Endothelial cells drive organ fibrosis in mice by inducing expression of the transcription factor SOX9.

Fibrosis is a hallmark of chronic disease. Although fibroblasts are involved, it is unclear to what extent endothelial cells also might contribute. We detected increased expression of the transcription factor Sox9 in endothelial cells in several different mouse fibrosis models. These models included systolic heart failure induced by pressure overload, diastolic heart failure induced by high-fat diet and nitric oxide synthase inhibition, pulmonary fibrosis induced by bleomycin treatment, and liver fibrosis due to a choline-deficient diet. We also observed up-regulation of endothelial SOX9 in cardiac tissue from patients with heart failure. To test whether SOX9 induction was sufficient to cause disease, we generated mice with endothelial cell-specific overexpression of Sox9, which promoted fibrosis in multiple organs and resulted in signs of heart failure. Endothelial Sox9 deletion prevented fibrosis and organ dysfunction in the two mouse models of heart failure as well as in the lung and liver fibrosis mouse models. Bulk and single-cell RNA sequencing of mouse endothelial cells across multiple vascular beds revealed that SOX9 induced extracellular matrix, growth factor, and inflammatory gene expression, leading to matrix deposition by endothelial cells. Moreover, mouse endothelial cells activated neighboring fibroblasts that then migrated and deposited matrix in response to SOX9, a process partly mediated by the secreted growth factor CCN2, a direct SOX9 target; endothelial cell-specific Sox9 deletion reversed these changes. These findings suggest a role for endothelial SOX9 as a fibrosis-promoting factor in different mouse organs during disease and imply that endothelial cells are an important regulator of fibrosis.

Metaheuristic optimization of data preparation and machine learning hyperparameters for prediction of dynamic methane production.

Machine learning algorithms provide detailed description of the anaerobic digestion process, but the impact of data preparation procedures and hyperparameter optimization has rarely been investigated. A genetic algorithm was developed for optimizing data preparation and model hyperparameters to simulate dynamic methane production from steady-state anaerobic digestion of agricultural residues at full-scale. A long short-term memory neural network was used as prediction model. Results indicate that batch size, learning rate and number of neurons are the most important model parameters for accurate description of methane production rates, whereas combination of hyperparameter and data preparation optimization shows best model efficiencies, with a root mean square scaled error of 76.5 %. Mass of solid feed, time and mass of volatile solids are the most relevant input features. This study provides fundamental steps for optimal prediction of dynamic biomethane production, as a reliable basis for improving bioconversion efficiency during anaerobic digestion of agricultural residues.

ALK1 controls hepatic vessel formation, angiodiversity, and angiocrine functions in hereditary hemorrhagic telangiectasia of the liver.

BACKGROUND AND AIMS: In hereditary hemorrhagic telangiectasia (HHT), severe liver vascular malformations are associated with mutations in the Activin A Receptor-Like Type 1 ( ACVRL1 ) gene encoding ALK1, the receptor for bone morphogenetic protein (BMP) 9/BMP10, which regulates blood vessel development. Here, we established an HHT mouse model with exclusive liver involvement and adequate life expectancy to investigate ALK1 signaling in liver vessel formation and metabolic function. APPROACH AND RESULTS: Liver sinusoidal endothelial cell (LSEC)-selective Cre deleter line, Stab2-iCreF3 , was crossed with Acvrl1 -floxed mice to generate LSEC-specific Acvrl1 -deficient mice ( Alk1HEC-KO ). Alk1HEC-KO mice revealed hepatic vascular malformations and increased posthepatic flow, causing right ventricular volume overload. Transcriptomic analyses demonstrated induction of proangiogenic/tip cell gene sets and arterialization of hepatic vessels at the expense of LSEC and central venous identities. Loss of LSEC angiokines Wnt2 , Wnt9b , and R-spondin-3 ( Rspo3 ) led to disruption of metabolic liver zonation in Alk1HEC-KO mice and in liver specimens of patients with HHT. Furthermore, prion-like protein doppel ( Prnd ) and placental growth factor ( Pgf ) were upregulated in Alk1HEC-KO hepatic endothelial cells, representing candidates driving the organ-specific pathogenesis of HHT. In LSEC in vitro , stimulation or inhibition of ALK1 signaling counter-regulated Inhibitors of DNA binding (ID)1-3, known Alk1 transcriptional targets. Stimulation of ALK1 signaling and inhibition of ID1-3 function confirmed regulation of Wnt2 and Rspo3 by the BMP9/ALK1/ID axis. CONCLUSIONS: Hepatic endothelial ALK1 signaling protects from development of vascular malformations preserving organ-specific endothelial differentiation and angiocrine signaling. The long-term surviving Alk1HEC-KO HHT model offers opportunities to develop targeted therapies for this severe disease.

