PFKFB3-mediated glycolysis in hepatic stellate cells promotes liver regeneration.

Hepatic stellate cells (HSCs) perform a significant function in liver regeneration (LR) by becoming active. We propose to investigate if activated HSCs enhance glycolysis via PFKFB3, an essential glycolytic regulator, and whether targeting this pathway could be beneficial for LR. The liver and isolated HSCs of mice subjected to 2/3 partial hepatectomy (PHx) exhibited a significant rise in PFKFB3 expression, as indicated by quantitative RT-PCR analyses and Western blotting. Also, the primary HSCs of mice subjected to PHx have a significant elevation of the glycolysis level. Knocking down PFKFB3 significantly diminished the enhancement of glycolysis by PDGF in human LX2 cells. The hepatocyte proliferation in mice treated with PHx was almost completely prevented when the PFKFB3 inhibitor 3PO was administered, emerging that PFKFB3 is essential in LR. Furthermore, there was a decline in mRNA expression of immediate early genes and proinflammatory cytokines. In terms of mechanism, both the p38 MAP kinase and ERK1/2 phosphorylation in LO2 cells and LO2 proliferation were significantly reduced by the conditioned medium (CM) obtained from LX2 cells with either PFKFB3 knockdown or inhibition. Compared to the control group, isolated hepatocytes from 3PO-treated mice showed decreased p38 MAP kinase and ERK1/2 phosphorylation and proliferation. Thus, LR after PHx involves the activation of PFKFB3 in HSCs, which enhances glycolysis and promotes lactate production, thereby facilitating hepatocyte proliferation via the p38/ERK MAPK signaling pathway.

Endothelial POFUT1 controls injury-induced liver fibrosis by repressing fibrinogen synthesis.

BACKGROUND & AIMS: NOTCH signaling in liver sinusoidal endothelial cells (LSECs) regulates liver fibrosis, a pathological feature of chronic liver diseases. POFUT1 is an essential regulator of NOTCH signaling. Here, we investigated the role of LSEC-expressed POFUT1 in liver fibrosis. METHODS: Endothelial-specific Pofut1 knockout mice were generated and experimental liver fibrosis was induced by chronic carbon tetrachloride exposure or common bile duct ligation. Liver samples were assessed by ELISA, histology, electron microscopy, immunostaining and RNA in situ hybridization. LSECs and hepatic stellate cells (HSCs) were isolated for gene expression analysis by RNA sequencing, qPCR, and western blotting. Signaling crosstalk between LSECs and HSCs was investigated by treating HSCs with supernatant from LSEC cultures. Liver single-cell RNA sequencing datasets from patients with cirrhosis and healthy individuals were analyzed to evaluate the clinical relevance of gene expression changes observed in mouse studies. RESULTS: POFUT1 loss promoted injury-induced LSEC capillarization and HSC activation, leading to aggravated liver fibrosis. RNA sequencing analysis revealed that POFUT1 deficiency upregulated fibrinogen expression in LSECs. Consistently, fibrinogen was elevated in LSECs of patients with cirrhosis. HSCs treated with supernatant from LSECs of Pofut1 null mice showed exacerbated activation compared to those treated with supernatant from control LSECs, and this effect was attenuated by knockdown of fibrinogen or by pharmacological inhibition of fibrinogen receptor signaling, altogether suggesting that LSEC-derived fibrinogen induced the activation of HSCs. Mechanistically, POFUT1 loss augmented fibrinogen expression by enhancing NOTCH/HES1/STAT3 signaling. CONCLUSIONS: Endothelial POFUT1 prevents injury-induced liver fibrosis by repressing the expression of fibrinogen, which functions as a profibrotic paracrine signal to activate HSCs. Therapies targeting the POFUT1/fibrinogen axis offer a promising strategy for the prevention and treatment of fibrotic liver diseases. IMPACT AND IMPLICATIONS: Paracrine signals produced by liver vasculature play a major role in the development of liver fibrosis, which is a pathological hallmark of most liver diseases. Identifying those paracrine signals is clinically relevant in that they may serve as therapeutic targets. In this study, we discovered that genetic deletion of Pofut1 aggravated experimental liver fibrosis in mouse models. Moreover, fibrinogen was identified as a downstream target repressed by Pofut1 in liver endothelial cells and functioned as a novel paracrine signal that drove liver fibrosis. In addition, fibrinogen was found to be relevant to cirrhosis and may serve as a potential therapeutic target for this devastating human disease.

