Resistin predicts disease severity and survival in patients with pulmonary arterial hypertension.

BACKGROUND: Abnormal remodeling of distal pulmonary arteries in patients with pulmonary arterial hypertension (PAH) leads to progressively increased pulmonary vascular resistance, followed by right ventricular hypertrophy and failure. Despite considerable advancements in PAH treatment prognosis remains poor. We aim to evaluate the potential for using the cytokine resistin as a genetic and biological marker for disease severity and survival in a large cohort of patients with PAH. METHODS: Biospecimens, clinical, and genetic data for 1121 adults with PAH, including 808 with idiopathic PAH (IPAH) and 313 with scleroderma-associated PAH (SSc-PAH), were obtained from a national repository. Serum resistin levels were measured by ELISA, and associations between resistin levels, clinical variables, and single nucleotide polymorphism genotypes were examined with multivariable regression models. Machine-learning (ML) algorithms were applied to develop and compare risk models for mortality prediction. RESULTS: Resistin levels were significantly higher in all PAH samples and PAH subtype (IPAH and SSc-PAH) samples than in controls (P < .0001) and had significant discriminative abilities (AUCs of 0.84, 0.82, and 0.91, respectively; P < .001). High resistin levels (above 4.54 ng/mL) in PAH patients were associated with older age (P = .001), shorter 6-min walk distance (P = .001), and reduced cardiac performance (cardiac index, P = .016). Interestingly, mutant carriers of either rs3219175 or rs3745367 had higher resistin levels (adjusted P = .0001). High resistin levels in PAH patients were also associated with increased risk of death (hazard ratio: 2.6; 95% CI: 1.27-5.33; P < .0087). Comparisons of ML-derived survival models confirmed satisfactory prognostic value of the random forest model (AUC = 0.70, 95% CI: 0.62-0.79) for PAH. CONCLUSIONS: This work establishes the importance of resistin in the pathobiology of human PAH. In line with its function in rodent models, serum resistin represents a novel biomarker for PAH prognostication and may indicate a new therapeutic avenue. ML-derived survival models highlighted the importance of including resistin levels to improve performance. Future studies are needed to develop multi-marker assays that improve noninvasive risk stratification.

Isoflurane Disrupts Postsynaptic Density-95 Protein Interactions Causing Neuronal Synapse Loss and Cognitive Impairment in Juvenile Mice via Canonical NO-mediated Protein Kinase-G Signaling.

BACKGROUND: Inhalational anesthetics are known to disrupt PDZ2 domain-mediated protein-protein interactions of the postsynaptic density (PSD)-95 protein. The aim of this study is to investigate the underlying mechanisms in response to early isoflurane exposure on synaptic PSD-95 PDZ2 domain disruption that altered spine densities and cognitive function. The authors hypothesized that activation of protein kinase-G by the components of nitric oxide (NO) signaling pathway constitutes a mechanism that prevents loss of early dendritic spines and synapse in neurons and cognitive impairment in mice in response to disruption of PDZ2 domain of the PSD-95 protein. METHODS: Postnatal day 7 mice were exposed to 1.5% isoflurane for 4 h or injected with 8 mg/kg active PSD-95 wild-type PDZ2 peptide or soluble guanylyl cyclase activator YC-1 along with their respective controls. Primary neurons at 7 days in vitro were exposed to isoflurane or PSD-95 wild-type PDZ2 peptide for 4 h. Coimmunoprecipitation, spine density, synapses, cyclic guanosine monophosphate-dependent protein kinase activity, and novel object recognition memory were assessed. RESULTS: Exposure of isoflurane or PSD-95 wild-type PDZ2 peptide relative to controls causes the following. First, there is a decrease in PSD-95 coimmunoprecipitate relative to N-methyl-d-aspartate receptor subunits NR2A and NR2B precipitate (mean ± SD [in percentage of control]: isoflurane, 54.73 ± 16.52, P = 0.001; and PSD-95 wild-type PDZ2 peptide, 51.32 ± 12.93, P = 0.001). Second, there is a loss in spine density (mean ± SD [spine density per 10 µm]: control, 5.28 ± 0.56 vs. isoflurane, 2.23 ± 0.67, P < 0.0001; and PSD-95 mutant PDZ2 peptide, 4.74 ± 0.94 vs. PSD-95 wild-type PDZ2 peptide, 1.47 ± 0.87, P < 0.001) and a decrease in synaptic puncta (mean ± SD [in percentage of control]: isoflurane, 41.1 ± 14.38, P = 0.001; and PSD-95 wild-type PDZ2 peptide, 50.49 ± 14.31, P < 0.001). NO donor or cyclic guanosine monophosphate analog prevents the spines and synapse loss and decline in the cyclic guanosine monophosphate-dependent protein kinase activity, but this prevention was blocked by soluble guanylyl cyclase or protein kinase-G inhibitors in primary neurons. Third, there were deficits in object recognition at 5 weeks (mean ± SD [recognition index]: male, control, 64.08 ± 10.57 vs. isoflurane, 48.49 ± 13.41, P = 0.001, n = 60; and female, control, 67.13 ± 11.17 vs. isoflurane, 53.76 ± 6.64, P = 0.003, n = 58). Isoflurane-induced impairment in recognition memory was preventable by the introduction of YC-1. CONCLUSIONS: Activation of soluble guanylyl cyclase or protein kinase-G prevents isoflurane or PSD-95 wild-type PDZ2 peptide-induced loss of dendritic spines and synapse. Prevention of recognition memory with YC-1, a NO-independent activator of guanylyl cyclase, supports a role for the soluble guanylyl cyclase mediated protein kinase-G signaling in countering the effects of isoflurane-induced cognitive impairment.

