Comparison of arterial storage conditions for delayed arterial ring testing.

Objective: Arterial ring testing is the gold standard for measuring arterial function. Increased arterial tone through arterial contraction and impaired endothelial relaxation (endothelial dysfunction) are key metrics of impaired arterial health in peripheral arterial disease (PAD). To allow for comparative testing of arteries during standard laboratory hours, storage buffers and conditions have been used to extend the functional life of arteries. Various storage conditions have been compared, but there has not been a robust comparison or validation in human arteries. The objective of this work is to optimize storage of arterial segments for endothelial cell (EC) testing in a murine model and to test EC function in human PAD arteries. We hypothesized that certain storage conditions would be superior to others. Methods: Healthy murine aortas were harvested from 10- to 14-week-old C57/Bl6J male and female mice and compared under different storage protocols (24 hours) to immediate arterial testing. The storage conditions tested were: Opti-MEM (37°C or 4°C), Krebs-HEPES with 1.8 mmol/L or 2.5 mmol/L calcium (4°C), or Wisconsin (WI) solution at 4°C. Vascular function was evaluated by isometric force testing. Endothelium-dependent and -independent relaxation were measured after precontraction with addition of methacholine or sodium nitroprusside, respectively. Arterial contraction was stimulated with potassium chloride or phenylephrine. Analysis of variance was used to determine significance compared with immediate testing with P < .05. Under institutional review board approval, 28 PAD arteries were collected at amputation and underwent vascular function testing as described. Disturbed flow conditions were determined by indirect (upstream occlusion) flow to the harvested tibial arteries. Stable flow arteries had in-line flow. Arterial calcification was quantified manually as present or not present. Results: We found that 4°C WI and 37°C Opti-MEM best preserved endothelium-dependent relaxation and performed similarly to immediately testing aortas (termed fresh for freshly tested) (P > .95). Other storage conditions were inferior to freshly tested aortas (P < .05). Vascular smooth muscle function was tested by endothelial-independent relaxation and contractility. All storage conditions preserved endothelial-independent relaxation and contractility similar to freshly tested arteries. However, 4°C WI and 37°C Opti-MEM storage conditions most closely approximated the maximum force of contraction of freshly tested arteries in response to potassium chloride (P > .39). For human arterial testing, 28 tibial arteries were tested for relaxation and contraction with 16 arteries with peripheral artery occlusive disease (PAD with disturbed flow) and 12 without peripheral artery occlusive disease (PAD with stable flow), of which 14 were calcified and 14 were noncalcified. Endothelial-dependent relaxation data was measurable in 9 arteries and arterial contraction data was measurable in 14 arteries. When comparing flow conditions, arteries exposed to disturbed flow (n = 4) had significantly less relaxation (2% vs 59%; P = .03) compared with stable flow conditions (n = 5). In contrast, presence the (n = 6) or absence of calcification (n = 3) did not impact arterial relaxation. Arterial contraction was not different between groups in either comparison by flow (n = 9 disturbed; n = 5 stable) or calcification (n = 6 present; n = 8 absent). Conclusions: In healthy murine aortas, arterial storage for 24 hours in 4°C WI or 37°C Opti-MEM both preserved endothelium-dependent relaxation and maximum force of contraction. In human PAD arteries stored in 4° WI, flow conditions before arterial harvest, but not arterial calcification, led to differences in arterial relaxation in human PAD arteries. Arterial contractility was more robust (11/28 arteries) compared with arterial relaxation (7/28 arteries), but was not significantly different under flow or calcification parameters. This work defines ideal storage conditions for arterial ring testing and identifies that EC dysfunction from disturbed flow may persist in delayed ex vivo arterial testing.

Polycomb Group Protein CBX7 Represses Cardiomyocyte Proliferation Through Modulation of the TARDBP/RBM38 Axis.