FTIR-Assisted Electroreduction of CO2 and H2O to CO and H2 by Electrochemically Deposited Copper on Oxidized Graphite Felt.

Obtaining CO and H2 from electrochemical CO2 reduction (CO2RR) offers a viable alternative to reduce CO2 emissions and produce chemicals and fuels. Herein, we report a simple strategy for obtaining polycrystalline copper deposited on oxidized graphite felt (Cu-OGF) and its performance on the selective conversion of CO2 and H2O to CO and H2. For the electrode obtaining, graphite felt (GF) was first oxidized (OGF) in order to make the substrate hydrophilic and then copper particles were electrochemically deposited onto OGF. The pH of deposition was investigated, and the CO2RR activity was assessed for the prepared electrodes at each pH (2.0, 4.0, 6.0, 8.0, and 10.0). It was found that pH 2.0 was the most promising for CO2RR due to the presence of hexagonal copper microparticles. Fourier transform infrared analysis of the produced gases showed that this is a low-cost catalyst capable of reducing CO2 and H2O to CO and H2, with Faradaic efficiencies between 0.50 and 5.21% for CO and 50.87 to 98.30% for H2, depending on the experimental conditions. Hence, it is possible for this gas mixture to be used as a fuel gas or to be enriched with CO for use in Fischer-Tropsch processes.

Angiogenic and molecular diversity determine hepatic melanoma metastasis and response to anti-angiogenic treatment.

BACKGROUND: Cutaneous melanoma exhibits heterogeneous metastatic patterns and prognosis. In this regard, liver metastasis, which is detected in ~ 10-20% of stage 4 patients, came to the fore of melanoma research, as it recently evolved as decisive indicator of treatment resistance to immune checkpoint inhibition. METHODS: Hepatic metastases were induced by intrasplenic injection of five different murine melanoma cell lines. The efficiencies of hepatic colonization, morphologic patterns, gene expression profiles and degree of vascularization were analyzed and Sorafenib was applied as anti-angiogenic treatment. RESULTS: WT31 melanoma showed the highest efficiency of hepatic colonization, while intermediate efficiencies were observed for B16F10 and RET, and low efficiencies for D4M and HCmel12. RNAseq-based gene expression profiles of high and intermediate metastatic melanomas in comparison to low metastatic melanomas indicated that this efficiency predominantly associates with gene clusters involved in cell migration and angiogenesis. Indeed, heterogeneous vascularization patterns were found in the five models. Although the degree of vascularization of WT31 and B16F10 metastases differed, both showed a strong response to Sorafenib with a successful abrogation of the vascularization. CONCLUSION: Our data indicate that molecular heterogeneity of melanomas can be associated with phenotypic and prognostic features of hepatic metastasis paving the way for organ-specific anti-angiogenic therapeutic approaches.

Rescue Blanket as a Provisional Seal for Penetrating Chest Wounds in a New Ex Vivo Porcine Model.