The inflammatory role of dysregulated IRS2 in pulmonary vascular remodeling under hypoxic conditions.

Pulmonary hypertension (PH) is a devastating disease characterized by progressive elevation of pulmonary vascular resistance, right ventricular failure, and ultimately death. We have shown previously that insulin receptor substrate 2 (IRS2), a molecule highly critical to insulin resistance and metabolism, has an anti-inflammatory role in Th2-skewed lung inflammation and pulmonary vascular remodeling. Here, we investigated the hypothesis that IRS2 has an immunomodulatory role in human and experimental PH. Expression analysis showed that IRS2 was significantly decreased in the pulmonary vasculature of patients with pulmonary arterial hypertension and in rat models of PH. In mice, genetic ablation of IRS2 enhanced the hypoxia-induced signaling pathway of Akt and Forkhead box O1 (FOXO1) in the lung tissue and increased pulmonary vascular muscularization, proliferation, and perivascular macrophage recruitment. Furthermore, mice with homozygous IRS2 gene deletion showed a significant gene dosage-dependent increase in pulmonary vascular remodeling and right ventricular hypertrophy in response to hypoxia. Functional studies with bone marrow-derived macrophages isolated from homozygous IRS2 gene-deleted mice showed that hypoxia exposure led to enhancement of the Akt and ERK signaling pathway followed by increases in the pro-PH macrophage activation markers, vascular endothelial growth factor-A and arginase 1. Our data suggest that IRS2 contributes to anti-inflammatory effects by regulating macrophage activation and recruitment, which may limit the vascular inflammation, remodeling, and right ventricular hypertrophy that are seen in PH pathology. Restoring the IRS2 pathway may be an effective therapeutic approach for the treatment of PH and right heart failure.

RELMα Licenses Macrophages for Damage-Associated Molecular Pattern Activation to Instigate Pulmonary Vascular Remodeling.

Pulmonary hypertension (PH) is a debilitating disease characterized by remodeling of the lung vasculature. In rodents, resistin-like molecule-α (RELMα, also known as HIMF or FIZZ1) can induce PH, but the signaling mechanisms are still unclear. In this study, we used human lung samples and a hypoxia-induced mouse model of PH. We found that the human homolog of RELMα, human (h) resistin, is upregulated in macrophage-like inflammatory cells from lung tissues of patients with idiopathic PH. Additionally, at PH onset in the mouse model, we observed RELMα-dependent lung accumulation of macrophages that expressed high levels of the key damage-associated molecular pattern (DAMP) molecule high-mobility group box 1 (HMGB1) and its receptor for advanced glycation end products (RAGE). In vitro, RELMα/hresistin-induced macrophage-specific HMGB1/RAGE expression and facilitated HMGB1 nucleus-to-cytoplasm translocation and extracellular secretion. Mechanistically, hresistin promoted HMGB1 posttranslational lysine acetylation by preserving the NAD+-dependent deacetylase sirtuin (Sirt) 1 in human macrophages. Notably, the hresistin-stimulated macrophages promoted apoptosis-resistant proliferation of human pulmonary artery smooth muscle cells in an HMGB1/RAGE-dependent manner. In the mouse model, RELMα also suppressed the Sirt1 signal in pulmonary macrophages in the early posthypoxic period. Notably, recruited macrophages in the lungs of these mice carried the RELMα binding partner Bruton tyrosine kinase (BTK). hResistin also mediated the migration of human macrophages by activating BTK in vitro. Collectively, these data reveal a vascular-immune cellular interaction in the early PH stage and suggest that targeting RELMα/DAMP-driven macrophages may offer a promising strategy to treat PH and other related vascular inflammatory diseases.

Resistin family proteins in pulmonary diseases.