BACKGROUND: Shortly after birth, cardiomyocytes exit the cell cycle and cease proliferation. At present, the regulatory mechanisms for this loss of proliferative capacity are poorly understood. CBX7 (chromobox 7), a polycomb group (PcG) protein, regulates the cell cycle, but its role in cardiomyocyte proliferation is unknown. METHODS: We profiled CBX7 expression in the mouse hearts through quantitative real-time polymerase chain reaction, Western blotting, and immunohistochemistry. We overexpressed CBX7 in neonatal mouse cardiomyocytes through adenoviral transduction. We knocked down CBX7 by using constitutive and inducible conditional knockout mice (Tnnt2-Cre;Cbx7fl/+ and Myh6-MCM;Cbx7fl/fl, respectively). We measured cardiomyocyte proliferation by immunostaining of proliferation markers such as Ki67, phospho-histone 3, and cyclin B1. To examine the role of CBX7 in cardiac regeneration, we used neonatal cardiac apical resection and adult myocardial infarction models. We examined the mechanism of CBX7-mediated repression of cardiomyocyte proliferation through coimmunoprecipitation, mass spectrometry, and other molecular techniques. RESULTS: We explored Cbx7 expression in the heart and found that mRNA expression abruptly increased after birth and was sustained throughout adulthood. Overexpression of CBX7 through adenoviral transduction reduced proliferation of neonatal cardiomyocytes and promoted their multinucleation. On the other hand, genetic inactivation of Cbx7 increased proliferation of cardiomyocytes and impeded cardiac maturation during postnatal heart growth. Genetic ablation of Cbx7 promoted regeneration of neonatal and adult injured hearts. Mechanistically, CBX7 interacted with TARDBP (TAR DNA-binding protein 43) and positively regulated its downstream target, RBM38 (RNA Binding Motif Protein 38), in a TARDBP-dependent manner. Overexpression of RBM38 inhibited the proliferation of CBX7-depleted neonatal cardiomyocytes. CONCLUSIONS: Our results demonstrate that CBX7 directs the cell cycle exit of cardiomyocytes during the postnatal period by regulating its downstream targets TARDBP and RBM38. This is the first study to demonstrate the role of CBX7 in regulation of cardiomyocyte proliferation, and CBX7 could be an important target for cardiac regeneration.

Myoglobinemia, Peripheral Arterial Disease, and Patient Mortality.

BACKGROUND: Peripheral arterial disease (PAD) causes leg muscle damage due to inadequate perfusion and increases cardiovascular events and mortality 2- to 3-fold. It is unclear if PAD is a biomarker for high-risk cardiovascular disease or if skeletal muscle injury harms arterial health. The objective of this work is to test if serum myoglobin levels (myoglobinemia) are a marker of PAD, and if so, whether myoglobin impairs vascular health. STUDY DESIGN: Patient blood samples were collected from PAD and control (no PAD) patients and interrogated for myoglobin concentrations and nitric oxide bioavailability. Patient mortality over time was captured from the medical record. Myoglobin activity was tested on endothelial cells and arterial function. RESULTS: Myoglobin is a biomarker for symptomatic PAD and was inversely related to nitric oxide bioavailability; 200 ng/mL myoglobin in vitro increased endothelial cell permeability in vitro and decreased nitrate bioavailability. Ex vivo, 100 ng/mL myoglobin increased vascular tone in naive murine aortas approximately 1.5 times, impairing absolute vessel relaxation. In vivo, we demonstrated that myoglobinemia caused impaired flow-mediated dilation in a porcine model. Patients presenting with myoglobin levels of 100 ng/mL or greater had significantly more deaths than those with myoglobin levels of less than 100 ng/mL. CONCLUSIONS: Using a combination of patient data, in vitro, ex vivo, and in vivo testing, we found that myoglobin is a biomarker for symptomatic PAD and a potent regulator of arterial health that can increase vascular tone, increase vascular permeability, and cause endothelial dysfunction, all of which may contribute to the vulnerability of PAD patients to cardiovascular events and death.

3'UTR shortening of HAS2 promotes hyaluronan hyper-synthesis and bioenergetic dysfunction in pulmonary hypertension.

