Upregulation of Robo4 expression by SMAD signaling suppresses vascular permeability and mortality in endotoxemia and COVID-19 models.

There is an urgent need to develop novel drugs to reduce the mortality from severe infectious diseases with the emergence of new pathogens, including Coronavirus disease 2019 (COVID-19). Although current drugs effectively suppress the proliferation of pathogens, immune cell activation, and inflammatory cytokine functions, they cannot completely reduce mortality from severe infections and sepsis. In this study, we focused on the endothelial cell-specific protein, Roundabout 4 (Robo4), which suppresses vascular permeability by stabilizing endothelial cells, and investigated whether enhanced Robo4 expression could be a novel therapeutic strategy against severe infectious diseases. Endothelial-specific overexpression of Robo4 suppresses vascular permeability and reduces mortality in lipopolysaccharide (LPS)-treated mice. Screening of small molecules that regulate Robo4 expression and subsequent analysis revealed that two competitive small mothers against decapentaplegic (SMAD) signaling pathways, activin receptor-like kinase 5 (ALK5)-SMAD2/3 and ALK1-SMAD1/5, positively and negatively regulate Robo4 expression, respectively. An ALK1 inhibitor was found to increase Robo4 expression in mouse lungs, suppress vascular permeability, prevent extravasation of melanoma cells, and decrease mortality in LPS-treated mice. The inhibitor suppressed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced endothelial barrier disruption and decreased mortality in mice infected with SARS-CoV-2. These results indicate that enhancing Robo4 expression is an efficient strategy to suppress vascular permeability and mortality in severe infectious diseases, including COVID-19, and that small molecules that upregulate Robo4 can be potential therapeutic agents against these diseases.

JAK inhibition during the early phase of SARS-CoV-2 infection worsens kidney injury by suppressing endogenous antiviral activity in mice.

Coronavirus disease 2019 (COVID-19) induces respiratory dysfunction as well as kidney injury. Although the kidney is considered a target organ of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and affected by the COVID-19-induced cytokine storm, the mechanisms of renal reaction in SARS-CoV-2 infection are unknown. In this study, a murine COVID-19 model was induced by nasal infection with mouse-adapted SARS-CoV-2 (MA10). MA10 infection induced body weight loss along with lung inflammation in mice 4 days after infection. Serum creatinine levels and the urinary albumin/creatinine ratio increased on day 4 after MA10 infection. Measurement of the urinary neutrophil gelatinase-associated lipocalin/creatinine ratio and hematoxylin and eosin staining revealed tubular damage in MA10-infected murine kidneys, indicating kidney injury in the murine COVID-19 model. Interferon (IFN)-γ and interleukin-6 upregulation in the sera of MA10-infected mice, along with the absence of MA10 in the kidneys, implied that the kidneys were affected by the MA10 infection-induced cytokine storm rather than by direct MA10 infection of the kidneys. RNA-sequencing analysis revealed that antiviral genes, such as the IFN/Janus kinase (JAK) pathway, were upregulated in MA10-infected kidneys. Upon administration of the JAK inhibitor baricitinib on days 1-3 after MA10 infection, an antiviral pathway was suppressed, and MA10 was detected more frequently in the kidneys. Notably, JAK inhibition upregulated the hypoxia response and exaggerated kidney injury. These results suggest that endogenous antiviral activity protects against SARS-CoV-2-induced kidney injury in the early phase of infection, providing valuable insights into the pathogenesis of COVID-19-associated nephropathy.NEW & NOTEWORTHY Patients frequently present with acute kidney injury or abnormal urinary findings after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Here, we investigated how the kidneys respond during SARS-CoV-2 infection using a murine coronavirus disease 2019 (COVID-19) model and showed that Janus kinase-mediated endogenous antiviral activity protects against kidney injury in the early phase of SARS-CoV-2 infection. These findings provide valuable insights into the renal pathophysiology of COVID-19.

Arid5a/IL-6/PAI-1 Signaling Is Involved in the Pathogenesis of Lipopolysaccharide-Induced Kidney Injury.