Epigenetic repression of Cend1 by lysine-specific demethylase 1 is essential for murine heart development.

Epigenetic regulation of heart development remains incompletely understood. Here we show that LSD1, a histone demethylase, plays a crucial role in regulating cardiomyocyte proliferation during heart development. Cardiomyocyte-specific deletion of Lsd1 in mice inhibited cardiomyocyte proliferation, causing severe growth defect of embryonic and neonatal heart. In vivo RNA-seq and in vitro functional studies identified Cend1 as a target suppressed by LSD1. Lsd1 loss resulted in elevated Cend1 transcription associated with increased active histone mark H3K4me2 at Cend1 promoter. Cend1 knockdown relieved the cell-cycle arrest and proliferation defect caused by LSD1 inhibition in primary rat cardiomyocytes. Moreover, genetic deletion of Cend1 rescued cardiomyocyte proliferation defect and embryonic lethality in Lsd1 null embryos. Consistently, LSD1 promoted the cell cycle of cardiomyocytes derived from human-induced pluripotent stem cells by repressing CEND1. Together, these findings reveal an epigenetic regulatory mechanism involving the LSD1-CEND1 axis that controls cardiomyocyte proliferation essential for murine heart development.

S-nitrosylation attenuates pregnane X receptor hyperactivity and acetaminophen-induced liver injury.

Drug-induced liver injury (DILI), especially acetaminophen overdose, is the leading cause of acute liver failure. Pregnane X receptor (PXR) is a nuclear receptor and the master regulator of drug metabolism. Aberrant activation of PXR plays a pathogenic role in the acetaminophen hepatotoxicity. Here, we aimed to examine the S-nitrosylation of PXR (SNO-PXR) in response to acetaminophen. We found that PXR was S-nitrosylated in hepatocytes and the mouse livers after exposure to acetaminophen or S-nitrosoglutathione (GSNO). Mass spectrometry and site-directed mutagenesis identified the cysteine 307 as the primary residue for S-nitrosylation (SNO) modification. In hepatocytes, SNO suppressed both agonist-induced (rifampicin and SR12813) and constitutively active PXR (VP-PXR, a human PXR fused to the minimal transactivator domain of the herpes virus transcription factor VP16) activations. Furthermore, in acetaminophen-overdosed mouse livers, PXR protein was decreased at the centrilobular regions overlapping with increased SNO. In PXR-/- mice, replenishing the livers with the SNO-deficient PXR significantly aggravated hepatic necrosis, increased HMGB1 release, and exacerbated liver injury and inflammation. Particularly, we demonstrated that S-nitrosoglutathione reductase (GSNOR) inhibitor N6022 promoted hepatoprotection by increasing the levels of SNO-PXR. In conclusion, PXR is posttranslationally modified by SNO in hepatocytes in response to acetaminophen. This modification mitigated the acetaminophen-induced PXR hyperactivity. It may serve as a target for therapeutical intervention.

221S-1a inhibits endothelial proliferation in pathological angiogenesis through ERK/c-Myc signaling.