The family of resistin-like molecules (RELMs) consists of four members in rodents (RELMα/FIZZ1/HIMF, RELMß/FIZZ2, Resistin/FIZZ3, and RELMγ/FIZZ4) and two members in humans (Resistin and RELMß), all of which exhibit inflammation-regulating, chemokine, and growth factor properties. The importance of these cytokines in many aspects of physiology and pathophysiology, especially in cardiothoracic diseases, is rapidly evolving in the literature. In this review article, we attempt to summarize the contribution of RELM signaling to the initiation and progression of lung diseases, such as pulmonary hypertension, asthma/allergic airway inflammation, chronic obstructive pulmonary disease, fibrosis, cancers, infection, and other acute lung injuries. The potential of RELMs to be used as biomarkers or risk predictors of these diseases also will be discussed. Better understanding of RELM signaling in the pathogenesis of pulmonary diseases may offer novel targets or approaches for the development of therapeutics to treat or prevent a variety of inflammation, tissue remodeling, and fibrosis-related disorders in respiratory, cardiovascular, and other systems.

Early postnatal exposure to isoflurane causes cognitive deficits and disrupts development of newborn hippocampal neurons via activation of the mTOR pathway.

Clinical and preclinical studies indicate that early postnatal exposure to anesthetics can lead to lasting deficits in learning and other cognitive processes. The mechanism underlying this phenomenon has not been clarified and there is no treatment currently available. Recent evidence suggests that anesthetics might cause persistent deficits in cognitive function by disrupting key events in brain development. The hippocampus, a brain region that is critical for learning and memory, contains a large number of neurons that develop in the early postnatal period, which are thus vulnerable to perturbation by anesthetic exposure. Using an in vivo mouse model we demonstrate abnormal development of dendrite arbors and dendritic spines in newly generated dentate gyrus granule cell neurons of the hippocampus after a clinically relevant isoflurane anesthesia exposure conducted at an early postnatal age. Furthermore, we find that isoflurane causes a sustained increase in activity in the mechanistic target of rapamycin pathway, and that inhibition of this pathway with rapamycin not only reverses the observed changes in neuronal development, but also substantially improves performance on behavioral tasks of spatial learning and memory that are impaired by isoflurane exposure. We conclude that isoflurane disrupts the development of hippocampal neurons generated in the early postnatal period by activating a well-defined neurodevelopmental disease pathway and that this phenotype can be reversed by pharmacologic inhibition.

Neonatal Isoflurane Anesthesia or Disruption of Postsynaptic Density-95 Protein Interactions Change Dendritic Spine Densities and Cognitive Function in Juvenile Mice.

BACKGROUND: Experimental evidence shows postnatal exposure to anesthesia negatively affects brain development. The PDZ2 domain, mediating protein-protein interactions of the postsynaptic density-95 protein, serves as a molecular target for several inhaled anesthetics. The authors hypothesized that early postnatal disruption of postsynaptic density-95 PDZ2 domain interactions has persistent effects on dendritic spines and cognitive function. METHODS: One-week-old mice were exposed to 1.5% isoflurane for 4 h or injected with 8 mg/kg active postsynaptic density-95 wild-type PDZ2 peptide along with their respective controls. A subset of these mice also received 4 mg/kg of the nitric oxide donor molsidomine. Hippocampal spine density, long-term potentiation, novel object recognition memory, and fear learning and memory were evaluated in mice. RESULTS: Exposure of 7-day-old mice to isoflurane or postsynaptic density-95 wild-type PDZ2 peptide relative to controls causes: (1) a long-term decrease in mushroom spines at 7 weeks (mean ± SD [spines per micrometer]): control (0.8 ± 0.2) versus isoflurane (0.4 ± 0.2), P < 0.0001, and PDZ2MUT (0.7 ± 0.2) versus PDZ2WT (0.4 ± 0.2), P < 0.001; (2) deficits in object recognition at 6 weeks (mean ± SD [recognition index]): naïve (70 ± 8) versus isoflurane (55 ± 14), P = 0.010, and control (65 ± 13) versus isoflurane (55 ± 14), P = 0.045, and PDZ2MUT (64 ±11) versus PDZ2WT (53 ± 18), P = 0.045; and (3) deficits in fear learning at 7 weeks and memory at 8 weeks (mean ± SD [% freezing duration]): Learning, control (69 ± 12) versus isoflurane (52 ± 13), P < 0.0001, and PDZ2MUT (65 ± 14) versus PDZ2WT (55 ± 14) P = 0.011, and Memory, control (80 ± 17) versus isoflurane (56 ± 23), P < 0.0001 and PDZ2MUT (73 ± 18) versus PDZ2WT (44 ± 19) P < 0.0001. Impairment in long-term potentiation has fully recovered here at 7 weeks (mean ± SD [% baseline]): control (140 ± 3) versus isoflurane (137 ± 8), P = 0.560, and PDZ2MUT (136 ± 17) versus PDZ2WT (128 ± 11), P = 0.512. The isoflurane induced decrease in mushroom spines was preventable by introduction of a nitric oxide donor. CONCLUSIONS: Early disruption of PDZ2 domain-mediated protein-protein interactions mimics isoflurane in decreasing mushroom spine density and causing learning and memory deficits in mice. Prevention of the decrease in mushroom spine density with a nitric oxide donor supports a role for neuronal nitric oxide synthase pathway in mediating this cellular change associated with cognitive impairment.

