mechanisms of PH development related to heme action at the early stage of the monocrotaline (MCT) PH model. METHODS: Rats received MCT injection (60 mg/kg, i.p.) and followed for 14 days to investigate early disease changes. Hemodynamic parameters were recorded at the end of the study; plasma, lung homogenates, and nuclear fractions were used for the evaluation of protein levels. RESULTS: Our data indicate that on day 14, rats did not show any significant increase in the Fulton index due to the early disease phase. However, the right ventricular systolic pressure was significantly increased in male rats, while female rats showed only a trend. Interestingly, only males demonstrated an increased lung-to-bodyweight ratio that indicated lung edema. Indeed, lung histology confirmed severe perivascular edema in males. Previously, we have reported that the increased perivascular edema in SU/Hx model correlated with intravascular hemolysis and activated heme signaling. Here, we found that elevated free hemoglobin levels and perivascular edema were increased, specifically in males showing more rapid progress of PH. A high level of heme carrier protein 1 (HCP-1), which is involved in heme uptake from the bloodstream into the cells, was also found elevated in the lungs of males. The upregulation of heme oxygenase in males indicated increased intracellular heme catabolism. Increased heme signaling resulted in the activation of heme-mediated barrier-disruptive mechanisms. Thus, hemolysis in males can be responsible for increased permeability of the lungs and early disease development. CONCLUSIONS: Our study indicates the importance of barrier-disruptive mechanisms as an earlier event in the induction of pulmonary hypertension. Importantly, males are more susceptible to hemolysis and develop PH earlier than females.

Antioxidant-Conjugated Peptide Attenuated Metabolic Reprogramming in Pulmonary Hypertension.

Pulmonary arterial hypertension (PAH) is a chronic cardiopulmonary disorder instigated by pulmonary vascular cell proliferation. Activation of Akt was previously reported to promote vascular remodeling. Also, the irreversible nitration of Y350 residue in Akt results in its activation. NitroAkt was increased in PAH patients and the SU5416/Hypoxia (SU/Hx) PAH model. This study investigated whether the prevention of Akt nitration in PAH by Akt targeted nitroxide-conjugated peptide (NP) could reverse vascular remodeling and metabolic reprogramming. Treatment of the SU/Hx model with NP significantly decreased nitration of Akt in lungs, attenuated right ventricle (RV) hypertrophy, and reduced RV systolic pressure. In the PAH model, Akt-nitration induces glycolysis by activation of the glucose transporter Glut4 and lactate dehydrogenase-A (LDHA). Decreased G6PD and increased GSK3ß in SU/Hx additionally shunted intracellular glucose via glycolysis. The increased glycolytic rate upregulated anaplerosis due to activation of pyruvate carboxylase in a nitroAkt-dependent manner. NP treatment resolved glycolytic switch and activated collateral pentose phosphate and glycogenesis pathways. Prevention of Akt-nitration significantly controlled pyruvate in oxidative phosphorylation by decreasing lactate and increasing pyruvate dehydrogenases activities. Histopathological studies showed significantly reduced pulmonary vascular proliferation. Based on our current observation, preventing Akt-nitration by using an Akt-targeted nitroxide-conjugated peptide could be a useful treatment option for controlling vascular proliferation in PAH.

Automated High-Throughput Damage Scoring of Zebrafish Lateral Line Hair Cells After Ototoxin Exposure.

Zebrafish have emerged as a powerful biological system for drug development against hearing loss. Zebrafish hair cells, contained within neuromasts along the lateral line, can be damaged with exposure to ototoxins, and therefore, pre-exposure to potentially otoprotective compounds can be a means of identifying promising new drug candidates. Unfortunately, anatomical assays of hair cell damage are typically low-throughput and labor intensive, requiring trained experts to manually score hair cell damage in fluorescence or confocal images. To enhance throughput and consistency, our group has developed an automated damage-scoring algorithm based on machine-learning techniques that produce accurate damage scores, eliminate potential operator bias, provide more fidelity in determining damage scores that are between two levels, and deliver consistent results in a fraction of the time required for manual analysis. The system has been validated against trained experts using linear regression, hypothesis testing, and the Pearson's correlation coefficient. Furthermore, performance has been quantified by measuring mean absolute error for each image and the time taken to automatically compute damage scores. Coupling automated analysis of zebrafish hair cell damage to behavioral assays for ototoxicity produces a novel drug discovery platform for rapid translation of candidate drugs into preclinical mammalian models of hearing loss.

A Fully Automated High-Throughput Zebrafish Behavioral Ototoxicity Assay.

