Corrigendum: Evaluation of Riboflavin Transporters as Targets for Drug Delivery and Theranostics.

[This corrects the article DOI: 10.3389/fphar.2019.00079.].

Non-activatable mutant of inhibitor of kappa B kinase α (IKKα) exerts vascular site-specific effects on atherosclerosis in Apoe-deficient mice.

BACKGROUND AND AIMS: IKKα is an important regulator of gene expression. As IKKα kinase inactivity in bone marrow-derived cells does not affect atherosclerosis, we here investigate the impact of a whole body-IKKα kinase inactivity on atherosclerosis. METHODS: Apolipoprotein E (Apoe)-deficient mice homozygous for an activation-resistant Ikkα-mutant (IkkαAA/AAApoe-/-) and Ikkα+/+Apoe-/- controls received a Western-type diet. Atherosclerotic lesion size and cellular content were analyzed using histology and immunofluorescence. Vascular protein expression and IKKα kinase activity were quantified by Luminex multiplex immuno-assay and ELISA. RESULTS: A vascular site-specific IKKα expression and kinase activation profile was revealed, with higher total IKKα protein levels in aortic root but increased IKKα phosphorylation, representing activated IKKα, in the aortic arch. This was associated with a vascular site-specific effect of IkkαAA/AA knock-in on atherosclerosis: in the aortic root, IkkαAA/AA knock-in decreased lesion size by 22.0 ±â€¯7.7% (p < 0.01), reduced absolute lesional smooth muscle cell numbers and lowered pro-atherogenic MMP2. In contrast, IkkαAA/AA knock-in increased lesion size in the aortic arch by 43.7 ±â€¯20.1% (p < 0.001), increased the abundance of lesional smooth muscle cells in brachiocephalic artery as main arch side branch and elevated MMP2. A similar profile was observed for MMP3. No effects were observed on necrotic core or collagen deposition in atherosclerotic lesions, nor on absolute lesional macrophage numbers. CONCLUSIONS: A non-activatable IKKα kinase differentially affects atherosclerosis in aortic root vs. aortic arch/brachiocephalic artery, associated with a differential vascular IKKα expression and kinase activation profile as well as with a vascular site-dependent impact on lesional smooth muscle cell accumulation and protein expression profiles.

Cardiac FGF23: new insights into the role and function of FGF23 after acute myocardial infarction.

OBJECTIVE: We aimed to elucidate the local role of FGF23 after myocardial infarction in a mouse model induced by left anterior descending artery (LAD) ligation. APPROACH AND RESULTS: (C57BL/6 N) mice underwent MI via LAD ligation and were sacrificed at different time-points post MI. The expression and influence of FGF23 on fibroblast and macrophages was also analyzed using isolated murine cells. We identified enhanced cardiac FGF23 mRNA expression in a time-dependent manner with an early increase, already on the first day after MI. FGF23 protein expression was abundantly detected in the infarcted area during the inflammatory phase. While described to be primarily produced in bone or macrophages, we identified cardiac fibroblasts as the only source of local FGF23 production after MI. Inflammatory mediators, such as IL-1ß, IL-6 and TNF-α, were able to induce FGF23 expression in these cardiac fibroblasts. Interestingly, we were not able to detect FGF23 at later time points after MI in mature scar tissue or remote myocardium, most likely due to TGF-ß1, which we have shown to inhibit the expression of FGF23. We identified FGFR1c to be the most abundant receptor for FGF23 in infarcted myocardium and cardiac macrophages and fibroblasts. FGF23 increased migration of cardiac fibroblast, as well as expression of Collagen 1, Periostin, Fibronectin and MMP8. FGF23 also increased expression of TGF-ß1 in M2 polarized macrophages. CONCLUSION: In conclusion, cardiac fibroblasts in the infarcted myocardium produce and express FGF23 as well as its respective receptors in a time-dependent manner, thus potentially influencing resident cell migration. The transitory local expression of FGF23 after MI points towards a complex role of FGF23 in myocardial ischemia and warrants further exploration, considering its role in ventricular remodeling.

