Schizophrenia interactome with 504 novel protein-protein interactions.

Genome-wide association studies of schizophrenia (GWAS) have revealed the role of rare and common genetic variants, but the functional effects of the risk variants remain to be understood. Protein interactome-based studies can facilitate the study of molecular mechanisms by which the risk genes relate to schizophrenia (SZ) genesis, but protein-protein interactions (PPIs) are unknown for many of the liability genes. We developed a computational model to discover PPIs, which is found to be highly accurate according to computational evaluations and experimental validations of selected PPIs. We present here, 365 novel PPIs of liability genes identified by the SZ Working Group of the Psychiatric Genomics Consortium (PGC). Seventeen genes that had no previously known interactions have 57 novel interactions by our method. Among the new interactors are 19 drug targets that are targeted by 130 drugs. In addition, we computed 147 novel PPIs of 25 candidate genes investigated in the pre-GWAS era. While there is little overlap between the GWAS genes and the pre-GWAS genes, the interactomes reveal that they largely belong to the same pathways, thus reconciling the apparent disparities between the GWAS and prior gene association studies. The interactome including 504 novel PPIs overall, could motivate other systems biology studies and trials with repurposed drugs. The PPIs are made available on a webserver, called Schizo-Pi at http://severus.dbmi.pitt.edu/schizo-pi with advanced search capabilities.

The HIV Protease Inhibitor Saquinavir Inhibits HMGB1-Driven Inflammation by Targeting the Interaction of Cathepsin V with TLR4/MyD88.

Extracellular high-mobility group box 1 (HMGB1) (disulfide form), via activation of toll-like receptor 4 (TLR4)-dependent signaling, is a strong driver of pathologic inflammation in both acute and chronic conditions. Identification of selective inhibitors of HMGB1-TLR4 signaling could offer novel therapies that selectively target proximal endogenous activators of inflammation. A cell-based screening strategy led us to identify first generation HIV-protease inhibitors (PI) as potential inhibitors of HMGB1-TLR4 driven cytokine production. Here we report that the first-generation HIV-PI saquinavir (SQV), as well as a newly identified mammalian protease inhibitor STO33438 (334), potently block disulfide HMGB1-induced TLR4 activation, as assayed by the production of TNF-α by human monocyte-derived macrophages (THP-1). We further report on the identification of mammalian cathepsin V, a protease, as a novel target of these inhibitors. Cellular as well as recombinant protein studies show that the mechanism of action involves a direct interaction between cathepsin V with TLR4 and its adaptor protein MyD88. Treatment with SQV, 334 or the known cathepsin inhibitor SID26681509 (SID) significantly improved survival in murine models of sepsis and reduced liver damage following warm liver ischemia/reperfusion (I/R) models, both characterized by strong HMGB1-TLR4 driven pathology. The current study demonstrates a novel role for cathepsin V in TLR4 signaling and implicates cathepsin V as a novel target for first-generation HIV-PI compounds. The identification of cathepsin V as a target to block HMGB1-TLR4-driven inflammation could allow for a rapid transition of the discovery from the bench to the bedside. Disulfide HMGB1 drives pathologic inflammation in many models by activating signaling through TLR4. Cell-based screening identified the mammalian protease cathepsin V as a novel therapeutic target to inhibit TLR4-mediated inflammation induced by extracellular HMGB1 (disulfide form). We identified two protease inhibitors (PIs) that block cathepsin V and thereby inhibit disulfide HMGB1-induced TLR4 activation: saquinavir (SQV), a first-generation PI targeting viral HIV protease and STO33438 (334), targeting mammalian proteases. We discovered that cathepsin V binds TLR4 under basal and HMGB1-stimulated conditions, but dissociates in the presence of SQV over time. Thus cathepsin V is a novel target for first-generation HIV PIs and represents a potential therapeutic target of pathologic inflammation.

Recombinant human interferon alpha 2b prevents and reverses experimental pulmonary hypertension.

