Yoshikazu Nakaoka

Enhanced Functional Gap Junction Neoformation by Protein Kinase A–Dependent and Epac-Dependent Signals Downstream of cAMP in Cardiac Myocytes

Gap junctions (GJs) constituted by neighboring cardiac myocytes are essential for gating ions and small molecules to coordinate cardiac contractions. cAMP is suggested to be a potent stimulus for enhancement of GJ function. However, it remains elusive how cAMP potentiates the GJ of cardiomyocytes. Here we demonstrated that the gating function of GJ is enhanced by the protein kinase A (PKA)–dependent signal, and that the accumulation of connexin43 (Cx43), the most abundant Cx in myocytes, is enhanced by an exchange protein directly activated by cAMP (Epac) (Rap1 activator)–dependent signal. The gating function of GJs was analyzed by microinjected dye transfer method. The accumulation of Cx43 was analyzed by quantitative immunostaining. Using the PKA-specific activator N6-benzoyladenosine-3′,5′-cyclic monophosphate (6Bnz) and Epac-specific activator 8-(4-chlorophenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate (8CPT), we could delineate the two important downstream signals of cAMP for enhanced GJ neoformation. Whereas 6Bnz potentiated gating function of GJs with slight accumulation of Cx43 at cell–cell contacts, 8CPT remarkably enhanced the accumulation of Cx43 with a slight effect on gating. We further noticed that adherens junctions (AJs) were maturated by 8CPT, as marked by increased neural-cadherin immunostaining. Because AJ formation precedes the GJ formation, AJ formation accelerated by Epac-Rap1 signal may result in enhanced GJ formation. The involvement of Epac-Rap1 signal in GJ neoformation was further confirmed by evidence that inactivation of Rap1 by overexpression of Rap1GAP1b perturbed the accumulation of Cx43 at cell–cell contacts. Collectively, PKA and Epac cooperatively enhance functional GJ neoformation in cardiomyocytes.

Activation of gp130 Transduces Hypertrophic Signal Through Interaction of Scaffolding/Docking Protein Gab1 With Tyrosine Phosphatase SHP2 in Cardiomyocytes

Abstract— Grb2-associated binder-1 (Gab1) is a scaffolding/docking protein and contains a Pleckstrin homology domain and potential binding sites for Src homology (SH) 2 and SH3 domains. Gab1 is tyrosine phosphorylated and associates with protein tyrosine phosphatase SHP2 and p85 phosphatidylinositol 3-kinase on stimulation with various cytokines and growth factors, including interleukin-6. We previously demonstrated that interleukin-6–related cytokine, leukemia inhibitory factor (LIF), induced cardiac hypertrophy through gp130. In this study, we report the role of Gab1 in gp130-mediated cardiac hypertrophy. Stimulation with LIF induced tyrosine phosphorylation of Gab1, and phosphorylated Gab1 interacted with SHP2 and p85 in cultured cardiomyocytes. We constructed three kinds of adenovirus vectors, those carrying wild-type Gab1 (AdGab1WT), mutated Gab1 lacking SHP2 binding site (AdGab1F627/659), and &bgr;-galactosidase (Ad&bgr;-gal). Compared with cardiomyocytes infected with Ad&bgr;-gal, longitudinal elongation of cardiomyocytes induced by LIF was enhanced in cardiomyocytes infected with AdGab1WT but inhibited in cardiomyocytes infected with AdGab1F627/659. Upregulation of BNP mRNA expression by LIF was evoked in cardiomyocytes infected with Ad&bgr;-gal and AdGab1WT but not in cardiomyocytes infected with AdGab1F627/659. In contrast, Gab1 repressed skeletal &agr;-actin mRNA expression through interaction with SHP2. Furthermore, activation of extracellular signal–regulated kinase 5 (ERK5) was enhanced in cardiomyocytes infected with AdGab1WT compared with cardiomyocytes infected with Ad&bgr;-gal but repressed in cardiomyocytes infected with AdGab1F627/659. Coinfection of AdGab1WT with adenovirus vector carrying dominant-negative ERK5 abrogated longitudinal elongation of cardiomyocytes induced by LIF. Taken together, these findings indicate that Gab1-SHP2 interaction plays a crucial role in gp130-dependent longitudinal elongation of cardiomyoctes through activation of ERK5.

