Mouse Pulmonary Arteries Research Articles

Abstract P258: Effect of MicroRNA-223 on Hypoxic Pulmonary Hypertension

Chronic hypoxia plays a key role in pulmonary hypertension and remodeling by inducing marked changes in gene expression. However, the regulatory effect of chronic hypoxia on microRNA (miR) expression in the lung has not yet been investigated. The purpose of this study was to determine which miRs are regulated by chronic hypoxia in the mouse lung and to determine their functional significance. We identified miRs altered in mouse lung after 3 weeks chronic hypoxia (10% O 2 (10 in 100) ) treatment by Microarray screening, verified miR target genes by reporter gene assays and Western blotting. Furthermore, we analyzed in vitro and in vivo functional significance for mouse pulmonary vascular artery smooth muscle cells. Among the miRs regulated, miR-223 was the most significantly decreased, a phenomenon verified by RT-qPCR. The insulin-like growth factor 1 receptor (IGF1R) is a known target of miR-223 and the binding of miR-223 to the 3’UTR of IGF1R was demonstrated. We found that IGF1R protein levels (but not RNA) were increased in lungs from hypoxic mice via a mechanism involving HIF1α/2α and reduced C/EBPα expression and transcription activity. Moreover, the expression of IGF1R was decreased following introduction of pre-mir-223 into pulmonary artery smooth muscle cells, an phenomenon coupled to attenuated IGF1-induced Akt phosphorylation, cell migration and proliferation. Decreasing endogenous miR-223 levels using a specific antagomir (3 weeks), increased pulmonary artery pressure and vessel muscularization. A similar phenomenon was observed in miR-223 -/y mice, which also demonstrated a more severe response to hypoxia than wild-type mice. In lungs from primary pulmonary hypertension (PPH) patients we also detected a decrease in miR-223 and increase in IGF1R expression. These data indicate that the downregulation of miR-223 may contribute to the IGF1-induced pulmonary smooth muscle migration by regulating the expression of its receptor and thus contributing to pulmonary remodeling and the development of pulmonary hypertension.

S101 Heterozygous loss of BMPR-II predisposes to inflammatory cytokine secretion and pulmonary vascular smooth muscle proliferation

IntroductionElevated levels of inflammatory cytokines are associated with idiopathic and heritable pulmonary hypertension (PAH) and predict mortality, but their role in the pathobiology of PAH and the underlying mechanisms remain...

Retracted: Mice lacking the Raf‐1 kinase inhibitor protein exhibit exaggerated hypoxia‐induced pulmonary hypertension

Increased pulmonary vascular remodelling, pulmonary arterial pressure and pulmonary vascular resistance characterize the development of pulmonary arterial hypertension (PAH). Activation of the Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK)1/2 is thought to play an important role in PAH and Raf-1 kinase inhibitor protein (RKIP), negatively regulates this pathway. This study investigated whether genetic deletion of RKIP (and hence ERK1/2 up-regulation) resulted in a pulmonary hypertensive phenotype in mice and investigated a role for RKIP in mitogen-regulated proliferative responses in lung fibroblasts. Pulmonary vascular haemodynamics and remodelling were assessed in mice genetically deficient in RKIP (RKIP-/-) after 2 weeks of either normoxia or hypoxia. Immunoblotting and immunohistochemistry were used to examine phosphorylation of Raf-1, RKIP and ERK1/2 in mouse pulmonary arteries. In vitro, RKIP inhibition of mitogen signalling was analysed in CCL39 hamster lung fibroblasts. RKIP-/- mice demonstrated elevated indices of PAH and ERK1/2 phosphorylation compared with wild-type (WT) mice. Hypoxic RKIP-/- mice exhibited exaggerated PAH indices. Hypoxia increased phosphorylation of Raf-1, RKIP and ERK1/2 in WT mouse pulmonary arteries and Raf-1 phosphorylation in RKIP-/- mouse pulmonary arteries. In CCL39 cells, inhibition of RKIP potentiated mitogen-induced proliferation and phosphorylation of RKIP, and Raf-1. The lack of RKIP protein resulted in a pulmonary hypertensive phenotype, exaggerated in hypoxia. Hypoxia induced phosphorylation of RKIP signalling elements in WT pulmonary arteries. RKIP inhibition potentiated mitogen-induced proliferation in lung fibroblasts. These results provide evidence for the involvement of RKIP in suppressing the development of hypoxia-induced PAH in mice.