Pathological angiogenesis plays a major role in many disease processes, including cancer and diabetic retinopathy. Antiangiogenic therapy is a potential management for pathologic angiogenesis. The novel synthetic compound 221S-1a, derived from captopril, tanshinol and borneol, may have antiangiogenic properties. On the basis of MS, NMR and HPLC analysis, the structure of 221S-1a was identified. The cellular uptake and metabolism of this compound was also observed. Next, the antiangiogenic properties of 221S-1a were evaluated in tumor-xenograft and OIR models in vivo. The inhibitory properties of 221S-1a on endothelial cell proliferation, migration, tube formation and sprouting were detected in vitro. Furthermore, 221S-1a induced G1/S phase arrest was detected by PI staining flow cytometry analysis and Cyclin D, Cyclin E expression. 221S-1a inhibited ERK1/2 activation and nuclear translocation, in addition to downregulation of c-Myc, a transcription factor that regulates cell cycle progression. Molecular docking indicated the interaction of 221S-1a with the ATP-binding site of ERK2, leading to the inhibition of ERK2 phosphorylation and a concomitant inhibition of ERK1 phosphorylation. In conclusion, 221S-1a inhibited the G1/S phase transition by blocking the ERK1/2/c-Myc pathway to reduce tumor and OIR retinal angiogenesis. These novel findings suggest that 221S-1a is a potential pharmacologic candidate for treating pathological angiogenesis.

YAP-galectin-3 signaling mediates endothelial dysfunction in angiotensin II-induced hypertension in mice.

BACKGROUND: Vascular endothelial dysfunction is regarded as an early event of hypertension. Galectin-3 (Gal-3) is known to participate in various pathological processes. Whilst previous studies showed that inhibition of Gal-3 effectively ameliorates angiotensin II (Ang II)-induced atherosclerosis or hypertension, it remains unclear whether Ang II regulates Gal-3 expression and actions in vascular endothelium. METHODS: Using techniques of molecular biology and myograph, we investigated Ang II-mediated changes in Gal-3 expression and activity in thoracic aortas and mesenteric arteries from wild-type and Gal-3 gene deleted (Gal-3-/-) mice and cultured endothelial cells. RESULTS: The serum level of Gal-3 was significantly higher in hypertensive patients or in mice with chronic Ang II-infusion. Ang II infusion to wild-type mice enhanced Gal-3 expression in the aortic and mesenteric arteries, elevated systolic blood pressure and impaired endothelium-dependent relaxation of the thoracic aortas and mesenteric arteries, changes that were abolished in Gal-3-/- mice. In human umbilical vein endothelial cells, Ang II significantly upregulated Gal-3 expression by promoting nuclear localization of Yes-associated protein (YAP) and its interaction with transcription factor Tead1 with enhanced YAP/Tead1 binding to Gal-3 gene promoter region. Furthermore, Gal-3 deletion augmented the bioavailability of nitric oxide, suppressed oxidative stress, and alleviated inflammation in the thoracic aorta of Ang II-infused mice or endothelial cells exposed to Ang II. CONCLUSIONS: Our results demonstrate for the first time that Ang II upregulates Gal-3 expression via increment in YAP nuclear localization in vascular endothelium, and that Gal-3 mediates endothelial dysfunction contributing to the development of hypertension.

Free fatty acids stabilize integrin ß1via S-nitrosylation to promote monocyte-endothelial adhesion.

Hyperlipidemia characterized by high blood levels of free fatty acids (FFAs) is important for the progression of inflammatory cardiovascular diseases. Integrin ß1 is a transmembrane receptor that drives various cellular functions, including differentiation, migration, and phagocytosis. However, the underlying mechanisms modifying integrin ß1 protein and activity in mediating monocyte/macrophage adhesion to endothelium remain poorly understood. In this study, we demonstrated that integrin ß1 protein underwent S-nitrosylation in response to nitrosative stress in macrophages. To examine the effect of elevated levels of FFA on the modulation of integrin ß1 expression, we treated the macrophages with a combination of oleic acid and palmitic acid (2:1) and found that FFA activated inducible nitric oxide synthase/nitric oxide and increased the integrin ß1 protein level without altering the mRNA level. FFA promoted integrin ß1 S-nitrosylation via inducible nitric oxide synthase/nitric oxide and prevented its degradation by decreasing binding to E3 ubiquitin ligase c-Cbl. Furthermore, we found that increased integrin α4ß1 heterodimerization resulted in monocyte/macrophage adhesion to endothelium. In conclusion, these results provided novel evidence that FFA-stimulated N--O stabilizes integrin ß1via S-nitrosylation, favoring integrin α4ß1 ligation to promote vascular inflammation.

