Purpose
Retinopathy of prematurity (ROP) is an ocular disorder that primarily occurs in premature infants and is the most common cause of vision impairment. This study examined the effect of ...desflurane on angiogenesis in a mouse model of oxygen-induced retinopathy (OIR).
Methods
Mice were randomly allocated to the control (
C
), ROP control (
R
c
), or ROP with desflurane exposure (
R
d
) group. To induce ROP, 7-day-old mice were exposed to 75% oxygen in a chamber for 5 days postnatal days (
P
) 7–12, and thereafter returned to room air. Age-matched mice exposed to room air formed the
C
group. The
R
d
group was exposed to 8% desflurane for 2 h on P12, P13, and P14 with 40% oxygen. To observe changes in angiogenesis of the retina, mice were sacrificed at P16.
Results
The ratio of avascular area/total retinal area was not changed significantly in the
R
d
group, compared to the
R
c
group. The expression of endothelial growth factor A (VEGF-A) and hypoxia inducible factor-1α (HIF-1α) in the
R
d
group and
R
c
group was not significantly different.
Conclusions
Desflurane does not have a significant influence on retinal angiogenesis via HIF-1α and VEGF-A expression in the OIR mouse model. However, these findings are not directly applicable to premature infants, and it is thus necessary to perform further studies to determine the effect of desflurane on angiogenesis.
Sphingosine kinase is a lipid kinase that converts sphingosine into sphingosine-1-phosphate, an important signaling molecule with intracellular and extracellular functions. Although diverse ...extracellular stimuli influence cellular sphingosine kinase activity, the molecular mechanisms underlying its regulation remain to be clarified. In this study, we investigated the phosphorylation-dependent regulation of mouse sphingosine kinase (mSK) isoforms 1 and 2. mSK1a was robustly phosphorylated in response to extracellular stimuli such as phorbol ester, whereas mSK2 exhibited a high basal level of phosphorylation in quiescent cells regardless of agonist stimulation. Interestingly, phorbol ester-induced phosphorylation of mSK1a correlated with suppression of its activity. Chemical inhibition of conventional PKCs (cPKCs) abolished mSK1a phosphorylation, while overexpression of PKCα, a cPKC isoform, potentiated the phosphorylation, in response to phorbol ester. Furthermore, an in vitro kinase assay showed that PKCα directly phosphorylated mSK1a. In addition, phosphopeptide mapping analysis determined that the S373 residue of mSK1a was the only site phosphorylated by cPKC. Interestingly, alanine substitution of S373 made mSK1a refractory to the inhibitory effect of phorbol esters, whereas glutamate substitution of the same residue resulted in a significant reduction in mSK1a activity, suggesting the significant role of this phosphorylation event. Taken together, we propose that mSK1a is negatively regulated through cPKC-dependent phosphorylation at S373 residue.
•27-Hydroxycholesterol enhances production of CCL2 by macrophages.•The secreted CCL2 induces migration of monocytic cells.•We report the mechanism underlying monocyte recruitment into atherosclerotic ...lesions.
Deposition of lipids in the intima is followed by infiltration of inflammatory cells, like monocytic cells and T lymphocytes, in atherosclerosis. However, roles of lipids in the infiltration of the inflammatory cells are not clearly defined. We investigated the possible involvement of cholesterol or its catabolites in recruitment of monocytic cells. Consumption of a high cholesterol-diet resulted in enhanced expression of CCL2 in arteries of ApoE−/− mice. 27-Hydroxycholesterol, the most abundant cholesterol oxide in atherosclerotic lesions, significantly induced the transcription of CCL2 and enhanced secretion of corresponding protein by THP-1 monocytic cells. However, cholesterol and 7-ketocholesterol did not influence expression of CCL2. Conditioned media containing CCL2 induced migration of monocytic cells, and migration was abrogated in the presence of CCL2-neutralizing antibody. TO-901317, a synthetic LXR agonist, inhibited both production of CCL2 and migration of monocytic cells induced by 27-hydroxycholesterol. Expression of CCL2 induced by 27-hydroxycholesterol was blocked when Akt inhibitor IV was added and when Akt1 was knocked down. We propose that 27-hydroxycholesterol will trigger a sequence of events leading to recruitment of monocytes into atherosclerotic lesions.
Acute kidney injury (AKI) is an underestimated yet important risk factor for the development of chronic kidney disease (CKD), characterized by tubulointerstitial fibrosis and tubular ...dedifferentiation. Tubular dedifferentiation, which is associated with the loss of epithelial markers and the gain of mesenchymal features, is thought to be involved in tubulointerstitial fibrosis. As protein kinase B/Akt is involved in the development of CKD, we investigated the role of Akt1, one of the three Akt isoforms, in a murine model of AKI-to-CKD progression.