Pulmonary hypertension (PH) is a progressive and fatal disease with no cure. Vascular remodeling in PH involves intraluminal growth of endothelial and smooth muscle cells, leading to obliterative vascular lesions. Cell growth in these lesions is quasi-neoplastic, with evidence of monoclonality, apoptosis resistance and cancer-like metabolic derangements. Herein we tested the effect of human interferon alpha 2b (IFNα), a pleiotropic cytokine and anti-cancer therapeutic, on the development and progression of PH in the rat SU5416/hypoxia (SUH) model and mouse hypoxia model of the disease. In both models IFNα attenuated the development of PH and reversed established PH as assessed by measuring right ventricular systolic pressure and right ventricular hypertrophy. The effect of IFNα was dependent on the type I interferon receptor (IFNAR) since mice lacking a subunit of the IFNAR were not protected by IFNα. Morphometric analysis of pulmonary aterioles from hypoxic mice or SUH rats showed that IFNα inhibited pulmonary vascular remodeling in both models and that IFNα reversed remodeling in SUH rats with established disease. Immunohistochemical staining revealed that IFNα decreased the number of PCNA and Tunel positive cells in the wall of pulmonary arterioles. In vitro, IFNα inhibited proliferation of human pulmonary artery smooth muscle cells and as well as human pulmonary artery endothelial cell proliferation and apoptosis. Together these findings demonstrate that IFNα reverses established experimental PH and provide a rationale for further exploration of the use of IFNα and other immunotherpies in PH.

Genetic deletion of toll-like receptor 4 on platelets attenuates experimental pulmonary hypertension.

RATIONALE: Recent studies demonstrate a role for toll-like receptor 4 (TLR4) in the pathogenesis of pulmonary hypertension (PH); however, the cell types involved in mediating the effects of TLR4 remain unknown. OBJECTIVES: The objective of this study was to determine the contribution of TLR4 expressed on nonparenchymal cells to the pathogenesis of PH. METHODS AND RESULTS: TLR4 bone marrow chimeric mice revealed an equal contribution of TLR4 on nonparenchymal and parenchymal cells in the pathogenesis of PH as determined by measuring right ventricular (RV) systolic pressure and RV hypertrophy. However, the deletion of TLR4 from myeloid lineage cells had no effect on the development of PH because we found no difference in RV systolic pressure or RV hypertrophy in wild-type versus LysM-TLR4(-/-) mice. To explore the potential role of platelet TLR4 in the pathogenesis of PH, platelet-specific TLR4(-/-) mice were generated (PF4-TLR4(-/-) mice). TLR4(-/-) platelets from either global TLR4(-/-) or PF4-TLR4(-/-) mice were functional but failed to respond to lipopolysaccharide, demonstrating a lack of TLR4. PF4-TLR4(-/-) mice demonstrated significant protection from hypoxia-induced PH, including attenuated increases in RV systolic pressure and RV hypertrophy, decreased platelet activation, and less pulmonary vascular remodeling. The deletion of TLR4 from platelets attenuated serotonin release after chronic hypoxia, and lipopolysaccharide-stimulated platelets released serotonin and promoted pulmonary artery smooth muscle cell proliferation in a serotonin-dependent manner. CONCLUSIONS: Our data demonstrate that TLR4 on platelets contributes to the pathogenesis of PH and further highlights the role of platelets in PH.

High mobility group Box 1 inhibits human pulmonary artery endothelial cell migration via a Toll-like receptor 4- and interferon response factor 3-dependent mechanism(s).

In pulmonary hypertension the loss of precapillary arterioles results from vascular injury causing endothelial dysfunction. Endothelial cell migration and proliferation are critical for vascular regeneration. This study focused on the effect of high mobility group box 1 protein (HMGB1) on these critical processes. HMGB1 had no effect on human pulmonary artery endothelial cell (HPAEC) proliferation. In contrast, treatment of HPAECs with HMGB1 dose-dependently inhibited VEGF-stimulated HPAEC migration. The effect of HMGB1 on HPAEC migration was TLR4-dependent because it was reversed by TLR4 siRNA or TLR4-neutralizing antibody. Exposure of HPAECs to hypoxia caused translocation and release of HMGB1 and inhibition of HPAEC migration. The effect of hypoxia on HPAEC migration was mediated by HMGB1 because HMGB1-neutralizing antibody but not control IgG restored HPAEC migration. Likewise, TLR4 siRNA but not control siRNA reversed the inhibitory effect of hypoxia in HPAECs. The canonical TLR4 signaling pathway requires the adaptor protein MyD88 and leads to downstream NFκB activation. Interestingly, HMGB1 failed to stimulate NFκB translocation to the nucleus, but instead activated an alternative pathway characterized by activation of interferon response factor 3 (IRF3). This was in contrast to human umbilical vein endothelial cells in which HMGB1 stimulated nuclear translocation of NFκB but not IRF3. IRF3 siRNA, but not MyD88 siRNA, reversed the inhibitory effect of HMGB1 on HPAEC migration. These data demonstrate that HMGB1 inhibits HPAEC migration, a critical process for vascular regeneration, via TLR4- and IRF3-dependent mechanisms.