Evaluation of Riboflavin Transporters as Targets for Drug Delivery and Theranostics.

The retention and cellular internalization of drug delivery systems and theranostics for cancer therapy can be improved by targeting molecules. Since an increased uptake of riboflavin was reported for various cancers, riboflavin and its derivatives may be promising binding moieties to trigger internalization via the riboflavin transporters (RFVT) 1, 2, and 3. Riboflavin is a vitamin with pivotal role in energy metabolism and indispensable for cellular growth. In previous preclinical studies on mice, we showed the target-specific accumulation of riboflavin-functionalized nanocarriers in cancer cells. Although the uptake mechanism of riboflavin has been studied for over a decade, little is known about the riboflavin transporters and their expression on cancer cells, tumor stroma, and healthy tissues. Furthermore, evidence is lacking concerning the representativeness of the preclinical findings to the situation in humans. In this study, we investigated the expression pattern of riboflavin transporters in human squamous cell carcinoma (SCC), melanoma and luminal A breast cancer samples, as well as in healthy skin, breast, aorta, and kidney tissues. Low constitutive expression levels of RFVT1-3 were found on all healthy tissues, while RFVT2 and 3 were significantly overexpressed in melanoma, RFVT1 and 3 in luminal A breast cancer and RFVT1-3 in SCC. Correspondingly, the SCC cell line A431 was highly positive for all RFVTs, thus qualifying as suitable in vitro model. In contrast, activated endothelial cells (HUVEC) only presented with a strong expression of RFVT2, and HK2 kidney cells only with a low constitutive expression of RFVT1-3. Functional in vitro studies on A431 and HK2 cells using confocal microscopy showed that riboflavin uptake is mostly ATP dependent and primarily driven by endocytosis. Furthermore, riboflavin is partially trafficked to the mitochondria. Riboflavin uptake and trafficking was significantly higher in A431 than in healthy kidney cells. Thus, this manuscript supports the hypothesis that addressing the riboflavin internalization pathway may be highly valuable for tumor targeted drug delivery.

Mif-deficiency favors an atheroprotective autoantibody phenotype in atherosclerosis.

The inflammatory cytokine macrophage migration-inhibitory factor (MIF) promotes atherosclerosis via lesional monocyte and T-cell recruitment. B cells have emerged as important components in atherogenesis, but the interaction between MIF and B cells in atherogenesis is unknown. Here, we investigated the atherosclerotic phenotype of Mif-gene deletion in Apoe-/- mice. Apoe-/- Mif-/- mice fed a Western diet exhibited strongly reduced atherosclerotic lesions in brachiocephalic artery (BC) and abdominal aorta compared with controls. This phenotype was accompanied by reduced circulating B cells. Flow cytometry revealed a B-cell developmental defect with increased premature and immature B-cell counts in bone marrow (BM) of Apoe-/- Mif-/- mice and diminished B-cell numbers in spleen. This finding was linked with a decreased expression of Baff-R and differentiation-driving transcription factors at the immature B-cell stage, whereas peritoneal B cells exhibited unchanged CD80 and CD86 expression but vastly decreased CD9 and elevated CD23 levels, indicating that the developmental block favors the generation of immature, egressing, and reactive B cells. Mif deficiency did not affect absolute B-cell numbers in the vessel wall but favored a relative increase of B cells in the atheroprone BC region and the appearance of periadventitial B-cell-rich clusters. Of note, Mif-/- mice exhibited a significant increase in oxidized low-density lipoprotein (oxLDL)-specific antibodies after the injection of oxLDL, indicating that Mif deficiency is associated with higher sensitivity of B cells against natural-occurring antigens such as oxLDL. Importantly, Apoe-/- mice adoptively transplanted with Apoe-/-Mif-/- BM showed reduced peripheral B cells compared with Apoe-/- BM transplantation but no atheroprotection in the BC; also, whereas there was a selective increase in atheroprotective IgM-anti-oxLDL-antibodies in global Mif deficiency, BM-specific Mif deficiency also led to elevated proatherogenic anti-oxLDL-IgG. Together, these findings reveal a novel link between MIF and B cells in atherogenesis. Protection from atherosclerosis by Mif deficiency is associated with enhanced B-cell hypersensitivity, which in global but not BM-restricted Mif deficiency favors an atheroprotective autoantibody profile in atherosclerotic mice. Targeting MIF may induce protective B-cell responses in atherosclerosis.-Schmitz, C., Noels, H., El Bounkari, O., Straussfeld, E., Megens, R. T. A., Sternkopf, M., Alampour-Rajabi, S., Krammer, C., Tilstam, P. V., Gerdes, N., Bürger, C., Kapurniotu, A., Bucala, R., Jankowski, J., Weber, C., Bernhagen, J. Mif-deficiency favors an atheroprotective autoantibody phenotype in atherosclerosis.