We subjected C57BL/6 male mice to unilateral ischemia-reperfusion injury (UIRI) and harvested their kidneys after 6 weeks. Mice were divided into four groups, namely, wild-type (WT) UIRI, Akt1-/- UIRI, WT sham, and Akt1-/- sham.
Akt1 (but not Akt2 or Akt3) was markedly activated in WT UIRI mice than in WT sham mice. Tubulointerstitial fibrosis and tubular dedifferentiation significantly increased in WT UIRI mice, but were attenuated in Akt1-/- UIRI mice. Both WT UIRI and Akt1-/- UIRI mice showed markedly upregulated transforming growth factor-β1 (TGF-β1)/Smad signaling compared with WT sham mice. However, TGF-β1/Smad expression did not differ between the two groups. The levels of phosphorylated GSK-3β, β-catenin, and Snail were attenuated in Akt1-/- UIRI mice compared with those in WT UIRI mice.
Deletion of Akt1 results in the attenuation of renal fibrosis and tubular dedifferentiation, independent of TGF-β1/Smad signaling, during AKI-to-CKD progression in a UIRI without contralateral nephrectomy model. Thus, Akt1 may serve as a therapeutic target in AKI-to-CKD progression.
Critical limb ischemia is a condition in which tissue necrosis occurs due to arterial occlusion, resulting in limb amputation in severe cases. Both endothelial cells (ECs) and vascular smooth muscle ...cells (SMCs) are needed for the regeneration of peripheral arteries in ischemic tissues. However, it is difficult to isolate and cultivate primary EC and SMC from patients for therapeutic angiogenesis. Induced pluripotent stem cells (iPSCs) are regarded as useful stem cells due to their pluripotent differentiation potential. In this study, we explored the therapeutic efficacy of human iPSC‐derived EC and iPSC‐derived SMC in peripheral artery disease model. After the induction of mesodermal differentiation of iPSC, CD34+ progenitor cells were isolated by magnetic‐activated cell sorting. Cultivation of the CD34+ progenitor cells in endothelial culture medium induced the expression of endothelial markers and phenotypes. Moreover, the CD34+ cells could be differentiated into SMC by cultivation in SMC culture medium. In a murine hindlimb ischemia model, cotransplantation of EC with SMC improved blood perfusion and increased the limb salvage rate in ischemic limbs compared to transplantation of either EC or SMC alone. Moreover, cotransplantation of EC and SMC stimulated angiogenesis and led to the formation of capillaries and arteries/arterioles in vivo. Conditioned medium derived from SMC stimulated the migration, proliferation, and tubulation of EC in vitro, and these effects were recapitulated by exosomes isolated from the SMC‐conditioned medium. Together, these results suggest that iPSC‐derived SMC enhance the therapeutic efficacy of iPSC‐derived EC in peripheral artery disease via an exosome‐mediated paracrine mechanism.
Co‐transplantation of the induced pluripotent stem cell (iPSC)‐derived endothelial cells and smooth muscle cells enhanced the therapeutic efficacies of endothelial cells and smooth muscle cells in a murine hindlimb ischemia model. Smooth muscle cells promoted angiogenesis and therapy of peripheral artery disease through exosome‐mediated paracrin mechanism.
Gastrin-releasing peptide (GRP) has been reported to be implicated in the pathogenesis of inflammatory disorders. The migration and proliferation of vascular smooth muscle cells (VSMCs) are key ...components of vascular inflammation that leads to the development of atherosclerosis. The present study aimed to investigate the molecular effect of GRP on VSMC proliferation and migration. We report that GRP significantly enhanced the proliferation and migration of rat VSMCs. GRP increased mRNA and protein expression of matrix metalloproteinase- 2 and -9 (MMP-2/9) in VSMCs. The induction of MMP-2/9 by GRP was regulated by the activation of the signal transducer and activator of transcription-3 (STAT3). In addition, STAT3-knockdown of VSMCs by siRNA or blockade of the GRP receptor inhibited GRP-induced migration of VSMCs. Taken together, our findings indicate that GRP promotes the migration of VSMCs through upregulation of MMP-2/9 via STAT3 activation. BMB Reports 2017; 50(12): 628-633
► 27OHChol or 7αOHChol induced transcription and intracellular expression of TNF-α in macrophages. ► Exposure of 27OHChol- or 7αOHChol-treated THP-1 macrophages to LPS resulted in augmentation in ...TNF-α production. ► TO-901317, pertussis toxin, PP2, and LY294002 attenuated expression of TNF-α induced by oxysterols.