CCN1/Integrin α5ß1 Instigates Free Fatty Acid-Induced Hepatocyte Lipid Accumulation and Pyroptosis through NLRP3 Inflammasome Activation.

Hyperlipidemia with high blood levels of free fatty acids (FFA) is the leading cause of non-alcoholic steatohepatitis. CCN1 is a secreted matricellular protein that drives various cellular functions, including proliferation, migration, and differentiation. However, its role in mediating FFA-induced pro-inflammatory cell death and its underlying molecular mechanisms have not been characterized. In this study, we demonstrated that CCN1 was upregulated in the livers of obese mice. The increase in FFA-induced CCN1 was evaluated in vitro by treating hepatocytes with a combination of oleic acid and palmitic acid (2:1). Gene silencing using specific small interfering RNAs (siRNA) revealed that CCN1 participated in FFA-induced intracellular lipid accumulation, caspase-1 activation, and hepatocyte pyroptosis. Next, we identified integrin α5ß1 as a potential receptor of CCN1. Co-immunoprecipitation demonstrated that the binding between CCN1 and integrin α5ß1 increased in hepatocytes upon FFA stimulation in the livers of obese mice. Similarly, the protein levels of integrin α5 and ß1 were increased in vitro and in vivo. Experiments with specific siRNAs confirmed that integrin α5ß1 played a part in FFA-induced intracellular lipid accumulation, NLRP3 inflammasome activation, and pyroptosis in hepatocytes. In conclusion, these results provide novel evidence that the CCN1/integrin α5ß1 is a novel mediator that drives hepatic lipotoxicity via NLRP3-dependent pyroptosis.

A pan-cancer analysis confirms PTPN11's potential as a prognostic and immunological biomarker.

Protein tyrosine phosphatase, non-receptor type 11 (PTPN11) is a multifunctional tyrosine phosphatase and has a significant part in many types of tumors. As of yet, neither the expression profile of PTPN11 nor its significance in pan-cancer diagnosis has been clarified. With the assistance of The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO), we have comprehensively mapped the expression profiles, prognostic significance, genetic alteration, phosphorylation status, infiltration of immune cells, and functional properties of PTPN11 in 33 human tumors. There was an inconsistent expression of PTPN11 in different tumors, and the alteration of PTPN11 expression predicted the survival outcomes of cancer patients. A significant association was found between the genetic alteration levels of PTPN11 and some tumor predictions. Besides, the reduced PTPN11 phosphorylation levels were observed in breast cancer, clear cell RCC, head and neck carcinoma, and lung adenocarcinoma (LUAD). Furthermore, there was a significant association between PTPN11 expression and infiltration of cancer-associated fibroblasts and endothelial cells, along with tumor mutation burden, microsatellite instability, mismatch repair genes, and immunoregulators. Finally, pathway enrichment analysis demonstrated that PTPN11-associated terms and pathways were involved in malignancy. Taken together, PTPN11 may become a new biomarker and target for cancer therapy.

Procyanidin B2 Attenuates Nicotine-Induced Hepatocyte Pyroptosis through a PPARγ-Dependent Mechanism.

Procyanidin B2 (PCB2), a natural flavonoid, has been demonstrated to exert anti-oxidation and anti-inflammatory effects on hepatic diseases. Increasing evidence shows the hepatoxicity of nicotine. However, whether PCB2 protects against nicotine-induced hepatoxicity and the underlying mechanisms remains uncharacterized. Here, we reported that nicotine promoted hepatocyte pyroptosis, as evidenced by the elevation of propidium iodide (PI)-positive cells, the activation of Caspase-1 and gasdermin D (GSDMD), the enhanced expression of NOD-like receptor containing pyrin domain 3 (NLRP3) and the increased release of lactate dehydrogenase (LDH), interleukin (IL)-1ß and IL-18. The silencing of GSDMD by small interfering RNA (siRNA) efficiently inhibited the release of LDH and the secretion of IL-1ß and IL-18. In addition, rosiglitazone (RGZ) prevented hepatocyte pyroptosis induced by nicotine. Furthermore, we showed that PCB2 attenuated nicotine-induced pyroptosis through the activation of peroxisome proliferator-activated receptor-γ (PPARγ) in hepatocytes. Moreover, administration of PCB2 ameliorated liver injury and hepatocyte pyroptosis in nicotine-treated mice. Hence, our findings demonstrated that PCB2 attenuated pyroptosis and liver damage in a PPARγ-dependent manner. Our results suggest a new mechanism by which PCB2 exerts its liver protective effects.