A systemic inflammatory response leads to widespread organ dysfunction, such as kidney dysfunction. Plasminogen activator inhibitor-1 (PAI-1) is involved in the pathogenesis of inflammatory kidney injury; however, the regulatory mechanism of PAI-1 in injured kidneys remains unclear. PAI-1 is induced by interleukin (IL)-6 in patients with sepsis. In addition, the stabilization of IL-6 is regulated by the adenine-thymine-rich interactive domain-containing protein 5a (Arid5a). Therefore, the aim of the present study was to examine the involvement of Arid5a/IL-6/PAI-1 signaling in lipopolysaccharide (LPS)-induced inflammatory kidney injury. LPS treatment to C57BL/6J mice upregulated Pai-1 mRNA in the kidneys. Enzyme-linked immunosorbent assay (ELISA) revealed that PAI-1 expression was induced in the culture supernatants of LPS-treated human umbilical vein endothelial cells, but not in those of LPS-treated human kidney 2 (HK-2) cells, a tubular cell line. Combined with single-cell analysis, endothelial cells were found to be responsible for PAI-1 elevation in LPS-treated kidneys. Administration of TM5441, a PAI-1 inhibitor, reduced the urinary albumin/creatinine ratio, concomitant with downregulation of Il-6 and Arid5a mRNA expressions. IL-6 treatment in LPS model mice further upregulated Pai-1 mRNA expression compared with LPS alone, accompanied by renal impairment. Furthermore, the expression of Il-6 and Pai-1 mRNA was lower in Arid5a knockout mice than in wild-type mice after LPS treatment. Taken together, the vicious cycle of Arid5a/IL-6/PAI-1 signaling is involved in LPS-induced kidney injury.

Yes-associated protein activation potentiates glycogen synthase kinase-3 inhibitor-induced proliferation of neonatal cardiomyocytes and iPS cell-derived cardiomyocytes.

Because mammalian cardiomyocytes largely cease to proliferate immediately after birth, the regenerative activity of the heart is limited. To date, much effort has been made to clarify the regulatory mechanism of cardiomyocyte proliferation because the amplification of cardiomyocytes could be a promising strategy for heart regenerative therapy. Recently, it was reported that the inhibition of glycogen synthase kinase (GSK)-3 promotes the proliferation of neonatal rat cardiomyocytes (NRCMs) and human iPS cell-derived cardiomyocytes (hiPSC-CMs). Additionally, Yes-associated protein (YAP) induces cardiomyocyte proliferation. The purpose of this study was to address the importance of YAP activity in cardiomyocyte proliferation induced by GSK-3 inhibitors (GSK-3Is) to develop a novel strategy for cardiomyocyte amplification. Immunofluorescent microscopic analysis using an anti-Ki-67 antibody demonstrated that the treatment of NRCMs with GSK-3Is, such as BIO and CHIR99021, increased the ratio of proliferative cardiomyocytes. YAP was localized in the nuclei of more than 95% of cardiomyocytes, either in the presence or absence of GSK-3Is, indicating that YAP was endogenously activated. GSK-3Is increased the expression of ß-catenin and promoted its translocation into the nucleus without influencing YAP activity. The knockdown of YAP using siRNA or pharmacological inhibition of YAP using verteporfin or CIL56 dramatically reduced GSK-3I-induced cardiomyocyte proliferation without suppressing ß-catenin activation. Interestingly, the inhibition of GSK-3 also induced the proliferation of hiPSC-CMs under sparse culture conditions, where YAP was constitutively activated. In contrast, under dense culture conditions, in which YAP activity was suppressed, the proliferative effects of GSK-3Is on hiPSC-CMs were not detected. Importantly, the activation of YAP by the knockdown of α-catenin restored the proproliferative activity of GSK-3Is. Collectively, YAP activation potentiates the GSK-3I-induced proliferation of cardiomyocytes. The blockade of GSK-3 in combination with YAP activation resulted in remarkable amplification of cardiomyocytes.

Identification of biomarkers of chronic kidney disease among kidney-derived proteins.