Activation Dynamics and Signaling Properties of Notch3 Receptor in the Developing Pulmonary Artery

Notch3 signaling is fundamental for arterial specification of systemic vascular smooth muscle cells (VSMCs). However, the developmental role and signaling properties of the Notch3 receptor in the mouse pulmonary artery remain unknown. Here, we demonstrate that Notch3 is expressed selectively in pulmonary artery VSMCs, is activated from late fetal to early postnatal life, and is required to maintain the morphological characteristics and smooth muscle gene expression profile of the pulmonary artery after birth. Using a conditional knock-out mouse model, we show that Notch3 receptor activation in VSMCs is Jagged1-dependent. In vitro VSMC lentivirus-mediated Jagged1 knockdown, confocal localization analysis, and co-culture experiments revealed that Notch3 activation is cell-autonomous and occurs through the physical engagement of Notch3 and VSMC-derived Jagged1 in the interior of the same cell. Although the current models of mammalian Notch signaling involve a two-cell system composed of a signal-receiving cell that expresses a Notch receptor on its surface and a neighboring signal-sending cell that provides membrane-bound activating ligand, our data suggest that pulmonary artery VSMC Notch3 activation is cell-autonomous. This unique mechanism of Notch activation may play an important role in the maturation of the pulmonary artery during the transition to air breathing.

Endothelin-1 contributes to increased NFATc3 activation by chronic hypoxia in pulmonary arteries

Chronic hypoxia (CH) activates the Ca(2+)-dependent transcription factor nuclear factor of activated T cells isoform c3 (NFATc3) in mouse pulmonary arteries. However, the mechanism of this response has not been explored. Since we have demonstrated that NFATc3 is required for CH-induced pulmonary arterial remodeling, establishing how CH activates NFATc3 is physiologically significant. The goal of this study was to test the hypothesis that endothelin-1 (ET-1) contributes to CH-induced NFATc3 activation. We propose that this mechanism requires increased pulmonary arterial smooth muscle cell (PASMC) intracellular Ca(2+) concentration ([Ca(2+)](i)) and stimulation of RhoA/Rho kinase (ROK), leading to calcineurin activation and actin cytoskeleton polymerization, respectively. We found that: 1) CH increases pulmonary arterial pre-pro-ET-1 mRNA expression and lung RhoA activity; 2) inhibition of ET receptors, calcineurin, L-type Ca(2+) channels, and ROK blunts CH-induced NFATc3 activation in isolated intrapulmonary arteries from NFAT-luciferase reporter mice; and 3) both ET-1-induced NFATc3 activation in isolated mouse pulmonary arteries ex vivo and ET-1-induced NFATc3-green fluorescence protein nuclear import in human PASMC depend on ROK and actin polymerization. This study suggests that CH increases ET-1 expression, thereby elevating PASMC [Ca(2+)](i) and RhoA/ROK activity. As previously demonstrated, elevated [Ca(2+)](i) is required to activate calcineurin, which dephosphorylates NFATc3, allowing its nuclear import. Here, we demonstrate that ROK increases actin polymerization, thus providing structural support for NFATc3 nuclear transport.