Molecular Ultrasound Imaging of Junctional Adhesion Molecule A Depicts Acute Alterations in Blood Flow and Early Endothelial Dysregulation.

OBJECTIVE: The junctional adhesion molecule A (JAM-A) is physiologically located in interendothelial tight junctions and focally redistributes to the luminal surface of blood vessels under abnormal shear and flow conditions accompanying atherosclerotic lesion development. Therefore, JAM-A was evaluated as a target for molecularly targeted ultrasound imaging of transient endothelial dysfunction under acute blood flow variations. APPROACH AND RESULTS: Flow-dependent endothelial dysfunction was induced in apolipoprotein E-deficient mice (n=43) by carotid partial ligation. JAM-A expression was investigated by molecular ultrasound using antibody-targeted poly(n-butyl cyanoacrylate) microbubbles and validated with immunofluorescence. Flow disturbance and arterial remodeling were assessed using functional ultrasound. Partial ligation led to an immediate drop in perfusion at the ligated side and a direct compensatory increase at the contralateral side. This was accompanied by a strongly increased JAM-A expression and JAM-A-targeted microbubbles binding at the partially ligated side and by a moderate and temporary increase in the contralateral artery (≈14× [P<0.001] and ≈5× [P<0.001] higher than control, respectively), both peaking after 2 weeks. Subsequently, although JAM-A expression and JAM-A-targeted microbubbles binding persisted at a higher level at the partially ligated side, it completely normalized within 4 weeks at the contralateral side. CONCLUSIONS: Temporary blood flow variations induce endothelial rearrangement of JAM-A, which can be visualized using JAM-A-targeted microbubbles. Thus, JAM-A may be considered as a marker of acute endothelial activation and dysfunction. Its imaging may facilitate the early detection of cardiovascular risk areas, and it enables the therapeutic prevention of their progression toward an irreversible pathological state.

Inhibition of atherogenesis by the COP9 signalosome subunit 5 in vivo.

Constitutive photomorphogenesis 9 (COP9) signalosome 5 (CSN5), an isopeptidase that removes neural precursor cell-expressed, developmentally down-regulated 8 (NEDD8) moieties from cullins (thus termed "deNEDDylase") and a subunit of the cullin-RING E3 ligase-regulating COP9 signalosome complex, attenuates proinflammatory NF-κB signaling. We previously showed that CSN5 is up-regulated in human atherosclerotic arteries. Here, we investigated the role of CSN5 in atherogenesis in vivo by using mice with myeloid-specific Csn5 deletion. Genetic deletion of Csn5 in Apoe-/- mice markedly exacerbated atherosclerotic lesion formation. This was broadly observed in aortic root, arch, and total aorta of male mice, whereas the effect was less pronounced and site-specific in females. Mechanistically, Csn5 KO potentiated NF-κB signaling and proinflammatory cytokine expression in macrophages, whereas HIF-1α levels were reduced. Inversely, inhibition of NEDDylation by MLN4924 blocked proinflammatory gene expression and NF-κB activation while enhancing HIF-1α levels and the expression of M2 marker Arginase 1 in inflammatory-elicited macrophages. MLN4924 further attenuated the expression of chemokines and adhesion molecules in endothelial cells and reduced NF-κB activation and monocyte arrest on activated endothelium in vitro. In vivo, MLN4924 reduced LPS-induced inflammation, favored an antiinflammatory macrophage phenotype, and decreased the progression of early atherosclerotic lesions in mice. On the contrary, MLN4924 treatment increased neutrophil and monocyte counts in blood and had no net effect on the progression of more advanced lesions. Our data show that CSN5 is atheroprotective. We conclude that MLN4924 may be useful in preventing early atherogenesis, whereas selectively promoting CSN5-mediated deNEDDylation may be beneficial in all stages of atherosclerosis.

