Discoidin domain receptor 2 signaling through PIK3C2α in fibroblasts promotes lung fibrosis.

Pulmonary fibrosis, especially idiopathic pulmonary fibrosis (IPF), portends significant morbidity and mortality, and current therapeutic options are suboptimal. We have previously shown that type I collagen signaling through discoidin domain receptor 2 (DDR2), a receptor tyrosine kinase expressed by fibroblasts, is critical for the regulation of fibroblast apoptosis and progressive fibrosis. However, the downstream signaling pathways for DDR2 remain poorly defined and could also be attractive potential targets for therapy. A recent phosphoproteomic approach indicated that PIK3C2α, a poorly studied member of the PI3 kinase family, could be a downstream mediator of DDR2 signaling. We hypothesized that collagen I/DDR2 signaling through PIK3C2α regulates fibroblast activity during progressive fibrosis. To test this hypothesis, we found that primary murine fibroblasts and IPF-derived fibroblasts stimulated with endogenous or exogenous type I collagen led to the formation of a DDR2/PIK3C2α complex, resulting in phosphorylation of PIK3C2α. Fibroblasts treated with an inhibitor of PIK3C2α or with deletion of PIK3C2α had fewer markers of activation after stimulation with TGFß and more apoptosis after stimulation with a Fas-activating antibody. Finally, mice with fibroblast-specific deletion of PIK3C2α had less fibrosis after bleomycin treatment than did littermate control mice with intact expression of PIK3Cα. Collectively, these data support the notion that collagen/DDR2/PIK3C2α signaling is critical for fibroblast function during progressive fibrosis, making this pathway a potential target for antifibrotic therapy. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Roles of Sphingosine Kinase and Sphingosine-1-Phosphate Receptor 2 in Endotoxin-Induced Acute Retinal Inflammation.

PURPOSE: To investigate the roles of sphingosine kinases (SphKs) and sphingosine-1-phosphate receptors (S1PRs) in endotoxin-induced uveitis (EIU) mice. METHODS: EIU model was induced using an intraperitoneal injection of lipopolysaccharide (LPS). The expression of SphKs and S1PRs in the retina was assessed using quantitative polymerase chain reaction (qPCR) and immunofluorescence. The effects of S1PR antagonists on the expression of inflammatory cytokines in the retina were evaluated using qPCR and western blotting. Effects of leukocyte infiltration of the retinal vessels were evaluated to determine the effects of the S1PR2 antagonist and genetic deletion of S1PR2 on retinal inflammation. RESULTS: Retinal SphK1 expression was significantly upregulated in EIU. SphK1 was expressed in the GCL, IPL, and OPL and S1PR2 was expressed in the GCL, INL, and OPL. Positive cells in IPL and OPL of EIU retina were identified as endothelial cells. S1PR2 antagonist and genetic deletion of S1PR2 significantly suppressed the expression of IL-1α, IL-6, TNF-α, and ICAM-1, whereas S1PR1/3 antagonist did not. Use of S1PR2 antagonist and S1PR2 knockout in mice significantly ameliorated leukocyte adhesion induced by LPS. CONCLUSION: SphK1/S1P/S1PR2 signaling was upregulated in EIU and S1PR2 inhibition suppressed inflammatory response. Targeting this signaling pathway has potential for treating retinal inflammatory diseases.

Sphingosine kinase 1 is involved in triglyceride breakdown by maintaining lysosomal integrity in brown adipocytes.