HIMF (Hypoxia-Induced Mitogenic Factor) Signaling Mediates the HMGB1 (High Mobility Group Box 1)-Dependent Endothelial and Smooth Muscle Cell Crosstalk in Pulmonary Hypertension.

OBJECTIVE: HIMF (hypoxia-induced mitogenic factor; also known as FIZZ1 [found in inflammatory zone-1] or RELM [resistin-like molecule-α]) is an etiological factor of pulmonary hypertension (PH) in rodents, but its underlying mechanism is unclear. We investigated the immunomodulatory properties of HIMF signaling in PH pathogenesis. Approach and Results: Gene-modified mice that lacked HIMF (KO [knockout]) or overexpressed HIMF human homolog resistin (hResistin) were used for in vivo experiments. The pro-PH role of HIMF was verified in HIMF-KO mice exposed to chronic hypoxia or sugen/hypoxia. Mechanistically, HIMF/hResistin activation triggered the HMGB1 (high mobility group box 1) pathway and RAGE (receptor for advanced glycation end products) in pulmonary endothelial cells (ECs) of hypoxic mouse lungs in vivo and in human pulmonary microvascular ECs in vitro. Treatment with conditioned medium from hResistin-stimulated human pulmonary microvascular ECs induced an autophagic response, BMPR2 (bone morphogenetic protein receptor 2) defects, and subsequent apoptosis-resistant proliferation in human pulmonary artery (vascular) smooth muscle cells in an HMGB1-dependent manner. These effects were confirmed in ECs and smooth muscle cells isolated from pulmonary arteries of patients with idiopathic PH. HIMF/HMGB1/RAGE-mediated autophagy and BMPR2 impairment were also observed in pulmonary artery (vascular) smooth muscle cells of hypoxic mice, effects perhaps related to FoxO1 (forkhead box O1) dampening by HIMF. Experiments in EC-specific hResistin-overexpressing transgenic mice confirmed that EC-derived HMGB1 mediated the hResistin-driven pulmonary vascular remodeling and PH. CONCLUSIONS: In HIMF-induced PH, HMGB1-RAGE signaling is pivotal for mediating EC-smooth muscle cell crosstalk. The humanized mouse data further support clinical implications for the HIMF/HMGB1 signaling axis and indicate that hResistin and its downstream pathway may constitute targets for the development of novel anti-PH therapeutics in humans.

Nitric Oxide Donor Prevents Neonatal Isoflurane-induced Impairments in Synaptic Plasticity and Memory.

WHAT WE ALREADY KNOW ABOUT THIS TOPIC: Some general anesthetics have been shown to have adverse effects on neuronal development that affect neural function and cognitive behavior.Clinically relevant concentrations of inhalational anesthetics inhibit the postsynaptic density (PSD)-95, discs large homolog, and zona occludens-1 (PDZ) domain-mediated protein-protein interaction between PSD-95 or PSD-93 and N-methyl-D-aspartate receptors or neuronal NO synthase. WHAT THIS ARTICLE TELLS US THAT IS NEW: Neonatal PSD-95 PDZ2WT peptide treatment mimics the effects of isoflurane (~1 minimum alveolar concentration) by altering dendritic spine morphology, neural plasticity, and memory without inducing detectable increases in apoptosis or changes in synaptic density.These results indicate that a single dose of isoflurane (~1 minimum alveolar concentration) or PSD-95 PDZ2WT peptide alters dendritic spine architecture and functions important for cognition in the developing brain. This impairment can be prevented by administration of the NO donor molsidomine. BACKGROUND: In humans, multiple early exposures to procedures requiring anesthesia constitute a significant risk factor for development of learning disabilities and disorders of attention. In animal studies, newborns exposed to anesthetics develop long-term deficits in cognition. Previously, our laboratory showed that postsynaptic density (PSD)-95, discs large homolog, and zona occludens-1 (PDZ) domains may serve as a molecular target for inhaled anesthetics. This study investigated a role for PDZ interactions in spine development, plasticity, and memory as a potential mechanism for early anesthetic exposure-produced cognitive impairment. METHODS: Postnatal day 7 mice were exposed to 1.5% isoflurane for 4 h or injected with 8 mg/kg active PSD-95 PDZ2WT peptide. Apoptosis, hippocampal dendritic spine changes, synapse density, long-term potentiation, and cognition functions were evaluated (n = 4 to 18). RESULTS: Exposure of postnatal day 7 mice to isoflurane or PSD-95 PDZ2WT peptide causes a reduction in long thin spines (median, interquartile range [IQR]: wild type control [0.54, 0.52 to 0.86] vs. wild type isoflurane [0.31, 0.16 to 0.38], P = 0.034 and PDZ2MUT [0.86, 0.67 to 1.0] vs. PDZ2WT [0.55, 0.53 to 0.59], P = 0.028), impairment in long-term potentiation (median, IQR: wild type control [123, 119 to 147] and wild type isoflurane [101, 96 to 118], P = 0.049 and PDZ2MUT [125, 119 to 131] and PDZ2WT [104, 97 to 107], P = 0.029), and deficits in acute object recognition (median, IQR: wild type control [79, 72 to 88] vs. wild type isoflurane [63, 55 to 72], P = 0.044 and PDZ2MUT [81, 69 to 84] vs. PDZ2WT [67, 57 to 77], P = 0.039) at postnatal day 21 without inducing detectable differences in apoptosis or changes in synaptic density. Impairments in recognition memory and long-term potentiation were preventable by introduction of a NO donor. CONCLUSIONS: Early disruption of PDZ domain-mediated protein-protein interactions alters spine morphology, synaptic function, and memory. These results support a role for PDZ interactions in early anesthetic exposure-produced cognitive impairment. Prevention of recognition memory and long-term potentiation deficits with a NO donor supports a role for the N-methyl-D-aspartate receptor/PSD-95/neuronal NO synthase pathway in mediating these aspects of isoflurane-induced cognitive impairment.