MIF interacts with CXCR7 to promote receptor internalization, ERK1/2 and ZAP-70 signaling, and lymphocyte chemotaxis.

Macrophage migration-inhibitory factor (MIF) is a pleiotropic cytokine with chemokine-like functions and is a mediator in numerous inflammatory conditions. Depending on the context, MIF signals through 1 or more of its receptors cluster of differentiation (CD)74, CXC-motif chemokine receptor (CXCR)2, and CXCR4. In addition, heteromeric receptor complexes have been identified. We characterized the atypical chemokine receptor CXCR7 as a novel receptor for MIF. MIF promoted human CXCR7 internalization up to 40%, peaking at 50-400 nM and 30 min, but CXCR7 internalization by MIF was not dependent on CXCR4. Yet, by coimmunoprecipitation, fluorescence microscopy, and a proximity ligation assay, CXCR7 was found to engage in MIF receptor complexes with CXCR4 and CD74, both after ectopic overexpression and in endogenous conditions in a human B-cell line. Receptor competition binding and coimmunoprecipitation studies combined with sulfo-SBED-biotin-transfer provided evidence for a direct interaction between MIF and CXCR7. Finally, we demonstrated MIF/CXCR7-mediated functional responses. Blockade of CXCR7 suppressed MIF-mediated ERK- and zeta-chain-associated protein kinase (ZAP)-70 activation (from 2.1- to 1.2-fold and from 2.5- to 1.6-fold, respectively) and fully abrogated primary murine B-cell chemotaxis triggered by MIF, but not by CXCL12. B cells from Cxcr7(-/-) mice exhibited an ablated transmigration response to MIF, indicating that CXCR7 is essential for MIF-promoted B-cell migration. Our findings provide biochemical and functional evidence that MIF is an alternative ligand of CXCR7 and suggest a functional role of the MIF-CXCR7 axis in B-lymphocyte migration.

Atheroprotective role of C5ar2 deficiency in apolipoprotein E-deficient mice.

Atherogenic processes and vascular remodelling after arterial injury are controlled and driven by a plethora of factors amongst which the activation of the complement system is pivotal. Recently, we reported a clear correlation between high expressions of the second receptor for complement anaphylatoxin C5a, the C5a receptor-like 2 (C5L2, C5aR2), with high pro-inflammatory cytokine expression in advanced human atherosclerotic plaques. This prompted us to speculate that C5aR2 might have a functional role in atherosclerosis. We, therefore, investigated the role of C5aR2 in atherosclerosis and vascular remodelling. Here, we demonstrate that C5ar2 deletion, in atherosclerosis-prone mice, attenuates atherosclerotic as well as neointimal plaque formation, reduces macrophages and CD3+ T cells and induces features of plaque stability, as analysed by histomorphometry and quantitative immunohistochemistry. As a possible underlying mechanism, C5ar2-deficient plaques showed significantly reduced expression of C5a receptor (C5ar1), Tnf-α as well as Vcam-1, as determined by qPCR and quantitative immunohistochemistry. In addition, in vitro mechanistic studies revealed a reduction of these pro-inflammatory and pro-atherosclerotic mediators in C5ar2-deficient macrophages. Finally, blocking C5ar1 with antagonist JPE1375, in C5ar2(-/-)/Apoe(-/-) mice, led to a further reduction in neointimal plaque formation with reduced inflammation. In conclusion, C5ar2 deficiency attenuates atherosclerosis and neointimal plaque formation after arterial injury. This identifies C5aR2 as a promising target to reduce atherosclerosis and restenosis after vascular interventions.