Sphingosine 1-phosphate (S1P) has been implicated in brown adipose tissue (BAT) formation and energy consumption; however, the mechanistic role of sphingolipids, including S1P, in BAT remains unclear. Here, we showed that, in mice, BAT activation by cold exposure upregulated mRNA and protein expression of the S1P-synthesizing enzyme sphingosine kinase 1 (SphK1) and S1P production in BAT. Treatment of wild-type brown adipocytes with exogenous S1P or S1P receptor subtype-selective agonists stimulated triglyceride (TG) breakdown only marginally, compared with noradrenaline. However, genetic deletion of Sphk1 resulted in hypothermia and diminished body weight loss upon cold exposure, suggesting that SphK1 is involved in thermogenesis through mechanisms different from receptor-mediated, extracellular action of S1P. In BAT of wild-type mice, SphK1 was localized largely in the lysosomes of brown adipocytes. In the brown adipocytes of Sphk1-/- mice, the number of lysosomes was reduced and lysosomal function, including proteolytic activity, acid esterase activity, and motility, was impaired. Concordantly, nuclear translocation of transcription factor EB, a master transcriptional regulator of lysosome biogenesis, was reduced, leading to decreased mRNA expression of the lysosome-related genes in Sphk1-/- BAT. Moreover, BAT of Sphk1-/- mice showed greater TG accumulation with dominant larger lipid droplets in brown adipocytes. Inhibition of lysosomes with chloroquine resulted in a less extent of triglyceride accumulation in Sphk1-/- brown adipocytes compared with wild-type brown adipocytes, suggesting a reduced lysosome-mediated TG breakdown in Sphk1-/- mice. Our results indicate a novel role of SphK1 in lysosomal integrity, which is required for TG breakdown and thermogenesis in BAT.

The ATG5 interactome links clathrin-mediated vesicular trafficking with the autophagosome assembly machinery.

Autophagosome formation involves the sequential actions of conserved ATG proteins to coordinate the lipidation of the ubiquitin-like modifier Atg8-family proteins at the nascent phagophore membrane. Although the molecular steps driving this process are well understood, the source of membranes for the expanding phagophore and their mode of delivery are only now beginning to be revealed. Here, we have used quantitative SILAC-based proteomics to identify proteins that associate with the ATG12-ATG5 conjugate, a crucial player during Atg8-family protein lipidation. Our datasets reveal a strong enrichment of regulators of clathrin-mediated vesicular trafficking, including clathrin heavy and light chains, and several clathrin adaptors. Also identified were PIK3C2A (a phosphoinositide 3-kinase involved in clathrin-mediated endocytosis) and HIP1R (a component of clathrin vesicles), and the absence of either of these proteins alters autophagic flux in cell-based starvation assays. To determine whether the ATG12-ATG5 conjugate reciprocally influences trafficking within the endocytic compartment, we captured the cell surface proteomes of autophagy-competent and autophagy-incompetent mouse embryonic fibroblasts under fed and starved conditions. We report changes in the relative proportions of individual cell surface proteins and show that cell surface levels of the SLC7A5-SLC3A2 amino acid transporter are influenced by autophagy capability. Our data provide evidence for direct regulatory coupling between the ATG12-ATG5 conjugate and the clathrin membrane trafficking system and suggest candidate membrane proteins whose trafficking within the cell may be modulated by the autophagy machinery. Abbreviations: ATG, autophagy related; BafA1, bafilomycin A1; GFP, green fluorescent protein; HIP1R, huntingtin interacting protein 1 related; MEF, mouse embryo fibroblast; PIK3C2A, phosphatidylinositol-4-phosphate 3-kinase catalytic subunit type 2 alpha; SILAC, stable isotope labelling with amino acids in culture; SQSTM1, sequestosome 1; STRING, search tool for the retrieval of interacting genes/proteins.

Class II phosphatidylinositol 3-kinase-C2α is essential for Notch signaling by regulating the endocytosis of γ-secretase in endothelial cells.

The class II α-isoform of phosphatidylinositol 3-kinase (PI3K-C2α) plays a crucial role in angiogenesis at least in part through participating in endocytosis and, thereby, endosomal signaling of several cell surface receptors including VEGF receptor-2 and TGFß receptor in vascular endothelial cells (ECs). The Notch signaling cascade regulates many cellular processes including cell proliferation, cell fate specification and differentiation. In the present study, we explored a role of PI3K-C2α in Delta-like 4 (Dll4)-induced Notch signaling in ECs. We found that knockdown of PI3K-C2α inhibited Dll4-induced generation of the signaling molecule Notch intracellular domain 1 (NICD1) and the expression of Notch1 target genes including HEY1, HEY2 and NOTCH3 in ECs but not in vascular smooth muscle cells. PI3K-C2α knockdown did not inhibit Dll4-induced endocytosis of cell surface Notch1. In contrast, PI3K-C2α knockdown as well as clathrin heavy chain knockdown impaired endocytosis of Notch1-cleaving protease, γ-secretase complex, with the accumulation of Notch1 at the perinuclear endolysosomes. Pharmacological blockage of γ-secretase also induced the intracellular accumulation of Notch1. Taken together, we conclude that PI3K-C2α is required for the clathrin-mediated endocytosis of γ-secretase complex, which allows for the cleavage of endocytosed Notch1 by γ-secretase complex at the endolysosomes to generate NICD1 in ECs.

