Cutaneous T-cell lymphomas (CTCL) are a group of rare hematological malignancies characterized by infiltration of malignant T-cells into the skin. Two main types of CTCL constitute of Mycosis Fungoides (MF), a more indolent form of the disease, and Sézary syndrome (SS), the aggressive and leukemic variant with blood involvement. Sézary syndrome presents a significant clinical challenge due to its very aggressive nature, poor prognosis, and treatment resistance, and to date, the disease remains incurable. Histone deacetylase inhibitors have gained attention in CTCL treatment with promising results, but they expose limited specificity and strong side effects. Recent genomic studies underscore the role of epigenetic modifiers in CTCL pathogenesis, prompting an investigation into HDAC10, a member of class IIb HDACs, in SS. HDAC10 was investigated in different cancers, revealing its involvement in cell cycle regulation, apoptosis, and autophagy, but its role in CTCL is unknown. In this study we aimed to determine the role of HDAC10 in SS, focusing on its cellular localization, role in cell growth, and therapeutic potential. We indicated that HDAC10 is overexpressed in SS patients and located mainly in the cytoplasm. Its overexpression leads to an inhibitory effect on apoptosis progression when exposed to the pro-apoptotic compound Camptothecin (CPT). Knockdown of HDAC10 resulted in reduced cell growth and induction of apoptosis and autophagy, highlighting its potential importance in CTCL pathogenesis. Whole transcriptome analysis indicated that HDAC10 is associated with crucial cancer-related pathways, for example, hematopoietic cell lineage, PI3K-Akt signaling pathway, Ras signaling pathway, MAPK signaling pathway or JAK-STAT signaling pathway, which are critical for the survival and proliferation of malignant T cells. Inhibition of HDAC10 with selective HDAC10i increased the sensitivity of Sézary cells to the pro-apoptotic CPT. Our findings demonstrate that HDAC10 plays a key role in the molecular background of Sézary syndrome, highlighting its importance in the cellular mechanisms of the disease.

BACKGROUND:Vascular calcification is highly prevalent in Chronic Kidney Disease (CKD) and is associated with markedly increased cardiovascular risk. High serum phosphate in CKD increases calcification propensity via generation of circulating calciprotein particles (CPP2), crystalline nanoaggregates composed of calcium, phosphate, and serum proteins. CPP2 induce vascular calcification in vascular smooth muscle cells (VSMCs) in vitro. In vivo, endothelial cells, rather than VSMCs are primarily exposed to CPP2, yet understanding the influence of endothelial cells on vascular calcification is limited. METHODS:We investigated calcification-promoting signalling by endothelial cells on VSMCs. Effects of CPP2 exposure to endothelial cells on CPP2 uptake, endothelial cell activation, and endothelial cell-derived secretome were studied. Effects of the secretome on VSMC calcification were investigated. Using NanoString nCounter analysis the effects of CPP2-activated endothelial cell-conditioned medium on VSMCs gene expression were mapped. RESULTS:Endothelial cells internalise CPP2 and elevate ICAM-1, E-selectin, and VCAM-1-mRNA expression, indicating endothelial activation. VSMCs cultured in conditioned medium from CPP2-activated endothelial cells demonstrated enhanced calcification, suggesting that CPP2-activated endothelial cells release pro-calcifying soluble factors. Mass spectrometry was utilized to identify 1171 proteins in the CPP2-activated endothelial cells' secretome. Among these, 76 proteins were differentially expressed compared to control endothelial cells' secretome, including proteins related to blood vessel development, extracellular matrix remodelling, and oxidative stress-related processes. Finally, endothelial cell-derived paracrine factors present in conditioned medium enhanced mRNA-expression of calcification-related factors in VSMCs. CONCLUSIONS:CPP2-activated endothelial cells promote VSMC calcification via paracrine signalling. In response to these paracrine factors, VSMCs increase the expression of pro-calcification genes.