Therapeutic Activation of Signal Transducer and Activator of Transcription 3 by Interleukin-11 Ameliorates Cardiac Fibrosis After Myocardial Infarction

Background— Glycoprotein 130 is the common receptor subunit for the interleukin (IL)-6 cytokine family. Previously, we reported that pretreatment of IL-11, an IL-6 family cytokine, activates the glycoprotein 130 signaling pathway in cardiomyocytes and prevents ischemia/reperfusion injury in vivo; however, its long-term effects on cardiac remodeling after myocardial infarction (MI) remain to be elucidated. Methods and Results— MI was generated by ligating the left coronary artery in C57BL/6 mice. Real-time reverse transcription polymerase chain reaction analyses showed that IL-11 mRNA was remarkably upregulated in the hearts exposed to MI. Intravenous injection of IL-11 activated signal transducer and activator of transcription 3 (STAT3), a downstream signaling molecule of glycoprotein 130, in cardiomyocytes in vivo, suggesting that cardiac myocytes are target cells of IL-11 in the hearts. Twenty-four hours after coronary ligation, IL-11 was administered intravenously, followed by consecutive administration every 24 hours for 4 days. IL-11 treatment reduced fibrosis area 14 days after MI, attenuating cardiac dysfunction. Consistent with a previous report that STAT3 exhibits antiapoptotic and angiogenic activity in the heart, IL-11 treatment prevented apoptotic cell death of the bordering myocardium adjacent to the infarct zone and increased capillary density at the border zone. Importantly, cardiac-specific ablation of STAT3 abrogated IL-11–mediated attenuation of fibrosis and was associated with left ventricular enlargement. Moreover, with the use of cardiac-specific transgenic mice expressing constitutively active STAT3, cardiac STAT3 activation was shown to be sufficient to prevent adverse cardiac remodeling. Conclusions— IL-11 attenuated cardiac fibrosis after MI through STAT3. Activation of the IL-11/glycoprotein 130/STAT3 axis may be a novel therapeutic strategy against cardiovascular diseases.

Lymphatic regulator PROX1 determines Schlemm’s canal integrity and identity

Schlemms canal (SC) is a specialized vascular structure in the eye that functions to drain aqueous humor from the intraocular chamber into systemic circulation. Dysfunction of SC has been proposed to underlie increased aqueous humor outflow (AHO) resistance, which leads to elevated ocular pressure, a factor for glaucoma development in humans. Here, using lymphatic and blood vasculature reporter mice, we determined that SC, which originates from blood vessels during the postnatal period, acquires lymphatic identity through upregulation of prospero homeobox protein 1 (PROX1), the master regulator of lymphatic development. SC expressed lymphatic valve markers FOXC2 and integrin α9 and exhibited continuous vascular endothelial-cadherin (VE-cadherin) junctions and basement membrane, similar to collecting lymphatics. SC notably lacked luminal valves and expression of the lymphatic endothelial cell markers podoplanin and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1). Using an ocular puncture model, we determined that reduced AHO altered the fate of SC both during development and under pathologic conditions; however, alteration of VEGF-C/VEGFR3 signaling did not modulate SC integrity and identity. Intriguingly, PROX1 expression levels linearly correlated with SC functionality. For example, PROX1 expression was reduced or undetectable under pathogenic conditions and in deteriorated SCs. Collectively, our data indicate that PROX1 is an accurate and reliable biosensor of SC integrity and identity.

Circulating interleukin-6 family cytokines and their receptors in patients with congestive heart failure.

Abstractgp130 is a common signal-transducing receptor subunit for the interleukin (IL)-6 cytokine family. Studies in genetically engineered animal models have demonstrated a critical role for the gp130-dependent cardiomyocyte survival pathway in the transition to heart failure. In the present study, we examined plasma levels of the IL-6 family of cytokines and the soluble form of their receptors in patients with congestive heart failure (CHF). Circulating levels of the IL-6 family of cytokines, soluble IL-6 receptor (sIL-6R), and soluble gp130 (sgp130) were examined in 48 patients with various degrees of CHF, including dilated cardiomyopathy (DCM), ischemic cardiomyopathy (ICM), and valvular cardiomyopathy (VCM). Circulating levels of IL-6, leukemia inhibitory factor (LIF), and sgp130 significantly increased in association with the severity of CHF. No significant difference was observed in the circulating levels of sIL-6R and IL-11 among these patients. Interestingly, DCM patients showed higher circulating sgp130 levels than patients with ICM or VCM. Our findings suggest that gp130 expression in the heart is likely to be dynamic, and that the IL-6 family of cytokines and their common receptor gp130 participates in the pathogenesis of CHF, especially in DCM.