Everolimus-Eluting Biodegradable Abluminal Coating Stent versus Durable Conformal Coating Stent: Termination of the Inflammatory Response Associated with Neointimal Healing in a Porcine Coronary Model.

OBJECTIVES: We evaluated the effect of the different carrier systems on early vascular response through histological analysis and scanning electron microscopy using a porcine model. BACKGROUND: Although Synergy™ and Promus PREMIER™ share an identical stent material and drug elution (everolimus), they use different drug carrier systems: biodegradable abluminal coating polymer or durable conformal coating polymer, respectively. However, data regarding the impact of the different coating systems on vessel healing are currently limited. METHODS: Twelve Synergy™ and Promus PREMIER™ were implanted in 12 swine. Histopathological analysis of the stented segments was performed on the 2nd and 14th days after implantation. Morphometric analysis of the inflammation and intimal fibrin content was also performed. RESULTS: On the 2nd day, neointimal thickness, percentage of neointimal area, and inflammatory and intimal fibrin content scores were not significantly different between the two groups. On the 14th day, the inflammatory and intimal fibrin content scores were significantly lower in Synergy™ versus those observed in Promus PREMIER™. In Synergy™, smooth muscle cells were found and the neointimal layers were smooth. In contrast, inflammatory cells were observed surrounding the struts of Promus PREMIER™. CONCLUSIONS: These results demonstrate that termination of reactive inflammation is accelerated after abluminal coating stent versus implantation of conformal coating stent.

Class II phosphatidylinositol 3-kinase α and ß isoforms are required for vascular smooth muscle Rho activation, contraction and blood pressure regulation in mice.

Class II phosphatidylinositol 3-kinases (PI3K), PI3K-C2α and PI3K-C2ß, are involved in cellular processes including endocytosis, cilia formation and autophagy. However, the role of PI3K-C2α and PI3K-C2ß at the organismal level is not well understood. We found that double knockout (KO) mice with both smooth muscle-specific KO of PI3K-C2α and global PI3K-C2ß KO, but not single KO mice of either PI3K-C2α or PI3K-C2ß, exhibited reductions in arterial blood pressure and substantial attenuation of contractile responses of isolated aortic rings. In wild-type vascular smooth muscle cells, double knockdown of PI3K-C2α and PI3K-C2ß but not single knockdown of either PI3K markedly inhibited contraction with reduced phosphorylation of 20-kDa myosin light chain and MYPT1 and Rho activation, but without inhibition of the intracellular Ca2+ mobilization. These data indicate that PI3K-C2α and PI3K-C2ß play the redundant but essential role for vascular smooth muscle contraction and blood pressure regulation mainly through their involvement in Rho activation.

TGFß receptor endocytosis and Smad signaling require synaptojanin1, PI3K-C2α-, and INPP4B-mediated phosphoinositide conversions.