BACKGROUND: Open pneumothorax after a penetrating thorax trauma is a life-threatening disease with high mortality. An emergency application of a chest seal (CS) allowing the release of trapped air is the optimum initial therapy until surgical chest drainage is available. METHODS: In a newly developed experimental porcine model of open pneumothorax, we tested 3 different materials regarding their applicability for acute treatment of sucking chest wounds in prehospital emergency care, namely a commonly used rescue blanket (RB), plastic foil from a gauze package (packing material), and a commercial CS. RESULTS: An ex vivo open pneumothorax model using a porcine chest wall and a vacuum-assisted drainage system was successfully established. RB segments sized 70 × 100 mm achieved significantly higher rates of successful sealing than plastic foils from a gauze package sized 100 × 100 mm when the devices were applied to the moistened chest wall and fixed on 3 sides (5/5 [100%] vs 0/5 [0%], respectively; P = .002). Loosely fixed RBs efficiently released injected air (10/10 [100%]) and consequently sealed the wound in all cases (10/10). CONCLUSIONS: RBs, applied wet, are appropriate CSs with good occlusive and adherence properties. Fixation on 2 sides of the dressing is sufficient to allow trapped air to exit while providing appropriate sealing of the chest wound. RBs were superior to plastic foils from a gauze package and were seen to function as a potent makeshift CS when no commercial CS is available.

Bone marrow sinusoidal endothelium controls terminal erythroid differentiation and reticulocyte maturation.

Within the bone marrow microenvironment, endothelial cells (EC) exert important functions. Arterial EC support hematopoiesis while H-type capillaries induce bone formation. Here, we show that BM sinusoidal EC (BM-SEC) actively control erythropoiesis. Mice with stabilized ß-catenin in BM-SEC (Ctnnb1OE-SEC) generated by using a BM-SEC-restricted Cre mouse line (Stab2-iCreF3) develop fatal anemia. While activation of Wnt-signaling in BM-SEC causes an increase in erythroblast subsets (PII-PIV), mature erythroid cells (PV) are reduced indicating impairment of terminal erythroid differentiation/reticulocyte maturation. Transplantation of Ctnnb1OE-SEC hematopoietic stem cells into wildtype recipients confirms lethal anemia to be caused by cell-extrinsic, endothelial-mediated effects. Ctnnb1OE-SEC BM-SEC reveal aberrant sinusoidal differentiation with altered EC gene expression and perisinusoidal ECM deposition and angiocrine dysregulation with de novo endothelial expression of FGF23 and DKK2, elevated in anemia and involved in vascular stabilization, respectively. Our study demonstrates that BM-SEC play an important role in the bone marrow microenvironment in health and disease.

Imbalanced Activation of Wnt-/ß-Catenin-Signaling in Liver Endothelium Alters Normal Sinusoidal Differentiation.

Endothelial wingless-related integration site (Wnt)-/ß-catenin signaling is a key regulator of the tightly sealed blood-brain barrier. In the hepatic vascular niche angiokine-mediated Wnt signaling was recently identified as an important regulator of hepatocyte function, including the determination of final adult liver size, liver regeneration, and metabolic liver zonation. Within the hepatic vasculature, the liver sinusoidal endothelial cells (LSECs) are morphologically unique and functionally specialized microvascular endothelial cells (ECs). Pathological changes of LSECs are involved in chronic liver diseases, hepatocarcinogenesis, and liver metastasis. To comprehensively analyze the effects of endothelial Wnt-/ß-catenin signaling in the liver, we used endothelial subtype-specific Clec4g-iCre mice to generate hepatic ECs with overexpression of Ctnnb1. In the resultant Clec4g-iCre tg/wt ;Ctnnb1(Ex3) fl/wt (Ctnnb1 OE-EC ) mice, activation of endothelial Wnt-/ß-catenin signaling resulted in sinusoidal transdifferentiation with disturbed endothelial zonation, that is, loss of midzonal LSEC marker lymphatic vessel endothelial hyaluronic acid receptor 1 (Lyve1) and enrichment of continuous EC genes, such as cluster of differentiation (CD)34 and Apln. Notably, gene set enrichment analysis revealed overrepresentation of brain endothelial transcripts. Activation of endothelial Wnt-/ß-catenin signaling did not induce liver fibrosis or alter metabolic liver zonation, but Ctnnb1 OE-EC mice exhibited significantly increased plasma triglyceride concentrations, while liver lipid content was slightly reduced. Ctnnb1 overexpression in arterial ECs of the heart has been reported previously to cause cardiomyopathy. As Clec4g-iCre is active in a subset of cardiac ECs, it was not unexpected that Ctnnb1 OE-EC mice showed reduced overall survival and cardiac dysfunction. Altogether, balanced endothelial Wnt-/ß-catenin signaling in the liver is required for normal LSEC differentiation and for maintenance of normal plasma triglyceride levels.