High mobility group box 1 contributes to the pathogenesis of experimental pulmonary hypertension via activation of Toll-like receptor 4.

Survival rates for patients with pulmonary hypertension (PH) remain low, and our understanding of the mechanisms involved are incomplete. Here we show in a mouse model of chronic hypoxia (CH)-induced PH that the nuclear protein and damage-associate molecular pattern molecule (DAMP) high mobility group box 1 (HMGB1) contributes to PH via a Toll-like receptor 4 (TLR4)-dependent mechanism. We demonstrate extranuclear HMGB1 in pulmonary vascular lesions and increased serum HMGB1 in patients with idiopathic pulmonary arterial hypertension. The increase in circulating HMGB1 correlated with mean pulmonary artery pressure. In mice, we similarly detected the translocation and release of HMGB1 after exposure to CH. HMGB1-neutralizing antibody attenuated the development of CH-induced PH, as assessed by measurement of right ventricular systolic pressure, right ventricular hypertrophy, pulmonary vascular remodeling and endothelial activation and inflammation. Genetic deletion of the pattern recognition receptor TLR4, but not the receptor for advanced glycation end products, likewise attenuated CH-induced PH. Finally, daily treatment of mice with recombinant human HMGB1 exacerbated CH-induced PH in wild-type (WT) but not Tlr4(-/-) mice. These data demonstrate that HMGB1-mediated activation of TLR4 promotes experimental PH and identify HMGB1 and/or TLR4 as potential therapeutic targets for the treatment of PH.

Chronic hypoxia induces right heart failure in caveolin-1-/- mice.

Caveolin-1 (Cav-1)-/- mice develop mild pulmonary hypertension as they age. In this study, we sought to determine the effect of chronic hypoxia, an established model of pulmonary hypertension, on young Cav-1-/- mice with no measurable signs of pulmonary hypertension. Exposure of Cav-1-/- mice to chronic hypoxia resulted in an initial rise in right ventricular (RV) systolic pressure (RVSP) similar to wild-type (WT) mice. By three weeks RVSP decreased in the Cav-1-/- mice, whereas it was maintained in WT mice. The drop in RVSP in Cav-1-/- mice was accompanied by decreased cardiac output, increased RV hypertrophy, RV interstitial fibrosis, decreased RV sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a mRNA and decreased RV function compared with WT mice. Importantly, minimal differences were noted in pulmonary vascular remodeling between WT and Cav-1-/- mice, and left ventricular function was normal in hypoxic Cav-1-/- mice. Mechanistically, increased endothelial nitric oxide synthase uncoupling and increased tyrosine nitration of protein kinase G were detected in the RV of Cav-1-/- mice. These hemodynamic, histological, and molecular changes were prevented in Cav-1-/- mice expressing an endothelial-specific Cav-1 transgene or by nitric oxide synthase inhibition. These data suggest that, in Cav-1-/- mice, increased oxidative/nitrosative stress due to endothelial nitric oxide synthase uncoupling modifies the response of the RV to pressure overload, accelerating the deterioration of RV function.

Activated CD47 promotes pulmonary arterial hypertension through targeting caveolin-1.

AIMS: Pulmonary arterial hypertension (PAH) is a progressive lung disease characterized by pulmonary vasoconstriction and vascular remodelling, leading to increased pulmonary vascular resistance and right heart failure. Loss of nitric oxide (NO) signalling and increased endothelial nitric oxide synthase (eNOS)-derived oxidative stress are central to the pathogenesis of PAH, yet the mechanisms involved remain incompletely determined. In this study, we investigated the role activated CD47 plays in promoting PAH. METHODS AND RESULTS: We report high-level expression of thrombospondin-1 (TSP1) and CD47 in the lungs of human subjects with PAH and increased expression of TSP1 and activated CD47 in experimental models of PAH, a finding matched in hypoxic human and murine pulmonary endothelial cells. In pulmonary endothelial cells CD47 constitutively associates with caveolin-1 (Cav-1). Conversely, in hypoxic animals and cell cultures activation of CD47 by TSP1 disrupts this constitutive interaction, promoting eNOS-dependent superoxide production, oxidative stress, and PAH. Hypoxic TSP1 null mice developed less right ventricular pressure and hypertrophy and markedly less arteriole muscularization compared with wild-type animals. Further, therapeutic blockade of CD47 activation in hypoxic pulmonary artery endothelial cells upregulated Cav-1, increased Cav-1CD47 co-association, decreased eNOS-derived superoxide, and protected animals from developing PAH. CONCLUSION: Activated CD47 is upregulated in experimental and human PAH and promotes disease by limiting Cav-1 inhibition of dysregulated eNOS.

