Single-Cell Gene-Regulatory Networks of Advanced Symptomatic Atherosclerosis.

BACKGROUND: While our understanding of the single-cell gene expression patterns underlying the transformation of vascular cell types during the progression of atherosclerosis is rapidly improving, the clinical and pathophysiological relevance of these changes remains poorly understood. METHODS: Single-cell RNA sequencing data generated with SmartSeq2 (≈8000 genes/cell) in 16 588 single cells isolated during atherosclerosis progression in Ldlr-/-Apob100/100 mice with human-like plasma lipoproteins and from humans with asymptomatic and symptomatic carotid plaques was clustered into multiple subtypes. For clinical and pathophysiological context, the advanced-stage and symptomatic subtype clusters were integrated with 135 tissue-specific (atherosclerotic aortic wall, mammary artery, liver, skeletal muscle, and visceral and subcutaneous, fat) gene-regulatory networks (GRNs) inferred from 600 coronary artery disease patients in the STARNET (Stockholm-Tartu Atherosclerosis Reverse Network Engineering Task) study. RESULTS: Advanced stages of atherosclerosis progression and symptomatic carotid plaques were largely characterized by 3 smooth muscle cells (SMCs), and 3 macrophage subtype clusters with extracellular matrix organization/osteogenic (SMC), and M1-type proinflammatory/Trem2-high lipid-associated (macrophage) phenotypes. Integrative analysis of these 6 clusters with STARNET revealed significant enrichments of 3 arterial wall GRNs: GRN33 (macrophage), GRN39 (SMC), and GRN122 (macrophage) with major contributions to coronary artery disease heritability and strong associations with clinical scores of coronary atherosclerosis severity. The presence and pathophysiological relevance of GRN39 were verified in 5 independent RNAseq data sets obtained from the human coronary and aortic artery, and primary SMCs and by targeting its top-key drivers, FRZB and ALCAM in cultured human coronary artery SMCs. CONCLUSIONS: By identifying and integrating the most gene-rich single-cell subclusters of atherosclerosis to date with a coronary artery disease framework of GRNs, GRN39 was identified and independently validated as being critical for the transformation of contractile SMCs into an osteogenic phenotype promoting advanced, symptomatic atherosclerosis.

SIX2 promotes cell plasticity via Wnt/ß-catenin signalling in androgen receptor independent prostate cancer.

The use of androgen receptor (AR) inhibitors in prostate cancer gives rise to increased cellular lineage plasticity resulting in resistance to AR-targeted therapies. In this study, we examined the chromatin landscape of AR-positive prostate cancer cells post-exposure to the AR inhibitor enzalutamide. We identified a novel regulator of cell plasticity, the homeobox transcription factor SIX2, whose motif is enriched in accessible chromatin regions after treatment. Depletion of SIX2 in androgen-independent PC-3 prostate cancer cells induced a switch from a stem-like to an epithelial state, resulting in reduced cancer-related properties such as proliferation, colony formation, and metastasis both in vitro and in vivo. These effects were mediated through the downregulation of the Wnt/ß-catenin signalling pathway and subsequent reduction of nuclear ß-catenin. Collectively, our findings provide compelling evidence that the depletion of SIX2 may represent a promising strategy for overcoming the cell plasticity mechanisms driving antiandrogen resistance in prostate cancer.

Comparison of early changes in tear film protein profiles after small incision lenticule extraction (SMILE) and femtosecond LASIK (FS-LASIK) surgery.

BACKGROUND: Small incision lenticule extraction (SMILE) and femtosecond laser-assisted in situ keratomileusis (LASIK) are widely used surgical methods to correct myopia with comparable efficacy, predictability, and safety. We examined and compared the early changes of tear protein profiles after SMILE and FS-LASIK surgery in order to find possible differences in the initial corneal healing process. METHODS: SMILE operations for 26 eyes were made with Visumax femtosecond laser. In FS-LASIK surgery for 30 eyes, the flaps were made with Ziemer FEMTO LDV Z6 femtosecond laser and stromal ablation with Wavelight EX500 excimer laser. Tear samples were collected preoperatively, and 1.5 h and 1 month postoperatively using glass microcapillary tubes. Tear protein identification and quantification were performed with sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS). RESULTS: Immediately (1.5 h) after we found differences in 89 proteins after SMILE and in 123 after FS-LASIK operation compared to preoperative protein levels. Of these differentially expressed proteins, 48 proteins were common for both surgery types. There were, however, quantitative differences between SMILE and FS-LASIK. Upregulated proteins were mostly connected to inflammatory response and migration of the cells connected to immune system. One month after the operation protein expressions levels were returned to baseline levels with both surgical methods. CONCLUSIONS: Our study showed that immediate changes in protein profiles after SMILE and FS-LASIK surgeries and differences between the methods are connected to inflammatory process, and the protein levels quickly return to the baseline within 1 month. The differences in protein profiles between the methods are probably associated with the different size of the epithelial wound induced.