Pulmonary hypertension (PH) comprises a diverse group of disorders that share a common pathway of pulmonary vascular remodeling leading to right ventricular failure. Development of anti-remodeling strategies is an emerging frontier in PH therapeutics that requires a greater understanding of the interactions between vascular wall cells and their extracellular matrices. The ubiquitous matrix glycan, hyaluronan (HA), is markedly elevated in lungs from patients and experimental models with PH. Herein, we identified HA synthase-2 (HAS2) in the pulmonary artery smooth muscle cell (PASMC) layer as a predominant locus of HA dysregulation. HA upregulation involves depletion of NUDT21, a master regulator of alternative polyadenylation, resulting in 3'UTR shortening and hyper-expression of HAS2. The ensuing increase of HAS2 and hyper-synthesis of HA promoted bioenergetic dysfunction of PASMC characterized by impaired mitochondrial oxidative capacity and a glycolytic shift. The resulting HA accumulation stimulated pro-remodeling phenotypes such as cell proliferation, migration, apoptosis-resistance, and stimulated pulmonary artery contractility. Transgenic mice, mimicking HAS2 hyper-synthesis in smooth muscle cells, developed spontaneous PH, whereas targeted deletion of HAS2 prevented experimental PH. Pharmacological blockade of HAS2 restored normal bioenergetics in PASMC, ameliorated cell remodeling phenotypes, and reversed experimental PH in vivo. In summary, our results uncover a novel mechanism of HA hyper-synthesis and downstream effects on pulmonary vascular cell metabolism and remodeling.

Probing the Intracellular Delivery of Nanoparticles into Hard-to-Transfect Cells.

Hard-to-transfect cells are cells that are known to present special difficulties in intracellular delivery of exogenous entities. However, the special transport behaviors underlying the special delivery problem in these cells have so far not been examined carefully. Here, we combine single-particle motion analysis, cell biology studies, and mathematical modeling to investigate nanoparticle transport in bone marrow-derived mesenchymal stem cells (BMSCs), a technologically important type of hard-to-transfect cells. Tat peptide-conjugated quantum dots (QDs-Tat) were used as the model nanoparticles. Two different yet complementary single-particle methods, namely, pair-correlation function and single-particle tracking, were conducted on the same cell samples and on the same viewing stage of a confocal microscope. Our results reveal significant differences in each individual step of transport of QDs-Tat in BMSCs vs a commonly used model cell line, HeLa cells. Single-particle motion analysis demonstrates that vesicle escape and cytoplasmic diffusion are dramatically more difficult in BMSCs than in HeLa cells. Cell biology studies show that BMSCs use different biological pathways for the cellular uptake, vesicular transport, and exocytosis of QDs-Tat than HeLa cells. A reaction-diffusion-advection model is employed to mathematically integrate the individual steps of cellular transport and can be used to predict and design nanoparticle delivery in BMSCs. This work provides dissective, quantitative, and mechanistic understandings of nanoparticle transport in BMSCs. The investigative methods described in this work can help to guide the tailored design of nanoparticle-based delivery in specific types and subtypes of hard-to-transfect cells.

MicroRNA-155 Modulates Macrophages' Response to Non-Tuberculous Mycobacteria through COX-2/PGE2 Signaling.

Non-tuberculous mycobacteria (NTM) have been recognized as a causative agent of various human diseases, including severe infections in immunocompromised patients, such as people living with HIV. The most common species identified is the Mycobacterium avium-intracellulare complex (MAI/MAC), accounting for a majority of infections. Despite abundant information detailing the clinical significance of NTM, little is known about host-pathogen interactions in NTM infection. MicroRNAs (miRs) serve as important post-transcriptional regulators of gene expression. Using a microarray profile, we found that the expression of miR-155 and cyclo-oxygenase 2 (COX-2) is significantly increased in bone-marrow-derived macrophages from mice and human monocyte-derived macrophages from healthy volunteers that are infected with NTM. Antagomir against miR-155 effectively suppressed expression of COX-2 and reduced Prostaglandin E2(PGE2) secretion, suggesting that COX-2/PGE2 expression is dependent on miR-155. Mechanistically, we found that inhibition of NF-κB activity significantly reduced miR-155/COX-2 expression in infected macrophages. Most importantly, blockade of COX-2, E-prostanoid receptors (EP2 and EP4) enhanced killing of MAI in macrophages. These findings provide novel mechanistic insights into the role of miR-155/COX-2/PGE2 signalling and suggest that induction of these pathways enhances survival of mycobacteria in macrophages. Defining host-pathogen interactions can lead to novel immunomodulatory therapies for NTM infections which are difficult to treat.

PPARγ increases HUWE1 to attenuate NF-κB/p65 and sickle cell disease with pulmonary hypertension.