Gab family proteins are essential for postnatal maintenance of cardiac function via neuregulin-1/ErbB signaling

Grb2-associated binder (Gab) family of scaffolding adaptor proteins coordinate signaling cascades downstream of growth factor and cytokine receptors. In the heart, among EGF family members, neuregulin-1beta (NRG-1beta, a paracrine factor produced from endothelium) induced remarkable tyrosine phosphorylation of Gab1 and Gab2 via erythroblastic leukemia viral oncogene (ErbB) receptors. We examined the role of Gab family proteins in NRG-1beta/ErbB-mediated signal in the heart by creating cardiomyocyte-specific Gab1/Gab2 double knockout mice (DKO mice). Although DKO mice were viable, they exhibited marked ventricular dilatation and reduced contractility with aging. DKO mice showed high mortality after birth because of heart failure. In addition, we noticed remarkable endocardial fibroelastosis and increase of abnormally dilated vessels in the ventricles of DKO mice. NRG-1beta induced activation of both ERK and AKT in the hearts of control mice but not in those of DKO mice. Using DNA microarray analysis, we found that stimulation with NRG-1beta upregulated expression of an endothelium-stabilizing factor, angiopoietin 1, in the hearts of control mice but not in those of DKO mice, which accounted for the pathological abnormalities in the DKO hearts. Taken together, our observations indicated that in the NRG-1beta/ErbB signaling, Gab1 and Gab2 of the myocardium are essential for both maintenance of myocardial function and stabilization of cardiac capillary and endocardial endothelium in the postnatal heart.

Angiopoietin-1 Guides Directional Angiogenesis Through Integrin αvβ5 Signaling for Recovery of Ischemic Retinopathy

Angiopoietin-1 supplementation stimulates guided angiogenesis and promotes the formation of a healthy vascular network in a mouse model of ischemic retinopathy. A Shot at Healthy Growth for Retinal Blood Vessels Abnormalities of retinal blood vessels, such as vascular leakage and inappropriate angiogenesis, contribute to a variety of retinal diseases such as diabetic retinopathy and retinopathy of prematurity. Current targeted treatments for these disorders include antibodies against vascular endothelial growth factor (VEGF) and specially designed proteins that can trap VEGF to inhibit excessive proliferation of abnormal blood vessels in the eye. Unfortunately, these treatments have multiple drawbacks in that they require repeated intraocular injections and may also interfere with the formation of healthy blood vessels in the eye. Now, Lee and coauthors have discovered that angiopoietin-1 (Ang1), another protein involved in angiogenesis, may present a viable target for more specific and longer-lasting treatment of retinopathies. In a mouse model of oxygen-induced retinopathy, the authors showed that a loss of Ang1 led to abnormalities in retinal vasculature, whereas an increase of Ang1 in the retina not only stimulated blood vessel formation but also promoted a normalization of vascular network structure. The authors achieved these results not only by genetic overexpression of Ang1 but also by treating the mice with an injectable form of this protein. The authors also confirmed that Ang1 treatment protected retinal neurons from damage and successfully preserved their function. Future studies will be necessary to confirm the long-term stability of Ang1-stimulated blood vessels and retinal neurons, as well as to translate these results into human patients. Nevertheless, these early findings on injectable Ang1 offer the possibility of a new and more effective treatment approach for retinopathy patients. Retinopathy of prematurity (ROP) and proliferative diabetic retinopathy (PDR) are ischemic retinal diseases caused by insufficient vascular network formation and vascular regression in addition to aberrant angiogenesis. We examined the role of angiopoietin-1 (Ang1) in retinal vascular network formation during postnatal development using Ang1 gain- and loss-of-function mouse models, and tested the effects of intraocular administration of Ang1 in an oxygen-induced retinopathy (OIR) mouse model that mimics cardinal features of ROP and PDR. We observed that Ang1 plays a substantial role in the formation of the retinal vascular network during postnatal development and that Ang1 supplementation can rescue vascular retinopathies by simultaneously promoting healthy vascular network formation and inhibiting subsequent abnormal angiogenesis, vascular leakage, and neuronal dysfunction in the retinas of the OIR model. We attribute these Ang1-induced effects to a dual signaling pathway—Tie2 signaling in the vascular region and integrin αvβ5 signaling in the astrocytes. The activation of integrin αvβ5 signaling promoted fibronectin accumulation and radial distribution along the sprouting endothelial cells, which consequently stimulated guided angiogenesis in the retina. These findings shed light on the role of Ang1 in the recovery of ischemic retinopathies such as ROP, PDR, and retinal vascular occlusive disease.