Guest Editorial — Novel Insights into the Pathology of Stachybotrys chartarum

The adverse health effects of indoor mold exposure gained much public attention in recent years. Although many studies support the involvement of fungi in building-related illnesses and emphasize the importance of mold exposure as an emerging public health issue, the role of toxigenic species such as Stachybotrys chartarum (SC) remains highly controversial [1]. Widespread litigation is making it more contentious, and the existing scientific information does not offer sufficient evidence that could lead to a better understanding of the problem and development of comprehensive public health policies. The article by Nagayoshi et al. [2] published in this issue of Mycopathologia describing for the first time the remodeling of pulmonary arteries in mice as a result of inhalation exposure to the spores of S. chartarum significantly extends our knowledge of the pathologic effects of this fungus. The ability to produce potent trichothecene mycotoxins became a source of great concern about S. chartarum. Approximately one-third of the isolates from the United States and Europe represent a chemotype that synthesizes macrocyclic trichothecenes [3]. Macrocyclic trichothecenes are not found in SC isolates belonging to the second chemotype which produces atranones. However, both chemotypes can synthesize much less potent simple trichothecenes. Molds can affect human health through mycotoxicosis, infection, allergy, and inflammation. The first category is often disputed, especially in the context of residential indoor air exposure [1, 4]. Arguments against the role of S. chartarum in building-related diseases are mostly based on the projection that the relatively low concentrations of the SC spores found in most indoor environments are insufficient to cause severe systemic toxicoses. Such opinions do not take into account local toxic effects at the site of deposition of fungal particles where the toxins can reach millimolar concentrations. Reports focusing on mycotoxins tend to present the ‘‘toxic black mold,’’ as S. chartarum is known to the public, as a species with unique properties compared with other fungi. While its high toxigenic potential clearly sets it apart, the effects of different fungal compounds common to all fungi such as b-D-glucan, proteins including hemolysin and proteases or various low-molecular-weight metabolites are likely to play a significant role in pathophysiology of this species [5–9]. In recent years, a number of animal studies of pulmonary effects of S. chartarum have been published, utilizing different animals/strains, different fungal isolates, and experimental protocols. The common finding was lung injury and inflammation. Some of those effects were paralleled in the experiments conducted with pure trichothecene toxins or atranones but toxin-independent mechanisms were also postulated [1, 10, 11]. It is I. Yike (&) D. Dearborn Department of Environmental Health Sciences, Case Western Reserve University, School of Medicine, Location Code 4940, Room WG19, 2109 Adelbert Rd., Cleveland, OH 44106, USA e-mail: ixy@case.edu

Role of G i/o-Src kinase-PI3K/Akt pathway and caveolin-1 in β 2-adrenoceptor coupling to endothelial NO synthase in mouse pulmonary artery

Activation of the β 2-adrenoceptor (β 2-AR) elicits an endothelial nitric oxide synthase (eNOS)-dependent relaxation in mouse pulmonary artery, which, contrary to the muscarinic receptor-dependent relaxation, is preserved in hypoxic pulmonary arterial hypertension. We therefore characterized the signaling pathways underlying the β 2-AR-mediated eNOS activation, with special focus on G i/o proteins, protein kinases and caveolae. Functional studies (for evaluation of vasorelaxant response), Western blotting (for assessment of eNOS and caveolin-1 phosphorylation) and transmission electron microscopy (for visualization of caveolae) were conducted in pulmonary arteries from wild-type or caveolin-1 knockout mice. In wild-type isolated arteries, relaxation to the selective β 2-AR agonist procaterol was reduced by inhibitors of G i/o proteins (pertussis toxin, PTX), phosphatidylinositol 3-kinase (PI3K; wortmannin or LY 294002), Akt (Akt inhibitor X) and Src-kinase (PP2) and by cholesterol depletion (using methyl-β-cyclodextrin). Procaterol induced eNOS phosphorylation at Ser 1177, which was prevented by PTX, PP2 or Akt inhibitor. Procaterol also promoted caveolin-1 phosphorylation at Tyr 14, which was decreased by PTX or PP2. Caveolin-1 gene deletion resulted in endothelial caveolae disruption in mouse pulmonary artery and in potentiation of procaterol-induced relaxation. Unlike procaterol, acetylcholine-induced relaxation was unaffected by PTX, methyl-β-cyclodextrin or caveolin-1 gene deletion. To conclude, the mouse pulmonary endothelial β 2-AR is coupled to a G i/o-Src kinase-PI3K/Akt pathway to promote eNOS phosphorylation at Ser 1177 leading to a NO-dependent vasorelaxation. Caveolin-1 exerts a negative control on this response that is abrogated by its phosphorylation at Tyr 14, through a G i/o-Src kinase pathway. Since pulmonary β 2-AR- and muscarinic receptor-mediated relaxations differentiate in their respective signaling pathways leading to eNOS activation and sensitivities during hypoxia-induced pulmonary arterial hypertension, mechanisms underlying eNOS activation might be key determinants of pulmonary endothelial dysfunction.