Sickle cell disease (SCD)-associated pulmonary hypertension (PH) causes significant morbidity and mortality. Here, we defined the role of endothelial specific peroxisome proliferator-activated receptor γ (PPARγ) function and novel PPARγ/HUWE1/miR-98 signaling pathways in the pathogenesis of SCD-PH. PH and right ventricular hypertrophy (RVH) were increased in chimeric Townes humanized sickle cell (SS) mice with endothelial-targeted PPARγ knockout (SSePPARγKO) compared with chimeric littermate control (SSLitCon). Lung levels of PPARγ, HUWE1, and miR-98 were reduced in SSePPARγKO mice compared with SSLitCon mice, whereas SSePPARγKO lungs were characterized by increased levels of p65, ET-1, and VCAM1. Collectively, these findings indicate that loss of endothelial PPARγ is sufficient to increase ET-1 and VCAM1 that contribute to endothelial dysfunction and SCD-PH pathogenesis. Levels of HUWE1 and miR-98 were decreased, and p65 levels were increased in the lungs of SS mice in vivo and in hemin-treated human pulmonary artery endothelial cells (HPAECs) in vitro. Although silencing of p65 does not regulate HUWE1 levels, the loss of HUWE1 increased p65 levels in HPAECs. Overexpression of PPARγ attenuated hemin-induced reductions of HUWE1 and miR-98 and increases in p65 and endothelial dysfunction. Similarly, PPARγ activation attenuated baseline PH and RVH and increased HUWE1 and miR-98 in SS lungs. In vitro, hemin treatment reduced PPARγ, HUWE1, and miR-98 levels and increased p65 expression, HPAEC monocyte adhesion, and proliferation. These derangements were attenuated by pharmacological PPARγ activation. Targeting these signaling pathways can favorably modulate a spectrum of pathobiological responses in SCD-PH pathogenesis, highlighting novel therapeutic targets in SCD pulmonary vascular dysfunction and PH.

MicroRNA-98 reduces nerve growth factor expression in nicotine-induced airway remodeling.

Evolving evidence suggests that nicotine may contribute to impaired asthma control by stimulating expression of nerve growth factor (NGF), a neurotrophin associated with airway remodeling and airway hyperresponsiveness. We explored the hypothesis that nicotine increases NGF by reducing lung fibroblast (LF) microRNA-98 (miR-98) and PPARγ levels, thus promoting airway remodeling. Levels of NGF, miR-98, PPARγ, fibronectin 1 (FN1), endothelin-1 (EDN1, herein referred to as ET-1), and collagen (COL1A1 and COL3A1) were measured in human LFs isolated from smoking donors, in mouse primary LFs exposed to nicotine (50 µg/ml), and in whole lung homogenates from mice chronically exposed to nicotine (100 µg/ml) in the drinking water. In selected studies, these pathways were manipulated in LFs with miR-98 inhibitor (anti-miR-98), miR-98 overexpression (miR-98 mimic), or the PPARγ agonist rosiglitazone. Compared with unexposed controls, nicotine increased NGF, FN1, ET-1, COL1A1, and COL3A1 expression in human and mouse LFs and mouse lung homogenates. In contrast, nicotine reduced miR-98 levels in LFs in vitro and in lung homogenates in vivo Treatment with anti-miR-98 alone was sufficient to recapitulate increases in NGF, FN1, and ET-1, whereas treatment with a miR-98 mimic significantly suppressed luciferase expression in cells transfected with a luciferase reporter linked to the putative seed sequence in the NGF 3'UTR and also abrogated nicotine-induced increases in NGF, FN1, and ET-1 in LFs. Similarly, rosiglitazone increased miR-98 and reversed nicotine-induced increases in NGF, FN1, and ET-1. Taken together, these findings demonstrate that nicotine-induced increases in NGF and other markers of airway remodeling are negatively regulated by miR-98.

Increased Transglutaminase 2 Expression and Activity in Rodent Models of Obesity/Metabolic Syndrome and Aging.