Phosphoinositide conversion regulates a diverse array of dynamic membrane events including endocytosis. However, it is not well understood which enzymes are involved in phosphoinositide conversions for receptor endocytosis. We found by small interfering RNA (siRNA)-mediated knockdown (KD) that class II PI3K α-isoform (PI3K-C2α), the 5'-phosphatase synaptojanin1 (Synj1), and the 4'-phosphatase INPP4B, but not PI3K-C2ß, Synj2, or INPP4A, were required for TGFß-induced endocytosis of TGFß receptor. TGFß induced rapid decreases in PI(4,5)P2 at the plasma membrane (PM) with increases in PI(4)P, followed by increases in PI(3,4)P2, in a TGFß receptor kinase ALK5-dependent manner. TGFß induced the recruitment of both synaptojanin1 and PI3K-C2α to the PM with their substantial colocalization. Knockdown of synaptojanin1 abolished TGFß-induced PI(4,5)P2 decreases and PI(4)P increases. Interestingly, PI3K-C2α KD abolished not only TGFß-induced PI(3,4)P2 increases but also TGFß-induced synaptojanin1 recruitment to the PM, PI(4,5)P2 decreases, and PI(4)P increases. Finally, the phosphoinositide conversions were necessary for TGFß-induced activation of Smad2 and Smad3. These observations demonstrate that the sequential phosphoinositide conversions mediated by Synj1, PI3K-C2α, and INPP4B are essential for TGFß receptor endocytosis and its signaling.

Sphingosine kinase-2 prevents macrophage cholesterol accumulation and atherosclerosis by stimulating autophagic lipid degradation.

Atherosclerosis is the major cause of ischemic coronary heart diseases and characterized by the infiltration of cholesterol-accumulating macrophages in the vascular wall. Although sphingolipids are implicated in atherosclerosis as both membrane components and lipid mediators, the precise role of sphingolipids in atherosclerosis remains elusive. Here, we found that genetic deficiency of sphingosine kinase-2 (SphK2) but not SphK1 aggravates the formation of atherosclerotic lesions in mice with ApoE deficiency. Bone marrow chimaera experiments show the involvement of SphK2 expressed in bone marrow-derived cells. In macrophages, deficiency of SphK2, a major SphK isoform in this cell type, results in increases in cellular sphingosine and ceramides. SphK2-deficient macrophages have increases in lipid droplet-containing autophagosomes and autolysosomes and defective lysosomal degradation of lipid droplets via autophagy with an impaired luminal acidic environment and proteolytic activity in the lysosomes. Transgenic overexpression of SphK1 in SphK2-deficient mice rescued aggravation of atherosclerosis and abnormalities of autophagosomes and lysosomes in macrophages with reductions of sphingosine, suggesting at least partial overlapping actions of two SphKs. Taken together, these results indicate that SphK2 is required for autophagosome- and lysosome-mediated catabolism of intracellular lipid droplets to impede the development of atherosclerosis; therefore, SphK2 may be a novel target for treating atherosclerosis.

Lysophosphatidic acid-induced YAP/TAZ activation promotes developmental angiogenesis by repressing Notch ligand Dll4.

Lysophosphatidic acid (LPA) is a potent lipid mediator with various biological functions mediated through six G protein-coupled receptors (GPCRs), LPA1-6. Previous studies have demonstrated that LPA-Gα12/Gα13 signaling plays an important role in embryonic vascular development. However, the responsible LPA receptors and underlying mechanisms are poorly understood. Here, we show a critical role of LPA4 and LPA6 in developmental angiogenesis. In mice, Lpa4;Lpa6 double knockout (DKO) embryos were lethal due to global vascular deficiencies, and endothelial cell (EC)-specific Lpa4;Lpa6 DKO retinas had impaired sprouting angiogenesis. Mechanistically, LPA activated the transcriptional regulators YAP and TAZ through LPA4/LPA6-mediated Gα12/Gα13-Rho-ROCK signaling in ECs. YAP/TAZ knockdown increased ß-catenin- and Notch intracellular domain (NICD)-mediated endothelial expression of the Notch ligand delta-like 4 (DLL4). Fibrin gel sprouting assay revealed that LPA4/LPA6, Gα12/Gα13, or YAP/TAZ knockdown consistently blocked EC sprouting, which was rescued by a Notch inhibitor. Of note, the inhibition of Notch signaling also ameliorated impaired retinal angiogenesis in EC-specific Lpa4;Lpa6 DKO mice. Overall, these results suggest that the Gα12/Gα13-coupled receptors LPA4 and LPA6 synergistically regulate endothelial Dll4 expression through YAP/TAZ activation. This could in part account for the mechanism of YAP/TAZ-mediated developmental angiogenesis. Our findings provide a novel insight into the biology of GPCR-activated YAP/TAZ.