S155 The role of ST2 in a model of pulmonary hypertension

BackgroundPulmonary arterial hypertension (PAH) is a fatal condition involving remodelling of the pulmonary vessel wall leading to elevated pulmonary arterial pressure and eventually right heart failure. ST2 is a transmembrane...

Treatment with the Kv7 potassium channel activator flupirtine is beneficial in two independent mouse models of pulmonary hypertension.

Voltage-gated potassium (K(v)) channels contribute to resting membrane potential in pulmonary artery smooth muscle cells and are down regulated in patients with pulmonary arterial hypertension (PAH) and a contribution from K(v)7 channels has been recently proposed. We investigated the effect of the K(v)7 channel activator, flupirtine, on PAH in two independent mouse models: PAH induced by hypoxia and spontaneous PAH in mice over-expressing the 5-HT transporter (SERT(+) mice). Right ventricular pressure was assessed in vivo in mice chronically treated with flupirtine (30 mg.kg(-1).day(-1)). In separate in vitro experiments, pulmonary arteries from untreated mice were mounted in a wire myograph. Relaxations to acute administration of flupirtine and contractions to K(v) channel blocking drugs, including the K(v)7 channel blocker linopirdine, were measured. In wild-type (WT) mice, hypoxia increased right ventricular pressure, pulmonary vascular remodelling and right ventricular hypertrophy. These effects were attenuated by flupirtine, which also attenuated these indices of PAH in SERT(+) mice. In the in vitro experiments, flupirtine induced a potent relaxant response in arteries from untreated WT and SERT(+) mice. The relaxation was fully reversed by linopirdine, which potently contracted mouse pulmonary arteries while other K(v) channel blockers did not. Flupirtine significantly attenuated development of chronic hypoxia-induced PAH in mice and reversed established PAH in SERT(+) mice, apparently via K(v)7 channel activation. These results provide the first direct evidence that drugs activating K(v)7 channels may be of benefit in the treatment of PAH with different aetiologies.

Regulation of soluble guanylyl cyclase-α1 expression in chronic hypoxia-induced pulmonary hypertension: role of NFATc3 and HuR

The nitric oxide/soluble guanylyl cyclase (sGC) signal transduction pathway plays an important role in smooth muscle relaxation and phenotypic regulation. However, the transcriptional regulation of sGC gene expression is largely unknown. It has been shown that sGC expression increases in pulmonary arteries from chronic hypoxia-induced pulmonary hypertensive animals. Since the transcription factor NFATc3 is required for the upregulation of the smooth muscle hypertrophic/differentiation marker alpha-actin in pulmonary artery smooth muscle cells from chronically hypoxic mice, we hypothesized that NFATc3 is required for the regulation of sGC-alpha1 expression during chronic hypoxia. Exposure to chronic hypoxia for 2 days induced a decrease in sGC-alpha1 expression in mouse pulmonary arteries. This reduction was independent of NFATc3 but mediated by nuclear accumulation of the mRNA-stabilizing protein human antigen R (HuR). Consistent with our hypothesis, chronic hypoxia (21 days) upregulated pulmonary artery sGC-alpha1 expression, bringing it back to the level of the normoxic controls. This response was prevented in NFATc3 knockout and cyclosporin (calcineurin/NFATc inhibitor)-treated mice. Furthermore, we identified effective binding sites for NFATc in the mouse sGC-alpha1 promoter. Activation of NFATc3 increased sGC-alpha1 promoter activity in human embryonic derived kidney cells, rat aortic-derived smooth muscle cells, and human pulmonary artery smooth muscle cells. Our results suggest that NFATc3 and HuR are important regulators of sGC-alpha1 expression in pulmonary vascular smooth muscle cells during chronic hypoxia-induced pulmonary hypertension.

