Riboflavin-targeted polymers improve tolerance of paclitaxel while maintaining therapeutic efficacy.

Active targeting can enhance precision and efficacy of drug delivery systems (DDS) against cancers. Riboflavin (RF) is a promising ligand for active targeting due to its biocompatibility and high riboflavin-receptor expression in cancers. In this study, RF-targeted 4-arm polyethylene glycol (PEG) stars conjugated with Paclitaxel (PTX), named PEG PTX RF, were evaluated as a targeted DDS. In vitro, PEG PTX RF exhibited higher toxicity against tumor cells compared to the non-targeted counterpart (PEG PTX), while free PTX displayed the highest acute toxicity. In vivo, all treatments were similarly effective, but PEG PTX RF-treated tumors showed fewer proliferating cells, pointing to sustained therapy effects. Moreover, PTX-treated animals' body and liver weights were significantly reduced, whereas both remained stable in PEG PTX and PEG PTX RF-treated animals. Overall, our targeted and non-targeted DDS reduced PTX's adverse effects, with RF targeting promoted drug uptake in cancer cells for sustained therapeutic effect.

Carbamylated sortilin associates with cardiovascular calcification in patients with chronic kidney disease.

Sortilin, an intracellular sorting receptor, has been identified as a cardiovascular risk factor in the general population. Patients with chronic kidney disease (CKD) are highly susceptible to develop cardiovascular complications such as calcification. However, specific CKD-induced posttranslational protein modifications of sortilin and their link to cardiovascular calcification remain unknown. To investigate this, we examined two independent CKD cohorts for carbamylation of circulating sortilin and detected increased carbamylated sortilin lysine residues in the extracellular domain of sortilin with kidney function decline using targeted mass spectrometry. Structure analysis predicted altered ligand binding by carbamylated sortilin, which was verified by binding studies using surface plasmon resonance measurement, showing an increased affinity of interleukin 6 to in vitro carbamylated sortilin. Further, carbamylated sortilin increased vascular calcification in vitro and ex vivo that was accelerated by interleukin 6. Imaging by mass spectrometry of human calcified arteries revealed in situ carbamylated sortilin. In patients with CKD, sortilin carbamylation was associated with coronary artery calcification, independent of age and kidney function. Moreover, patients with carbamylated sortilin displayed significantly faster progression of coronary artery calcification than patients without sortilin carbamylation. Thus, carbamylated sortilin may be a risk factor for cardiovascular calcification and may contribute to elevated cardiovascular complications in patients with CKD.

Guanidinylated Apolipoprotein C3 (ApoC3) Associates with Kidney and Vascular Injury.

BACKGROUND: Coexistent CKD and cardiovascular diseases are highly prevalent in Western populations and account for substantial mortality. We recently found that apolipoprotein C-3 (ApoC3), a major constituent of triglyceride-rich lipoproteins, induces sterile systemic inflammation by activating the NOD-like receptor protein-3 (NLRP3) inflammasome in human monocytes via an alternative pathway. METHODS: To identify posttranslational modifications of ApoC3 in patients with CKD, we used mass spectrometry to analyze ApoC3 from such patients and from healthy individuals. We determined the effects of posttranslationally modified ApoC3 on monocyte inflammatory response in vitro, as well as in humanized mice subjected to unilateral ureter ligation (a kidney fibrosis model) and in a humanized mouse model for vascular injury and regeneration. Finally, we conducted a prospective observational trial of 543 patients with CKD to explore the association of posttranslationally modified ApoC3 with renal and cardiovascular events in such patients. RESULTS: We identified significant posttranslational guanidinylation of ApoC3 (gApoC3) in patients with CKD. We also found that mechanistically, guanidine and urea induce guanidinylation of ApoC3. A 2D-proteomic analysis revealed that gApoC3 accumulated in kidneys and plasma in a CKD mouse model (mice fed an adenine-rich diet). In addition, gApoC3 augmented the proinflammatory effects of ApoC3 in monocytes in vitro . In humanized mice, gApoC3 promoted kidney tissue fibrosis and impeded vascular regeneration. In CKD patients, higher gApoC3 plasma levels (as determined by mass spectrometry) were associated with increased mortality as well as with renal and cardiovascular events. CONCLUSIONS: Guanidinylation of ApoC3 represents a novel pathogenic mechanism in CKD and CKD-associated vascular injury, pointing to gApoC3 as a potential therapeutic target.