Introduction PIs form the backbone of most first-line regimens for MM, both in transplant-eligible and ineligible patients. Despite advances, MM remains incurable primarily due to the development of drug resistance. Many studies on this phenomenon in PI-resistant cells have been conducted in recent years; nevertheless, we still lack an explanation that would lead to clinically translatable findings. In this study, to achieve better insight into the process of acquiring resistance to bortezomib (BTZ) and carfilzomib (CFZ), we conducted comparative proteomic profiling of sensitive MM cell lines and their resistant counterparts at different stages of acquiring the resistant phenotype. To further enhance the generalizability of these findings, we performed the experiments on two different MM cell lines. Methods Resistant cell lines were generated by continuously culturing sensitive RPMI8226 and U266B1 MM cells with increasing concentrations of BTZ or CFZ. Resistance was defined by the ability to remain viable in continuous culture with increasing concentrations of the drugs. Comparative proteomic profiling of sensitive cells and cells resistant to increasing concentrations of both drugs was performed as described previously (Kubicki, Am J of Cancer Res 2022). The Western blot (WB) method was used to validate the proteomic results. Proteasome activity was measured by the Proteasome-Glo Assays (Promega, USA). Apoptosis was measured by BH3 profiling as in previous reports (Langedonk, Int J Mol Sci 2022). Results RPMI8226 cells included in this analysis were able to remain viable in culture with 3, 4, 6 and 13 nM of BTZ and 2, 4, 6 and 23 nM of CFZ. For U266B1 cell lines, the concentrations were 1, 2, 5 and 11 nM of BTZ and 2, 5, 6, 11 and 17 nM of CFZ. Both drugs were able to similarly suppress chymotrypsin-like proteasome activity in both sensitive and resistant RPMI8226 and U266B1 cells. Additionally, the baseline activity of all three proteasome catalytic domains was the same in all cell lines. Subsequently, we assessed the cells' global adaptations through comparative proteomic profiling. The top-ranked canonical pathway was oxidative phosphorylation inhibited in BTZ-resistant (B-H p < 10-21; z-score = 5.7), CFZ-resistant RPMI8226 (B-H p < 10-30; z-score = 5.9), BTZ-resistant (B-H p < 10-2; z-score = 5.0), and CFZ-resistant U266B1 (B-H p < 10-2; z-score = 5.3) cells. Accumulation of almost all proteins involved in this process was linearly downregulated with increasing drugs concentrations. In line with the well-described Warburg effect, cytoplasmic proteins responsible for gluconeogenesis and glycolysis were also consistently upregulated in the resistant U266B1 and RPMI8226 cells, whereas mitochondrial proteins involved in the tricarboxylic acid cycle were downregulated. These finding were further supported by the downregulation of mitochondrial proteins that may contribute to mitochondrial dysfunction in all resistant cell lines. Based on these results, we selected the mitochondrial transcription factor A (TFAM) protein as a potential marker of resistance and confirmed its lower accumulation in both resistant RPMI8226 and U266B1 cells using WB. We also identified significant differences in accumulation of proteins associated with apoptosis in BTZ-resistant RPMI8226 cells (B-H p < 10-29; z-score = -2.9). After priming during BH3 profiling, mitochondrial outer membrane permeabilization was lower in BTZ-resistant cells compared to the sensitive ones. Synergy between bortezomib and different BH3-mimetics was confirmed (5.1 for 1 µM of venetoclax, 2.1 for 1 µM of navitoclax, 2.7 for 0.1 µM of BCL-XL inhibitor, 1.5 for 0.01 µM of MCL1 inhibitor); the combination treatment was capable of partially restoring sensitivity to BTZ. Of note, RPMI8226 cells do not harbor the translocation t(11;14). Intriguingly, we did not observe similar pattern for CFZ-resistant cells and for U266B1. Conclusions CFZ- and BTZ-resistant RPMI8226 and U266B1 MM cells exhibit a Warburg effect phenotype and signs of mitochondrial dysfunction. We identified TFAM as a potential biomarker of resistance to PIs. Combining BH3-mimetics with BTZ restored sensitivityin the resistant RPMI8226 cells. Further studies are needed to confirm the clinical significance of these results, explore the potential of targeting mitochondrial metabolism and identify patients who may benefit from BH3-mimetics.