Sphigosine-1-phosphate receptor 1 promotes neointimal hyperplasia in a mouse model of carotid artery injury.

Vascular remodeling, resulting from proliferation and migration of vascular smooth muscle cells (VSMCs), is a major cause of atherosclerosis and restenosis. The lysophospholipid mediator sphingosine-1-phosphate (S1P) regulates proliferation and migration of VSMCs via S1P-specific G protein-coupled receptors, including S1P receptor 1 (S1PR1) to S1PR3. However, the role of S1PR1 in vascular remodeling is not well understood. Therefore, in this study, we aimed to investigate the effect of S1PR1 on neointimal hyperplasia in a carotid artery ligation mouse model using transgenic C57Bl/6 mice that overexpressed S1PR1 (Tg-S1PR1) under the control of α-smooth muscle actin promoter. We found that S1PR1 expression in carotid artery was upregulated after carotid artery ligation in non-transgenic (nTg) mice. Tg-S1PR1 mice showed enhanced ligation-induced neointimal hyperplasia with increased neointimal cell proliferation, compared with control nTg mice. VSMCs isolated from Tg-S1PR1 mice showed enhanced proliferation and migration in response to S1P stimulation. VSMCs from Tg-S1PR1 mice showed greater expression of interleukin-6 (IL-6) compared with nTg mouse-derived VSMCs, and administration of IL-6-neutralizing antibody into Tg-S1PR1 mice suppressed neointimal hyperplasia. These results suggest that S1P-S1PR1 signaling plays an important role in neointimal hyperplasia after vascular injury via IL-6 production.

The class II phosphoinositide 3-kinases PI3K-C2α and PI3K-C2ß differentially regulate clathrin-dependent pinocytosis in human vascular endothelial cells.

Pinocytosis is an important fundamental cellular process that is used by the cell to transport fluid and solutes. Phosphoinositide 3-kinases (PI3Ks) regulate a diverse array of dynamic membrane events. However, it is not well-understood which PI3K isoforms are involved in specific mechanisms of pinocytosis. We performed knockdown studies of endogenous PI3K isoforms and clathrin heavy chain (CHC) mediated by small interfering RNA (siRNA). The results demonstrated that the class II PI3K PI3K-C2α and PI3K-C2ß, but not the class I or III PI3K, were required for pinocytosis, based on an evaluation of fluorescein-5-isothiocyanate (FITC)-dextran uptake in endothelial cells. Pinocytosis was partially dependent on both clathrin and dynamin, and both PI3K-C2α and PI3K-C2ß were required for clathrin-mediated-but not clathrin-non-mediated-FITC-dextran uptake at the step leading up to its delivery to early endosomes. Both PI3K-C2α and PI3K-C2ß were co-localized with clathrin-coated pits and vesicles. However, PI3K-C2ß, but not PI3K-C2α, was highly co-localized with actin filament-associated clathrin-coated structures and required for actin filament formation at the clathrin-coated structures. These results indicate that PI3K-C2α and PI3K-C2ß play differential, indispensable roles in clathrin-mediated pinocytosis.

Class II PI3Ks α and ß Are Required for Rho-Dependent Uterine Smooth Muscle Contraction and Parturition in Mice.

Class II phosphoinositide 3-kinases (PI3Ks), PI3K-C2α and PI3K-C2ß, are highly homologous and distinct from class I and class III PI3Ks in catalytic products and domain structures. In contrast to class I and class III PI3Ks, physiological roles of PI3K-C2α and PI3K-C2ß are not fully understood. Because we previously demonstrated that PI3K-C2α is involved in vascular smooth muscle contraction, we studied the phenotypes of smooth muscle-specific knockout (KO) mice of PI3K-C2α and PI3K-C2ß. The pup numbers born from single PI3K-C2α-KO and single PI3K-C2ß-KO mothers were similar to those of control mothers, but those from double KO (DKO) mothers were smaller compared with control mice. However, the number of intrauterine fetuses in pregnant DKO mothers was similar to that in control mice. Both spontaneous and oxytocin-induced contraction of isolated uterine smooth muscle (USM) strips was diminished in DKO mice but not in either of the single KO mice, compared with control mice. Furthermore, contraction of USM of DKO mice was less sensitive to a Rho kinase inhibitor. Mechanistically, the extent of oxytocin-induced myosin light chain phosphorylation was greatly reduced in USM from DKO mice compared with control mice. The oxytocin-induced rise in the intracellular Ca2+ concentration in USM was similar in DKO and control mice. However, Rho activation in the intracellular compartment was substantially attenuated in DKO mice compared with control mice, as evaluated by fluorescence resonance energy transfer imaging technique. These data indicate that both PI3K-C2α and PI3K-C2ß are required for normal USM contraction and parturition mainly through their involvement in Rho activation.