Diastolic dysfunction of the heart and decreased compliance of the vasculature and lungs (i.e., increased organ tissue stiffness) are known features of obesity and the metabolic syndrome. Similarly, cardiac diastolic dysfunction is associated with aging. Elevation of the enzyme transglutaminase 2 (TG2) leads to protein cross-linking and enhanced collagen synthesis and participates as a candidate pathway for development of tissue stiffness. With these observations in mind we hypothesized that TG2 may be elevated in tissues of a rat model of obesity/metabolic syndrome (the ZSF 1 rat) and a mouse model of aging, i.e., the senescent SAMP8 mouse. In the experiments reported here, TG2 expression and activity were found for the first time to be spontaneously elevated in organs from both the ZSF1 rat and the SAMP8 mouse. These observations are consistent with a hypothesis that a TG2-related pathway may participate in the known tissue stiffness associated with cardiac diastolic dysfunction in these two rodent models. The potential TG2 pathway needs better correlation with physiologic dysfunction and may eventually provide novel therapeutic insights to improve tissue compliance.

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.

Macrophage Signaling Pathways in Pulmonary Nontuberculous Mycobacteria Infections.

The incidence and prevalence of nontuberculous mycobacteria (NTM) lung disease is rising worldwide and accounts for most clinical cases of NTM disease. NTM infections occur in both immunocompetent and immunocompromised hosts. Macrophages are the primary host cells that initiate an immune response to NTM. Defining the molecular events that govern the control of infection within macrophages is fundamental to understanding the pathogenesis of NTM disease. Here, we review key macrophage host signaling pathways that contribute to the host immune response to pulmonary NTM infections. In this review, we focus primarily on NTM that are known to cause lung disease, including Mycobacterium avium intracellulare, M. abscessus, and M. kansasii.

Lovastatin attenuates hypertension induced by renal tubule-specific knockout of ATP-binding cassette transporter A1, by inhibiting epithelial sodium channels.

BACKGROUND AND PURPOSE: We have shown that cholesterol is synthesized in the principal cells of renal cortical collecting ducts (CCD) and stimulates the epithelial sodium channels (ENaC). Here we have determined whether lovastatin, a cholesterol synthesis inhibitor, can antagonize the hypertension induced by activated ENaC, following deletion of the cholesterol transporter (ATP-binding cassette transporter A1; ABCA1). EXPERIMENTAL APPROACH: We selectively deleted ABCA1 in the principal cells of mouse CCD and used the cell-attached patch-clamp technique to record ENaC activity. Western blot and immunofluorescence staining were used to evaluate protein expression levels. Systolic BP was measured with the tail-cuff method. KEY RESULTS: Specific deletion of ABCA1 elevated BP and ENaC single-channel activity in the principal cells of CCD in mice. These effects were antagonized by lovastatin. ABCA1 deletion elevated intracellular cholesterol levels, which was accompanied by elevated ROS, increased expression of serum/glucocorticoid regulated kinase 1 (Sgk1), phosphorylated neural precursor cell-expressed developmentally down-regulated protein 4-2 (Nedd4-2) and furin, along with shorten the primary cilium, and reduced ATP levels in urine. CONCLUSIONS AND IMPLICATIONS: These data suggest that specific deletion of ABCA1 in principal cells increases BP by stimulating ENaC channels via a cholesterol-dependent pathway which induces several secondary responses associated with oxidative stress, activated Sgk1/Nedd4-2, increased furin expression, and reduced cilium-mediated release of ATP. As ABCA1 can be blocked by cyclosporine A, these results suggest further investigation of the possible use of statins to treat CsA-induced hypertension.

The cofilin phosphatase slingshot homolog 1 restrains angiotensin II-induced vascular hypertrophy and fibrosis in vivo.