DPYSL5 is highly expressed in treatment-induced neuroendocrine prostate cancer and promotes lineage plasticity via EZH2/PRC2.

Treatment-induced neuroendocrine prostate cancer (t-NEPC) is a lethal subtype of castration-resistant prostate cancer resistant to androgen receptor (AR) inhibitors. Our study unveils that AR suppresses the neuronal development protein dihydropyrimidinase-related protein 5 (DPYSL5), providing a mechanism for neuroendocrine transformation under androgen deprivation therapy. Our unique CRPC-NEPC cohort, comprising 135 patient tumor samples, including 55 t-NEPC patient samples, exhibits a high expression of DPYSL5 in t-NEPC patient tumors. DPYSL5 correlates with neuroendocrine-related markers and inversely with AR and PSA. DPYSL5 overexpression in prostate cancer cells induces a neuron-like phenotype, enhances invasion, proliferation, and upregulates stemness and neuroendocrine-related markers. Mechanistically, DPYSL5 promotes prostate cancer cell plasticity via EZH2-mediated PRC2 activation. Depletion of DPYSL5 decreases proliferation, induces G1 phase cell cycle arrest, reverses neuroendocrine phenotype, and upregulates luminal genes. In conclusion, DPYSL5 plays a critical role in regulating prostate cancer cell plasticity, and we propose the AR/DPYSL5/EZH2/PRC2 axis as a driver of t-NEPC progression.

Oncogenic KEAP1 mutations activate TRAF2-NFκB signaling to prevent apoptosis in lung cancer cells.

The Kelch-like ECH-associated protein 1 (KEAP1) - Nuclear factor erythroid 2 -related factor 2 (NRF2) pathway is the major transcriptional stress response system in cells against oxidative and electrophilic stress. NRF2 is frequently constitutively active in many cancers, rendering the cells resistant to chemo- and radiotherapy. Loss-of-function (LOF) mutations in the repressor protein KEAP1 are common in non-small cell lung cancer, particularly adenocarcinoma. While the mutations can occur throughout the gene, they are enriched in certain areas, indicating that these may have unique functional importance. In this study, we show that in the GSEA analysis of TCGA lung adenocarcinoma RNA-seq data, the KEAP1 mutations in R320 and R470 were associated with enhanced Tumor Necrosis Factor alpha (TNFα) - Nuclear Factor kappa subunit B (NFκB) signaling as well as MYC and MTORC1 pathways. To address the functional role of these hotspot mutations, affinity purification and mass spectrometry (AP-MS) analysis of wild type (wt) KEAP1 and its mutation forms, R320Q and R470C were employed to interrogate differences in the protein interactome. We identified TNF receptor associated factor 2 (TRAF2) as a putative protein interaction partner. Both mutant KEAP1 forms showed increased interaction with TRAF2 and other anti-apoptotic proteins, suggesting that apoptosis signalling could be affected by the protein interactions. A549 lung adenocarcinoma cells overexpressing mutant KEAP1 showed high TRAF2-mediated NFκB activity and increased protection against apoptosis, XIAP being one of the key proteins involved in anti-apoptotic signalling. To conclude, KEAP1 R320Q and R470C and its interaction with TRAF2 leads to activation of NFκB pathway, thereby protecting against apoptosis.

Protective Alzheimer's disease-associated APP A673T variant predominantly decreases sAPPß levels in cerebrospinal fluid and 2D/3D cell culture models.