Enhanced production of TNF-α from macrophages promotes development and instability of atherosclerotic plaques, but involvement of lipid component in TNF-α production has not been clarified in atherosclerosis. We attempted to determine whether cholesterol oxidation products (oxysterols) could modify TNF-α production. Treatment of THP-1 cells with 27-hydroxycholesterol (27OHChol) or 7α-hydroxycholesterol (7αOHChol) resulted in a profound increase in TNF-α transcription, while treatment with an identical concentration of cholesterol and 7-ketochoelsterol did not lead to any change in TNF-α expression. Treatment with 27OHChol resulted in increased synthesis, as well as secretion, of TNF-α, while 7αOHChol led to increased synthesis of TNF-α without affecting secretion of the cytokine. Co-treatment with 7αOHChol or 27OHChol and LPS resulted in synergistically enhanced or augmented secretion of TNF-α. Treatment with TO-901317, pertussis toxin, PP2, and LY294002 resulted not only in attenuated transcription of TNF-α induced by 27OHChol and 7αOHChol, but also secretion of TNF-α enhanced by 27OHChol. This is the first report demonstrating enhanced production of TNF-α in macrophages by treatment with oxysterols which are detected in abundance in atheromatous lesions; in addition, results of the current study provide evidence indicating that certain types of oxysterols contribute to development of atherosclerosis by promoting production of proinflammatory cytokines.
Immunoglobulin A nephropathy (IgAN) is the most prevalent form of glomerulonephritis worldwide. Prediction of disease progression in IgAN can help to provide individualized treatment based on ...accurate risk stratification.
We performed proton nuclear magnetic resonance-based metabolomics analyses of serum and urine samples from healthy controls, non-progressor (NP), and progressor (P) groups to identify metabolic profiles of IgAN disease progression. Metabolites that were significantly different between the NP and P groups were selected for pathway analysis. Subsequently, we analyzed multivariate area under the receiver operating characteristic (ROC) curves to evaluate the predictive power of metabolites associated with IgAN progression.
We observed several distinct metabolic fingerprints of the P group involving the following metabolic pathways: glycolipid metabolism; valine, leucine, and isoleucine biosynthesis; aminoacyl-transfer RNA biosynthesis; glycine, serine, and threonine metabolism; and glyoxylate and dicarboxylate metabolism. In multivariate ROC analyses, the combinations of serum glycerol, threonine, and proteinuria (area under the curve AUC, 0.923; 95% confidence interval CI, 0.667-1.000) and of urinary leucine, valine, and proteinuria (AUC, 0.912; 95% CI, 0.667-1.000) showed the highest discriminatory ability to predict IgAN disease progression.
This study identified serum and urine metabolites profiles that can aid in the identification of progressive IgAN and proposed perturbed metabolic pathways associated with the identified metabolites.
Akt (also called protein kinase B) is one of the major downstream targets of the phosphatidylinositol 3-kinase pathway. This protein kinase has been implicated in insulin signaling, stimulation of ...cellular growth, and inhibition of apoptosis as well as transformation of cells. Although a number of cellular proteins have been identified as putative targets of the enzyme, additional substrates may play a role in the varied responses elicited by this enzyme. We have used a combination of 14-3-3 binding and recognition by an antibody to the phosphorylation consensus of the enzyme to identify and isolate one of the major substrates of Akt, which is also a 14-3-3 binding protein. This 40-kDa protein, designated PRAS40, is a proline-rich Akt substrate. Demonstration that it is a substrate of Akt was accomplished by showing that 1) PRAS40 was phosphorylated in vitro by purified Akt on the same site that was phosphorylated in insulin-treated cells; 2) activation of an inducible Akt was alone sufficient to stimulate the phosphorylation of PRAS40; and 3) cells lacking Akt1 and Akt2 exhibit a diminished ability to phosphorylate this protein. Thus, PRAS40 is a novel substrate of Akt, the phosphorylation of which leads to the binding of this protein to 14-3-3.
NHERF1/EBP50 (Na
/H
exchanger regulating factor 1; Ezrin-binding phosphoprotein of 50 kDa) organizes stable protein complexes beneath the apical membrane of polar epithelial cells. By contrast, in ...cancer cells without any fixed polarity, NHERF1 often localizes in the cytoplasm. The regulation of cytoplasmic NHERF1 and its role in cancer progression remain unclear. In this study, we found that, upon lysophosphatidic acid (LPA) stimulation, cytoplasmic NHERF1 rapidly translocated to the plasma membrane, and subsequently to cortical protrusion structures, of ovarian cancer cells. This movement depended on direct binding of NHERF1 to C-terminally phosphorylated ERM proteins (cpERMs). Moreover, NHERF1 depletion downregulated cpERMs and further impaired cpERM-dependent remodeling of the cell cortex, suggesting reciprocal regulation between these proteins. The LPA-induced protein complex was highly enriched in migratory pseudopodia, whose formation was impaired by overexpression of NHERF1 truncation mutants. Consistent with this, NHERF1 depletion in various types of cancer cells abolished chemotactic cell migration toward a LPA gradient. Taken together, our findings suggest that the high dynamics of cytosolic NHERF1 provide cancer cells with a means of controlling chemotactic migration. This capacity is likely to be essential for ovarian cancer progression in tumor microenvironments containing LPA.
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