The dual specificity phosphatase slingshot homolog 1 (SSH1) contributes to actin remodeling by dephosphorylating and activating the actin-severing protein cofilin. The reorganization of the actin cytoskeleton has been implicated in chronic hypertension and the subsequent mechano-adaptive rearrangement of vessel wall components. Therefore, using a novel Ssh1-/- mouse model, we investigated the potential role of SSH1 in angiotensin II (Ang II)-induced hypertension, and vascular remodeling. We found that loss of SSH1 did not produce overt phenotypic changes and that baseline blood pressures as well as heart rates were comparable between Ssh1+/+ and Ssh1-/- mice. Although 14 days of Ang II treatment equally increased systolic blood pressure in both genotypes, histological assessment of aortic samples indicated that medial thickening was exacerbated by the loss of SSH1. Consequently, reverse-transcription quantitative PCR analysis of the transcripts from Ang II-infused animals confirmed increased aortic expression levels of fibronectin, and osteopontin in Ssh1-/- when compared to wild-type mice. Mechanistically, our data suggest that fibrosis in SSH1-deficient mice occurs by a process that involves aberrant responses to Ang II-induced TGFß1. Taken together, our work indicates that Ang II-dependent fibrotic gene expression and vascular remodeling, but not the Ang II-induced pressor response, are modulated by SSH1-mediated signaling pathways and SSH1 activity is protective against Ang II-induced remodeling in the vasculature.

Redox Biology of Peroxisome Proliferator-Activated Receptor-γ in Pulmonary Hypertension.

Significance: Peroxisome proliferator-activated receptor-gamma (PPARγ) maintains pulmonary vascular health through coordination of antioxidant defense systems, inflammation, and cellular metabolism. Insufficient PPARγ contributes to pulmonary hypertension (PH) pathogenesis, whereas therapeutic restoration of PPARγ activity attenuates PH in preclinical models. Recent Advances: Numerous studies in the past decade have elucidated the complex mechanisms by which PPARγ in the pulmonary vasculature and right ventricle (RV) protects against PH. The scope of PPARγ-interconnected pathways continues to expand and includes induction of antioxidant genes, transrepression of inflammatory signaling, regulation of mitochondrial biogenesis and bioenergetic integrity, control of cell cycle and proliferation, and regulation of vascular tone through interactions with nitric oxide and endogenous vasoactive molecules. Furthermore, PPARγ interacts with an extensive regulatory network of transcription factors and microRNAs leading to broad impact on cell signaling. Critical Issues: Abundant evidence suggests that targeting PPARγ exerts diverse salutary effects in PH and represents a novel and potentially translatable therapeutic strategy. However, progress has been slowed by an incomplete understanding of how specific PPARγ pathways are critically disrupted across PH disease subtypes and lack of optimal pharmacological ligands. Future Directions: Recent studies indicate that ligand-induced post-translational modifications of the PPARγ receptor differentially induce therapeutic benefits versus adverse side effects of PPARγ receptor activation. Strategies to selectively target PPARγ activity in diseased cells of pulmonary circulation and RV, coupled with development of ligands designed to specifically regulate post-translational PPARγ modifications, may unlock the full therapeutic potential of this versatile master transcriptional and metabolic regulator in PH.

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.

The Role of Intercalated Cell Nedd4-2 in BP Regulation, Ion Transport, and Transporter Expression.

BackgroundNedd4-2 is an E3 ubiquitin-protein ligase that associates with transport proteins, causing their ubiquitylation, and then internalization and degradation. Previous research has suggested a correlation between Nedd4-2 and BP. In this study, we explored the effect of intercalated cell (IC) Nedd4-2 gene ablation on IC transporter abundance and function and on BP.Methods We generated IC Nedd4-2 knockout mice using Cre-lox technology and produced global pendrin/Nedd4-2 null mice by breeding global Nedd4-2 null (Nedd4-2-/- ) mice with global pendrin null (Slc26a4-/- ) mice. Mice ate a diet with 1%-4% NaCl; BP was measured by tail cuff and radiotelemetry. We measured transepithelial transport of Cl- and total CO2 and transepithelial voltage in cortical collecting ducts perfused in vitro Transporter abundance was detected with immunoblots, immunohistochemistry, and immunogold cytochemistry.Results IC Nedd4-2 gene ablation markedly increased electroneutral Cl-/HCO3- exchange in the cortical collecting duct, although benzamil-, thiazide-, and bafilomycin-sensitive ion flux changed very little. IC Nedd4-2 gene ablation did not increase the abundance of type B IC transporters, such as AE4 (Slc4a9), H+-ATPase, barttin, or the Na+-dependent Cl-/HCO3- exchanger (Slc4a8). However, IC Nedd4-2 gene ablation increased CIC-5 total protein abundance, apical plasma membrane pendrin abundance, and the ratio of pendrin expression on the apical membrane to the cytoplasm. IC Nedd4-2 gene ablation increased BP by approximately 10 mm Hg. Moreover, pendrin gene ablation eliminated the increase in BP observed in global Nedd4-2 knockout mice.Conclusions IC Nedd4-2 regulates Cl-/HCO3- exchange in ICs., Nedd4-2 gene ablation increases BP in part through its action in these cells.