Endothelial MicroRNA-483-3p Is Hypertension-Protective.

Hypertension is a high-risk factor for developing coronary heart disease and stroke. Endothelial dysfunction and arterial remodeling can lead to increased vascular wall thickness and arterial stiffness. Previous studies showed that microRNA-483 (miR-483) enhances endothelial cell (EC) function. Here, we investigated the protective role of miR-483 in hypertension. Data collected from two patient cohorts showed that the serum miR-483-3p level was associated with the progression of hypertension and positively correlated with vascular function. In cultured ECs, miR-483 targets a number of endothelial dysfunction-related genes, such as transforming growth factor-ß (TGF-ß), connective tissue growth factor (CTGF), angiotensin-converting enzyme 1 (ACE1), and endothelin-1 (ET-1). Overexpression of miR-483-3p in ECs inhibited Ang II-induced endothelial dysfunction, revealed by the decreased expression of TGF-ß, CTGF, ACE1, and ET-1. Furthermore, miR-483-3p secreted from ECs was taken up by smooth muscle cells (SMCs) via the exosome pathway, which also decreased these genes in SMCs. Additionally, telmisartan could increase the aortic and serum levels of miR-483-3p in hypertension patients and spontaneous hypertension rats (SHR). These findings suggest that miR-483-3p exerts a protective effect on EC function during the onset of hypertension and thus may be considered a potential therapeutic target for hypertension-related cardiovascular diseases.

Xenobiotic Receptor CAR Is Highly Induced in Psoriasis and Promotes Keratinocyte Proliferation.

Psoriasis is a chronic inflammatory skin disease with abnormal epidermal proliferation. Xenobiotics contribute to the pathogenesis of psoriasis. The mechanism linking xenobiotic stimuli with epidermal proliferation remains largely unknown. In this study, we investigated the role of CAR, a nuclear receptor (NR1I3) responsible for xenobiotics detoxification. We showed that CAR and its target genes were induced in the lesions from patients with psoriasis and imiquimod-treated mice. Proinflammatory cytokines (IL-17A, IL-22, oncostatin M, IL-1α, and TNF-α) synergistically increased the expressions of CAR and its target genes in both human and mouse keratinocytes. Overexpression of CAR promoted the G1/S transition by regulating cyclin E and c-Myc expressions, whereas the silencing of CAR attenuated it. Importantly, a selective CAR agonist 6-(4-chlorophenyl)imidazo(2,1-b)(1,3)thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime or the proinflammatory cytokines induced cyclin E and c-Myc, which were largely blocked by clotrimazole, a selective CAR antagonist, or CAR small interfering RNA. In addition, we showed that topical application of 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene, a selective agonist for mouse CAR, exacerbated the IMQ-induced psoriasis lesions with increased expressions of proliferative and inflammatory markers. In contrast, Car-knockout mice developed significantly milder lesions. In conclusion, these results showed that CAR plays a pathogenic role and, potentially, may be a target for the treatment of psoriasis.

AMPK upregulates KCa2.3 channels and ameliorates endothelial dysfunction in diet-induced obese mice.