BACKGROUND: Chronic kidney disease (CKD) has few objective symptoms, and it is difficult to make an early diagnosis by using existing methods. Therefore, new biomarkers enabling diagnosis of renal dysfunction at an early stage need to be developed. Here, we searched for new biomarkers of CKD by focusing on kidney-derived proteins that could sensitively reflect that organ's disease state. METHODS: To identify candidate marker proteins, we performed a proteomics analysis on renal influx and efflux blood collected from the same individual. RESULTS: Proteomics analysis revealed 662 proteins in influx blood and 809 in efflux. From these identified proteins, we selected complement C1q as a candidate; the plasma C1q level was significantly elevated in the renal efflux of donors. Moreover, the plasma concentration of C1q in a mouse model of diabetic nephropathy was significantly increased, in association with increases in blood glucose concentration and urinary protein content. Importantly, we demonstrated that the tendency of C1q to increase in the plasma of CKD patients was correlated with a decrease in their estimated glomerular filtration rate. CONCLUSION: Overall, our results indicate that our approach of focusing on kidney-derived proteins is useful for identifying new CKD biomarkers and that C1q has potential as a biomarker of renal function.

Transcription factor old astrocyte specifically induced substance is a novel regulator of kidney fibrosis.

Prevention of kidney fibrosis is an essential requisite for effective therapy in preventing chronic kidney disease (CKD). Here, we identify Old astrocyte specifically induced substance (OASIS)/cAMP responsive element-binding protein 3-like 1 (CREB3l1), a CREB/ATF family transcription factor, as a candidate profibrotic gene that drives the final common pathological step along the fibrotic pathway in CKD. Although microarray data from diseased patient kidneys and fibrotic mouse model kidneys both exhibit OASIS/Creb3l1 upregulation, the pathophysiological roles of OASIS in CKD remains unknown. Immunohistochemistry revealed that OASIS protein was overexpressed in human fibrotic kidney compared with normal kidney. Moreover, OASIS was upregulated in murine fibrotic kidneys, following unilateral ureteral obstruction (UUO), resulting in an increase in the number of OASIS-expressing pathological myofibroblasts. In vitro assays revealed exogenous TGF-ß1 increased OASIS expression coincident with fibroblast-to-myofibroblast transition and OASIS contributed to TGF-ß1-mediated myofibroblast migration and increased proliferation. Significantly, in vivo kidney fibrosis induced via UUO or ischemia/reperfusion injury was ameliorated by systemic genetic knockout of OASIS, accompanied by reduced myofibroblast proliferation. Microarrays revealed that the transmembrane glycoprotein Bone marrow stromal antigen 2 (Bst2) expression was reduced in OASIS knockout myofibroblasts. Interestingly, a systemic anti-Bst2 blocking antibody approach attenuated kidney fibrosis in normal mice but not in OASIS knockout mice after UUO, signifying Bst2 functions downstream of OASIS. Finally, myofibroblast-restricted OASIS conditional knockouts resulted in resistance to kidney fibrosis. Taken together, OASIS in myofibroblasts promotes kidney fibrosis, at least in part, via increased Bst2 expression. Thus, we have identified and demonstrated that OASIS signaling is a novel regulator of kidney fibrosis.

Maresin-1 induces cardiomyocyte hypertrophy through IGF-1 paracrine pathway.

The resolution of inflammation is closely linked with tissue repair. Recent studies have revealed that macrophages suppress inflammatory reactions by producing lipid mediators, called specialized proresolving mediators (SPMs); however, the biological significance of SPMs in tissue repair remains to be fully elucidated in the heart. In this study, we focused on maresin-1 (MaR1) and examined the reparative effects of MaR1 in cardiomyocytes. The treatment with MaR1 increased cell size in cultured neonatal rat cardiomyocytes. Since the expression of fetal cardiac genes was unchanged by MaR1, physiological hypertrophy was induced by MaR1. SR3335, an inhibitor of retinoic acid-related orphan receptor α (RORα), mitigated MaR1-induced cardiomyocyte hypertrophy, consistent with the recent report that RORα is one of MaR1 receptors. Importantly, in response to MaR1, cardiomyocytes produced IGF-1 via RORα. Moreover, MaR1 activated phosphoinositide 3-kinase (PI3K)/Akt signaling pathway and wortmannin, a PI3K inhibitor, or triciribine, an Akt inhibitor, abrogated MaR1-induced cardiomyocyte hypertrophy. Finally, the blockade of IGF-1 receptor by NVP-AEW541 inhibited MaR-1-induced cardiomyocyte hypertrophy as well as the activation of PI3K/Akt pathway. These data indicate that MaR1 induces cardiomyocyte hypertrophy through RORα/IGF-1/PI3K/Akt pathway. Considering that MaR1 is a potent resolving factor, MaR1 could be a key mediator that orchestrates the resolution of inflammation with myocardial repair.