Protective actions of epoxyeicosatrienoic acid: Dual targeting of cardiovascular PI3K and K ATP channels

Epoxyeicosatrienoic acid(s) (EETs) have been shown to protect cardiovascular tissue against apoptosis dependent on activation of targets such as ATP-sensitive K + (K ATP) channels (sarcolemmal and mitochondrial), calcium-activated K + channels, extracellular signal-regulated kinase or phosphoinositide 3-kinase (PI3K). We tested if EETs protect human atrial tissue ex vivo from hypoxia/reoxygenation (H/R) injury, and compared our results with myocardium from two rodent species, rats and mice. EETs reduced myocardial caspase 3 activity in all three species and protected against loss of mitochondrial membrane potential in primary cultures of neonatal rat ventricular myocytes submitted to H/R. In addition, EETs protected mouse pulmonary arteries ex vivo exposed to H/R. Myocardium and pulmonary arteries from genetically engineered mice having elevated plasma levels of EETs ( Ephx2 −/− ) exhibited protection from H/R-induced injury over that of wild type controls, suggesting that endogenously produced EETs may have pro-survival effects. Electrophysiological studies in myocytes demonstrated that EETs can stimulate K ATP currents even when PI3K is inhibited. Similarly, activation of PI3K/Akt occurred in the presence of the K ATP channel blocker glibenclamide. Based upon loss of protection with EETs in the presence of either wortmannin (a PI3K inhibitor) or glibenclamide, simultaneous activation of at least 2 pathways, PI3K and K ATP channels respectively, appears to be required for protection. In conclusion, we demonstrate that exogenous and endogenous EETs have powerful pro-survival effects in cardiovascular tissues including diseased human myocardium, mediated by activation of not only one but at least two pathways, PI3K and K ATP channels.

In vivo quantification of blood velocity in mouse carotid and pulmonary arteries by ECG‐triggered 3D time‐resolved magnetic resonance angiography

Blood flow velocity is a functional parameter of fundamental importance in diagnosis and follow-up of various vascular diseases. Vascular pathologies can be efficiently studied in animal models, especially in small rodents. ECG-gated magnetic resonance imaging (MRI) assessment of blood velocity in small animals is a challenge because of limited spatial resolution and high-frequency physiological parameters. Here it is shown that a bright-blood cine-3D-MRI method can be used to measure blood velocity at specific times of the cardiac cycle in mouse pulmonary and carotid arteries. The method used a series of time-of-flight (TOF) acquisitions in a volume of interest at different times after signal cancellation in the same volume. This scheme was repeated at different periods of the cardiac cycle by varying the delay between the ECG R-wave peak and signal cancellation. Velocity values in mouse pulmonary artery varied from 35 cm/s in systole to 0-10 cm/s in diastole. A similar pattern was displayed in carotid arteries (18 and 2.5 cm/s, in systole and diastole, respectively). Results are discussed in terms of efficiency, limitation, and comparison with other methods.

P75 neurotrophin receptor regulates agonist-induced pulmonary vasoconstriction

A member of the TNF receptor family, the p75 neurotrophin receptor (p75(NTR)) has been previously shown to play a role in the regulation of fibrin deposition in the lung. However, the role of p75(NTR) in the regulation of pulmonary vascular tone in the lung is unknown. In the present study, we evaluated the expression of p75(NTR) in mouse pulmonary arteries and the putative role of p75(NTR) in modulating pulmonary vascular tone and agonist responsiveness using wild-type (WT) and p75(NTR) knockout (p75(-/-)) mice. Our data indicated that p75(NTR) is expressed in both smooth muscle and endothelial cells within the pulmonary vascular wall in WT mice. Pulmonary artery rings from p75(-/-) mice exhibited significantly elevated active tension due to endothelin-1-mediated Ca(2+) influx. Furthermore, the contraction due to capacitative Ca(2+) entry (CCE) in response to phenylephrine-mediated active depletion of intracellular Ca(2+) stores was significantly enhanced compared with WT rings. The contraction due to CCE induced by passive store depletion, however, was comparable between WT and p75(-/-) rings. Active tension induced by serotonin, U-46619 (a thromboxane A(2) analog), thrombin, 4-aminopyridine (a K(+) channel blocker), and high extracellular K(+) in p75(-/-) rings was similar to that in WT rings. Deletion of p75(NTR) did not alter pulmonary vasodilation to sodium nitroprusside (a nitric oxide donor). These data suggest that intact p75(NTR) signaling may play a role in modulating pulmonary vasoconstriction induced by endothelin-1 and by active store depletion.