The rare A673T variant was the first variant found within the amyloid precursor protein (APP) gene conferring protection against Alzheimer's disease (AD). Thereafter, different studies have discovered that the carriers of the APP A673T variant show reduced levels of amyloid beta (Aß) in the plasma and better cognitive performance at high age. Here, we analyzed cerebrospinal fluid (CSF) and plasma of APP A673T carriers and control individuals using a mass spectrometry-based proteomics approach to identify differentially regulated targets in an unbiased manner. Furthermore, the APP A673T variant was introduced into 2D and 3D neuronal cell culture models together with the pathogenic APP Swedish and London mutations. Consequently, we now report for the first time the protective effects of the APP A673T variant against AD-related alterations in the CSF, plasma, and brain biopsy samples from the frontal cortex. The CSF levels of soluble APPß (sAPPß) and Aß42 were significantly decreased on average 9-26% among three APP A673T carriers as compared to three well-matched controls not carrying the protective variant. Consistent with these CSF findings, immunohistochemical assessment of cortical biopsy samples from the same APP A673T carriers did not reveal Aß, phospho-tau, or p62 pathologies. We identified differentially regulated targets involved in protein phosphorylation, inflammation, and mitochondrial function in the CSF and plasma samples of APP A673T carriers. Some of the identified targets showed inverse levels in AD brain tissue with respect to increased AD-associated neurofibrillary pathology. In 2D and 3D neuronal cell culture models expressing APP with the Swedish and London mutations, the introduction of the APP A673T variant resulted in lower sAPPß levels. Concomitantly, the levels of sAPPα were increased, while decreased levels of CTFß and Aß42 were detected in some of these models. Our findings emphasize the important role of APP-derived peptides in the pathogenesis of AD and demonstrate the effectiveness of the protective APP A673T variant to shift APP processing towards the non-amyloidogenic pathway in vitro even in the presence of two pathogenic mutations.

Intranasal administration of adenoviral vaccines expressing SARS-CoV-2 spike protein improves vaccine immunity in mouse models.

The ongoing SARS-CoV-2 pandemic is controlled but not halted by public health measures and mass vaccination strategies which have exclusively relied on intramuscular vaccines. Intranasal vaccines can prime or recruit to the respiratory epithelium mucosal immune cells capable of preventing infection. Here we report a comprehensive series of studies on this concept using various mouse models, including HLA class II-humanized transgenic strains. We found that a single intranasal (i.n.) dose of serotype-5 adenoviral vectors expressing either the receptor binding domain (Ad5-RBD) or the complete ectodomain (Ad5-S) of the SARS-CoV-2 spike protein was effective in inducing i) serum and bronchoalveolar lavage (BAL) anti-spike IgA and IgG, ii) robust SARS-CoV-2-neutralizing activity in the serum and BAL, iii) rigorous spike-directed T helper 1 cell/cytotoxic T cell immunity, and iv) protection of mice from a challenge with the SARS-CoV-2 beta variant. Intramuscular (i.m.) Ad5-RBD or Ad5-S administration did not induce serum or BAL IgA, and resulted in lower neutralizing titers in the serum. Moreover, prior immunity induced by an intramuscular mRNA vaccine could be potently enhanced and modulated towards a mucosal IgA response by an i.n. Ad5-S booster. Notably, Ad5 DNA was found in the liver or spleen after i.m. but not i.n. administration, indicating a lack of systemic spread of the vaccine vector, which has been associated with a risk of thrombotic thrombocytopenia. Unlike in otherwise genetically identical HLA-DQ6 mice, in HLA-DQ8 mice Ad5-RBD vaccine was inferior to Ad5-S, suggesting that the RBD fragment does not contain a sufficient collection of helper-T cell epitopes to constitute an optimal vaccine antigen. Our data add to previous promising preclinical results on intranasal SARS-CoV-2 vaccination and support the potential of this approach to elicit mucosal immunity for preventing transmission of SARS-CoV-2.

ApoA-I Nanotherapy Rescues Postischemic Vascular Maladaptation by Modulating Endothelial Cell and Macrophage Phenotypes in Type 2 Diabetic Mice.