PKNOX2 regulates myofibroblast functions and tubular cell survival during kidney fibrosis.

The number of patients with chronic kidney disease (CKD) is increasing worldwide. When kidneys are exposed to severe injury, tubular cell death occurs and kidney fibrosis progresses by activating fibroblasts and myofibroblasts (referred to as (myo)fibroblasts), leading to CKD; however, the pathological and molecular mechanisms underlying CKD, including kidney fibrosis, remain obscure. In the present study, we focused on a transcription factor PBX/Knotted Homeobox 2 (PKNOX2) in kidney fibrosis. The transcript and protein expression of PKNOX2 was upregulated in fibrotic kidneys after unilateral ureteral obstruction (UUO). Importantly, immunofluorescence microscopic analysis revealed that the number of PKNOX2-expressing myofibroblasts was increased, whereas the expression of PKNOX2 was decreased in proximal tubular epithelial cells after UUO. In (myo)fibroblasts, PKNOX2 was induced by TGF-ß1. Knockdown of PKNOX2 using shRNA lentiviral system reduced the viability of (myo)fibroblasts either in the presence or absence of TGF-ß1, accompanied by increased apoptosis. Moreover, PKNOX2 knockdown decreased TGF-ß1-induced migration of myofibroblasts and differentiation of fibroblasts into myofibroblasts. Significantly, knockdown of PKNOX2 also decreased the viability and increased apoptosis of tubular epithelial cells. Collectively, PKNOX2 regulates the function of (myo)fibroblasts and the viability of proximal tubular epithelial cells in progression of kidney fibrosis.

Mouse model of Epstein-Barr virus LMP1- and LMP2A-driven germinal center B-cell lymphoproliferative disease.

Epstein-Barr virus (EBV) is a major cause of immunosuppression-related B-cell lymphomas and Hodgkin lymphoma (HL). In these malignancies, EBV latent membrane protein 1 (LMP1) and LMP2A provide infected B cells with surrogate CD40 and B-cell receptor growth and survival signals. To gain insights into their synergistic in vivo roles in germinal center (GC) B cells, from which most EBV-driven lymphomas arise, we generated a mouse model with conditional GC B-cell LMP1 and LMP2A coexpression. LMP1 and LMP2A had limited effects in immunocompetent mice. However, upon T- and NK-cell depletion, LMP1/2A caused massive plasmablast outgrowth, organ damage, and death. RNA-sequencing analyses identified EBV oncoprotein effects on GC B-cell target genes, including up-regulation of multiple proinflammatory chemokines and master regulators of plasma cell differentiation. LMP1/2A coexpression also up-regulated key HL markers, including CD30 and mixed hematopoietic lineage markers. Collectively, our results highlight synergistic EBV membrane oncoprotein effects on GC B cells and provide a model for studies of their roles in immunosuppression-related lymphoproliferative diseases.

Myofibroblast ß2 adrenergic signaling amplifies cardiac hypertrophy in mice.