Thermo- and pH-Responsive Gelatin/Polyphenolic Tannin/Graphene Oxide Hydrogels for Efficient Methylene Blue Delivery.

Gelatin (GE), amino-functionalized polyphenolic tannin derivative (TN), and graphene oxide (GO) were associated to yield thermo- and pH-responsive hydrogels for the first time. Durable hydrogel assemblies for drug delivery purposes were developed using the photosensitizer methylene blue (MB) as a drug model. The cooling GE/TN blends provide brittle physical assemblies. To overcome this disadvantage, different GO contents (between 0.31% and 1.02% wt/wt) were added to the GE/TN blend at 89.7/10.3 wt/wt. FTIR and RAMAN spectroscopy analyses characterized the materials, indicating GO presence in the hydrogels. Incorporation studies revealed a total MB (0.50 mg/mL) incorporation into the GE/TN-GO hydrogel matrices. Additionally, the proposed systems present a mechanical behavior similar to gel. The GO presence in the hydrogel matrices increased the elastic modulus from 516 to 1650 Pa. SEM revealed that hydrogels containing MB present higher porosity with interconnected pores. Dissolution and swelling degree studies revealed less stability of the GE/TN-GO-MB hydrogels in SGF medium (pH 1.2) than SIF (pH 6.8). The degradation increased in SIF with the GO content, making the polymeric matrices more hydrophilic. MB release studies revealed a process controlled by Fickian diffusion. Our results point out the pH-responsible behavior of mechanically reinforced GE/TN-GO-MB hydrogels for drug delivery systems purposes.

Exploring the transcriptomic network of multi-ligand scavenger receptor Stabilin-1- and Stabilin-2-deficient liver sinusoidal endothelial cells.

The Class H scavenger receptors Stabilin-1 (Stab1) and Stabilin-2 (Stab2) are two of the most highly expressed genes in liver sinusoidal endothelial cells (LSECs). While Stab1-deficient (Stab1KO) and Stab2-deficient (Stab2KO) mice are phenotypically unremarkable, Stab1/2-double-deficient (StabDKO) mice exhibit perisinusoidal liver fibrosis, glomerulofibrotic nephropathy and a reduced life expectancy. These conditions are caused by insufficiently scavenged circulating noxious blood factors. The effects of either Stab-single- or double-deficiency on LSEC differentiation and function, however, have not yet been thoroughly investigated. Therefore, we performed comprehensive transcriptomic analyses of primary LSECs from Stab1KO, Stab2KO and StabDKO mice. Microarray analysis revealed dysregulation of pathways and genes involved in established LSEC functions while sinusoidal endothelial marker gene expression was grossly unchanged. 82 genes were significantly altered in Stab1KO, 96 genes in Stab2KO and 238 genes in StabDKO compared with controls; 42 genes were found to be commonly dysregulated in all three groups and all of these genes were downregulated. These commonly downregulated genes (CDGs) were categorized as "potential scavengers," "cell adhesion molecules," "TGF-ß/BMP-signaling" or "collagen and extracellular matrix (ECM) components". Among CDGs, Colec10, Lumican and Decorin, were the most strongly down-regulated genes and the corresponding proteins impact on the interaction of LSECs with chemokines, ECM components and carbohydrate structures. Similarly, "chemokine signaling," "cytokine-cytokine receptor interaction" and "ECM-receptor interaction," were the GSEA categories which represented most of the downregulated genes in Stab1KO and Stab2KO LSECs. In summary, our data show that loss of a single Stabilin scavenger receptor - and to a greater extent of both receptors - profoundly alters the transcriptomic repertoire of LSECs. These alterations may affect LSEC-specific functions, especially interactions of LSECs with the ECM and during inflammation as well as clearance of the peripheral blood.