BACKGROUND: Diabetes is a major risk factor for peripheral arterial disease. Clinical and preclinical studies suggest an impaired collateral remodeling and angiogenesis in response to atherosclerotic arterial occlusion in diabetic conditions, although the underlying mechanisms are poorly understood. OBJECTIVE: To clarify the cellular and molecular mechanisms underlying impaired postischemic adaptive vascular responses and to evaluate rHDL (reconstituted HDL)-ApoA-I nanotherapy to rescue the defect in type 2 diabetic mouse model of hindlimb ischemia. METHODS AND RESULTS: Hindlimb ischemia was induced by unilateral femoral artery ligation. Collateral and capillary parameters together with blood flow recovery were analyzed from normoxic adductor and ischemic gastrocnemius muscles, respectively, at day 3 and 7 post-ligation. In response to femoral artery ligation, collateral lumen area was significantly reduced in normoxic adductor muscles. Distally, ischemic gastrocnemius muscles displayed impaired perfusion recovery and angiogenesis paralleled with persistent inflammation. Muscle-specific mRNA sequencing revealed differential expression of genes critical for smooth muscle proliferation and sprouting angiogenesis in normoxic adductor and ischemic gastrocnemius, respectively, at day 7 post-ligation. Genes typical for macrophage (Mϕ) subsets were differentially expressed across both muscle types. Cell-specific gene expression, flow cytometry, and immunohistochemistry revealed persistent IFN-I response gene upregulation in arterial endothelial cells, ECs and Mϕs from T2DM mice associated with impaired collateral remodeling, angiogenesis and perfusion recovery. Furthermore, rHDL nanotherapy rescued impaired collateral remodeling and angiogenesis through dampening EC and Mϕ inflammation in T2DM mice. CONCLUSIONS: Our results suggest that an impaired collateral remodeling and sprouting angiogenesis in T2DM mice is associated with persistent IFN-I response in ECs and Mϕs. Dampening persistent inflammation and skewing ECs and Mϕ phenotype toward less inflammatory ones using rHDL nanotherapy may serve as a potential therapeutic target for T2DM peripheral arterial disease.

Nuclear microRNA-466c regulates Vegfa expression in response to hypoxia.

MicroRNAs are well characterized in their role in silencing gene expression by targeting 3´-UTR of mRNAs in cytoplasm. However, recent studies have shown that miRNAs have a role in the regulation of genes in the nucleus, where they are abundantly located. We show here that in mouse endothelial cell line (C166), nuclear microRNA miR-466c participates in the regulation of vascular endothelial growth factor a (Vegfa) gene expression in hypoxia. Upregulation of Vegfa expression in response to hypoxia was significantly compromised after removal of miR-466c with CRISPR-Cas9 genomic deletion. We identified a promoter-associated long non-coding RNA on mouse Vegfa promoter and show that miR-466c directly binds to this transcript to modulate Vegfa expression. Collectively, these observations suggest that miR-466c regulates Vegfa gene transcription in the nucleus by targeting the promoter, and expands on our understanding of the role of miRNAs well beyond their canonical role.

Citrate-Saline-Formulated mRNA Delivery into the Heart Muscle with an Electromechanical Mapping and Injection Catheter Does Not Lead to Therapeutic Effects in a Porcine Chronic Myocardial Ischemia Model.

Based on recent success in using modified RNA in clinical applications, we tested the safety, feasibility, and efficacy of direct delivery of citrate-saline-formulated mRNA into an hibernating ischemic heart muscle using an electromechanical mapping and injection catheter system (NOGA/Myostar) in a porcine chronic myocardial ischemia model. Chronic ischemia was induced in domestic pigs (n = 24) using a bottleneck stent placed in the left anterior descending coronary artery. Low (1 mg) and high (7.5 mg) doses of citrate-saline-formulated vascular endothelial growth factor (VEGF)-A165 mRNA were administered in the study. LacZ mRNA and citrate-saline buffer were used as controls. Ten intramyocardial injections (200 µL each) of the mRNAs or citrate-saline buffer were given endovascularly into the hibernating ischemic myocardium using the NOGA catheter. Positron emission tomography 15O-radiowater imaging was performed 7 days after the induction of ischemia and 28 days after the mRNA delivery to measure quantitative myocardial blood perfusion. Coronary angiography, left ventricular function measurements, and clinical chemistry were obtained at each time point. Thirty-five days after the mRNA transfers, pigs were sacrificed, and infarct size and general histology were analyzed. LacZ mRNA pigs were sacrificed 24 h after the transduction. Citrate-saline-formulated mRNA delivery into the ischemic myocardium with endovascular injection catheter did not lead to meaningful transduction with the translation of VEGF-A165, nor therapeutic effects in the heart. VEGF-A165 mRNA showed no statistically significant improvements in left ventricular ejection fraction (LVEF), cardiac output, myocardial perfusion, infarct size, collateral growth, or capillary area in the study groups. However, there was a trend in the high-dose group toward an improved LVEF and cardiac output at rest. No significant adverse effects were observed. In conclusion, the NOGA/Myostar injection catheter system is ineffective in delivering citrate-saline-formulated mRNAs into the heart muscle with the doses and methods used in a porcine chronic myocardial ischemia model.