The YB-1:Notch-3 axis modulates immune cell responses and organ damage in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease and lupus nephritis is a major risk factor for morbidity and mortality. Notch-3 signaling induced by membrane-bound or soluble ligands such as YB-1 constitutes an evolutionarily conserved pathway that determines major decisions in cell fate. Mass spectrometry of extracellular YB-1 in sera from patients with SLE and lupus-prone mice revealed specific post-translational guanidinylation of two lysine residues within the highly conserved cold-shock domain of YB-1 (YB-1-G). These modifications highly correlated with SLE disease activity, especially in patients with lupus nephritis and resulted in enhanced activation of Notch-3 signaling in T lymphocytes. The importance of YB-1:Notch-3 interaction in T cells was further evidenced by increased interleukin (Il)10 expression following YB-1-G stimulation and detection of both, YB-1-G and Notch-3, in kidneys of MRL.lpr mice by mass spectrometry imaging. Notch-3 expression and activation was significantly up-regulated in kidneys of 20-week-old MRL.lpr mice. Notably, lupus-prone mice with constitutional Notch-3 depletion (B6.Faslpr/lprNotch3-/-) exhibited an aggravated lupus phenotype with significantly increased mortality, enlarged lymphoid organs and aggravated nephritis. Additionally, these mice displayed fewer regulatory T cells and reduced amounts of anti-inflammatory IL-10. Thus, our results indicate that the YB-1:Notch-3 axis exerts protective effects in SLE and that Notch-3 deficiency exacerbates the SLE phenotype.

CXCR6 Inhibits Hepatocarcinogenesis by Promoting Natural Killer T- and CD4+ T-Cell-Dependent Control of Senescence.

BACKGROUND & AIMS: Inflammation in the liver provokes fibrosis, but inflammation is also important for tumor surveillance. Inhibitors of chemokine pathways, such as CXCL16 and CXCR6 regulation of lymphocyte trafficking, are being tested as antifibrotic agents, but their effects on the development of hepatocellular carcinoma (HCC) are unclear. We assessed the roles of CXCR6-dependent immune mechanisms in hepatocarcinogenesis. METHODS: C57BL/6J wild-type (WT) mice and CXCR6-deficient mice (Cxcr6eGfp/eGfp) were given injections of diethylnitrosamine (DEN) to induce liver cancer and α-galactosylceramide to activate natural killer T (NKT) cells. We also performed studies in mice with conditional, hepatocyte-specific deletion of NEMO, which develop inflammation-associated liver tumors (NemoLPC-KO and NemoLPC-KOCxcr6eGfp/eGfp mice). We collected liver tissues from patients with cirrhosis (n = 43), HCC (n = 35), and neither of these diseases (control individuals, n = 25). Human and mouse liver tissues were analyzed by histology, immunohistochemistry, flow cytometry, RNA expression arrays (from sorted hepatic lymphocytes), and matrix-assisted laser desorption/ionization imaging. Bone marrow was transferred from Cxcr6eGfp/eGfp or WT mice to irradiated C57BL/6J mice, and spleen and liver cells were analyzed by flow cytometry. CD4+ T cells or NKT cells were isolated from the spleen and liver of CD45.1+ WT mice and transferred into CXCR6-deficient mice after DEN injection. RESULTS: After DEN injection, CXCR6-deficient mice had a significantly higher tumor burden than WT mice and increased tumor progression, characterized by reduced intrahepatic numbers of invariant NKT and CD4+ T cells that express tumor necrosis factor and interferon gamma. Livers of NemoLPC-KOCxcr6eGfp/eGfp mice had significantly more senescent hepatocytes than livers of NemoLPC-KO mice. In studies of bone-marrow chimeras, adoptive cell transfer experiments, and analyses of NemoLPC-KO mice, we found that NKT and CD4 T cells promote the removal of senescent hepatocytes to prevent hepatocarcinogenesis, and that this process required CXCR6. Injection of WT with α-galactosylceramide increased removal of senescent hepatocytes by NKT cells. We observed peritumoral accumulation of CXCR6-associated lymphocytes in human HCC, which appeared reduced compared with cirrhosis tissues. CONCLUSIONS: In studies of mice with liver tumors, we found that CXCR6 mediated NKT-cell and CD4+ T-cell removal of senescent hepatocytes. Antifibrotic strategies to reduce CXCR6 activity in liver, or to reduce inflammation or modulate the immune response, should be tested for their effects on hepatocarcinogenesis.

Sample preparation of formalin-fixed paraffin-embedded tissue sections for MALDI-mass spectrometry imaging.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MALDI MSI) has become a powerful tool with a high potential relevance for the analysis of biomolecules in tissue samples in the context of diseases like cancer and cardiovascular or cardiorenal diseases. In recent years, significant progress has been made in the technology of MALDI MSI. However, a more systematic optimization of sample preparation would likely achieve an increase in the molecular information derived from MALDI MSI. Therefore, we have employed a systematic approach to develop, establish and validate an optimized "standard operating protocol" (SOP) for sample preparation in MALDI MSI of formalin-fixed paraffin-embedded (FFPE) tissue sample analyses within this study. The optimized parameters regarding the impact on the resulting signal-to-noise (S/N) ratio were as follows: (i) trypsin concentration, solvents, deposition method, and incubation time; (ii) tissue washing procedures and drying processes; and (iii) spray flow rate, number of layers of trypsin deposition, and grid size. The protocol was evaluated on interday variability and its applicability for analyzing the mouse kidney, aorta, and heart FFPE tissue samples. In conclusion, an optimized SOP for MALDI MSI of FFPE tissue sections was developed to generate high sensitivity, to enhance spatial resolution and reproducibility, and to increase its applicability for various tissue types. This optimized SOP will further increase the molecular information content and intensify the use of MSI in future basic research and diagnostic applications. Graphical Abstract.