Hypoxia activates NADPH oxidase to increase [ROS] i and [Ca 2+] i through the mitochondrial ROS-PKCɛ signaling axis in pulmonary artery smooth muscle cells

The importance of NADPH oxidase (Nox) in hypoxic responses in hypoxia-sensing cells, including pulmonary artery smooth muscle cells (PASMCs), remains uncertain. In this study, using Western blot analysis we found that the major Nox subunits Nox1, Nox4, p22 phox, p47 phox, and p67 phox were equivalently expressed in mouse pulmonary and systemic (mesenteric) arteries. However, acute hypoxia significantly increased Nox activity and translocation of p47 phox protein to the plasma membrane in pulmonary, but not mesenteric, arteries. The Nox inhibitor apocynin and p47 phox gene deletion attenuated the hypoxic increase in intracellular concentrations of reactive oxygen species and Ca 2+ ([ROS] i and [Ca 2+] i), as well as contractions in mouse PASMCs, and abolished the hypoxic activation of Nox in pulmonary arteries. The conventional/novel protein kinase C (PKC) inhibitor chelerythrine, specific PKCɛ translocation peptide inhibitor, and PKCɛ gene deletion, but not the conventional PKC inhibitor GÖ6976, prevented the hypoxic increase in Nox activity in pulmonary arteries and [ROS] i in PASMCs. The PKC activator phorbol 12-myristate 13-acetate could increase Nox activity in pulmonary and mesenteric arteries. Inhibition of mitochondrial ROS generation with rotenone or myxothiazol prevented hypoxic activation of Nox. Glutathione peroxidase-1 (Gpx1) gene overexpression to enhance H 2O 2 removal significantly inhibited the hypoxic activation of Nox, whereas Gpx1 gene deletion had the opposite effect. Exogenous H 2O 2 increased Nox activity in pulmonary and mesenteric arteries. These findings suggest that acute hypoxia may distinctively activate Nox to increase [ROS] i through the mitochondrial ROS-PKCɛ signaling axis, providing a positive feedback mechanism to contribute to the hypoxic increase in [ROS] i and [Ca 2+] i as well as contraction in PASMCs.

Protection of mouse pulmonary arteries from hypoxia‐induced apoptosis: Cross talk between phosphoinositide 3‐kinase (PI3K) and ATP‐sensitive potassium (KATP) channels

We have demonstrated that epoxyeicosatrienoic acids (EETs), the cytochrome P450 epoxygenase metabolites of arachidonic acid, inhibit both intrinsic and extrinsic pathways of apoptosis of human endothelial cells, through activation of phosphatidyl inositol 3‐kinase (PI3K). EETs are also potent sarcolemmal and mitochondrial ATP‐sensitive K+ (KATP) channel activators. In this study, we examined the effects of EETs on hypoxia/reoxygenation (HR)‐induced apoptosis (as determined by measuring increase in caspase 3 activity) in mouse pulmonary arteries maintained ex vivo, by organ culture. Mouse pulmonary arteries subjected to HR for 8/16 h demonstrated increased apoptosis. This was inhibited by pretreatment with either 14,15‐EET (300 nM), the non‐specific KATP channel opener pinacidil (100 microM) or the mitochondrial KATP channel opener, diazoxide (100 microM). Pretreatment with the PI3K inhibitor, wortmannin abolished the protective effect of EET, pinacidil or diazoxide. There was no cumulative effect of wortmannin and glibenclamide when applied together, on the apoptotic indices during HR. Thus, cross‐talk between PI3K and KATP channels, two targets of signaling by EET, mediate protection of the pulmonary vasculature. Funding: NIH/NHLBI 69996 (MMM), 68627 (ERJ), 49294 (ERJ), GM 31278 (JRF).