Single cell characterization of B-lymphoid differentiation and leukemic cell states during chemotherapy in ETV6-RUNX1-positive pediatric leukemia identifies drug-targetable transcription factor activities.

BACKGROUND: Tight regulatory loops orchestrate commitment to B cell fate within bone marrow. Genetic lesions in this gene regulatory network underlie the emergence of the most common childhood cancer, acute lymphoblastic leukemia (ALL). The initial genetic hits, including the common translocation that fuses ETV6 and RUNX1 genes, lead to arrested cell differentiation. Here, we aimed to characterize transcription factor activities along the B-lineage differentiation trajectory as a reference to characterize the aberrant cell states present in leukemic bone marrow, and to identify those transcription factors that maintain cancer-specific cell states for more precise therapeutic intervention. METHODS: We compared normal B-lineage differentiation and in vivo leukemic cell states using single cell RNA-sequencing (scRNA-seq) and several complementary genomics profiles. Based on statistical tools for scRNA-seq, we benchmarked a workflow to resolve transcription factor activities and gene expression distribution changes in healthy bone marrow lymphoid cell states. We compared these to ALL bone marrow at diagnosis and in vivo during chemotherapy, focusing on leukemias carrying the ETV6-RUNX1 fusion. RESULTS: We show that lymphoid cell transcription factor activities uncovered from bone marrow scRNA-seq have high correspondence with independent ATAC- and ChIP-seq data. Using this comprehensive reference for regulatory factors coordinating B-lineage differentiation, our analysis of ETV6-RUNX1-positive ALL cases revealed elevated activity of multiple ETS-transcription factors in leukemic cells states, including the leukemia genome-wide association study hit ELK3. The accompanying gene expression changes associated with natural killer cell inactivation and depletion in the leukemic immune microenvironment. Moreover, our results suggest that the abundance of G1 cell cycle state at diagnosis and lack of differentiation-associated regulatory network changes during induction chemotherapy represent features of chemoresistance. To target the leukemic regulatory program and thereby overcome treatment resistance, we show that inhibition of ETS-transcription factors reduced cell viability and resolved pathways contributing to this using scRNA-seq. CONCLUSIONS: Our data provide a detailed picture of the transcription factor activities characterizing both normal B-lineage differentiation and those acquired in leukemic bone marrow and provide a rational basis for new treatment strategies targeting the immune microenvironment and the active regulatory network in leukemia.

Early changes in tear film protein profiles after femtosecond LASIK surgery.

BACKGROUND: Femtosecond laser-assisted in situ keratomileusis (LASIK) has proven to be an efficacious, predictable, and safe procedure for the correction of refractive errors. We examined the early tear protein changes of patients undergoing LASIK surgery in order to better understand the mechanisms and proteins related to laser corneal surgery and initial recovery. METHODS: Corneal flaps were created with Ziemer FEMTO LDV Z6 I femtosecond laser and stroma was ablated using Wavelight EX500 excimer laser. Tear samples were collected preoperatively as well as 1.5 h and 1 month after LASIK treatment using glass microcapillary tubes. Relative quantification of tear proteins was performed with sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS). RESULTS: SWATH-MS revealed that 158 proteins had altered expression levels 1.5 h after the operation. Two-thirds of these proteins, mostly connected to migration and inflammation response, returned to preoperative levels within the first postoperative month. The other proteins, which did not return to baseline levels, included proteins connected to for example epithelial barrier function. We also identified several proteins, which correlated with surgical variables, such as the amount of correction, flap thickness and flap diameter. CONCLUSIONS: The present study showed that an uneventful femtosecond LASIK refractive surgery induced a significant immune cell migration and inflammation-associated changes in tear proteomics profile quickly after the operation, but the expression of most proteins recovered almost completely to the preoperative levels within the first month. The individual proteins identified in our study are potential targets for the follow-up and modification of LASIK-induced biochemical processes.

Susceptibility to Cardiac Arrhythmias and Sympathetic Nerve Growth in VEGF-B Overexpressing Myocardium.