In vitro and in vivo evaluation of biohybrid tissue-engineered vascular grafts with transformative 1H/19F MRI traceable scaffolds.

Biohybrid tissue-engineered vascular grafts (TEVGs) promise long-term durability due to their ability to adapt to hosts' needs. However, the latter calls for sensitive non-invasive imaging approaches to longitudinally monitor their functionality, integrity, and positioning. Here, we present an imaging approach comprising the labeling of non-degradable and degradable TEVGs' components for their in vitro and in vivo monitoring by hybrid 1H/19F MRI. TEVGs (inner diameter 1.5 mm) consisted of biodegradable poly(lactic-co-glycolic acid) (PLGA) fibers passively incorporating superparamagnetic iron oxide nanoparticles (SPIONs), non-degradable polyvinylidene fluoride scaffolds labeled with highly fluorinated thermoplastic polyurethane (19F-TPU) fibers, a smooth muscle cells containing fibrin blend, and endothelial cells. 1H/19F MRI of TEVGs in bioreactors, and after subcutaneous and infrarenal implantation in rats, revealed that PLGA degradation could be faithfully monitored by the decreasing SPIONs signal. The 19F signal of 19F-TPU remained constant over weeks. PLGA degradation was compensated by cells' collagen and α-smooth-muscle-actin deposition. Interestingly, only TEVGs implanted on the abdominal aorta contained elastin. XTT and histology proved that our imaging markers did not influence extracellular matrix deposition and host immune reaction. This concept of non-invasive longitudinal assessment of cardiovascular implants using 1H/19F MRI might be applicable to various biohybrid tissue-engineered implants, facilitating their clinical translation.

Identification and characterization of post-translational modifications: Clinical implications.

Post-translational modifications (PTMs) generate marginally modified isoforms of native peptides, proteins and lipoproteins thereby regulating protein functions, molecular interactions, and localization. With a key role in functional proteomics, post-translational modifications are recently also associated with the onsets and progressions of various diseases, such as cancer, cardiovascular, renal, and metabolic diseases. With the impact of post-translational modifications becoming increasingly clear, its reliable detection and quantification remain a major obstacle in the translation of these novel pathological markers into clinical diagnosis. While current antibody-based clinical diagnostics struggle to detect and quantify these marginal protein and lipoprotein alterations, state-of-the-art mass spectrometric, proteomic approaches provide the mass accuracy and resolving power necessary to isolate, identify and quantify novel and pathological post-translational modifications; however clinical translation of mass spectrometric applications are still facing major challenges. Here we review the status quo of the clinical translation of mass-spectrometric applications as novel diagnostic tools for the identification and quantification of post-translational modifications and focus on the emerging role of mass spectrometric methods in the clinical assessment of PTMs in disease states.

Glutamine prevents acute kidney injury by modulating oxidative stress and apoptosis in tubular epithelial cells.

Acute kidney injury (AKI) represents a common complication in critically ill patients that is associated with increased morbidity and mortality. In a murine AKI model induced by ischemia/reperfusion injury (IRI), we show that glutamine significantly decreases kidney damage and improves kidney function. We demonstrate that glutamine causes transcriptomic and proteomic reprogramming in murine renal tubular epithelial cells (TECs), resulting in decreased epithelial apoptosis, decreased neutrophil recruitment, and improved mitochondrial functionality and respiration provoked by an ameliorated oxidative phosphorylation. We identify the proteins glutamine gamma glutamyltransferase 2 (Tgm2) and apoptosis signal-regulating kinase (Ask1) as the major targets of glutamine in apoptotic signaling. Furthermore, the direct modulation of the Tgm2-HSP70 signalosome and reduced Ask1 activation resulted in decreased JNK activation, leading to diminished mitochondrial intrinsic apoptosis in TECs. Glutamine administration attenuated kidney damage in vivo during AKI and TEC viability in vitro under inflammatory or hypoxic conditions.

Registration of Image Modalities for Analyses of Tissue Samples Using 3D Image Modelling.

PURPOSE: Biopsies are a diagnostic tool for the diagnosis of histopathological, molecular biological, proteomic, and imaging data, to narrow down disease patterns or identify diseases. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) provides an emerging state-of-the-art technique for molecular imaging of biological tissue. The aim of this study is the registration of MALDI MSI data sets and data acquired from different histological stainings to create a 3D model of biopsies and whole organs. EXPERIMENTAL DESIGN: The registration of the image modalities is achieved by using a variant of the authors' global, deformable Schatten-q-Norm registration approach. Utilizing a connected-component segmentation for background removal followed by a principal-axis based linear pre-registration, the images are adjusted into a homogeneous alignment. This registration approach is accompanied by the 3D reconstruction of histological and MALDI MSI data. RESULTS: With this, a system of automatic registration for cross-process evaluation, as well as for creating 3D models, is developed and established. The registration of MALDI MSI data with different histological image data is evaluated by using the established global image registration system. CONCLUSIONS AND CLINICAL RELEVANCE: In conclusion, this multimodal image approach offers the possibility of molecular analyses of tissue specimens in clinical research and diagnosis.