Hypoxia-Inducible Factor 1α Is a Critical Downstream Mediator for Hypoxia-Induced Mitogenic Factor (FIZZ1/RELMα)-Induced Pulmonary Hypertension.

OBJECTIVE: Pulmonary hypertension (PH) is characterized by progressive elevation of pulmonary vascular resistance, right ventricular failure, and ultimately death. We have shown that in rodents, hypoxia-induced mitogenic factor (HIMF; also known as FIZZ1 or resistin-like molecule-ß) causes PH by initiating lung vascular inflammation. We hypothesized that hypoxia-inducible factor-1 (HIF-1) is a critical downstream signal mediator of HIMF during PH development. APPROACH AND RESULTS: In this study, we compared the degree of HIMF-induced pulmonary vascular remodeling and PH development in wild-type (HIF-1α(+/+)) and HIF-1α heterozygous null (HIF-1α(+/-)) mice. HIMF-induced PH was significantly diminished in HIF-1α(+/-) mice and was accompanied by a dysregulated vascular endothelial growth factor-A-vascular endothelial growth factor receptor 2 pathway. HIF-1α was critical for bone marrow-derived cell migration and vascular tube formation in response to HIMF. Furthermore, HIMF and its human homolog, resistin-like molecule-ß, significantly increased interleukin (IL)-6 in macrophages and lung resident cells through a mechanism dependent on HIF-1α and, at least to some extent, on nuclear factor κB. CONCLUSIONS: Our results suggest that HIF-1α is a critical downstream transcription factor for HIMF-induced pulmonary vascular remodeling and PH development. Importantly, both HIMF and human resistin-like molecule-ß significantly increased IL-6 in lung resident cells and increased perivascular accumulation of IL-6-expressing macrophages in the lungs of mice. These data suggest that HIMF can induce HIF-1, vascular endothelial growth factor-A, and interleukin-6, which are critical mediators of both hypoxic inflammation and PH pathophysiology.

Stress induces pain transition by potentiation of AMPA receptor phosphorylation.

Chronic postsurgical pain is a serious issue in clinical practice. After surgery, patients experience ongoing pain or become sensitive to incident, normally nonpainful stimulation. The intensity and duration of postsurgical pain vary. However, it is unclear how the transition from acute to chronic pain occurs. Here we showed that social defeat stress enhanced plantar incision-induced AMPA receptor GluA1 phosphorylation at the Ser831 site in the spinal cord and greatly prolonged plantar incision-induced pain. Interestingly, targeted mutation of the GluA1 phosphorylation site Ser831 significantly inhibited stress-induced prolongation of incisional pain. In addition, stress hormones enhanced GluA1 phosphorylation and AMPA receptor-mediated electrical activity in the spinal cord. Subthreshold stimulation induced spinal long-term potentiation in GluA1 phosphomimetic mutant mice, but not in wild-type mice. Therefore, spinal AMPA receptor phosphorylation contributes to the mechanisms underlying stress-induced pain transition.

Resistin-Like Molecule α in Allergen-Induced Pulmonary Vascular Remodeling.