VEGF-B gene therapy is a promising proangiogenic treatment for ischemic heart disease, but, unexpectedly, we found that high doses of VEGF-B promote ventricular arrhythmias (VAs). VEGF-B knockout, alpha myosin heavy-chain promoter (αMHC)-VEGF-B transgenic mice, and pigs transduced intramyocardially with adenoviral (Ad)VEGF- B186 were studied. Immunostaining showed a 2-fold increase in the number of nerves per field (76 vs. 39 in controls, p < 0.001) and an abnormal nerve distribution in the hypertrophic hearts of 11- to 20-month-old αMHC-VEGF-B mice. AdVEGF-B186 gene transfer (GT) led to local sprouting of nerve endings in pig myocardium (141 vs. 78 nerves per field in controls, p < 0.05). During dobutamine stress, 60% of the αMHC-VEGF-B hypertrophic mice had arrhythmias as compared to 7% in controls, and 20% of the AdVEGF-B186-transduced pigs and 100% of the combination of AdVEGF-B186- and AdsVEGFR-1-transduced pigs displayed VAs and even ventricular fibrillation. AdVEGF-B186 GT significantly increased the risk of sudden cardiac death in pigs when compared to any other GT with different VEGFs (hazard ratio, 500.5; 95% confidence interval [CI] 46.4-5,396.7; p < 0.0001). In gene expression analysis, VEGF-B induced the upregulation of Nr4a2, ATF6, and MANF in cardiomyocytes, molecules previously linked to nerve growth and differentiation. Thus, high AdVEGF-B186 overexpression induced nerve growth in the adult heart via a VEGFR-1 signaling-independent mechanism, leading to an increased risk of VA and sudden cardiac death.

HAS3-induced extracellular vesicles from melanoma cells stimulate IHH mediated c-Myc upregulation via the hedgehog signaling pathway in target cells.

Intercellular communication is fundamental to the survival and maintenance of all multicellular systems, whereas dysregulation of communication pathways can drive cancer progression. Extracellular vesicles (EVs) are mediators of cell-to-cell communication that regulate a variety of cellular processes involved in tumor progression. Overexpression of a specific plasma membrane enzyme, hyaluronan synthase 3 (HAS3), is one of the factors that can induce EV shedding. HAS3, and particularly its product hyaluronan (HA), are carried by EVs and are known to be associated with the tumorigenic properties of cancer cells. To elucidate the specific effects of cancerous, HAS3-induced EVs on target cells, normal human keratinocytes and melanoma cells were treated with EVs derived from GFP-HAS3 expressing metastatic melanoma cells. We found that the HA receptor CD44 participated in the regulation of EV binding to target cells. Furthermore, GFP-HAS3-positive EVs induced HA secretion, proliferation and invasion of target cells. Our results suggest that HAS3-EVs contains increased quantities of IHH, which activates the target cell hedgehog signaling cascade and leads to the activation of c-Myc and regulation of claspin expression. This signaling of IHH in HAS3-EVs resulted in increased cell proliferation. Claspin immunostaining correlated with HA content in human cutaneous melanocytic lesions, supporting our in vitro findings and suggesting a reciprocal regulation between claspin expression and HA synthesis. This study shows for the first time that EVs originating from HAS3 overexpressing cells carry mitogenic signals that induce proliferation and epithelial-to-mesenchymal transition in target cells. The study also identifies a novel feedback regulation between the hedgehog signaling pathway and HA metabolism in melanoma, mediated by EVs carrying HA and IHH.

Age-associated changes in human tear proteome.

BACKGROUND: Prevalence of many eye and ocular surface diseases increases with age. While the clinical characteristics and pathophysiologic mechanisms of these conditions are often either known or extensively studied, the effects of normal aging on tear film and ocular surface have not been as widely researched. METHODS: In order to examine the effects of aging on tear fluid proteomics, tear fluid samples were collected preoperatively from 115 subjects undergoing strabismus or refractive surgery using glass microcapillary tubes. In addition to their refractive error or strabismus, the subjects did not have any other current, known eye diseases. The non-pooled samples were analysed using NanoLC-TripleTOF implementing a sequential window acquisition of all theoretical fragment ion spectra mass spectrometry, resulting in quantified data of 849 proteins. RESULTS: According to correlation results, 17 tear proteins correlated significantly with increased age and many of these proteins were connected to inflammation, immune response and cell death. According to enrichment analysis, growth and survival of cells decreased while immune response and inflammation increased with aging. We also discovered several well-known, activated and inhibited upstream regulators, e.g. NF-κB, which has been previously connected to aging in numerous previous studies. CONCLUSIONS: Overall, the results show the common age-dependent alterations in tear fluid protein profile, which demonstrate similar age-associated alterations of biological functions previously shown in other tissue and sample types.