Endothelial GATA4 controls liver fibrosis and regeneration by preventing a pathogenic switch in angiocrine signaling.

BACKGROUND & AIMS: Angiocrine signaling by liver sinusoidal endothelial cells (LSECs) regulates hepatic functions such as growth, metabolic maturation, and regeneration. Recently, we identified GATA4 as the master regulator of LSEC specification during development. Herein, we studied the role of endothelial GATA4 in the adult liver and in hepatic pathogenesis. METHODS: We generated adult Clec4g-icretg/0xGata4fl/fl (Gata4LSEC-KO) mice with LSEC-specific depletion of Gata4. Livers were analyzed by histology, electron microscopy, immunohistochemistry/immunofluorescence, in situ hybridization, and LSECs were isolated for gene expression profiling, ChIP- and ATAC-sequencing. Partial hepatectomy was performed to assess regeneration. We used choline-deficient, l-amino acid-defined (CDAA) diet and chronic carbon tetrachloride exposure to model liver fibrosis. Human single cell RNA-seq data sets were analyzed for endothelial alterations in healthy and cirrhotic livers. RESULTS: Genetic Gata4 deficiency in LSECs of adult mice caused perisinusoidal liver fibrosis, hepatopathy and impaired liver regeneration. Sinusoidal capillarization and LSEC-to-continuous endothelial transdifferentiation were accompanied by a profibrotic angiocrine switch involving de novo endothelial expression of hepatic stellate cell-activating cytokine PDGFB. Increased chromatin accessibility and amplification by activated MYC mediated angiocrine Pdgfb expression. As observed in Gata4LSEC-KO livers, CDAA diet-induced perisinusoidal liver fibrosis was associated with GATA4 repression, MYC activation and a profibrotic angiocrine switch in LSECs. Comparison of CDAA-fed Gata4LSEC-KO and control mice demonstrated that endothelial GATA4 indeed protects against dietary-induced perisinusoidal liver fibrosis. In human cirrhotic livers, GATA4-positive LSECs and endothelial GATA4 target genes were reduced, while non-LSEC endothelial cells and MYC target genes including PDGFB were enriched. CONCLUSIONS: Endothelial GATA4 protects against perisinusoidal liver fibrosis by repressing MYC activation and profibrotic angiocrine signaling at the chromatin level. Therapies targeting the GATA4/MYC/PDGFB/PDGFRß axis offer a promising strategy for prevention and treatment of liver fibrosis. LAY SUMMARY: The liver vasculature is supposed to play a major role in the development of liver fibrosis and cirrhosis, which can lead to liver failure and liver cancer. Herein, we discovered that structural and transcriptional changes induced by genetic deletion of the transcription factor GATA4 in the hepatic endothelium were sufficient to cause liver fibrosis. Activation of the transcription factor MYC and de novo expression of the "angiocrine" growth factor PDGFB were identified as downstream drivers of fibrosis and as potential therapeutic targets for this potentially fatal disease.

Single-Cell RNA Sequencing of Tumor-Infiltrating NK Cells Reveals that Inhibition of Transcription Factor HIF-1α Unleashes NK Cell Activity.

Enhancing immune cell functions in tumors remains a major challenge in cancer immunotherapy. Hypoxia is a common feature of solid tumors, and cells adapt by upregulating the transcription factor HIF-1α. Here, we defined the transcriptional landscape of mouse tumor-infiltrating natural killer (NK) cells by using single-cell RNA sequencing. Conditional deletion of Hif1a in NK cells resulted in reduced tumor growth, elevated expression of activation markers, effector molecules, and an enriched NF-κB pathway in tumor-infiltrating NK cells. Interleukin-18 (IL-18) from myeloid cells was required for NF-κB activation and the enhanced anti-tumor activity of Hif1a-/- NK cells. Extended culture with an HIF-1α inhibitor increased human NK cell responses. Low HIF1A expression was associated with high expression of IFNG in human tumor-infiltrating NK cells, and an enriched NK-IL18-IFNG signature in solid tumors correlated with increased overall patient survival. Thus, inhibition of HIF-1α unleashes NK cell anti-tumor activity and could be exploited for cancer therapy.