Resistin-like molecule α (RELMα) has mitogenic, angiogenic, vasoconstrictive, and chemokine-like properties and is highly relevant in lung pathology. Here, we used RELMα knockout (Retnla(-/-)) mice to investigate the role of RELMα in pulmonary vascular remodeling after intermittent ovalbumin (OVA) challenge. We compared saline- and OVA-exposed wild-type (WT) mice and found that OVA induced significant increases in right ventricular systolic pressure, cardiac hypertrophy, pulmonary vascular remodeling of intra-alveolar arteries, goblet cell hyperplasia in airway epithelium, and intensive lung inflammation, especially perivascular inflammation. Genetic ablation of Retnla prevented the OVA-induced increase in pulmonary pressure and cardiac hypertrophy seen in WT mice. Histological analysis showed that Retnla(-/-) mice exhibited less vessel muscularization, less perivascular inflammation, reduced medial thickness of intra-alveolar vessels, and fewer goblet cells in upper airway epithelium (250-600 µm) than did WT animals after OVA challenge. Gene expression profiles showed that genes associated with vascular remodeling, including those related to muscle protein, contractile fibers, and actin cytoskeleton, were expressed at a lower level in OVA-challenged Retnla(-/-) mice than in similarly treated WT mice. In addition, bronchoalveolar lavage from OVA-challenged Retnla(-/-) mice had lower levels of cytokines, such as IL-1ß, -1 receptor antagonist, and -16, chemokine (C-X-C motif) ligand 1, -2, -9, -10, and -13, monocyte chemoattractant protein-1, macrophage colony-stimulating factor, TIMP metallopeptidase inhibitor-1, and triggering receptor expressed on myeloid cells-1, than did that from WT mice when analyzed by cytokine array dot blots. Retnla knockout inhibited the OVA-induced T helper 17 response but not the T helper 2 response. Altogether, our results suggest that RELMα is involved in immune response-induced pulmonary vascular remodeling and the associated increase in inflammation typically observed after OVA challenge.

Inhalational anesthetics disrupt postsynaptic density protein-95, Drosophila disc large tumor suppressor, and zonula occludens-1 domain protein interactions critical to action of several excitatory receptor channels related to anesthesia.

BACKGROUND: The authors have shown previously that inhaled anesthetics disrupt the interaction between the second postsynaptic density protein-95, Drosophila disc large tumor suppressor, and zonula occludens-1 (PDZ) domain of postsynaptic density protein-95 (PSD-95) and the C-terminus of N-methyl-D-aspartate receptor subunits NR2A and NR2B. The study data indicate that PDZ domains may serve as a molecular target for inhaled anesthetics. However, the underlying molecular mechanisms remain to be illustrated. METHODS: Glutathione S-transferase pull-down assay, coimmunoprecipitation, and yeast two-hybrid analysis were used to assess PDZ domain-mediated protein-protein interactions in different conditions. Nuclear magnetic resonance spectroscopy was used to investigate isoflurane-induced chemical shift changes in the PDZ1-3 domains of PSD-95. A surface plasmon resonance-based BIAcore (Sweden) assay was used to examine the ability of isoflurane to inhibit the PDZ domain-mediated protein-protein interactions in real time. RESULTS: Halothane and isoflurane dose-dependently inhibited PDZ domain-mediated interactions between PSD-95 and Shaker-type potassium channel Kv1.4 and between α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunit GluA2 and its interacting proteins-glutamate receptor-interacting protein or protein interacting with c kinase 1. However, halothane and isoflurane had no effect on PDZ domain-mediated interactions between γ-aminobutyric acid type B receptor and its interacting proteins. The inhaled anesthetic isoflurane mostly affected the residues close to or in the peptide-binding groove of PSD-95 PDZ1 and PDZ2 (especially PDZ2), while barely affecting the peptide-binding groove of PSD-95 PDZ3. CONCLUSION: These results suggest that inhaled anesthetics interfere with PDZ domain-mediated protein-protein interactions at several receptors important to neuronal excitation, anesthesia, and pain processing.

Hypoxia-induced mitogenic factor (FIZZ1/RELMα) induces endothelial cell apoptosis and subsequent interleukin-4-dependent pulmonary hypertension.

Pulmonary hypertension (PH) is characterized by elevated pulmonary artery pressure that leads to progressive right heart failure and ultimately death. Injury to endothelium and consequent wound repair cascades have been suggested to trigger pulmonary vascular remodeling, such as that observed during PH. The relationship between injury to endothelium and disease pathogenesis in this disorder remains poorly understood. We and others have shown that, in mice, hypoxia-induced mitogenic factor (HIMF, also known as FIZZ1 or RELMα) plays a critical role in the pathogenesis of lung inflammation and the development of PH. In this study, we dissected the mechanism by which HIMF and its human homolog resistin (hRETN) induce pulmonary endothelial cell (EC) apoptosis and subsequent lung inflammation-mediated PH, which exhibits many of the hallmarks of the human disease. Systemic administration of HIMF caused increases in EC apoptosis and interleukin (IL)-4-dependent vascular inflammatory marker expression in mouse lung during the early inflammation phase. In vitro, HIMF, hRETN, and IL-4 activated pulmonary microvascular ECs (PMVECs) by increasing angiopoietin-2 expression and induced PMVEC apoptosis. In addition, the conditioned medium from hRETN-treated ECs had elevated levels of endothelin-1 and caused significant increases in pulmonary vascular smooth muscle cell proliferation. Last, HIMF treatment caused development of PH that was characterized by pulmonary vascular remodeling and right heart failure in wild-type mice but not in IL-4 knockout mice. These data suggest that HIMF contributes to activation of vascular inflammation at least in part by inducing EC apoptosis in the lung. These events lead to subsequent PH.