The opening of endothelial small-conductance calcium-activated potassium channels (KCa2.3) is essential for endothelium-dependent hyperpolarization (EDH), which predominantly occurs in small resistance arteries. Adenosine monophosphate-activated protein kinase (AMPK), an important metabolic regulator, has been implicated in regulating endothelial nitric oxide synthase activity. However, it was unclear whether AMPK regulated endothelial KCa2.3-mediated EDH-type vasodilation. Using bioinformatics analysis and myograph system, we investigated the regulation by AMPK of KCa2.3 in human umbilical vein endothelial cells (HUVECs) or mouse second-order mesenteric resistance arteries. In HUVECs, AMPK activation either by activators (AICAR, A769662 and MK-8722) or expression of the constitutively active form of AMPK significantly upregulated KCa2.3 expression. Such effects were abolished by AMPK inhibitor (compound C) or AMPK α1-/α2-siRNA, extracellular-signal-regulated-kinase 5 (ERK5) inhibitor (ERK5-IN-1), and specific siRNA to myocyte-enhancer factor 2 (MEF2) or krüppel-like factor 2/4 (KLF2/4). KCa2.3 expression was significantly reduced in mesenteric resistance arteries in AMPKα2 knockout mice when compared with littermate control mice. Furthermore, in high-fat diet fed mice, 2-week treatment with AICAR restored endothelial KCa2.3 expression in mesenteric resistance arteries with improved endothelial dysfunction. Our results demonstrate that activation of AMPK upregulates KCa2.3 channel expression through the ERK5-MEF2-KLF2/4 signaling pathway in vascular endothelium, which contributes to benefits through KCa2.3-mediated EDH-type vasodilation in mesenteric resistance arteries.

PPARγ induces NEDD4 gene expression to promote autophagy and insulin action.

The E3 ubiquitin ligase neural precursor cell-expressed developmentally down-regulated protein 4 (NEDD4) plays a crucial role in governing a number of signaling pathways, including insulin and autophagy signaling. However, the molecular mechanism by which NEDD4 gene is transcriptionally regulated has not been fully elucidated. Here, we reported that NEDD4 mRNA and protein levels were increased by peroxisome proliferator-activated receptor-γ (PPARγ) in HepG2 hepatocytes. PPARγ antagonist GW9662 abolished thiazolidinedione (TZD)-induced NEDD4 expression. ChIP and luciferase reporter assays showed that PPARγ directly bound to the potential PPAR-responsive elements (PPREs) within the promoter region of the human NEDD4 gene. In addition, TZDs increased Akt phosphorylation and glucose uptake, which were abrogated through NEDD4 depletion. Furthermore, we showed that NEDD4-mediated autophagy induction and Akt phosphorylation were suppressed by oleic acid and high glucose treatment, activation of PPARγ successfully prevented this suppression. In conclusion, these results suggest that PPARγ plays a novel role in linking glucose metabolism and protein homeostasis through NEDD4-mediated effects on the autophagy machinery.

APC/Cdh1 targets PECAM-1 for ubiquitination and degradation in endothelial cells.

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a member of the immunoglobulin superfamily and is expressed by hematopoietic and endothelial cells (ECs). Recent studies have shown that PECAM-1 plays a crucial role in promoting the development of the EC inflammatory response in the context of disturbed flow. However, the mechanistic pathways that control PECAM-1 protein stability remain largely unclear. Here, we identified PECAM-1 as a novel substrate of the APC/Cdh1 E3 ubiquitin ligase. Specifically, lentivirus-mediated Cdh1 depletion stabilized PECAM-1 in ECs. Conversely, overexpression of Cdh1 destabilized PECAM-1. The proteasome inhibitor MG132 blocked Cdh1-mediated PECAM-1 degradation. In addition, Cdh1 promoted K48-linked polyubiquitination of PECAM-1 in a destruction box-dependent manner. Furthermore, we demonstrated that compared with pulsatile shear stress (PS), oscillatory shear stress decreased the expression of Cdh1 and the ubiquitination of PECAM-1, therefore stabilizing PECAM-1 to promote inflammation in ECs. Hence, our study revealed a novel mechanism by which fluid flow patterns regulate EC homeostasis via Cdh1-dependent ubiquitination and subsequent degradation of PECAM-1.

Gal-3 (Galectin-3) and KCa3.1 Mediate Heterogeneous Cell Coupling and Myocardial Fibrogenesis Driven by ßAR (ß-Adrenoceptor) Activation.

