Panjamaporn Sangwung

Antigen-specific, antibody-coated, exosome-like nanovesicles deliver suppressor T-cell microRNA-150 to effector T cells to inhibit contact sensitivity

BACKGROUND T-cell tolerance of allergic cutaneous contact sensitivity (CS) induced in mice by high doses of reactive hapten is mediated by suppressor cells that release antigen-specific suppressive nanovesicles. OBJECTIVE We sought to determine the mechanism or mechanisms of immune suppression mediated by the nanovesicles. METHODS T-cell tolerance was induced by means of intravenous injection of hapten conjugated to self-antigens of syngeneic erythrocytes and subsequent contact immunization with the same hapten. Lymph node and spleen cells from tolerized or control donors were harvested and cultured to produce a supernatant containing suppressive nanovesicles that were isolated from the tolerized mice for testing in active and adoptive cell-transfer models of CS. RESULTS Tolerance was shown due to exosome-like nanovesicles in the supernatants of CD8(+) suppressor T cells that were not regulatory T cells. Antigen specificity of the suppressive nanovesicles was conferred by a surface coat of antibody light chains or possibly whole antibody, allowing targeted delivery of selected inhibitory microRNA (miRNA)-150 to CS effector T cells. Nanovesicles also inhibited CS in actively sensitized mice after systemic injection at the peak of the responses. The role of antibody and miRNA-150 was established by tolerizing either panimmunoglobulin-deficient JH(-/-) or miRNA-150(-/-) mice that produced nonsuppressive nanovesicles. These nanovesicles could be made suppressive by adding antigen-specific antibody light chains or miRNA-150, respectively. CONCLUSIONS This is the first example of T-cell regulation through systemic transit of exosome-like nanovesicles delivering a chosen inhibitory miRNA to target effector T cells in an antigen-specific manner by a surface coating of antibody light chains.

Reticulon 4B (Nogo‐B) is a novel regulator of hepatic fibrosis

Nogo‐B, also known as Reticulon 4B, plays important roles in vascular injuries. Its function in the liver is not understood. The aim of this study was to characterize Nogo‐B in liver fibrosis and cirrhosis. Nogo‐B distribution was assessed in normal and cirrhotic human liver sections. We also determined the levels of liver fibrosis in wild‐type (WT) and Nogo‐A/B knockout (NGB KO) mice after sham operation or bile duct ligation (BDL). To investigate the mechanisms of Nogo‐Bs involvement in fibrosis, hepatic stellate cells were isolated from WT and NGB KO mice and transformed into myofibroblasts. Portal pressure was measured to test whether Nogo‐B gene deletion could ameliorate portal hypertension. In normal livers, Nogo‐B expression was found in nonparenchymal cells, whereas its expression in hepatocytes was minimal. Nogo‐B staining was significantly elevated in cirrhotic livers. Fibrosis was significantly increased in WT mice 4 weeks after BDL compared with NGB KO mice. The absence of Nogo‐B significantly reduced phosphorylation of Smad2 levels upon transforming growth factor β (TGF‐β) stimulation. Reconstitution of the Nogo‐B gene into NGB KO fibroblasts restored Smad2 phosphorylation. Four weeks after BDL, portal pressure was significantly increased in WT mice by 47%, compared with sham‐operated controls (P = 0.03), whereas such an increase in portal pressure was not observed in NGB KO mice (P = NS). Conclusion: Nogo‐B regulates liver fibrosis, at least in part, by facilitating the TGFβ/Smad2 signaling pathway in myofibroblasts. Because absence of Nogo‐B ameliorates liver fibrosis and portal hypertension, Nogo‐B blockade may be a potential therapeutic target in fibrosis/cirrhosis. (HEPATOLOGY 2011;)

Kruppel-like factor 15 is critical for vascular inflammation

Activation of cells intrinsic to the vessel wall is central to the initiation and progression of vascular inflammation. As the dominant cellular constituent of the vessel wall, vascular smooth muscle cells (VSMCs) and their functions are critical determinants of vascular disease. While factors that regulate VSMC proliferation and migration have been identified, the endogenous regulators of VSMC proinflammatory activation remain incompletely defined. The Kruppel-like family of transcription factors (KLFs) are important regulators of inflammation. In this study, we identified Kruppel-like factor 15 (KLF15) as an essential regulator of VSMC proinflammatory activation. KLF15 levels were markedly reduced in human atherosclerotic tissues. Mice with systemic and smooth muscle-specific deficiency of KLF15 exhibited an aggressive inflammatory vasculopathy in two distinct models of vascular disease: orthotopic carotid artery transplantation and diet-induced atherosclerosis. We demonstrated that KLF15 alters the acetylation status and activity of the proinflammatory factor NF-κB through direct interaction with the histone acetyltransferase p300. These studies identify a previously unrecognized KLF15-dependent pathway that regulates VSMC proinflammatory activation.