MTMR4, a phosphoinositide-specific 3'-phosphatase, regulates TFEB activity and the endocytic and autophagic pathways.

Phosphatidylinositol 3-phosphate (PI(3)P) is the predominant phosphoinositide species in early endosomes and autophagosomes, in which PI(3)P dictates traffic of these organelles. Phosphoinositide levels are tightly regulated by lipid-kinases and -phosphatases; however, a phosphatase that converts PI(3)P back to phosphatidylinositol in the endosomal and autophagosomal compartments is not fully understood. We investigated the subcellular distribution and functions of myotubularin-related protein-4 (MTMR4), which is distinct among other MTMRs in that it possesses a PI(3)P-binding FYVE domain, in lung alveolar epithelium-derived A549 cells. MTMR4 was localized mainly in late endosomes and autophagosomes. MTMR4 knockdown markedly suppressed the motility, fusion, and fission of PI(3)P-enriched structures, resulting in decreases in late endosomes, autophagosomes, and lysosomes, and enlargement of PI(3)P-enriched early and late endosomes. In amino acid- and serum-starved cells, MTMR4 knockdown decreased both autophagosomes and autolysosomes and markedly increased PI(3)P-containing autophagosomes and late endosomes, suggesting that the fusion with lysosomes of autophagosomes and late endosomes might be impaired. Notably, MTMR4 knockdown inhibited the nuclear translocation of starvation stress responsive transcription factor-EB (TFEB) with reduced expression of lysosome-related genes in starved cells. These findings indicate that MTMR4 is essential for the integrity of endocytic and autophagic pathways.

Sphingosine-1-phosphate receptor-2 facilitates pulmonary fibrosis through potentiating IL-13 pathway in macrophages.

Idiopathic pulmonary fibrosis is a devastating disease with poor prognosis. The pathogenic role of the lysophospholipid mediator sphingosine-1-phosphate and its receptor S1PR2 in lung fibrosis is unknown. We show here that genetic deletion of S1pr2 strikingly attenuated lung fibrosis induced by repeated injections of bleomycin in mice. We observed by using S1pr2LacZ/+ mice that S1PR2 was expressed in alveolar macrophages, vascular endothelial cells and alveolar epithelial cells in the lung and that S1PR2-expressing cells accumulated in the fibrotic legions. Bone marrow chimera experiments suggested that S1PR2 in bone marrow-derived cells contributes to the development of lung fibrosis. Depletion of macrophages greatly attenuated lung fibrosis. Bleomycin administration stimulated the mRNA expression of the profibrotic cytokines IL-13 and IL-4 and the M2 markers including arginase 1, Fizz1/Retnla, Ccl17 and Ccl24 in cells collected from broncho-alveolar lavage fluids (BALF), and S1pr2 deletion markedly diminished the stimulated expression of these genes. BALF cells from bleomycin-administered wild-type mice showed a marked increase in phosphorylation of STAT6, a transcription factor which is activated downstream of IL-13, compared with saline-administered wild-type mice. Interestingly, in bleomycin-administered S1pr2-/- mice, STAT6 phosphorylation in BALF cells was substantially diminished compared with wild-type mice. Finally, pharmacological S1PR2 blockade in S1pr2+/+ mice alleviated bleomycin-induced lung fibrosis. Thus, S1PR2 facilitates lung fibrosis through the mechanisms involving augmentation of IL-13 expression and its signaling in BALF cells, and represents a novel target for treating lung fibrosis.