Role of neuregulin-1/ErbB signaling in stem cell therapy for spinal cord injury-induced chronic neuropathic pain.

Chronic neuropathic pain is a common and debilitating consequence of spinal cord injury (SCI). In a rat contusion injury model, we observed that chronic neuropathic pain is present on day 7 after SCI and persists for the entire 56-day observation period. However, currently available pain therapies are inadequate for SCI-induced neuropathic pain. In this study, we show that spinal transplantation of mouse embryonic stem cell-derived oligodendrocyte progenitor cells (OPCs) enhances remyelination in the injured spinal cord and reduces SCI-induced chronic neuropathic pain. Moreover, we found that SCI reduces the protein level of neuregulin-1 and ErbB4 in the injured spinal cord and that OPC transplantation enhances the spinal expression of both proteins after SCI. Finally, intrathecal injection of neuregulin-1 small interfering RNA, but not the control nontarget RNA, diminishes OPC transplantation-produced remyelination and reverses the antinociceptive effect of OPC transplantation. Our findings suggest that the transplantation of embryonic stem cell-derived OPCs is an appropriate therapeutic intervention for treatment of SCI-induced chronic neuropathic pain, and that neuregulin-1/ErbB signaling plays an important role in central remyelination under pathological conditions and contributes to the alleviation of such pain.

Resistin Pathway as Novel Mechanism of Post-lung Transplantation Bronchial Stenosis.

BACKGROUND: Bronchial stenosis remains a significant source of morbidity among lung transplant recipients. Though infection and anastomotic ischemia have been proposed etiologies of the development of bronchial stenosis, the pathophysiologic mechanism has not been well elucidated. METHODS: In this single-centered prospective study, from January 2013 through September 2015, we prospectively collected bronchoalveolar lavage (BAL) and endobronchial epithelial brushings from the direct anastomotic site of bronchial stenosis of bilateral lung transplant recipients who developed unilateral post-transplant bronchial stenosis. Endobronchial epithelial brushings from the contralateral anastomotic site without bronchial stenosis and BAL from bilateral lung transplant recipients who did not develop post-transplant bronchial stenosis were used as controls. Total RNA was isolated from the endobronchial brushings and real-time polymerase chain reaction reactions were performed. Electrochemiluminescence biomarker assay was used to measure 10 cytokines from the BAL. RESULTS: Out of 60 bilateral lung transplant recipients, 9 were found to have developed bronchial stenosis with 17 samples adequate for analysis. We observed a 1.56 to 70.8 mean-fold increase in human resistin gene expression in the anastomotic bronchial stenosis epithelial cells compared with nonstenotic airways. Furthermore, IL-1ß (21.76±10.96 pg/mL; control 0.86±0.44 pg/mL; P <0.01) and IL-8 levels (990.56±326.60 pg/mL; control 20.33±1.17 pg/mL; P <0.01) were significantly elevated in the BAL of the lung transplant patients who developed anastomotic bronchial stenosis. CONCLUSION: Our data suggest that the development of postlung transplantation bronchial stenosis may be in part mediated through the human resistin pathway by IL-1ß induced transcription factor nuclear factor-κß activation and downstream upregulation of IL-8 in alveolar macrophages. Further study is needed in the larger patient cohorts and to determine its potential therapeutic role in the management of post-transplant bronchial stenosis.

Choosing the right antibody for resistin-like molecule (RELM/FIZZ) family members.

The family of resistin-like molecules (RELM), also known as found in inflammatory zone (FIZZ), consists of four members in mouse (RELMα/FIZZ1/HIMF, RELMß/FIZZ2, Resistin/FIZZ3, and RELMγ/FIZZ4) and two members in human (resistin and RELMß). The importance of these proteins in many aspects of physiology and pathophysiology, especially inflammatory processes, is rapidly evolving in the literature, and many investigators are beginning to work in this field. Most published studies focus on only one isoform, do not evaluate other isoforms that might be present, and have not tested for the specificity of the antibody used. Because RELM isoforms have high sequence and structural similarity and both distinct and overlapping functions, it is important to use a specific antibody to distinguish each isoform in the study. We constructed and established HEK 293 cell lines that constitutively express each isoform. Using these cell lines, we determined the specificity of antibodies (both commercially available and laboratory-made) to each isoform by Western blot and immunofluorescence. Some of the antibodies showed specificity in Western blotting but were not applicable in immunofluorescence. Others showed cross reactivity in Western blot assays. Our results indicate that RELM antibody specificity should be taken into account when using them in research and interpreting data obtained with them.