PPARγ Regulates Mitochondrial Structure and Function and Human Pulmonary Artery Smooth Muscle Cell Proliferation.

Pulmonary hypertension (PH) is a progressive disorder that causes significant morbidity and mortality despite existing therapies. PH pathogenesis is characterized by metabolic derangements that increase pulmonary artery smooth muscle cell (PASMC) proliferation and vascular remodeling. PH-associated decreases in peroxisome proliferator-activated receptor γ (PPARγ) stimulate PASMC proliferation, and PPARγ in coordination with PPARγ coactivator 1α (PGC1α) regulates mitochondrial gene expression and biogenesis. To further examine the impact of decreases in PPARγ expression on human PASMC (HPASMC) mitochondrial function, we hypothesized that depletion of either PPARγ or PGC1α perturbs mitochondrial structure and function to stimulate PASMC proliferation. To test this hypothesis, HPASMCs were exposed to hypoxia and treated pharmacologically with the PPARγ antagonist GW9662 or with siRNA against PPARγ or PGC1α for 72 hours. HPASMC proliferation (cell counting), target mRNA levels (qRT-PCR), target protein levels (Western blotting), mitochondria-derived H2O2 (confocal immunofluorescence), mitochondrial mass and fragmentation, and mitochondrial bioenergetic profiling were determined. Hypoxia or knockdown of either PPARγ or PGC1α increased HPASMC proliferation, enhanced mitochondria-derived H2O2, decreased mitochondrial mass, stimulated mitochondrial fragmentation, and impaired mitochondrial bioenergetics. Taken together, these findings provide novel evidence that loss of PPARγ diminishes PGC1α and stimulates derangements in mitochondrial structure and function that cause PASMC proliferation. Overexpression of PGC1α reversed hypoxia-induced HPASMC derangements. This study identifies additional mechanistic underpinnings of PH, and provides support for the notion of activating PPARγ as a novel therapeutic strategy in PH.

PPARγ attenuates hypoxia-induced hypertrophic transcriptional pathways in the heart.

Chronic hypoxia-induced pulmonary hypertension (PH) is characterized by increased pressure and resistance in the pulmonary vasculature and hypertrophy of the right ventricle (RV). The transcription factors, nuclear factor activated T-cells (NFAT), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB/p65) contribute to RV hypertrophy (RVH). Because peroxisome proliferator-activated receptor gamma (PPARγ) activation attenuates hypoxia-induced PH and RVH, we hypothesized that PPARγ inhibits activation of RV hypertrophic transcriptional signaling mechanisms. C57BL/6J mice were exposed to normoxia (21% O2) or hypoxia (10% O2) for 21 days. During the final 10 days of exposure, selected mice were treated with the PPARγ ligand, pioglitazone. RV systolic pressure (RVSP) and RVH were measured, and NFATc2 and NF-kB/p65 protein levels were measured in RV and LV nuclear and cytosolic fractions. Cardiomyocyte hypertrophy was assessed with wheatgerm agglutinin staining. NFAT activation was also examined with luciferase reporter mice and analysis of protein levels of selected transcriptional targets. Chronic-hypoxia increased: (1) RVH, RVSP, and RV cardiomyocyte hypertrophy; (2) NFATc2 and NF-κB activation in RV nuclear homogenates; (3) RV and LV NFAT luciferase activity; and (4) RV protein levels of brain natriuretic peptide (BNP) and ß-myosin heavy chain (ß-MyHC). Treatment with pioglitazone attenuated hypoxia-induced increases in both RV and LV NFAT luciferase activity. Chronic hypoxia caused sustained RV NFATc2 and NF-κB activation. Pioglitazone attenuated PH, RVH, cardiomyocyte hypertrophy, and activation of RV hypertrophic signaling and also attenuated LV NFAT activation. PPARγ favorably modulates signaling derangements in the heart as well as in the pulmonary vascular wall.

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.