Photoacoustic imaging of tumour vascular permeability with indocyanine green in a mouse model.

BACKGROUND: We analysed the haemodynamics of indocyanine green (ICG) in mouse organs and tumours and evaluated responses to anti-angiogenic agents in an allograft tumour mouse model by photoacoustic imaging. METHODS: Thirty-six male mice (aged 10-14 weeks; body weight 20-25 g) were used. Real-time photoacoustic imaging of organs and tumours after intravenous injection of ICG was conducted in mice until 10 min after ICG injection. ICG distribution in tumour tissues was assessed by immunohistochemical staining and observation of ICG-derived fluorescence. Vascular permeability changes induced by the vascular endothelial growth factor (VEGF)-blocking agent VEGF-trap on tumour photoacoustic signals were studied. RESULTS: The photoacoustic signals in salivary glands and tumours after intravenous injection of iCG (0.604 ± 0.011 and 0.994 ± 0.175 [mean ± standard deviation], respectively) were significantly increased compared with those in the liver, kidney, and great vessel (0.234 ± 0.043, 0.204 ± 0.058 and 0.127 ± 0.040, respectively; p < 0.010). In tumours, the photoacoustic signal increased within 30 s after ICG injection in a dose-dependent manner (r2 = 0.899) and then decreased gradually. ICG was found to extravasate in tumour tissues. In VEGF-trap-treated mice, the photoacoustic signal in the tumour decreased at the early phase before inhibition of tumour growth was detected (0.297 ± 0.052 vs 1.011 ± 0.170 in the control; p < 0.001). CONCLUSIONS: Photoacoustic imaging with ICG administration demonstrated extravasation of ICG in mouse organs and tumours, indicating the potential for early detection of changes in vascular permeability during cancer therapy.

Neuronal SphK1 acetylates COX2 and contributes to pathogenesis in a model of Alzheimer's Disease.

Although many reports have revealed the importance of defective microglia-mediated amyloid ß phagocytosis in Alzheimer's disease (AD), the underlying mechanism remains to be explored. Here we demonstrate that neurons in the brains of patients with AD and AD mice show reduction of sphingosine kinase1 (SphK1), leading to defective microglial phagocytosis and dysfunction of inflammation resolution due to decreased secretion of specialized proresolving mediators (SPMs). Elevation of SphK1 increased SPMs secretion, especially 15-R-Lipoxin A4, by promoting acetylation of serine residue 565 (S565) of cyclooxygenase2 (COX2) using acetyl-CoA, resulting in improvement of AD-like pathology in APP/PS1 mice. In contrast, conditional SphK1 deficiency in neurons reduced SPMs secretion and abnormal phagocytosis similar to AD. Together, these results uncover a novel mechanism of SphK1 pathogenesis in AD, in which impaired SPMs secretion leads to defective microglial phagocytosis, and suggests that SphK1 in neurons has acetyl-CoA-dependent cytoplasmic acetyltransferase activity towards COX2.

Peretinoin, an acyclic retinoid, inhibits hepatocarcinogenesis by suppressing sphingosine kinase 1 expression in vitro and in vivo.

Sphingosine-1-phospate is a potent bioactive lipid metabolite that regulates cancer progression. Because sphingosine kinase 1 and sphingosine kinase 2 (SPHK 1/2) are both essential for sphingosine-1-phospate production, they could be a therapeutic target in various cancers. Peretinoin, an acyclic retinoid, inhibits post-therapeutic recurrence of hepatocellular carcinoma via unclear mechanisms. In this study, we assessed effects of peretinoin on SPHK expression and liver cancer development in vitro and in vivo. We examined effects of peretinoin on expression, enzymatic and promoter activity of SPHK1 in a human hepatoma cell line, Huh-7. We also investigated effects of SPHK1 on hepatocarcinogenesis induced by diethylnitrosamine using SPHK1 knockout mice. Peretinoin treatment of Huh-7 cells reduced mRNA levels, protein expression and enzymatic activity of SPHK1. Peretinoin reduced SPHK1 promoter activity; this effect of peretinoin was blocked by overexpression of Sp1, a transcription factor. Deletion of all Sp1 binding sites within the SPHK1 promoter region abolished SPHK1 promoter activity, suggesting that peretinoin reduced mRNA levels of SPHK1 via Sp1. Additionally, diethylnitrosamine-induced hepatoma was fewer and less frequent in SPHK1 knockout compared to wild-type mice. Our data showed crucial roles of SPHK1 in hepatocarcinogenesis and suggests that peretinoin prevents hepatocarcinogenesis by suppressing mRNA levels of SPHK1.

