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.

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.

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.

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.

Caveolin-1 knockout mice exhibit airway hyperreactivity.

Caveolae are flask-shaped plasma membrane invaginations expressing the scaffolding caveolin proteins. Although caveolins have been found in endothelium and epithelium (where they regulate nitric oxide synthase activity), their role in smooth muscle is still under investigation. We and others have previously shown that caveolae of human airway smooth muscle (ASM), which express caveolin-1, contain Ca(2+) and force regulatory proteins and are involved in mediating the effects of inflammatory cytokines such as TNF-α on intracellular Ca(2+) concentration responses to agonist. Accordingly, we tested the hypothesis that in vivo, absence of caveolin-1 leads to reduced airway hyperresponsiveness, using a knockout (KO) (Cav1 KO) mouse and an ovalbumin-sensitized/challenged (OVA) model of allergic airway hyperresponsiveness. Surprisingly, airway responsiveness to methacholine, tested by use of a FlexiVent system, was increased in Cav1 KO control (CTL) as well as KO OVA mice, which could not be explained by a blunted immune response to OVA. In ASM of wild-type (WT) OVA mice, expression of caveolin-1, the caveolar adapter proteins cavins 1-3, and caveolae-associated Ca(2+) and force regulatory proteins such as Orai1 and RhoA were all increased, effects absent in Cav1 KO CTL and OVA mice. However, as with WT OVA, both CTL and OVA Cav1 KO airways showed signs of enhanced remodeling, with high expression of proliferation markers and increased collagen. Separately, epithelial cells from airways of all three groups displayed lower endothelial but higher inducible nitric oxide synthase and arginase expression. Arginase activity was also increased in these three groups, and the inhibitor nor-NOHA (N-omega-nor-l-arginine) enhanced sensitivity of isolated tracheal rings to ACh, especially in Cav1 KO mice. On the basis of these data disproving our original hypothesis, we conclude that caveolin-1 has complex effects on ASM vs. epithelium, resulting in airway hyperreactivity in vivo mediated by altered airway remodeling and bronchodilation.

Estrogen increases nitric-oxide production in human bronchial epithelium.

Although sex differences in asthma severity are recognized, the mechanisms by which sex steroids such as estrogen influence the airway are still under investigation. Airway tone, a key aspect of asthma, represents a balance between bronchoconstriction and dilation. Nitric oxide (NO) from the bronchial epithelium is an endogenous bronchodilator. We hypothesized that estrogens facilitate bronchodilation by generating NO in bronchial epithelium. In acutely dissociated human bronchial epithelial cells from female patients exposure to 17ß-estradiol (E(2); 10 pM-100 nM) resulted in rapid increase of diaminofluorescein fluorescence (NO indicator) within minutes, comparable with that induced by ATP (20 µM). Estrogen receptor (ER) isoform-specific agonists (R,R)-5,11-diethyl-5,6,11,12-tetrahydro-2,8-chrysenediol (THC) (ERα) and diaryl-propionitrile (DPN) (ERß) stimulated NO production to comparable levels and at comparable rates, whereas the ER antagonist 7α,17ß-[9-[(4,4,5,5,5-pentafluoropentyl)sulfinyl]nonyl]estra-1,3,5(10)-triene-3,17-diol (ICI 182,780) (1 µM) was inhibitory. Estrogen effects on NO were mediated via caveolin-1 (blocked using the caveolin-1 scaffolding domain peptide) and by increased intracellular calcium concentration [prevented by 20 µM 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester but not by blocking Ca(2+) influx using LaCl(3)]. Estrogen increased endothelial NO synthase activation (inhibited by 100 µM N(G)-nitro-l-arginine methyl ester) and phosphorylated Akt. In epithelium-intact human bronchial rings contracted with acetylcholine (1 µM), E(2), THC, and DPN all produced acute bronchodilation in a dose-dependent fashion. Such bronchodilatory effects were substantially reduced by epithelial denudation. Overall, these data indicate that estrogens, acting via ERα or ERß, can acutely produce NO in airway epithelium (akin to vascular endothelium). Estrogen-induced NO and its impairment may contribute to altered bronchodilation in women with asthma.

Neurotrophins induce nitric oxide generation in human pulmonary artery endothelial cells.

AIMS: Members of the growth factor family of neurotrophins [NTs; e.g. brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3)] and their high-affinity receptors (tropomyosin-related kinase; Trk) and low-affinity receptors p75 neurotrophin receptor (p75NTR) have been localized to pulmonary artery (PA) in humans. However, their role is unclear. Based on previous findings of NTs and their receptors within the pulmonary endothelium, we tested the hypothesis that NTs induce nitric oxide (NO) production in pulmonary endothelial cells (ECs), thus contributing to vasodilation. METHODS AND RESULTS: In human pulmonary artery ECs loaded with the NO-sensitive fluorescent dye diaminofluorescein-2, both BDNF and NT3 (100 pM, 1 nM, and 10 nM) acutely (<10 min) and substantially increased fluorescence levels in a concentration-dependent fashion (to levels comparable to that induced by 1 µM acetylcholine). NT-induced elevation of NO levels was blunted by the tyrosine kinase inhibitor K252a, the nitric oxide synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester, the Ca(2+) chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, and the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. Suppression of TrkB or TrkC expression via siRNA as well as functional blockade of p75NTR prevented NT-induced NO elevation. Both BDNF and NT3 increased phosphorylation of Akt and endothelial NO synthase (eNOS). In endothelium-intact porcine PA rings, NTs increased cGMP and induced vasodilation in pre-contracted arteries. CONCLUSION: These results indicate that NTs acutely modulate pulmonary endothelial NO production and contribute to relaxation of the pulmonary vasculature.