Complement C3 deficiency attenuates chronic hypoxia-induced pulmonary hypertension in mice.

BACKGROUND: Evidence suggests a role of both innate and adaptive immunity in the development of pulmonary arterial hypertension. The complement system is a key sentry of the innate immune system and bridges innate and adaptive immunity. To date there are no studies addressing a role for the complement system in pulmonary arterial hypertension. METHODOLOGY/PRINCIPAL FINDINGS: Immunofluorescent staining revealed significant C3d deposition in lung sections from IPAH patients and C57Bl6/J wild-type mice exposed to three weeks of chronic hypoxia to induce pulmonary hypertension. Right ventricular systolic pressure and right ventricular hypertrophy were increased in hypoxic vs. normoxic wild-type mice, which were attenuated in C3-/- hypoxic mice. Likewise, pulmonary vascular remodeling was attenuated in the C3-/- mice compared to wild-type mice as determined by the number of muscularized peripheral arterioles and morphometric analysis of vessel wall thickness. The loss of C3 attenuated the increase in interleukin-6 and intracellular adhesion molecule-1 expression in response to chronic hypoxia, but not endothelin-1 levels. In wild-type mice, but not C3-/- mice, chronic hypoxia led to platelet activation as assessed by bleeding time, and flow cytometry of platelets to determine cell surface P-selectin expression. In addition, tissue factor expression and fibrin deposition were increased in the lungs of WT mice in response to chronic hypoxia. These pro-thrombotic effects of hypoxia were abrogated in C3-/- mice. CONCLUSIONS: Herein, we provide compelling genetic evidence that the complement system plays a pathophysiologic role in the development of PAH in mice, promoting pulmonary vascular remodeling and a pro-thrombotic phenotype. In addition we demonstrate C3d deposition in IPAH patients suggesting that complement activation plays a role in the development of PAH in humans.

Bone morphogenetic protein receptor II is a novel mediator of endothelial nitric-oxide synthase activation.

Activation of bone morphogenetic protein (BMP) receptor II (BMPRII) promotes pulmonary artery endothelial cell (PAEC) survival, proliferation, and migration. Mutations to BMPRII are associated with the development of pulmonary arterial hypertension (PAH). Endothelial dysfunction, including decreased endothelial nitric-oxide synthase (eNOS) activity and loss of bioactive nitric oxide (NO), plays a prominent role in the development of PAH. We hypothesized that stimulation of BMPRII promotes normal PAEC function by activating eNOS. We report that BMPRII ligands, BMP2 and BMP4, (i) stimulate eNOS phosphorylation at a critical regulatory site, (ii) increase eNOS activity, and (iii) result in canonical changes in eNOS protein-protein interactions. The stimulation of eNOS activity by BMPRII ligands was largely dependent on protein kinase A (PKA) activation, as demonstrated using the PKA inhibitors H89 and myristoylated PKI(6-22) amide. PAEC migration stimulated by BMP2 and BMP4 was inhibited by the NOS inhibitor l-nitroarginine methyl ester, providing functional evidence of eNOS activation. Furthermore, BMP2 and BMP4 failed to stimulate eNOS phosphorylation when BMPRII was knocked down by siRNA. Most important to the pathophysiology of the disease, BMP2 and BMP4 failed to stimulate eNOS phosphorylation in PAECs isolated from patients with mutations in the BMPR2 gene. These data demonstrate a new action of BMPs/BMPRII in the pulmonary endothelium and provide novel mechanistic insight into the pathogenesis of PAH.

A role for zinc in regulating hypoxia-induced contractile events in pulmonary endothelium.