Aims Calciprotein particles (CPPs) are circulating calcium and phosphate nanoparticles associated with the development of vascular calcification (VC) in chronic kidney disease (CKD). Although recent studies have been focusing on associations of CPPs with the presence of VC in CKD, insights in the underlying processes and mechanisms by which CPPs might aggravate VC and vascular dysfunction in vivo are currently lacking. Here, we assessed the overall burden of abdominal VC in healthy kidney donors and CKD patients and subsequently performed transcriptome profiling in the vascular tissue obtained from these subjects, linking outcome to CPP counts and calcification propensity. Methods and results Calcification scores were quantified in renal arteries, iliac arteries, and abdominal aorta using computed tomography (CT) scans of kidney donors and CKD patients. The vascular tissue was collected from kidney donors (renal artery) and CKD patients (iliac artery), after which bulk RNA sequencing and gene set enrichment analysis (GSEA) were performed on a subset of patients. Calcification propensity (crystallization time, T50) was measured using nephelometry and CPP counts with microparticle flow cytometric analysis. Increased calcification scores (based on CT) were found in CKD patients compared to kidney donors. Transcriptome profiling revealed enrichment for processes related to endothelial activation, inflammation, extracellular matrix (ECM) remodelling, and ossification in CKD vascular biopsies compared to kidney donors. Calcification propensity was increased in CKD, as well as CPP counts, with the latter being significantly associated with markers of vascular remodelling. Conclusion Our findings reveal that CKD is characterized by systemic VC with increased calcification propensity and CPP counts. Transcriptome profiling showed altered vascular gene expression with enrichment for endothelial activation, inflammation, ECM remodelling, and ossification. Moreover, we demonstrate, for the first time, that vascular remodelling processes are associated with increased circulating CPP counts. Interventions targeting CPPs are promising avenues for alleviating vascular remodelling and VC in CKD.

Mesenchymal stromal cells (MSCs) may provide crucial support in the regeneration of destructed alveolar tissue (emphysema) in chronic obstructive pulmonary disease (COPD). We hypothesized that lung-derived MSCs (LMSCs) from patients with emphysema are hampered in their repair capacity, either intrinsically or due to their interaction with the damaged microenvironment. LMSCs were isolated from the lung tissue of controls and patients with severe emphysema and characterized at baseline. In addition, LMSCs were seeded onto control and emphysematous decellularized lung tissue scaffolds and assessed for deposition of extracellular matrix (ECM). We observed no differences in surface markers, differentiation/proliferation potential, and expression of ECM genes between control- and COPD-derived LMSCs. Notably, COPD-derived LMSCs displayed lower expression of FGF10 and HGF messenger RNA (mRNA) and hepatocyte growth factor (HGF) and decorin protein. When seeded on control decellularized lung tissue scaffolds, control- and COPD-derived LMSCs showed no differences in engraftment, proliferation, or survival within 2 wk, with similar ability to deposit new matrix on the scaffolds. Moreover, LMSC numbers and the ability to deposit new matrix were not compromised on emphysematous scaffolds. Collectively, our data show that LMSCs from patients with COPD compared with controls show less expression of FGF10 mRNA, HGF mRNA and protein, and decorin protein, whereas other features including the mRNA expression of various ECM molecules are unaffected. Furthermore, COPD-derived LMSCs are capable of engraftment, proliferation, and functioning on native lung tissue scaffolds. The damaged, emphysematous microenvironment as such does not hamper the potential of LMSCs. Thus, specific intrinsic deficiencies in growth factor production by diseased LMSCs may contribute to impaired alveolar repair in emphysema.