Pianp deficiency links GABAB receptor signaling and hippocampal and cerebellar neuronal cell composition to autism-like behavior.

Pianp (also known as Leda-1) is a type I transmembrane protein with preferential expression in the mammalian CNS. Its processing is characterized by proteolytic cleavage by a range of proteases including Adam10, Adam17, MMPs, and the γ-secretase complex. Pianp can interact with Pilrα and the GB1a subunit of the GABAB receptor (GBR) complex. A recent case description of a boy with global developmental delay and homozygous nonsense variant in PIANP supports the hypothesis that PIANP is involved in the control of behavioral traits in mammals. To investigate the physiological functions of Pianp, constitutive, global knockout mice were generated and comprehensively analyzed. Broad assessment did not indicate malformation or malfunction of internal organs. In the brain, however, decreased sizes and altered cellular compositions of the dentate gyrus as well as the cerebellum, including a lower number of cerebellar Purkinje cells, were identified. Functionally, loss of Pianp led to impaired presynaptic GBR-mediated inhibition of glutamate release and altered gene expression in the cortex, hippocampus, amygdala, and hypothalamus including downregulation of Erdr1, a gene linked to autism-like behavior. Behavioral phenotyping revealed that Pianp deficiency leads to context-dependent enhanced anxiety and spatial learning deficits, an altered stress response, severely impaired social interaction, and enhanced repetitive behavior, which all represent characteristic features of an autism spectrum disorder-like phenotype. Altogether, Pianp represents a novel candidate gene involved in autism-like behavior, cerebellar and hippocampal pathology, and GBR signaling.

Effect of phase composition on the photocatalytic activity of titanium dioxide obtained from supercritical antisolvent.

Photocatalytic activity of TiO2 nanoparticles is highly dependent on their phase composition. The coexistence of anatase and rutile phases in a single nanoparticle eases the electron transfer process between the phases, and favors the separation of photogenerated pairs. In this work, highly photoactive mixed-phase TiO2 nanostructures were prepared by supercritical antisolvent precipitation (SAS), an environmentally friendly technology. It is shown here that this methodology has the remarkable ability to produce highly porous (515 m2/g) and crystalline TiO2 nanoparticles. The phase composition of as-prepared TiO2 samples can be tailored through annealing process. Several mixed-phase TiO2 samples were tested to assess the correlation between photocatalytic activity and phase composition. The photocatalytic performance is strongly affected by the anatase-rutile ratio, since the synergism between phases enhances the charge separation, reducing the recombination effect of the photogenerated pairs (e-/h+). It was found that the nanocatalyst composed by 7.0 wt% of rutile phase and 93.0 wt% of anatase phase, named as TiO2_650, presented the highest photodegradation for both methyl orange (MO) and methylene blue (MB) dyes. Interestingly, TiO2 samples prepared by SAS have superior photoactivity than the benchmark photocatalyst names as P25, which is a widely used TiO2 material composed of anatase and rutile phases.

Hepatic Endothelial Notch Activation Protects against Liver Metastasis by Regulating Endothelial-Tumor Cell Adhesion Independent of Angiocrine Signaling.