Neurokinin-neurotrophin interactions in airway smooth muscle.

Neurally derived tachykinins such as substance P (SP) play a key role in modulating airway contractility (especially with inflammation). Separately, the neurotrophin brain-derived neurotrophic factor (BDNF; potentially derived from nerves as well as airway smooth muscle; ASM) and its tropomyosin-related kinase receptor, TrkB, are involved in enhanced airway contractility. In this study, we hypothesized that neurokinins and neurotrophins are linked in enhancing intracellular Ca(2+) concentration ([Ca(2+)](i)) regulation in ASM. In rat ASM cells, 24 h exposure to 10 nM SP significantly increased BDNF and TrkB expression (P < 0.05). Furthermore, [Ca(2+)](i) responses to 1 µM ACh as well as BDNF (30 min) effects on [Ca(2+)](i) regulation were enhanced by prior SP exposure, largely via increased Ca(2+) influx (P < 0.05). The enhancing effect of SP on BDNF signaling was blunted by the neurokinin-2 receptor antagonist MEN-10376 (1 µM, P < 0.05) to a greater extent than the neurokinin-1 receptor antagonist RP-67580 (5 nM). Chelation of extracellular BDNF (chimeric TrkB-F(c); 1 µg/ml), as well as tyrosine kinase inhibition (100 nM K252a), substantially blunted SP effects (P < 0.05). Overnight (24 h) exposure of ASM cells to 50% oxygen increased BDNF and TrkB expression and potentiated both SP- and BDNF-induced enhancement of [Ca(2+)](i) (P < 0.05). These results suggest a novel interaction between SP and BDNF in regulating agonist-induced [Ca(2+)](i) regulation in ASM. The autocrine mechanism we present here represents a new area in the development of bronchoconstrictive reflex response and airway hyperreactive disorders.

Caveolin-1 and force regulation in porcine airway smooth muscle.

Caveolae are specialized membrane microdomains expressing the scaffolding protein caveolin-1. We recently demonstrated the presence of caveolae in human airway smooth muscle (ASM) and the contribution of caveolin-1 to intracellular calcium ([Ca(2+)](i)) regulation. In the present study, we tested the hypothesis that caveolin-1 regulates ASM contractility. We examined the role of caveolins in force regulation of porcine ASM under control conditions as well as TNF-α-induced airway inflammation. In porcine ASM strips, exposure to 10 mM methyl-ß-cyclodextrin (CD) or 5 µM of the caveolin-1 specific scaffolding domain inhibitor peptide (CSD) resulted in time-dependent decrease in force responses to 1 µM ACh. Overnight exposure to the cytokine TNF-α (50 ng/ml) accelerated and increased caveolin-1 expression and enhanced force responses to ACh. Suppression of caveolin-1 with small interfering RNA mimicked the effects of CD or CSD. Regarding mechanisms by which caveolae contribute to contractile changes, inhibition of MAP kinase with 10 µM PD98059 did not alter control or TNF-α-induced increases in force responses to ACh. However, inhibiting RhoA with 100 µM fasudil or 10 µM Y27632 resulted in significant decreases in force responses, with lesser effects in TNF-α exposed samples. Furthermore, Ca(2+) sensitivity for force generation was substantially reduced by fasudil or Y27632, an effect even more enhanced in the absence of caveolin-1 signaling. Overall, these results indicate that caveolin-1 is a critical player in enhanced ASM contractility with airway inflammation.

Cutaneous sympathetic neural responses to body cooling in type 2 diabetes mellitus.

In humans, sympathetic vasoconstrictor nerves in the skin contribute to resting vascular tone and mediate reflex vasoconstrictor responses to body cooling. Although it is well recognized that type 2 diabetes mellitus (T2DM) is associated with peripheral neurovascular changes, it is unclear to what extent the thermal responsiveness of the cutaneous vasoconstrictor system is altered in individuals with relatively uncomplicated T2DM. We tested the hypothesis that skin sympathetic nerve activity (SSNA) is decreased at baseline and during body cooling in individuals with T2DM compared to healthy controls (C) of similar age and body size. We measured SSNA (microneurography) and skin blood flow (laser-Doppler flowmetry) in the innervated area in 8 T2DM and 12 C subjects at baseline and during 3-4min of rapid whole body cooling via a water-perfused suit. SSNA (total integrated activity) increased, and cutaneous vascular conductance decreased in both groups during body cooling (P<0.01 for both). However, SSNA was not different between groups during either baseline or body cooling conditions (P=NS). The deltas in SSNA between baseline and body cooling were similar between groups: T2DM: 55±27 and C: 57±12 units (P=NS). We conclude that reflex cutaneous sympathetic and vascular responses to rapid whole body cooling are preserved in relatively healthy individuals with T2DM.