Doxycycline modulates VEGF-A expression: Failure of doxycycline-inducible lentivirus shRNA vector to knockdown VEGF-A expression in transgenic mice.

Vascular endothelial growth factor-A (VEGF-A) is the master regulator of angiogenesis, vascular permeability and growth. However, its role in mature blood vessels is still not well understood. To better understand the role of VEGF-A in the adult vasculature, we generated a VEGF-A knockdown mouse model carrying a doxycycline (dox)-regulatable short hairpin RNA (shRNA) transgene, which silences VEGF-A. The aim was to find the critical level of VEGF-A reduction for vascular well-being in vivo. In vitro, the dox-inducible lentiviral shRNA vector decreased VEGF-A expression efficiently and dose-dependently in mouse endothelial cells and cardiomyocytes. In the generated transgenic mice plasma VEGF-A levels decreased shortly after the dox treatment but returned back to normal after two weeks. VEGF-A expression decreased shortly after the dox treatment only in some tissues. Surprisingly, increasing the dox exposure time and dose led to elevated VEGF-A expression in some tissues of both wildtype and knockdown mice, suggesting that dox itself has an effect on VEGF-A expression. When the effect of dox on VEGF-A levels was further tested in naïve/non-transduced cells, the dox administration led to a decreased VEGF-A expression in endothelial cells but to an increased expression in cardiomyocytes. In conclusion, the VEGF-A knockdown was achieved in a dox-regulatable fashion with a VEGF-A shRNA vector in vitro, but not in the knockdown mouse model in vivo. Dox itself was found to regulate VEGF-A expression explaining the unexpected results in mice. The effect of dox on VEGF-A levels might at least partly explain its previously reported beneficial effects on myocardial and brain ischemia. Also, this effect on VEGF-A should be taken into account in all studies using dox-regulated vectors.

The effect of subcellular localization on the efficiency of EGFR-targeted VHH photosensitizer conjugates.

Photodynamic therapy (PDT) is an emerging method to treat light-accessible malignancies. To increase specificity and allow dose reduction, conjugates of photosensitizers (PS) with antibodies against tumor-associated antigens have been developed for photoimmunotherapy (PIT). However, so far it is unclear whether cellular internalization of these conjugates after binding affects PIT efficacy. The use of low molecular weight llama single domain antibodies (VHHs, nanobodies) for PIT is preferred above full size antibodies because of better tumor penetration. Therefore, we functionalized the VHH 7D12, directed against the epidermal growth factor receptor (EGFR), with a PS (IRDye700DX). To assess the impact of cellular internalization on activity, the VHHs were additionally conjugated to a cell-penetrating peptide (VHH[PS]-CPP). Here we show that upon illumination with near-infrared (NIR) light, both VHH[PS] and VHH[PS]-CPP conjugates specifically induce cell death of EGFR expressing cancer cell lines and of EGFR-expressing cells derived from surgically obtained ascites from patients with high-grade serous ovarian cancer. However, VHH[PS] conjugates were significantly more effective compared to internalizing VHH[PS]-CPP suggesting that cell surface association is required for optimal therapeutic activity.

Macrophage selective photodynamic therapy by meta-tetra(hydroxyphenyl)chlorin loaded polymeric micelles: A possible treatment for cardiovascular diseases.