Purpose 2-deoxy-2-[ 18 F]fluoro- d -glucose ([ 18 F]FDG) uptake is a marker of metabolic activity and is therefore used to measure the inflammatory state of several tissues. This radionuclide marker is transported through the cell membrane via glucose transport proteins (GLUTs). The aim of this study is to investigate whether insulin resistance (IR) or inflammation plays a role in [ 18 F]FDG uptake in adipose tissue (AT). Procedures This study consisted of an in vivo clinical part and an ex vivo mechanistic part. In the clinical part, [ 18 F]FDG uptake in abdominal visceral AT (VAT) and subcutaneous AT (SAT) was determined using PET/CT imaging in 44 patients with early type 2 diabetes mellitus (T2DM) (age 63 [54–66] years, HbA1c [6.3 ± 0.4 %], HOMA-IR 5.1[3.1–8.5]). Plasma levels were measured with ELISA. In the mechanistic part, AT biopsies obtained from 8 patients were ex vivo incubated with [ 18 F]FDG followed by autoradiography. Next, a qRT-PCR analysis was performed to determine GLUT and cytokine mRNA expression levels. Immunohistochemistry was performed to determine CD68 + macrophage infiltration and GLUT4 protein expression in AT. Results In vivo VAT [ 18 F]FDG uptake in patients with T2DM was inversely correlated with HOMA-IR ( r = − 0.32, p = 0.034), and positively related to adiponectin plasma levels ( r = 0.43, p = 0.003). Ex vivo [ 18 F]FDG uptake in VAT was not related to CD68 + macrophage infiltration, and IL-1ß and IL-6 mRNA expression levels. Ex vivo VAT [ 18 F]FDG uptake was positively related to GLUT4 ( r = 0.83, p = 0.042), inversely to GLUT3 ( r = − 0.83, p = 0.042) and not related to GLUT1 mRNA expression levels. Conclusions In vivo [ 18 F]FDG uptake in VAT from patients with T2DM is positively correlated with adiponectin levels and inversely with IR. Ex vivo [ 18 F]FDG uptake in AT is associated with GLUT4 expression but not with pro-inflammatory markers. The effect of IR should be taken into account when interpreting data of [ 18 F]FDG uptake as a marker for AT inflammation.

Background: Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease, characterized by high levels of genomic instability and the activation of DNA damage repair pathways. We previously found high expression of the cell cycle regulator WEE1 in DLBCL cell lines. Here, we investigated the combination of the WEE1 inhibitor, AZD1775, with cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP) and radiation therapy (RT), with the aim of improving first-line treatment. Methods: Cell viability experiments were performed to determine synergistic combinations. Levels of DNA damage were established using flow cytometry for γH2AX and protein analysis for DNA damage response proteins CHK1 and CHK2. Flow cytometry analysis for cell cycle and pH3 were performed to determine cell cycle distribution and premature mitotic entry. Results: Treatment with either RT or CHOP led to enhanced sensitivity to AZD1775 in several DLBCL cell lines. Treatment of cells with AZD1775 induced unscheduled mitotic progression, resulting in abnormal cell cycle distribution in combination with RT or CHOP treatment. In addition, a significant increase in DNA damage was observed compared with CHOP or RT alone. Of the single CHOP components, doxorubicin showed the strongest effect together with AZD1775, reducing viability and increasing DNA damage. Conclusion: In conclusion, the combination of RT or CHOP with AZD1775 enhances sensitivity to WEE1 inhibition through unscheduled G2/M progression, leading to increased DNA damage. Based on these results, WEE1 inhibition has great potential together with other G2/M arresting or DNA damaging (chemo) therapeutic compounds and should be further explored in clinical trials.