Abnormal ß-adrenergic signaling plays a central role in human heart failure. In mice, chronic ß-adrenergic receptor (ßAR) stimulation elicits cardiac hypertrophy. It has been reported that cultured cardiac fibroblasts express ßAR; however, the functional in vivo requirement of ßAR signaling in cardiac fibroblasts during the development of cardiac hypertrophy remains elusive. ß2AR null mice exhibited attenuated hypertrophic responses to chronic ßAR stimulation upon continuous infusion of an agonist, isoprenaline (ISO), compared to those in wildtype controls, suggesting that ß2AR activation in the heart induces pro-hypertrophic effects in mice. Since ß2AR signaling is protective in cardiomyocytes, we focused on ß2AR signaling in cardiac myofibroblasts. To determine whether ß2AR signaling in myofibroblasts affects cardiac hypertrophy, we generated myofibroblast-specific transgenic mice (TG) with the catalytic subunit of protein kinase A (PKAcα) using Cre-loxP system. Myofibroblast-specific PKAcα overexpression resulted in enhanced heart weight normalized to body weight ratio, associated with an enlargement of cardiomyocytes at 12 weeks of age, indicating that myofibroblast-specific activation of PKA mediates cardiac hypertrophy in mice. Neonatal rat cardiomyocytes stimulated with conditioned media from TG cardiac fibroblasts likewise exhibited significantly more growth than those from controls. Thus, ß2AR signaling in myofibroblasts plays a substantial role in ISO-induced cardiac hypertrophy, possibly due to a paracrine effect. ß2AR signaling in cardiac myofibroblasts may represent a promising target for development of novel therapies for cardiac hypertrophy.

Caveolae-specific activation loop between CaMKII and L-type Ca2+ channel aggravates cardiac hypertrophy in α1-adrenergic stimulation.

Activation of CaMKII induces a myriad of biological processes and plays dominant roles in cardiac hypertrophy. Caveolar microdomain contains many calcium/calmodulin-dependent kinase II (CaMKII) targets, including L-type Ca2+ channel (LTCC) complex, and serves as a signaling platform. The location of CaMKII activation is thought to be critical; however, the roles of CaMKII in caveolae are still elusive due to lack of methodology for the assessment of caveolae-specific activation. Our aim was to develop a novel tool for the specific analysis of CaMKII activation in caveolae and to determine the functional role of caveolar CaMKII in cardiac hypertrophy. To assess the caveolae-specific activation of CaMKII, we generated a fusion protein composed of phospholamban and caveolin-3 (cPLN-Cav3) and GFP fusion protein with caveolin-binding domain fused to CaMKII inhibitory peptide (CBD-GFP-AIP), which inhibits CaMKII activation specifically in caveolae. Caveolae-specific activation of CaMKII was detected using phosphospecific antibody for PLN (Thr17). Furthermore, adenoviral overexpression of LTCC ß2a-subunit (ß2a) in NRCMs showed its constitutive phosphorylation by CaMKII, which induces hypertrophy, and that both phosphorylation and hypertrophy are abolished by CBD-GFP-AIP expression, indicating that ß2a phosphorylation occurs specifically in caveolae. Finally, ß2a phosphorylation was observed after phenylephrine stimulation in ß2a-overexpressing mice, and attenuation of cardiac hypertrophy after chronic phenylephrine stimulation was observed in nonphosphorylated mutant of ß2a-overexpressing mice. We developed novel tools for the evaluation and inhibition of caveolae-specific activation of CaMKII. We demonstrated that phosphorylated ß2a dominantly localizes to caveolae and induces cardiac hypertrophy after α1-adrenergic stimulation in mice.NEW & NOTEWORTHY While signaling in caveolae is thought to be important in cardiac hypertrophy, direct evidence is missing due to lack of tools to assess caveolae-specific signaling. This is the first study to demonstrate caveolae-specific activation of CaMKII signaling in cardiac hypertrophy induced by α1-adrenergic stimulation using an originally developed tool.

Sustained Activation of Guanylate Cyclase-A with TDT, a Natriuretic Peptide Derivative, Exhibits Cardiorenal Protection in Dahl Salt-Sensitive Hypertensive Rats.