Selective elimination of macrophages by photodynamic therapy (PDT) is a new and promising therapeutic modality for the reduction of atherosclerotic plaques. m-Tetra(hydroxyphenyl)chlorin (mTHPC, or Temoporfin) may be suitable as photosensitizer for this application, as it is currently used in the clinic for cancer PDT. In the present study, mTHPC was encapsulated in polymeric micelles based on benzyl-poly(ε-caprolactone)-b-methoxy poly(ethylene glycol) (Ben-PCL-mPEG) using a film hydration method, with loading capacity of 17%. Because of higher lipase activity in RAW264.7 macrophages than in C166 endothelial cells, the former cells degraded the polymers faster, resulting in faster photosensitizer release and higher in vitro photocytotoxicity of mTHPC-loaded micelles in those macrophages. However, we observed release of mTHPC from the micelles in 30min in blood plasma in vitro which explains the observed similar in vivo pharmacokinetics of the mTHPC micellar formulation and free mTHPC. Therefore, we could not translate the beneficial macrophage selectivity from in vitro to in vivo. Nevertheless, we observed accumulation of mTHPC in atherosclerotic lesions of mice aorta's which is probably the result of binding to lipoproteins upon release from the micelles. Therefore, future experiments will be dedicated to increase the stability and thus allow accumulation of intact mTHPC-loaded Ben-PCL-mPEG micelles to macrophages of atherosclerotic lesions.

Low interleukin-2 concentration favors generation of early memory T cells over effector phenotypes during chimeric antigen receptor T-cell expansion.

BACKGROUND: Adoptive T-cell therapy offers new options for cancer treatment. Clinical results suggest that T-cell persistence, depending on T-cell memory, improves efficacy. The use of interleukin (IL)-2 for in vitro T-cell expansion is not straightforward because it drives effector T-cell differentiation but does not promote the formation of T-cell memory. We have developed a cost-effective expansion protocol for chimeric antigen receptor (CAR) T cells with an early memory phenotype. METHODS: Lymphocytes were transduced with third-generation lentiviral vectors and expanded using CD3/CD28 microbeads. The effects of altering the IL-2 supplementation (0-300 IU/mL) and length of expansion (10-20 days) on the phenotype of the T-cell products were analyzed. RESULTS: High IL-2 levels led to a decrease in overall generation of early memory T cells by both decreasing central memory T cells and augmenting effectors. T memory stem cells (TSCM, CD95+CD45RO-CD45RA+CD27+) were present variably during T-cell expansion. However, their presence was not IL-2 dependent but was linked to expansion kinetics. CD19-CAR T cells generated in these conditions displayed in vitro antileukemic activity. In summary, production of CAR T cells without any cytokine supplementation yielded the highest proportion of early memory T cells, provided a 10-fold cell expansion and the cells were functionally potent. DISCUSSION: The number of early memory T cells in a T-cell preparation can be increased by simply reducing the amount of IL-2 and limiting the length of T-cell expansion, providing cells with potentially higher in vivo performance. These findings are significant for robust and cost-effective T-cell manufacturing.

Primary and metastatic ovarian cancer: Characterization by 3.0T diffusion-weighted MRI.

OBJECTIVES: We aimed to investigate whether apparent diffusion coefficients (ADCs) measured by 3.0T diffusion-weighted magnetic resonance imaging (DWI) associate with histological aggressiveness of ovarian cancer (OC) or predict the clinical outcome. This prospective study enrolled 40 patients with primary OC, treated 2011-2014. METHODS: DWI was performed prior to surgery. Two observers used whole lesion single plane region of interest (WLsp-ROI) and five small ROIs (S-ROI) to analyze ADCs. Samples from tumours and metastases were collected during surgery. Immunohistochemistry and quantitative reverse transcription polymerase chain reaction (qRT-PCR) were used to measure the expression of vascular endothelial growth factor (VEGF) and its receptors. RESULTS: The interobserver reliability of ADC measurements was excellent for primary tumours ICC 0.912 (WLsp-ROI). Low ADCs significantly associated with poorly differentiated OC (WLsp-ROI P = 0.035). In primary tumours, lower ADCs significantly associated with high Ki-67 (P = 0.001) and low VEGF (P = 0.001) expression. In metastases, lower ADCs (WLsp-ROI) significantly correlated with low VEGF receptors mRNA levels. ADCs had predictive value; 3-year overall survival was poorer in patients with lower ADCs (WLsp-ROI P = 0.023, S-ROI P = 0.038). CONCLUSION: Reduced ADCs are associated with histological severity and worse outcome in OC. ADCs measured with WLsp-ROI may serve as a prognostic biomarker of OC. KEY POINTS: • Reduced ADCs correlate with prognostic markers: poor differentiation and high Ki-67 expression • ADCs also significantly correlated with VEGF protein expression in primary tumours • Lower ADC values are associated with poorer survival in ovarian cancer • Whole lesion single plane-ROI ADCs may be used as a prognostic biomarker in OC.