Emphysema is characterized by irreversible loss of alveolar tissue with an urgent need for novel, regenerative treatment strategies. The use of autologous mesenchymal stromal cells (MSCs) is promising, but treatment with bone marrow-derived MSCs (BM-MSCs) showed little beneficial effect in clinical studies. MSCs can also be derived from other sources e.g. lung (LMSCs) and adipose tissue (ADSCs). We hypothesized that LMSCs are better equipped for lung repair than BM-MSCs or ADSCs, but may have lower regenerative capacity in emphysema. Therefore, we compared the regenerative potential of LMSCs, BM-MSCs and ADSCs from emphysema patients and controls. MSCs were isolated from lung, fat and bone marrow of emphysema patients and controls and expanded in vitro. Expression of growth factors and extracellular matrix (ECM) molecules involved in lung repair was measured by qPCR. Overall, LMSCs displayed higher FGF10 and HGF expression compared to BM-MSCs and ADSCs, and higher WNT-5A expression compared to ADSCs. By contrast, BM-MSCs expressed considerably higher levels of collagen1α1, elastin and periostin than both LMSCs and ADSCs, while ADSCs showed higher periostin expression than LMSCs. When comparing emphysema to control, LMSCs showed lower expression of FGF2 , HGF and WNT-5A, while BM-MSCs expressed less collagen1α1, elastin and periostin in emphysema, without significant differences in ADSCs. These results suggest that LMSCs are more beneficial for tissue repair in emphysema than BM-MSC and ADSCs. LMSCs express higher levels of growth factors involved in alveolar repair, although LMSCs from emphysema patients may need in vitro boosting.

Background Chronic obstructive pulmonary disease (COPD) is a chronic lung disease characterized by chronic airway inflammation and emphysema, and is caused by exposure to noxious particles or gases, e.g. cigarette smoke. Smoking and oxidative stress lead to accelerated formation and accumulation of advanced glycation end products (AGEs), causing local tissue damage either directly or by binding the receptor for AGEs (RAGE). This study assessed the association of AGEs or RAGE in plasma, sputum, bronchial biopsies and skin with COPD and lung function, and their variance between these body compartments. Methods Healthy smoking and never-smoking controls ( n = 191) and COPD patients ( n = 97, GOLD stage I-IV) were included. Autofluorescence (SAF) was measured in the skin, AGEs (pentosidine, CML and CEL) and sRAGE in blood and sputum by ELISA, and in bronchial biopsies by immunohistochemistry. eQTL analysis was performed in bronchial biopsies. Results COPD patients showed higher SAF values and lower plasma sRAGE levels compared to controls and these values associated with decreased lung function ( p <0.001; adjusting for relevant covariates). Lower plasma sRAGE levels significantly and independently predicted higher SAF values ( p < 0.001). One SNP (rs2071278) was identified within a region of 50 kB flanking the AGER gene, which was associated with the gene and protein expression levels of AGER and another SNP (rs2071278) which was associated with the accumulation of AGEs in the skin. Conclusion In COPD, AGEs accumulate differentially in body compartments, i.e. they accumulate in the skin, but not in plasma, sputum and bronchial biopsies. The association between lower sRAGE and higher SAF levels supports the hypothesis that the protective mechanism of sRAGE as a decoy-receptor is impaired in COPD.