Heart failure often presents with prognosis-relevant impaired renal function. To investigate whether the chronic activation of guanylate cyclase-A (GC-A) protects both heart and kidney, we examined the effects of TDT, a neprilysin (NEP)-resistant natriuretic peptide (NP) derivative, on cardiac and renal dysfunction in Dahl salt-sensitive hypertensive (DS) rats. Pretreatment with NEP or NEP inhibitor did not influence GC-A activation by TDT both in vitro and in vivo, resulting in a long-acting profile of TDT compared with native human atrial NP (hANP). The repeated administration of TDT to DS rats suppressed the progress of cardiac hypertrophy, systolic/diastolic dysfunction, and proteinuria in a dose-dependent manner. Compared with vehicle and hANP, salt diet-induced podocyte injury was reduced by TDT, as analyzed by urinary podocalyxin concentration, renal expression of nephrin mRNA, and glomerular expression of desmin protein. Since glomerular TRPC6 plays detrimental roles in podocyte homeostasis, we examined the renal expression of TRPC6 in DS rats and found that salt diet upregulated the expression of TRPC6. Importantly, TRPC6 induction was significantly decreased in TDT-treated rats, compared with vehicle and hANP. Consistently, in primary-culture podocytes from DS rats, TDT inhibited ATP-induced calcium influx, similar to TRPC inhibitor SKF96365. Finally, TDT-mediated protection of podocytes was abolished by protein kinase G inhibitor KT5823. In conclusion, TDT treatment attenuated heart and kidney dysfunction, accompanied by podocyte protection through inhibition of TRPC6. Thus, long-acting NPs could be a new avenue for treatment of heart failure.

Moesin is activated in cardiomyocytes in experimental autoimmune myocarditis and mediates cytoskeletal reorganization with protrusion formation.

Acute myocarditis is a self-limiting disease. Most patients with myocarditis recover without cardiac dysfunction in spite of limited capacity of myocardial regeneration. Therefore, to address intrinsic reparative machinery of inflamed hearts, we investigated the cellular dynamics of cardiomyocytes in response to inflammation using experimental autoimmune myocarditis (EAM) model. EAM was induced by immunization of BALB/c mice with α-myosin heavy chain peptides twice. The inflammatory reaction was evoked with myocardial damage with the peak at 3 wk after the first immunization (EAM3w). Morphological and functional restoration started from EAM3w, when active protrusion formation, a critical process of myocardial healing, was observed in cardiomyocytes. Shotgun proteomics revealed that cytoskeletal proteins were preferentially increased in cardiomyocytes at EAM3w, compared with preimmunized (EAM0w) hearts, and that moesin was the most remarkably upregulated among them. Immunoblot analyses demonstrated that the expression of both total and phosphorylated moesin was upregulated in isolated cardiomyocytes from EAM3w hearts. Immunofluorescence staining showed that moesin was localized at cardiomyocyte protrusions at EAM3w. Adenoviral vectors expressing wild-type, constitutively active and inactive form of moesin (wtMoesin, caMoesin, and iaMoesin, respectively) were transfected in neonatal rat cardiomyocytes. The overexpression of wtMoesin and caMoesin resulted in protrusion formation, while not iaMoesin. Finally, we found that cardiomyocyte protrusions were accompanied by cell-cell contact formation. The expression of moesin was upregulated in cardiomyocytes under inflammation, inducing protrusion formation in a phosphorylation-dependent fashion. Moesin signal could be a novel therapeutic target that stimulates myocardial repair by promoting contact formation of cardiomyocytes.

Cardiac-specific ablation of the STAT3 gene in the subacute phase of myocardial infarction exacerbated cardiac remodeling.

STAT3 is a cardioprotective molecule against acute myocardial injury; however, recent studies have suggested that chronic STAT3 activation in genetically modified mice was detrimental after myocardial infarction (MI). In the present study, we assessed the biological significance of STAT3 activity in subacute MI using tamoxifen (TM)-inducible cardiac-specific STAT3 knockout (STAT3 iCKO) mice. After coronary ligation, STAT3 was rapidly activated in hearts, and its activation was sustained to the subacute phase. To make clear the pathophysiological roles of STAT3 activation specifically in subacute MI, MI was generated in STAT3 iCKO mice followed by TM treatment for 14 consecutive days beginning from day 11 after MI, which ablated the STAT3 gene in the subacute phase. Intriguingly, mortality was increased by TM treatment in STAT3 iCKO mice, accompanied by an increased heart weight-to-body weight ratio. Masson's trichrome staining demonstrated that cardiac fibrosis was dramatically exacerbated in STAT3 iCKO mice 24 days after MI (fibrotic circumference: 58.3 ± 6.7% in iCKO mice and 40.8 ± 9.3% in control mice), concomitant with increased expressions of fibrosis-related gene transcripts, including matrix metalloproteinase 9, procollagen 1, and procollagen 3. Echocardiography clarified that cardiac function was deteriorated in STAT3 iCKO mice (fractional shortening: 20.6 ± 4.1% in iCKO mice and 29.1 ± 6.0% in control mice). Dihydroethidium fluorescence analysis revealed that superoxide production was increased in STAT3 iCKO mice. Moreover, immunohistochemical analyses revealed that capillary density was decreased in STAT3 iCKO mice. Finally, STAT3 deletion in subacute MI evoked severe cardiac hypertrophy in the border zone. In conclusion, the intrinsic activity of STAT3 in the myocardium confers the resistance to cardiac remodeling in subacute MI.