Augmented sphingosine 1 phosphate receptor-1 signaling in cardiac fibroblasts induces cardiac hypertrophy and fibrosis through angiotensin II and interleukin-6.

BACKGROUND: Cardiac fibroblasts, together with cardiomyocytes, occupy the majority of cells in the myocardium and are involved in myocardial remodeling. The lysophospholipid mediator sphigosine-1-phosphate (S1P) regulates functions of cardiovascular cells through multiple receptors including S1PR1-S1PR3. S1PR1 but not other S1P receptors was upregulated in angiotensin II-induced hypertrophic hearts. Therefore, we investigated a role of S1PR1 in fibroblasts for cardiac remodeling by employing transgenic mice that overexpressed S1PR1 under the control of α-smooth muscle actin promoter. In S1PR1-transgenic mouse heart, fibroblasts and/or myofibroblasts were hyperplastic, and those cells as well as vascular smooth muscle cells overexpressed S1PR1. Transgenic mice developed bi-ventricular hypertrophy by 12-week-old and diffuse interstitial fibrosis by 24-week-old without hemodynamic stress. Cardiac remodeling in transgenic mice was associated with greater ERK phosphorylation, upregulation of fetal genes, and systolic dysfunction. Transgenic mouse heart showed increased mRNA expression of angiotensin-converting enzyme and interleukin-6 (IL-6). Isolated fibroblasts from transgenic mice exhibited enhanced generation of angiotensin II, which in turn stimulated IL-6 release. Either an AT1 blocker or angiotensin-converting enzyme inhibitor prevented development of cardiac hypertrophy and fibrosis, systolic dysfunction and increased IL-6 expression in transgenic mice. Finally, administration of anti-IL-6 antibody abolished an increase in tyrosine phosphorylation of STAT3, a major signaling molecule downstream of IL-6, in the transgenic mouse heart and prevented development of cardiac hypertrophy in transgenic mice. These results demonstrate a promoting role of S1PR1 in cardiac fibroblasts for cardiac remodeling, in which angiotensin II-AT1 and IL-6 are involved.

Early endothelialization associated with a biolimus A9 bioresorbable polymer stent in a porcine coronary model.

Although Nobori®, with a bioresorbable polymer and biolimus A9 abluminal coating, has unique characteristics, few data exist regarding endothelialization early after implantation. Fifteen Nobori® and 14 control bare-metal stents (S-stent™) were implanted in 12 pigs. Histopathology of stented segments, inflammation, and intimal fibrin content was evaluated on the 2nd and 14th day after implantation. On the 2nd day, endothelial cells were morphologically and immunohistologically confirmed on the surface of both stents, although some inflammatory cells might be involved. Stent surface endothelialization evaluated with a scanning electron microscope showed partial cellular coverage in both stents. On the 14th day, neointimal thickness and percentage of the neointimal area were significantly lower in Nobori® than in S-stent™ (51.4 ± 4.5 vs. 76.4 ± 23.6 µm, p < 0.05 and 10.8 ± 2.6 vs. 14.1 ± 4.2%, p < 0.01). No significant differences were found in these parameters on the 2nd day (17.3 ± 14.9 vs. 26.7 ± 13.6 µm and 3.7 ± 3.0 vs. 6.7 ± 3.7%), in inflammatory and intimal fibrin content scores. These results demonstrate that endothelialization could occur early after Nobori® implantation with similar inflammatory reaction to bare-metal stents, probably contributing to low frequency of in-stent thrombosis and restenosis.