Characterization of agonist-induced vasoconstriction in mouse pulmonary artery

In recent years, transgenic mouse models have been developed to examine the underlying cellular and molecular mechanisms of lung disease and pulmonary vascular disease, such as asthma, pulmonary thromboembolic disease, and pulmonary hypertension. However, there has not been systematic characterization of the basic physiological pulmonary vascular reactivity in normal and transgenic mice. This represents an intellectual "gap", since it is important to characterize basic murine pulmonary vascular reactivity in response to various contractile and relaxant factors to which the pulmonary vasculature is exposed under physiological conditions. The present study evaluates excitation- and pharmacomechanical-contraction coupling in pulmonary arteries (PA) isolated from wild-type BALB/c mice. We demonstrate that both pharmaco- and electromechanical coupling mechanisms exist in mice PA. These arteries are also reactive to stimulation by alpha(1)-adrenergic agonists, serotonin, endothelin-1, vasopressin, and U-46619 (a thromboxane A(2) analog). We conclude that the basic vascular responsiveness of mouse PA is similar to those observed in PA of other species, including rat, pig, and human, albeit on a different scale and to varying amplitudes.

Myoferlin Regulates Vascular Endothelial Growth Factor Receptor-2 Stability and Function

Myoferlin and dysferlin are members of the ferlin family of membrane proteins. Recent studies have shown that mutation or genetic disruption of myoferlin or dysferlin promotes muscular dystrophy-related phenotypes in mice, which are the result of impaired plasma membrane integrity. However, no biological functions have been ascribed to myoferlin in non-muscle tissues. Herein, using a proteomic analysis of endothelial cell (EC) caveolae/lipid raft microdomains we identified myoferlin in these domains and show that myoferlin is highly expressed in ECs and vascular tissues. The loss of myoferlin results in lack of proliferation, migration, and nitric oxide (NO) release in response to vascular endothelial growth factor (VEGF). Western blotting and surface biotinylation experiments show that loss of myoferlin reduces the expression level and autophosphorylation of VEGF receptor-2 (VEGFR-2) in native ECs. In a reconstituted cell system, transfection of myoferlin increases VEGFR-2 membrane expression and autophosphorylation in response to VEGF. In vivo, VEGFR-2 levels and VEGF-induced permeability are impaired in myoferlin-deficient mice. Mechanistically, myoferlin forms a complex with dynamin-2 and VEGFR-2, which prevents CBL-dependent VEGFR-2 polyubiquitination and proteasomal degradation. These data are the first to report novel biological activities for myoferlin and reveal the role of membrane integrity to VEGF signaling.

MIP-2 causes differential activation of RhoA in mouse aortic versus pulmonary artery endothelial cells

Previously, we have shown that endothelial cell chemotaxis to the proangiogenic chemokine MIP-2 (macrophage inflammatory protein-2) is much greater in mouse aortic endothelial cells (EC) than pulmonary arterial endothelial cells (PA EC). This was true despite the observation that both cell types display comparable levels of the ligand receptor, CXCR 2 (8). Since the systemic arterial circulation is proangiogenic in the adult lung and the pulmonary circulation is relatively resistant to neovascularization, we questioned whether the observed functional heterogeneity is related to inherent differences in cell signaling cascades of the two EC subtypes. Specifically, we measured activation of Rac1 and RhoA, both thought to be involved in EC cell migration. Rac1 showed inconsistent and minimal changes in both cell types after MIP-2 treatment ( p > 0.05). However, activated RhoA was increased upon exposure to MIP-2 only in aortic EC (61% increase; p < 0.05). Decreased RhoA activation after treatment of aortic EC with specific siRNA for RhoA resulted in a functional decrease in EC chemotaxis to MIP-2 (17% increase; p < 0.05). Additionally, increased RhoA activation in PA EC with adenoviral infection of RhoA caused an increase in PA EC chemotaxis to MIP-2 (46% increase; p < 0.05). Inhibition of RhoA activity with the Rho kinase inhibitor, Y27632, blocked aortic EC chemotaxis and stress fiber formation. Thus, RhoA activation is increased after MIP-2 treatment in mouse aortic endothelial cells but not in pulmonary artery endothelial cells. We conclude that RhoA is part of a signaling pathway essential for aortic cell migration after CXCR 2 ligation. This result provides one explanation for the difference in chemotaxis observed in these two endothelial subtypes that express similar levels of CXCR 2.