Interleukin-27 induces the endothelial differentiation in Sca-1+ cardiac resident stem cells.

Cytokines play important roles in cardiac repair and regeneration. Recently, we demonstrated that interleukin (IL)-6 family cytokines induce the endothelial differentiation of Sca-1+ cardiac resident stem cells through STAT3/Pim-1 signaling pathway. In contrast, the biological functions of IL-12 family cytokines in heart remain to be elucidated, though they show structural homology with IL-6. In the present study, we examined the effects of IL-12 family cytokines on the transdifferentiation of cardiac Sca-1+ cells into cardiac cells. RT-PCR analyses revealed that IL-27 receptor α (IL-27Rα), but not IL-12R or IL-23R, was expressed in cardiac Sca-1+ cells. The transcript expression of IL-27 was elevated in murine hearts in cardiac injury models. Intriguingly, IL-27 stimulation for 14 days induced the endothelial cell (EC) marker genes, such as CD-31 and VE-cadherin. Immunoblot analyses clarified that IL-27 treatment rapidly phosphorylated STAT3. IL-27 upregulated the expression of Pim-1, but the overexpression of dominant negative STAT3 abrogated the induction of Pim-1 by IL-27. Finally, adenoviral transfection of dominant negative Pim-1 inhibited IL-27-induced EC differentiation of cardiac Sca-1+ cells. These findings demonstrated that IL-27 promoted the commitment of cardiac stem cells into the EC lineage, possibly leading to neovascularization as a novel biological function. IL-27 could not only regulate the inflammation but also contribute to the maintenance of the tissue homeostasis through stem cell differentiation at inflammatory sites.

Endothelial ROBO4 suppresses PTGS2/COX-2 expression and inflammatory diseases.

Accumulating evidence suggests that endothelial cells can be useful therapeutic targets. One of the potential targets is an endothelial cell-specific protein, Roundabout4 (ROBO4). ROBO4 has been shown to ameliorate multiple diseases in mice, including infectious diseases and sepsis. However, its mechanisms are not fully understood. In this study, using RNA-seq analysis, we found that ROBO4 downregulates prostaglandin-endoperoxide synthase 2 (PTGS2), which encodes cyclooxygenase-2. Mechanistic analysis reveals that ROBO4 interacts with IQ motif-containing GTPase-activating protein 1 (IQGAP1) and TNF receptor-associated factor 7 (TRAF7), a ubiquitin E3 ligase. In this complex, ROBO4 enhances IQGAP1 ubiquitination through TRAF7, inhibits prolonged RAC1 activation, and decreases PTGS2 expression in inflammatory endothelial cells. In addition, Robo4-deficiency in mice exacerbates PTGS2-associated inflammatory diseases, including arthritis, edema, and pain. Thus, we reveal the molecular mechanism by which ROBO4 suppresses the inflammatory response and vascular hyperpermeability, highlighting its potential as a promising therapeutic target for inflammatory diseases.

Myeloid-derived suppressor cells exacerbate poly(I:C)-induced lung inflammation in mice with renal injury and older mice.