Hypoxia-induced mitogenic factor (HIMF/FIZZ1/RELMα) in chronic hypoxia- and antigen-mediated pulmonary vascular remodeling.

BACKGROUND: Both chronic hypoxia and allergic inflammation induce vascular remodeling in the lung, but only chronic hypoxia appears to cause PH. We investigate the nature of the vascular remodeling and the expression and role of hypoxia-induced mitogenic factor (HIMF/FIZZ1/RELMα) in explaining this differential response. METHODS: We induced pulmonary vascular remodeling through either chronic hypoxia or antigen sensitization and challenge. Mice were evaluated for markers of PH and pulmonary vascular remodeling throughout the lung vascular bed as well as HIMF expression and genomic analysis of whole lung. RESULTS: Chronic hypoxia increased both mean pulmonary artery pressure (mPAP) and right ventricular (RV) hypertrophy; these changes were associated with increased muscularization and thickening of small pulmonary vessels throughout the lung vascular bed. Allergic inflammation, by contrast, had minimal effect on mPAP and produced no RV hypertrophy. Only peribronchial vessels were significantly thickened, and vessels within the lung periphery did not become muscularized. Genomic analysis revealed that HIMF was the most consistently upregulated gene in the lungs following both chronic hypoxia and antigen challenge. HIMF was upregulated in the airway epithelial and inflammatory cells in both models, but only chronic hypoxia induced HIMF upregulation in vascular tissue. CONCLUSIONS: The results show that pulmonary vascular remodeling in mice induced by chronic hypoxia or antigen challenge is associated with marked increases in HIMF expression. The lack of HIMF expression in the vasculature of the lung and no vascular remodeling in the peripheral resistance vessels of the lung is likely to account for the failure to develop PH in the allergic inflammation model.

S100A11 mediates hypoxia-induced mitogenic factor (HIMF)-induced smooth muscle cell migration, vesicular exocytosis, and nuclear activation.

Hypoxia-induced mitogenic factor (HIMF) is a newly discovered protein that is up-regulated in murine models of pulmonary arterial hypertension and asthma. Our previous study shows that HIMF is a potent mitogenic, angiogenic, and vasoconstrictive chemokine associated with pulmonary arterial hypertension. Two-dimensional gel electrophoresis was used to investigate downstream molecules in HIMF-induced cell signaling, demonstrating that S100A11, an EF-hand calcium-binding protein, was exclusively altered and was decreased (2.7±0.2-fold, p<0.05) in pulmonary artery smooth muscle cells (SMCs) treated with HIMF for 5 min compared with untreated cells (n=4). Immunofluorescence showed that in control cells S100A11 is a cytosolic protein, which then aggregates and translocates both to the plasma membrane with subsequent exocytosis and to the nucleus upon HIMF stimulation. Annexin A2, a known S100A11 binding partner, also colocalized with S100A11 during HIMF-induced membrane trafficking. To investigate the intracellular function of S100A11, siRNA was used to knock down S100A11 expression in SMCs. The S100A11 knockdown significantly reduced HIMF-induced SMC migration but did not affect the SMC mitogenic action of HIMF. Our data show that S100A11 mediates HIMF-induced smooth muscle cell migration, vesicular exocytosis, and nuclear activation.

Human Resistin Induces Cardiac Dysfunction in Pulmonary Hypertension.

Background Cardiac failure is the primary cause of death in most patients with pulmonary arterial hypertension (PH). As pleiotropic cytokines, human resistin (Hresistin) and its rodent homolog, resistin-like molecule α, are mechanistically critical to pulmonary vascular remodeling in PH. However, it is still unclear whether activation of these resistin-like molecules can directly cause PH-associated cardiac dysfunction and remodeling. Methods and Results In this study, we detected Hresistin protein in right ventricular (RV) tissue of patients with PH and elevated resistin-like molecule expression in RV tissues of rodents with RV hypertrophy and failure. In a humanized mouse model, cardiac-specific Hresistin overexpression was sufficient to cause cardiac dysfunction and remodeling. Dilated hearts exhibited reduced force development and decreased intracellular Ca2+ transients. In the RV tissues overexpressing Hresistin, the impaired contractility was associated with the suppression of protein kinase A and AMP-activated protein kinase. Mechanistically, Hresistin activation triggered the inflammation mediated by signaling of the key damage-associated molecular pattern molecule high-mobility group box 1, and subsequently induced pro-proliferative Ki67 in RV tissues of the transgenic mice. Intriguingly, an anti-Hresistin human antibody that we generated protected the myocardium from hypertrophy and failure in the rodent PH models. Conclusions Our data indicate that Hresistin is expressed in heart tissues and plays a role in the development of RV dysfunction and maladaptive remodeling through its immunoregulatory activities. Targeting this signaling to modulate cardiac inflammation may offer a promising strategy to treat PH-associated RV hypertrophy and failure in humans.