Zebrafish animal models lend themselves to behavioral assays that can facilitate rapid screening of ototoxic, otoprotective, and otoregenerative drugs. Structurally similar to human inner ear hair cells, the mechanosensory hair cells on their lateral line allow the zebrafish to sense water flow and orient head-to-current in a behavior called rheotaxis. This rheotaxis behavior deteriorates in a dose-dependent manner with increased exposure to the ototoxin cisplatin, thereby establishing itself as an excellent biomarker for anatomic damage to lateral line hair cells. Building on work by our group and others, we have built a new, fully automated high-throughput behavioral assay system that uses automated image analysis techniques to quantify rheotaxis behavior. This novel system consists of a custom-designed swimming apparatus and imaging system consisting of network-controlled Raspberry Pi microcomputers capturing infrared video. Automated analysis techniques detect individual zebrafish, compute their orientation, and quantify the rheotaxis behavior of a zebrafish test population, producing a powerful, high-throughput behavioral assay. Using our fully automated biological assay to test a standardized ototoxic dose of cisplatin against varying doses of compounds that protect or regenerate hair cells may facilitate rapid translation of candidate drugs into preclinical mammalian models of hearing loss.

Zebrafish swimming behavior as a biomarker for ototoxicity-induced hair cell damage: a high-throughput drug development platform targeting hearing loss.

Hearing loss is one of the most common human sensory disabilities, adversely affecting communication, socialization, mood, physical functioning, and quality of life. In addition to age and noise-induced damage, ototoxicity is a common cause of sensorineural hearing loss with chemotherapeutic agents, for example, cisplatin, being a major contributor. Zebrafish (Danio rerio) are an excellent model to study hearing loss as they have neurosensory hair cells on their body surface that are structurally similar to those within the human inner ear. Anatomic assays of toxin-mediated hair cell damage in zebrafish have been established; however, using fish swimming behavior--rheotaxis--as a biomarker for this anatomic damage was only recently described. We hypothesized that, in parallel, multilane measurements of rheotaxis could be used to create a high-throughput platform for drug development assessing both ototoxic and potentially otoprotective compounds in real time. Such a device was created, and results demonstrated a clear dose response between cisplatin exposure, progressive hair cell damage, and reduced rheotaxis in zebrafish. Furthermore, pre-exposure to the otoprotective medication dexamethasone, before cisplatin exposure, partially rescued rheotaxis swimming behavior and hair cell integrity. These results provide the first evidence that rescued swimming behavior can serve as a biomarker for rescued hair cell function. Developing a drug against hearing loss represents an unmet clinical need with global implications. Because hearing loss from diverse etiologies may result from common end-effects at the hair cell level, lessons learned from the present study may be broadly used.

Development and swimming behavior of Medaka fry in a spaceflight aboard the Space Shuttle Columbia (STS-107).

A space experiment aimed at closely observing the development and swimming activity of medaka fry under microgravity was carried out as a part of the S*T*A*R*S Program, a space shuttle mission, in STS-107 in January 2003. Four eggs laid on earth in an artificially controlled environment were put in a container with a functionally closed ecological system and launched on the Space Shuttle Columbia. Each egg was held in place by a strip of Velcro in the container to be individually monitored by close-up CCD cameras. In the control experiment, four eggs prepared using the same experimental set-up remained on the ground. There was no appreciable difference in the time course of development between space- and ground-based embryos. In the ground experiment, embryos were observed to rotate in place enclosed with the egg membrane, whereas those in the flight unit did not rotate. One of the four eggs hatched on the 8th day after being launched into space. All four eggs hatched in the ground unit. The fry hatched in space was mostly motionless, but with occasional control of its posture with respect to references in the experimental chamber. The fry hatched on ground were observed to move actively, controlling their posture with respect to the gravity vector. These findings suggest that the absence of gravity affects the initiation process of motility of embryos and hatched fry.

Embryogenesis and swimming behavior of Medaka fish in JUSTSAP STARS Program (STS-107).

JUSTSAP (Japan-US Science, Technology and Space Application Program) Medaka fish experiment was carried out as a part of STARS (Space Technology and Research for Student) experiment, a space shuttle mission, STS-107 in January 2003. Four eggs laid on earth under artificially controlled environment were put in a closed ecological system, AHAB (Aquatic Habitat), and launched by Space Shuttle Columbia. For the control experiment, four eggs were put in the AHAB and remained on the ground. There was no remarkable difference in the time course of the development. In ground experiment embryos were observed to rotate in the egg membrane, whereas in flight unit they did not rotate. One egg hatched out on L (Launch) +8 days in flight unit. Four eggs hatched out in ground unit. Fry in flight unit was observed to face its back usually to the camera with little swimming movement. Fry in ground unit were observed to move actively and also to control their posture with respect to gravity vector.