Functional characterization of voltage-gated K+ channels in mouse pulmonary artery smooth muscle cells

Mice are useful animal models to study pathogenic mechanisms involved in pulmonary vascular disease. Altered expression and function of voltage-gated K(+) (K(V)) channels in pulmonary artery smooth muscle cells (PASMCs) have been implicated in the development of pulmonary arterial hypertension. K(V) currents (I(K(V))) in mouse PASMCs have not been comprehensively characterized. The main focus of this study was to determine the biophysical and pharmacological properties of I(K(V)) in freshly dissociated mouse PASMCs with the patch-clamp technique. Three distinct whole cell I(K(V)) were identified based on the kinetics of activation and inactivation: rapidly activating and noninactivating currents (in 58% of the cells tested), rapidly activating and slowly inactivating currents (23%), and slowly activating and noninactivating currents (17%). Of the cells that demonstrated the rapidly activating noninactivating current, 69% showed I(K(V)) inhibition with 4-aminopyridine (4-AP), while 31% were unaffected. Whole cell I(K(V)) were very sensitive to tetraethylammonium (TEA), as 1 mM TEA decreased the current amplitude by 32% while it took 10 mM 4-AP to decrease I(K(V)) by a similar amount (37%). Contribution of Ca(2+)-activated K(+) (K(Ca)) channels to whole cell I(K(V)) was minimal, as neither pharmacological inhibition with charybdotoxin or iberiotoxin nor perfusion with Ca(2+)-free solution had an effect on the whole cell I(K(V)). Steady-state activation and inactivation curves revealed a window K(+) current between -40 and -10 mV with a peak at -31.5 mV. Single-channel recordings revealed large-, intermediate-, and small-amplitude currents, with an averaged slope conductance of 119.4 +/- 2.7, 79.8 +/- 2.8, 46.0 +/- 2.2, and 23.6 +/- 0.6 pS, respectively. These studies provide detailed electrophysiological and pharmacological profiles of the native K(V) currents in mouse PASMCs.

Repulsive Guidance Molecule RGMa Alters Utilization of Bone Morphogenetic Protein (BMP) Type II Receptors by BMP2 and BMP4

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta superfamily of multifunctional ligands that transduce their signals through type I and II serine/threonine kinase receptors and intracellular Smad proteins. Recently, we identified the glycosylphosphatidylinositol-anchored repulsive guidance molecules RGMa, DRAGON (RGMb), and hemojuvelin (RGMc) as coreceptors for BMP signaling (Babbit, J. L., Huang, F. W., Wrighting, D. W., Xia, Y., Sidis, Y., Samad, T. A., Campagna, J. A., Chung, R., Schneyer, A., Woolf, C. J., Andrews, N. C., and Lin, H. Y. (2006) Nat. Genet. 38, 531-539; Babbit, J. L., Zhang, Y., Samad, T. A., Xia, Y., Tang, J., Schneyer, A., Woolf, C. J., and Lin, H. Y. (2005) J. Biol. Chem. 280, 29820-29827; Samad, T. A., Rebbapragada, A., Bell, E., Zhang, Y., Sidis, Y., Jeong, S. J., Campagna, J. A., Perusini, S., Fabrizio, D. A., Schneyer, A. L., Lin, H. Y., Brivanlou, A. H., Attisano, L., and Woolf, C. J. (2005) J. Biol. Chem. 280, 14122-14129). However, the mechanism by which RGM family members enhance BMP signaling remains unknown. Here, we report that RGMa bound to radiolabeled BMP2 and BMP4 with Kd values of 2.4+/-0.2 and 1.4+/-0.1 nm, respectively. In KGN human ovarian granulosa cells and mouse pulmonary artery smooth muscle cells, BMP2 and BMP4 signaling required BMP receptor type II (BMPRII), but not activin receptor type IIA (ActRIIA) or ActRIIB, based on changes in BMP signaling by small interfering RNA inhibition of receptor expression. In contrast, cells transfected with RGMa utilized both BMPRII and ActRIIA for BMP2 or BMP4 signaling. Furthermore, in BmpRII-null pulmonary artery smooth muscle cells, BMP2 and BMP4 signaling was reduced by inhibition of endogenous RGMa expression, and RGMa-mediated BMP signaling required ActRIIA expression. These findings suggest that RGMa facilitates the use of ActRIIA by endogenous BMP2 and BMP4 ligands that otherwise prefer signaling via BMPRII and that increased utilization of ActRIIA leads to generation of an enhanced BMP signal.