Macrophage migration inhibitory factor limits activation-induced apoptosis of platelets via CXCR7-dependent Akt signaling.

RATIONALE: Macrophage migration inhibitory factor (MIF) is released on platelet activation. Circulating MIF could potentially regulate platelets and thereby platelet-mediated inflammatory and regenerative mechanisms. However, the effect of MIF on platelets is unknown. OBJECTIVE: The present study evaluated MIF in regulating platelet survival and thrombotic potential. METHODS AND RESULTS: MIF interacted with CXCR4-CXCR7 on platelets, defining CXCR7 as a hitherto unrecognized receptor for MIF on platelets. MIF internalized CXCR4, but unlike CXCL12 (SDF-1α), it did not phosphorylate Erk1/2 after CXCR4 ligation because of the lack of CD74 and failed in subsequent CXCR7 externalization. MIF did not alter the activation status of platelets. However, MIF rescued platelets from activation and BH3 mimetic ABT-737-induced apoptosis in vitro via CXCR7 and enhanced circulating platelet survival when administered in vivo. The antiapoptotic effect of MIF was absent in Cxcr7(-/-) murine embryonic cells but pronounced in CXCR7-transfected Madin-Darby canine kidney cells. This prosurvival effect was attributed to the MIF-CXCR7-initiated PI3K-Akt pathway. MIF induced CXCR7-Akt-dependent phosphorylation of BCL-2 antagonist of cell death (BAD) both in vitro and in vivo. Consequentially, MIF failed to rescue Akt(-/-) platelets from thrombin-induced apoptosis when challenged ex vivo, also in prolonging platelet survival and in inducing BAD phosphorylation among Akt(-/-) mice in vivo. MIF reduced thrombus formation under arterial flow conditions in vitro and retarded thrombotic occlusion after FeCl3-induced arterial injury in vivo, an effect mediated through CXCR7. CONCLUSION: MIF interaction with CXCR7 modulates platelet survival and thrombotic potential both in vitro and in vivo and thus could regulate thrombosis and inflammation.

Calcineurin-mediated YB-1 dephosphorylation regulates CCL5 expression during monocyte differentiation.

Y-box (YB) protein-1 serves as a master regulator in gene transcription and mRNA translation. YB-1 itself is regulated at various levels, e.g. through post-translational modifications. In our previous work, we identified RANTES/CCL5 as a transcriptional target of YB-1. We previously demonstrated that YB-1 protein is transiently up-regulated during monocyte/macrophage differentiation evidenced in monocytic cells (THP-1 cells) that were differentiated using phorbol myristate acetate (PMA). Here we provide evidence that YB-1 phosphorylation, specifically at its serine residue 102 (Ser-102), increases early on in THP-1 cells following PMA treatment as well as in differentiated primary human monocytes. This process is mediated through the Akt signaling pathway. Ser-102-phosphorylated YB-1 displays stronger binding affinity and trans-activating capacity at the CCL5 gene promoter. Notably, Ser-102-phosphorylated YB-1 disappears at later stages of the monocyte/macrophage differentiation process. We demonstrate that serine-threonine phosphatase calcineurin (CN) dephosphorylates YB-1 preventing it from binding to and trans-activating the CCL5 promoter. Co-immunoprecipitation assays prove a direct YB-1/CN interaction. Furthermore, analyses in kidney tissues from mice that were treated with the CN inhibitor cyclosporine A revealed an in vivo effect of CN on the YB-1 phosphorylation status. We conclude that YB-1 phosphorylation at Ser-102 is an important prerequisite for CCL5 promoter activation during macrophage differentiation. Our findings point to a critical role of YB-1 in the resolution of inflammatory processes which may largely be due to CN-mediated dephosphorylation.