Background Interleukin (IL)-17 plays a critical role in numerous immune and inflammatory responses and was recently suggested to contribute to the pathogenesis of nonatopic (non-eosinophil/neutrophil-dominant) asthma. We aimed to compare expression of IL-17 in bronchial airways between atopic and nonatopic asthmatics, with/without inhaled corticosteroid (ICS) use and to identify its major cellular source. Methods Bronchial biopsies from 114 patients with mild-to-moderate asthma were investigated: 33 nonatopic, 63 non-corticosteroid users, 90 nonsmokers. IL-17 expression was correlated with atopy and inflammatory cell counts (EPX, NP57, CD3, CD4, CD8, CD20, CD68), taking ICS use and smoking into account. Multiple linear regression analyses were used to determine the independent factors as well as the most relevant inflammatory cells contributing to IL-17 expression. Double immunostainings were performed to confirm the major cellular source of IL-17. Results In non-ICS users, nonatopic asthmatics had more IL-17+ cells in the airway wall than atopic asthmatics. In both atopic and nonatopic asthmatics, ICS use was associated with lower numbers of IL-17+ cells, independent of smoking. The number of IL-17+ cells was associated with the number of neutrophils (B: 0.26, 95% CI: 0.17–0.35) and eosinophils (B: 0.18, 95% CI: 0.07–0.29). The majority of IL-17+ cells were neutrophils, as confirmed by double immunostaining. Conclusions We show for the first time that atopy and ICS use are associated with lower numbers of IL-17+ cells in asthmatic airways. Importantly, IL-17+ cells were mostly neutrophils which conflicts with the paradigm that lymphocytes (Th17) are the main source of IL-17.

Cigarette smoking is the main risk factor for COPD (Chronic Obstructive Pulmonary Disease), yet only a subset of smokers develops COPD. Family members of patients with severe early-onset COPD have an increased risk to develop COPD and are therefore defined as "susceptible individuals". Here we perform unbiased analyses of proteomic profiles to assess how "susceptible individuals" differ from age-matched "non-susceptible individuals" in response to cigarette smoking. Epithelial lining fluid (ELF) was collected at baseline and 24 hours after smoking 3 cigarettes in young individuals susceptible or non-susceptible to develop COPD and older subjects with established COPD. Controls at baseline were older healthy smoking and non-smoking individuals. Five samples per group were pooled and analysed by stable isotope labelling (iTRAQ) in duplicate. Six proteins were selected and validated by ELISA or immunohistochemistry. After smoking, 23 proteins increased or decreased in young susceptible individuals, 7 in young non-susceptible individuals, and 13 in COPD in the first experiment; 23 proteins increased or decreased in young susceptible individuals, 32 in young non-susceptible individuals, and 11 in COPD in the second experiment. SerpinB3 and Uteroglobin decreased after acute smoke exposure in young non-susceptible individuals exclusively, whereas Peroxiredoxin I, S100A9, S100A8, ALDH3A1 (Aldehyde dehydrogenase 3A1) decreased both in young susceptible and non-susceptible individuals, changes being significantly different between groups for Uteroglobin with iTRAQ and for Serpin B3 with iTRAQ and ELISA measures. Peroxiredoxin I, SerpinB3 and ALDH3A1 increased in COPD patients after smoking. We conclude that smoking induces a differential protein response in ELF of susceptible and non-susceptible young individuals, which differs from patients with established COPD. This is the first study applying unbiased proteomic profiling to unravel the underlying mechanisms that induce COPD. Our data suggest that SerpinB3 and Uteroglobin could be interesting proteins in understanding the processes leading to COPD.

Microfluidics-based nanoLC-MS/MS (chipLC-MS/MS) was used to identify and quantify proteins in epithelial lining fluid (ELF), collected during bronchoscopy from the main bronchi of chronic obstructive pulmonary disease (COPD) patients and healthy controls using microprobes. ELF is a biofluid that is well suited to study pathophysiological processes in the lung, because it contains high concentrations of biologically active molecules. 1D-PAGE followed by in-gel tryptic digestion and chipLC-MS/MS resulted in identification of approximately 300 proteins. A comparative study of ELF from COPD patients and non-COPD controls using chemical stable isotope labeling (iTRAQ®-8Plex) showed that the levels of lactotransferrin, high-mobility group protein B1 (HMGB 1), alpha 1-antichymotrypsin and cofilin-1 differed significantly in ELF from COPD patients and non-COPD controls (p-values < 0.05). These results were reproduced in another, independent set of ELF samples from COPD patients and non-COPD controls and further validated by immunohistochemistry. This study shows the feasibility of performing chipLC-MS/MS and quantitative proteomics in human ELF.