Viral pneumonia is a global health burden with a high mortality rate, especially in the elderly and in patients with underlying diseases. Recent studies have found that myeloid-derived suppressor cells (MDSCs) are abundant in these patient groups; however, their roles in the progression of viral pneumonia remain unclear. In this study, we observed a substantial increase in MDSCs in a mouse model of renal ischemia/reperfusion (I/R) injury and in older mice. When intranasal polyinosinic-polycytidylic acid (poly(I:C)) administration was used to mimic viral pneumonia, mice with renal I/R injury exhibited more severe lung inflammation than sham mice challenged with poly(I:C). In addition, MDSC depletion attenuated lung inflammation in mice with I/R injury. Similar results were obtained in older mice compared with those in young mice. Furthermore, adoptive transfer of in vitro-differentiated MDSCs exacerbated poly(I:C)-induced lung inflammation. Taken together, these experimental results suggest that the increased proportion of MDSCs in mice with renal I/R injury and in older mice exacerbates poly(I:C)-induced lung inflammation. These findings have important implications for the treatment and prevention of severe lung inflammation caused by viral pneumonia.

Cardioprotective effect of Interleukin-11 against warm ischemia-reperfusion injury in a rat heart donor model.

Shortage of donor organs for heart transplantation is a worldwide problem. Donation after circulatory death (DCD) has been proposed to expand the donor pool. However, in contrast to the donation after brain death that undergoes immediate cold preservation, warm ischemia and subsequent reperfusion injury are inevitable in DCD. It has been reported that interleukin-11 (IL-11) mitigates ischemia-reperfusion injury in rodent models of myocardial infarction and donation after brain death heart transplantation. We hypothesized that IL-11 also offers benefit to warm ischemia in an experimental model of cardiac transplantation that resembles DCD. The hearts of naïve male Sprague Dawley rats (n = 15/group) were procured, subjected to 25-min warm ischemia, and reperfused for 60 min using Langendorff apparatus. IL-11 or saline was administered intravenously before the procurement, added to maintenance buffer, and infused via perfusion during reperfusion. IL-11 group exhibited significantly better cardiac function post-reperfusion. Severely damaged mitochondria was found in the electron microscopic analysis of control hearts whereas the mitochondrial structure was better preserved in the IL-11 treated hearts. Immunoblot analysis using neonatal rat cardiomyocytes revealed increased signal transducer and activator of transcription 3 (STAT3) phosphorylation at Ser727 after IL-11 treatment, suggesting its role in mitochondrial protection. Consistent with expected activation of mitochondrial respiration by mitochondrial STAT3, immunohistochemical staining demonstrated a higher mitochondrial cytochrome c oxidase subunit 2 expression. In summary, IL-11 protects the heart from warm ischemia reperfusion injury by alleviating mitochondrial injury and could be a viable therapeutic option for DCD heart transplantation.

Runx1 is upregulated by STAT3 and promotes proliferation of neonatal rat cardiomyocytes.

Though it is well known that mammalian cardiomyocytes exit cell cycle soon after birth, the mechanisms that regulate proliferation remain to be fully elucidated. Recent studies reported that cardiomyocytes undergo dedifferentiation before proliferation, indicating the importance of dedifferentiation in cardiomyocyte proliferation. Since Runx1 is expressed in dedifferentiated cardiomyocytes, Runx1 is widely used as a dedifferentiation marker of cardiomyocytes; however, little is known about the role of Runx1 in the proliferation of cardiomyocytes. The purpose of this study was to clarify the functional significance of Runx1 in cardiomyocyte proliferation. qRT-PCR analysis and immunoblot analysis demonstrated that Runx1 expression was upregulated in neonatal rat cardiomyocytes when cultured in the presence of FBS. Similarly, STAT3 was activated in the presence of FBS. Interestingly, knockdown of STAT3 significantly decreased Runx1 expression, indicating Runx1 is regulated by STAT3. We next investigated the effect of Runx1 on proliferation. Immunofluorescence microscopic analysis using an anti-Ki-67 antibody revealed that knockdown of Runx1 decreased the ratio of proliferating cardiomyocytes. Conversely, Runx1 overexpression using adenovirus vector induced cardiomyocyte proliferation in the absence of FBS. Finally, RNA-sequencing analysis revealed that Runx1 overexpression induced upregulation of cardiac fetal genes and downregulation of genes associated with fatty acid oxidation. Collectively, Runx1 is regulated by STAT3 and induces cardiomyocyte proliferation by juvenilizing cardiomyocytes.