We previously reported that zinc thiolate signaling contributes to hypoxic contraction of small, nonmuscularized arteries of the lung. The present studies were designed to investigate mechanisms by which hypoxia-released zinc induces contraction in isolated pulmonary endothelial cells and to delineate the signaling pathways involved in zinc-mediated changes in the actin cytoskeleton. We used fluorescence-based imaging to show that hypoxia induced time-dependent increases in actin stress fibers that were reversed by the zinc chelator, N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN). We further showed that hypoxia-induced phosphorylation of the contractile protein myosin light chain (MLC) and assembly of actin stress fibers were each TPEN sensitive. Hypoxia and zinc-induced inhibition of MLC phosphatase (MLCP) were independent of the regulatory subunit (MYPT1) of MLCP, and therefore hypoxia-released zinc likely inhibits MLCP at its catalytic (PP1) subunit. Inhibition of PKC by Ro-31-8220 and a dominant-negative construct of PKC-ε attenuated hypoxia-induced contraction of isolated pulmonary endothelial cells. Furthermore, zinc-induced phosphorylation of MLC (secondary to inhibition of MLCP) was PKC dependent, and hypoxia-released zinc promoted the phosphorylation of the PKC substrate, CPI-17. Collectively, these data suggest a link between hypoxia, elevations in labile zinc, and activation of PKC, which in turn acts through CPI-17 to inhibit MLCP activity and promote MLC phosphorylation, ultimately inducing stress fiber formation and endothelial cell contraction.

Thrombospondin-1 supports blood pressure by limiting eNOS activation and endothelial-dependent vasorelaxation.

AIMS: Thrombospondin-1 (TSP1), via its necessary receptor CD47, inhibits nitric oxide (NO)-stimulated soluble guanylate cyclase activation in vascular smooth muscle cells, and TSP1-null mice have increased shear-dependent blood flow compared with wild-type mice. Yet, the endothelial basement membrane should in theory function as a barrier to diffusion of soluble TSP1 into the arterial smooth muscle cell layer. These findings suggested that endothelial-dependent differences in blood flow in TSP1-null mice may be the result of direct modulation of endothelial NO synthase (eNOS) activation by circulating TSP1. Here we tested the hypothesis that TSP1 inhibits eNOS activation and endothelial-dependent arterial relaxation. METHODS AND RESULTS: Acetylcholine (ACh)-stimulated activation of eNOS and agonist-driven calcium transients in endothelial cells were inhibited by TSP1. TSP1 also inhibited eNOS phosphorylation at serine(1177). TSP1 treatment of the endothelium of wild-type and TSP1-null but not CD47-null arteries inhibited ACh-stimulated relaxation. TSP1-null vessels demonstrated greater endothelial-dependent vasorelaxation compared with the wild type. Conversely, TSP1-null arteries demonstrated less vasoconstriction to phenylephrine compared with the wild type, which was corrected upon inhibition of eNOS. In TSP1-null mice, intravenous TSP1 blocked ACh-stimulated decreases in blood pressure, and both intravenous TSP1 and a CD47 agonist antibody acutely elevated blood pressure in mice. CONCLUSION: TSP1, via CD47, inhibits eNOS activation and endothelial-dependent arterial relaxation and limits ACh-driven decreases in blood pressure. Conversely, intravenous TSP1 and a CD47 antibody increase blood pressure. These findings suggest that circulating TSP1, by limiting endogenous NO production, functions as a pressor agent supporting blood pressure.

Use of spectral fluorescence resonance energy transfer to detect nitric oxide-based signaling events in isolated perfused lung.

Fluorescence resonance energy transfer (FRET) is a fluorescence microscopy technique suitable for live cells and capable of detecting changes in the conformational state of a single protein or the distance between two interacting proteins when the proteins are conjugated with appropriate donor and acceptor fluorophores. Confocal-based spectral detection systems enable the resolution of fluorescent images by providing full spectral information for each voxel of the image without switching of optical filters. Furthermore, using calibration spectra, it is possible to unambiguously separate the cross-talk between overlapping donor and acceptor emissions. This unit describes the use of confocal-based spectral imaging of nitric oxide (NO) sensitive FRET reporters in the vasculature of the intact, isolated perfused mouse lung. This type of in situ imaging approach allows the visualization and study of temporal molecular signaling events within the appropriate physiologic microenvironment of the intact, living organ.