The interaction of tumor cells with organ-specific endothelial cells (EC) is an important step during metastatic progression. Notch signaling in organ-specific niches has been implicated in mediating opposing effects on organotropic metastasis to the lungs and the liver, respectively. In this study, we scrutinized the role of endothelial Notch activation during liver metastasis. To target hepatic EC (HEC), a novel EC subtype-specific Cre driver mouse was generated. Clec4g-Cretg/wt mice were crossed to Rosa26N1ICD-IRES-GFP to enhance Notch signaling in HEC (NICDOE-HEC). In NICDOE-HEC mice, hepatic metastasis of malignant melanoma and colorectal carcinoma was significantly reduced. These mice revealed reduced liver growth and impaired metabolic zonation due to suppression of hepatic angiocrine Wnt signaling. Hepatic metastasis, however, was not controlled by angiocrine Wnt signaling, as deficiency of the Wnt cargo receptor Wls in HEC of WlsHEC-KO mice did not affect hepatic metastasis. In contrast, the hepatic microvasculature in NICDOE-HEC mice revealed a special form of sinusoidal capillarization, with effacement of endothelial zonation functionally paralleled by reduced tumor cell adhesion in vivo. Notably, expression of endothelial adhesion molecule ICAM1 by HEC was significantly reduced. Treatment with an anti-ICAM1 antibody significantly inhibited tumor cell adhesion to HEC in wild-type mice confirming that Notch controls hepatic metastasis via modulation of HEC adhesion molecules. As endothelial Notch activation in the lung has been shown to promote lung metastasis, tumor therapy will require approaches that target Notch in an organ-, cell type-, and context-specific manner. SIGNIFICANCE: Manipulation of Notch signaling in the endothelium has opposing, organ-specific effects on metastasis to the lung and the liver, demonstrating that this pathway should be targeted in a cell- and context-specific fashion.

Influence of Gadolinium-Based Contrast Agents on Tissue Sodium Quantification in Sodium Magnetic Resonance Imaging.

OBJECTIVES: Sodium magnetic resonance (MR) imaging provides noninvasive insights to cellular processes by measuring tissue sodium concentration (TSC). Many clinical studies combine sodium MR imaging with clinical standard MR procedures, in which contrast media is frequently administered. This work investigates the influence of gadolinium-based contrast agents on quantification of TSC. Thus, either scan pauses between early and late contrast-enhanced acquisitions can be used efficiently or sodium imaging can be performed as the final scan after dynamic contrast-enhanced acquisition. MATERIALS AND METHODS: For this study, 2 gadolinium-based contrast agents, Dotarem and Gadovist, were diluted with saline solution covering contrast agent concentrations in a clinical range. In addition, agarose-based sample series were created to simulate tissue relaxation time behavior. In vivo, the influence of Dotarem on sodium acquisition and TSC quantification was investigated in 1 ischemic stroke patient. RESULTS: Proton relaxation times decreased for increasing contrast agent concentrations as hyperbolic functions. Sodium relaxation times displayed a negative slope in regression analysis in most cases. The largest influence (-1.52 milliseconds per mmol/L contrast agent) was measured for sodium T1. Worst case calculations in ultrashort echo time sequence signal analysis showed a signal drop of (1.21% ± 0.56%) on tissue sodium quantification. In vivo sodium brain acquisitions of a stroke patient before and after Dotarem injection resulted in statistically nonsignificant differences in TSC quantification of relevant tissues and stroke areas (P > 0.05). CONCLUSIONS: Our study showed a quantitative influence of Dotarem and Gadovist on sodium relaxation times. However, quantification of TSC was not impaired, which was proven by worst case calculations and nonsignificant differences in vivo in an ischemic stroke patient. We suggest performing sodium imaging in useful clinical positions in protocols regardless of included Dotarem or Gadovist administrations. Being flexible in the study protocol design will strengthen ongoing sodium imaging investigations for various pathologies.

Posttranslational proteolytic processing of Leda-1/Pianp involves cleavage by MMPs, ADAM10/17 and gamma-secretase.