Interleukin-6/interleukin-21 signaling axis is critical in the pathogenesis of pulmonary arterial hypertension

Significance Pulmonary arterial hypertension (PAH) is a serious disease characterized by vascular remodeling in pulmonary arteries. Although an elevated IL-6 serum level correlates with poor prognosis of PAH patients, it is unclear how IL-6 promotes PAH. Here we identified IL-21 as a downstream target of IL-6 signaling in PAH. In mice with hypoxia-induced pulmonary hypertension (HPH), Th17 cells and M2 macrophages accumulate in the lungs after hypoxia exposure. IL-21 primarily derived from Th17 cells promotes M2 macrophage polarization. Consistently, IL-21 receptor-deficient mice show resistance to HPH with no accumulation of M2 macrophages in the lungs. IL-21 and M2 macrophage markers were upregulated in the lungs of patients with end-stage idiopathic PAH. These findings suggest promising therapeutic strategies for PAH targeting IL-6/IL-21–signaling axis. IL-6 is a multifunctional proinflammatory cytokine that is elevated in the serum of patients with pulmonary arterial hypertension (PAH) and can predict the survival of patients with idiopathic PAH (IPAH). Previous animal experiments and clinical human studies indicate that IL-6 is important in PAH; however, the molecular mechanisms of IL-6–mediated pathogenesis of PAH have been elusive. Here we identified IL-21 as a downstream target of IL-6 signaling in PAH. First, we found that IL-6 blockade by the monoclonal anti-IL-6 receptor antibody, MR16-1, ameliorated hypoxia-induced pulmonary hypertension (HPH) and prevented the hypoxia-induced accumulation of Th17 cells and M2 macrophages in the lungs. Consistently, the expression levels of IL-17 and IL-21 genes, one of the signature genes for Th17 cells, were significantly up-regulated after hypoxia exposure in the lungs of mice treated with control antibody but not in the lungs of mice treated with MR16-1. Although IL-17 blockade with an anti–IL-17A neutralizing antibody had no effect on HPH, IL-21 receptor-deficient mice were resistant to HPH and exhibited no significant accumulation of M2 macrophages in the lungs. In accordance with these findings, IL-21 promoted the polarization of primary alveolar macrophages toward the M2 phenotype. Of note, significantly enhanced expressions of IL-21 and M2 macrophage markers were detected in the lungs of IPAH patients who underwent lung transplantation. Collectively, these findings suggest that IL-21 promotes PAH in association with M2 macrophage polarization, downstream of IL-6-signaling. The IL-6/IL-21–signaling axis may be a potential target for treating PAH.

Amelioration of sepsis by TIE2 activation–induced vascular protection

Vascular protection through TIE2 activation is a potential treatment strategy to ameliorate sepsis. Antibody TIEs sepsis up in knots Sepsis, or severe systemic inflammation caused by infection, has a high mortality despite the availability of antibiotic treatment, and more specific therapies are urgently needed. One of the difficult-to-treat and potentially life-threatening components of sepsis is vascular disintegration and leakage. Han et al. have discovered an antibody, called ABTAA, which binds to a ligand called angiopoietin 2 (ANG2) in the vasculature, but then activates it instead of blocking its activity like standard antibodies. When ABTAA binds to ANG2, it causes clustering of ANG2 and subsequently its receptor TIE2 at the site, and the resulting signaling cascade protects the vascular walls and blunts the damaging effects of sepsis, greatly increasing survival in mouse models of the disease. Protection of endothelial integrity has been recognized as a frontline approach to alleviating sepsis progression, yet no effective agent for preserving endothelial integrity is available. Using an unusual anti–angiopoietin 2 (ANG2) antibody, ABTAA (ANG2-binding and TIE2-activating antibody), we show that activation of the endothelial receptor TIE2 protects the vasculature from septic damage and provides survival benefit in three sepsis mouse models. Upon binding to ANG2, ABTAA triggers clustering of ANG2, assembling an ABTAA/ANG2 complex that can subsequently bind and activate TIE2. Compared with a conventional ANG2-blocking antibody, ABTAA was highly effective in augmenting survival from sepsis by strengthening the endothelial glycocalyx, reducing cytokine storms, vascular leakage, and rarefaction, and mitigating organ damage. Together, our data advance the role of TIE2 activation in ameliorating sepsis progression and open a potential therapeutic avenue for sepsis to address the lack of sepsis-specific treatment.

Signals through gp130 upregulate Wnt5a and contribute to cell adhesion in cardiac myocytes

Glycoprotein 130 (gp130), a common receptor of IL‐6 family cytokines, plays critical roles in cardiac functions. Here, we demonstrate that the stimulation of gp130 with leukemia inhibitory factor (LIF) promoted cell adhesion in a cadherin‐dependent manner in cultured cardiomyocytes. Wnt5a was upregulated by the stimulation of gp130 with IL‐6 family cytokines, accompanied by N‐cadherin protein upregulation. Wnt5a was not induced by LIF in cardiomyocytes expressing dominant‐negative STAT3. Ablation of Wnt5a by antisense cDNA inhibited LIF‐induced cell adhesion. Collectively, signals through gp130 upregulate Wnt5a through STAT3, promoting the N‐cadherin‐mediated cell adhesion.