Kruppel-like factor 4 is critical for transcriptional control of cardiac mitochondrial homeostasis

Mitochondrial homeostasis is critical for tissue health, and mitochondrial dysfunction contributes to numerous diseases, including heart failure. Here, we have shown that the transcription factor Kruppel-like factor 4 (KLF4) governs mitochondrial biogenesis, metabolic function, dynamics, and autophagic clearance. Adult mice with cardiac-specific Klf4 deficiency developed cardiac dysfunction with aging or in response to pressure overload that was characterized by reduced myocardial ATP levels, elevated ROS, and marked alterations in mitochondrial shape, size, ultrastructure, and alignment. Evaluation of mitochondria isolated from KLF4-deficient hearts revealed a reduced respiration rate that is likely due to defects in electron transport chain complex I. Further, cardiac-specific, embryonic Klf4 deletion resulted in postnatal premature mortality, impaired mitochondrial biogenesis, and altered mitochondrial maturation. We determined that KLF4 binds to, cooperates with, and is requisite for optimal function of the estrogen-related receptor/PPARγ coactivator 1 (ERR/PGC-1) transcriptional regulatory module on metabolic and mitochondrial targets. Finally, we found that KLF4 regulates autophagy flux through transcriptional regulation of a broad array of autophagy genes in cardiomyocytes. Collectively, these findings identify KLF4 as a nodal transcriptional regulator of mitochondrial homeostasis.

Circadian control of bile acid synthesis by a KLF15- Fgf15 axis

Recent studies have shown that starburst dwarf galaxies have steeply rising rotation curves in their inner parts, pointing to a close link between the intense star formation and a centrally concentrated mass distribution (baryons and dark matter). More quiescent dwarf irregulars typically have slowly rising rotation curves, although some “compact” irregulars with steep, inner rotation curves exist. We analyze archival Hubble Space Telescope images of two nearby “compact” irregular galaxies (NGC 4190 and NGC 5204), which were selected solely on the basis of their dynamical properties and their proximity. We derive their recent star-formation histories by fitting colormagnitude diagrams of resolved stellar populations, and find that the star-formation properties of both galaxies are consistent with those of known starburst dwarfs. Despite the small sample, this strongly reinforces the notion that the starburst activity is closely related to the inner shape of the potential well.Circadian control of nutrient availability is critical to efficiently meet the energetic demands of an organism. Production of bile acids (BAs), which facilitate digestion and absorption of nutrients, is a major regulator of this process. Here we identify a KLF15-Fgf15 signalling axis that regulates circadian BA production. Systemic Klf15 deficiency disrupted circadian expression of key BA synthetic enzymes, tissue BA levels and triglyceride/cholesterol absorption. Studies in liver-specific Klf15-knockout mice suggested a non-hepatic basis for regulation of BA production. Ileal Fgf15 is a potent inhibitor of BA synthesis. Using a combination of biochemical, molecular and functional assays (including ileectomy and bile duct catheterization), we identify KLF15 as the first endogenous negative regulator of circadian Fgf15 expression. Elucidation of this novel pathway controlling circadian BA production has important implications for physiologic control of nutrient availability and metabolic homeostasis.

miR-483 Targeting of CTGF Suppresses Endothelial-to-Mesenchymal Transition: Therapeutic Implications in Kawasaki Disease.

Rationale: Endothelial–mesenchymal transition (EndoMT) is implicated in myofibroblast-like cell–mediated damage to the coronary arterial wall in acute Kawasaki disease (KD) patients, as evidenced by positive staining for connective tissue growth factor (CTGF) and EndoMT markers in KD autopsy tissues. However, little is known about the molecular basis of EndoMT involved in KD. Objective: We investigated the microRNA (miRNA) regulation of CTGF and the consequent EndoMT in KD pathogenesis. As well, the modulation of this process by statin therapy was studied. Methods and Results: Sera from healthy children and KD subjects were incubated with human umbilical vein endothelial cells. Cardiovascular disease–related miRNAs, CTGF, and EndoMT markers were quantified using reverse transcriptase quantitative polymerase chain reaction, ELISA, and Western blotting. Compared with healthy controls, human umbilical vein endothelial cell incubated with sera from acute KD patients had decreased miR-483, increased CTGF, and increased EndoMT markers. Bioinformatics analysis followed by functional validation demonstrated that Krüppel-like factor 4 (KLF4) transactivates miR-483, which in turn targets the 3′ untranslated region of CTGF mRNA. Overexpression of KLF4 or pre-miR-483 suppressed, whereas knockdown of KLF4 or anti-miR-483 enhanced, CTGF expression in endothelial cells in vitro and in vivo. Furthermore, atorvastatin, currently being tested in a phase I/IIa clinical trial in KD children, induced KLF4-miR-483, which suppressed CTGF and EndoMT in endothelial cells. Conclusions: KD sera suppress the KLF4-miR-483 axis in endothelial cells, leading to increased expression of CTGF and induction of EndoMT. This detrimental process in the endothelium may contribute to coronary artery abnormalities in KD patients. Statin therapy may benefit acute KD patients, in part, through the restoration of KLF4-miR-483 expression. Clinical Trial Registration: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01431105.