Leda-1/Pianp is a type I transmembrane protein expressed by CNS cells, murine melanoma cell line B16F10 and rat liver sinusoidal endothelial cells. The early steps of posttranslational modifications of Leda-1/Pianp have been described to include glycosylation and processing by proprotein convertases. Here, we comprehensively characterized the subsequent steps of proteolytic processing of Leda-1/Pianp. For this purpose specific protease inhibitors and cell lines deficient in PS1, PS2, PS1/PS2 and ADAM10/17 were deployed. Leda-1/Pianp was cleaved at numerous cleavage sites within the N-terminal extracellular domain. The sheddases involved included MMPs and ADAM10/17. Ectodomain shedding yielded C-terminal fragments (CTF) of ∼15 kDa. The CTF was further processed by the γ (gamma)-secretase complex to generate the intracellular domain (ICD) of ∼10 kDa. Although PS1 was the dominant intramembrane protease, PS2 was also able to cleave Leda-1/Pianp in the absence of PS1. Thus, Leda-1/Pianp is constitutively processed by proprotein convertases, sheddases including MMPs and ADAM10/17 and intramembrane protease γ-secretase.

Leda-1/Pianp is targeted to the basolateral plasma membrane by a distinct intracellular juxtamembrane region and modulates barrier properties and E-Cadherin processing.

Leda-1/Pianp is a type-I transmembrane protein which is sorted to the basolateral membrane domain of polarized epithelial cells. Here, we investigated trafficking mechanisms and functions of Leda-1/Pianp in MDCK and MCF-7 cells. Basolateral sorting and posttranslational modifications depended on the intracellular juxtamembrane region. Functionally, Leda-1/Pianp increased the transepithelial electrical resistance generated by a polarized cell sheet. Furthermore, resistance to junctional destabilization by tumor cells was enhanced by Leda-1/Pianp indicating increased stability and tightness of intercellular junctions. While Claudin 1 and 4 expression and activities of small GTPases were not affected, γ-Secretase-mediated cleavage of E-Cadherin was attenuated by Leda-1/Pianp. Regulation of proteolytic processing is thus a molecular mechanism by which Leda-1/Pianp can affect junctional integrity and function.

Counter-regulation of the ligand-receptor pair Leda-1/Pianp and Pilrα during the LPS-mediated immune response of murine macrophages.

Liver endothelial differentiation-associated protein-1 (Leda-1/Pianp) is a type-I-transmembrane protein that is able to bind and activate immune inhibitory receptor Pilrα. Here we show that Leda-1/Pianp is strain-specifically expressed in lymphoid organs and macrophages of Th2-prone BALB/c mice but not of Th1-prone C57BL/6J mice. LPS stimulation of BALB/c bone marrow-derived macrophages (BMM) and macrophage-like Raw 264.7 cells conversely regulated Leda-1/Pianp and Pilrα expression. Pilrα induction was caused by LPS-mediated transcriptional modulation and increased mRNA expression. On the other hand, the LPS-mediated decline of Leda-1/Pianp expression was the result of proteolytic degradation by matrix metalloproteinases. In summary, these findings demonstrate that counter-regulation of the ligand-receptor pair Leda-1/Pianp and Pilrα is part of the complex innate immune response of macrophages and its genetically determined strain-specific modulation.

Proteolytic cleavage of LEDA-1/PIANP by furin-like proprotein convertases precedes its plasma membrane localization.

Liver endothelial differentiation-associated protein-1 (LEDA-1/PIANP) is a type-I-transmembrane protein first identified by us as a putative junctional protein in liver sinusoidal endothelial cells. Others have shown that LEDA-1/PIANP binds and activates immune inhibitory receptor PILRα in trans, a process that requires sialidation of LEDA-1/PIANP. Here we show that LEDA-1/PIANP is subject to O-glycosylation and sialidation as demonstrated in brain tissue as well as in LEDA-1 expressing cell lines by using anti-LEDA-1/PIANP C-terminal antibodies. In addition, analysis of LEDA-1/PIANP processing with His-tags inserted at different positions in the extracellular domain revealed that multiple steps of proteolytic cleavage occur during maturation of the protein. Proteolytic cleavage between aa59 and aa83 preceded sorting of the protein to the plasma membrane. Deletion of aa75-79 and inhibition with Furin inhibitor I confirmed that LEDA-1/PIANP is processed by a Furin-like proprotein convertase. In summary, these findings show that Furin-like proprotein convertase-dependent processing precedes plasma membrane localization of LEDA-1/PIANP that is a pre-requisite of functional receptor-ligand interactions in vitro and in vivo.