Heart failure is associated with sympatho-ßAR (ß-adrenoceptor) activation and cardiac fibrosis. Gal-3 (galectin-3) and KCa3.1 channels that are upregulated in diverse cells of diseased heart are implicated in mediating myocardial inflammation and fibrosis. It remains unclear whether Gal-3 interacts with KCa3.1 leading to cardiac fibrosis in the setting of ßAR activation. We tested the effect of KCa3.1 blocker TRAM-34 on cardiac fibrosis and inflammation in cardiac-restricted ß2-TG (ß2AR overexpressed transgenic) mice and determined KCa3.1 expression in ß2-TG×Gal-3-/- mouse hearts. Mechanisms of KCa3.1 in mediating Gal-3 induced fibroblast activation were studied ex vivo. Expression of Gal-3 and KCa3.1 was elevated in ß2-TG hearts. Gal-3 gene deletion in ß2-TG mice decreased KCa3.1 expression in inflammatory cells but not in fibroblasts. Treatment of ß2-TG mice with TRAM-34 for 1 or 2 months significantly ameliorated cardiac inflammation and fibrosis and reduced Gal-3 level. In cultured fibroblasts, Gal-3 upregulated KCa3.1 expression and channel currents with enhanced membrane potential and Ca2+ entry through TRPV4 (transient receptor potential V4) and TRPC6 (transient receptor potential C6) channels leading to fibroblast activation. In conclusion, ßAR stimulation promotes Gal-3 production that upregulates KCa3.1 channels in noncardiomyocyte cells and activates KCa3.1 channels in fibroblasts leading to hyperpolarization of membrane potential and Ca2+ entry via TRP channels. Gal-3-KCa3.1 signaling mobilizes diverse cells facilitating regional inflammation and fibroblast activation and hence myocardial fibrosis.

KCa3.1 channel mediates inflammatory signaling of pancreatic ß cells and progression of type 2 diabetes mellitus.

Chronic islet inflammation is associated with development of type 2 diabetes mellitus (T2DM). Intermediate-conductance calcium-activated K+ (KCa3.1) channel plays an important role in inflammatory diseases. However, the role and regulation of KCa3.1 in pancreatic ß cells in progression of T2DM remain unclarified. In the present study, we evaluated the effect of the specific KCa3.1 channel blocker 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34) on diabetic phenotype in the db/db model. In diabetic mice, blockade of KCa3.1 significantly improved glucose tolerance, enhanced secretion of postprandial insulin level, and reduced loss of ß-cell mass through attenuating the expression and secretion of inflammatory mediators. Furthermore, in cultured pancreatic ß cells, exposure to high levels of glucose or palmitic acid significantly increased expression and current density of the KCa3.1 channel as well as secretion of proinflammatory chemokines, and the effects were similarly reversed by preincubation with TRAM-34 or a NF-κB inhibitor pyrrolidinedithiocarbamate. Additionally, expression of KCa3.1 in pancreas islet cells was up-regulated by activation of NF-κB with IL-1ß stimulation. In summary, up-regulated KCa3.1 due to activation of NF-κB pathway leads to pancreatic inflammation via expression and secretion of chemokines and cytokines by pancreatic ß cells, thereby facilitating progression of T2DM.-Pang, Z.-D., Wang, Y., Wang, X.-J., She, G., Ma, X.-Z., Song, Z., Zhao, L.-M., Wang, H.-F., Lai, B.-C., Gou, W., Du, X.-J., Deng, X.-L. KCa3.1 channel mediates inflammatory signaling of pancreatic ß cells and progression of type 2 diabetes mellitus.

Procyanidin B2 Activates PPARγ to Induce M2 Polarization in Mouse Macrophages.