KLF2 and KLF4 control endothelial identity and vascular integrity

Maintenance of vascular integrity in the adult animal is needed for survival, and it is critically dependent on the endothelial lining, which controls barrier function, blood fluidity, and flow dynamics. However, nodal regulators that coordinate endothelial identity and function in the adult animal remain poorly characterized. Here, we show that endothelial KLF2 and KLF4 control a large segment of the endothelial transcriptome, thereby affecting virtually all key endothelial functions. Inducible endothelial-specific deletion of Klf2 and/or Klf4 reveals that a single allele of either gene is sufficient for survival, but absence of both (EC-DKO) results in acute death from myocardial infarction, heart failure, and stroke. EC-DKO animals exhibit profound compromise in vascular integrity and profound dysregulation of the coagulation system. Collectively, these studies establish an absolute requirement for KLF2/4 for maintenance of endothelial and vascular integrity in the adult animal.

Regulation of an Inflammatory Disease

This invited review summarizes work presented in the Russell Ross lecture delivered at the 2012 proceedings of the American Heart Association. We begin with a brief overview of the structural, cellular, and molecular biology of Kruppel-like factors. We then focus on discoveries during the past decade, implicating Kruppel-like factors as key determinants of vascular cell function in atherosclerotic vascular disease. # ATVB Named Lecture Review—Insight into Author {#article-title-115}This invited review summarizes work presented in the Russell Ross lecture delivered at the 2012 proceedings of the American Heart Association. We begin with a brief overview of the structural, cellular, and molecular biology of Krüppel-like factors. We then focus on discoveries during the past decade, implicating Krüppel-like factors as key determinants of vascular cell function in atherosclerotic vascular disease.

Regulation of an inflammatory disease: Krüppel-like factors and atherosclerosis.

This invited review summarizes work presented in the Russell Ross lecture delivered at the 2012 proceedings of the American Heart Association. We begin with a brief overview of the structural, cellular, and molecular biology of Kruppel-like factors. We then focus on discoveries during the past decade, implicating Kruppel-like factors as key determinants of vascular cell function in atherosclerotic vascular disease. # ATVB Named Lecture Review—Insight into Author {#article-title-115}This invited review summarizes work presented in the Russell Ross lecture delivered at the 2012 proceedings of the American Heart Association. We begin with a brief overview of the structural, cellular, and molecular biology of Krüppel-like factors. We then focus on discoveries during the past decade, implicating Krüppel-like factors as key determinants of vascular cell function in atherosclerotic vascular disease.

Proteomic identification of S-nitrosylated Golgi proteins: new insights into endothelial cell regulation by eNOS-derived NO.

Background Endothelial nitric oxide synthase (eNOS) is primarily localized on the Golgi apparatus and plasma membrane caveolae in endothelial cells. Previously, we demonstrated that protein S-nitrosylation occurs preferentially where eNOS is localized. Thus, in endothelial cells, Golgi proteins are likely to be targets for S-nitrosylation. The aim of this study was to identify S-nitrosylated Golgi proteins and attribute their S-nitrosylation to eNOS-derived nitric oxide in endothelial cells. Methods Golgi membranes were isolated from rat livers. S-nitrosylated Golgi proteins were determined by a modified biotin-switch assay coupled with mass spectrometry that allows the identification of the S-nitrosylated cysteine residue. The biotin switch assay followed by Western blot or immunoprecipitation using an S-nitrosocysteine antibody was also employed to validate S-nitrosylated proteins in endothelial cell lysates. Results Seventy-eight potential S-nitrosylated proteins and their target cysteine residues for S-nitrosylation were identified; 9 of them were Golgi-resident or Golgi/endoplasmic reticulum (ER)-associated proteins. Among these 9 proteins, S-nitrosylation of EMMPRIN and Golgi phosphoprotein 3 (GOLPH3) was verified in endothelial cells. Furthermore, S-nitrosylation of these proteins was found at the basal levels and increased in response to eNOS stimulation by the calcium ionophore A23187. Immunofluorescence microscopy and immunoprecipitation showed that EMMPRIN and GOLPH3 are co-localized with eNOS at the Golgi apparatus in endothelial cells. S-nitrosylation of EMMPRIN was notably increased in the aorta of cirrhotic rats. Conclusion Our data suggest that the selective S-nitrosylation of EMMPRIN and GOLPH3 at the Golgi apparatus in endothelial cells results from the physical proximity to eNOS-derived nitric oxide.