Procyanidins, a subclass of flavonoids found in commonly consumed foods, possess potential anti-inflammatory activity. Manipulation of M1/M2 macrophage homeostasis is an effective strategy for the treatment of metabolic inflammatory diseases. The objective of this study was to determine the effect of procyanidins on macrophage polarization. Procyanidin B2 (PCB2), the most widely distributed natural procyanidins, enhanced the expressions of M2 macrophage markers (Arg1, Ym1, and Fizz1). PCB2 activated peroxisome proliferator-activated receptor γ (PPARγ) activity and increased the expressions of PPARγ target genes (CD36 and ABCG1) in macrophages. Inhibition of PPARγ using siRNA or antagonist GW9662 attenuated the PCB2-induced expressions of M2 macrophage markers. In addition, we identified cognate PPAR-responsive elements (PPREs) within the 5'-flanking regions of the mouse Arg1, Ym1, and Fizz1 genes. Furthermore, macrophages isolated from db/db diabetic mice showed lower expressions of M2 markers. PCB2 effectively restored the Arg1, Ym1, and Fizz1 expressions in a PPARγ-dependent manner. These findings support the notion that PCB2 regulated macrophage M2 polarization via the activation of PPARγ. Our results provide a new mechanism by which procyanidins exert their beneficial anti-inflammatory effects.

Xenobiotic pregnane X receptor promotes neointimal formation in balloon-injured rat carotid arteries.

Pregnane X receptor (PXR) is a member of nuclear receptor superfamily and responsible for the detoxification of xenobiotics. Recent studies demonstrated that PXR was also expressed in the vasculature and protected the vessels from endogenous and exogenous insults, thus representing a novel gatekeeper in vascular defense. In this study, we examined the potential function of PXR in the neointimal formation following vascular injury. In the rat carotid artery after balloon injury, overexpression of a constitutively active PXR increased the intima-to-media ratio in the injured region. PXR increased cell proliferation and migration in cultured rat aortic smooth muscle cells (SMCs) by inducing the expressions of cyclins (cyclin A, D1, and E) and cyclin-dependent kinase 2. In addition, PXR increased the phosphorylation and activation of extracellular-signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK). Inactivation of ERK1/2 and p38 MAPK pathways using selective inhibitors (U0126 and SB203580) abrogated PXR-induced SMC proliferation and migration. Furthermore, cigarette smoke particles (CSP) activated PXR in SMCs. Knockdown of PXR by small interfering RNA suppressed the cell proliferation, migration, and activation of the MAPK pathways by CSP. These findings suggested a novel role for PXR in promoting SMC proliferation and migration, and neointimal hyperplasia. Therefore, PXR may be a potential therapeutic target for vascular disease related to xenobiotics such as cigarette smoking and other environmental pollutants.

KCa3.1 Channels Promote Cardiac Fibrosis Through Mediating Inflammation and Differentiation of Monocytes Into Myofibroblasts in Angiotensin II -Treated Rats.

Background Cardiac fibrosis is a core pathological process associated with heart failure. The recruitment and differentiation of primitive fibroblast precursor cells of bone marrow origin play a critical role in pathological interstitial cardiac fibrosis. The KCa3.1 channels are expressed in both ventricular fibroblasts and circulating mononuclear cells in rats and are upregulated by angiotensin II . We hypothesized that KCa3.1 channels mediate the inflammatory microenvironment in the heart, promoting the infiltrated bone marrow-derived circulating mononuclear cells to differentiate into myofibroblasts, leading to myocardial fibrosis. Methods and Results We established a cardiac fibrosis model in rats by infusing angiotensin II to evaluate the impact of the specific KCa3.1 channel blocker TRAM -34 on cardiac fibrosis. At the same time, mouse CD 4+ T cells and rat circulating mononuclear cells were separated to investigate the underlying mechanism of the TRAM -34 anti-cardiac fibrosis effect. TRAM -34 significantly attenuated cardiac fibrosis and the inflammatory reaction and reduced the number of fibroblast precursor cells and myofibroblasts. Inhibition of KCa3.1 channels suppressed angiotensin II -stimulated expression and secretion of interleukin-4 and interleukin-13 in CD 4+ T cells and interleukin-4- or interleukin-13-induced differentiation of monocytes into fibrocytes. Conclusions KCa3.1 channels facilitate myocardial inflammation and the differentiation of bone marrow-derived monocytes into myofibroblasts in cardiac fibrosis caused by angiotensin II infusion.