Autophagy-related genes analysis reveals potential biomarkers for prediction of the impaired walking capacity of peripheral arterial disease.

BACKGROUND: The role of autophagy and autophagy-related genes in peripheral arterial disease (PAD) remains unknown and may be of diagnostic and prognostic value. The aim of this study is to investigate the relationship between autophagy and PAD, and identify potential diagnostic or prognostic biomarkers for medical practice. METHODS: Differentially expressed autophagy-related genes in PAD were explored from GSE57691 and validated in our WalkByLab registry participants by quantitative real-time polymerase chain reaction (qRT-PCR). The level of autophagy in peripheral blood mononuclear cells (PBMCs) of WalkByLab participants was assessed by analyzing autophagic marker proteins (beclin-1, P62, LC3B). Single sample gene set enrichment analysis (ssGSEA) was used to evaluate the immune microenvironment within the artery wall of PAD patients and healthy persons. Chemokine antibody array and enzyme-linked immunosorbent assay were used to assess the chemokines in participants' plasma. Treadmill testing with Gardner protocol was used to evaluate participants' walking capacity. Pain-free walking distance, maximum walking distance, and walking time were recorded. Finally, a nomogram model based on logistic regression was built to predict impaired walking performance. RESULTS: A total of 20 relevant autophagy-related genes were identified, and these genes were confirmed to be expressed at low levels in our PAD participants. Western blotting demonstrated that the expression of autophagic marker proteins beclin-1 and LC3BII were significantly reduced in PAD patients' PBMCs. ssGSEA revealed that most of the autophagy-related genes were strongly correlated with immune function, with the largest number of associated genes showing interaction between cytokine-and-cytokine receptors (CCR). In this context, the chemokines growth-related oncogene (GRO) and neutrophil activating protein2 (NAP2) are highly expressed in the plasma of WalkByLab PAD patients and were significantly negatively correlated with the walking distance assessed by Gardner treadmill testing. Finally, the plasma NAP2 level (AUC: 0.743) and derived nomogram model (AUC: 0.860) has a strong predictive potential to identify a poor walking capacity. CONCLUSIONS: Overall, these data highlight both the important role of autophagy and autophagy-related genes in PAD and link them to vascular inflammation (expression of chemokines). In particular, chemokine NAP2 emerged as a novel biomarker that can be used to predict the impaired walking capacity in PAD patients.

Effect of ACEI and ARB treatment on nitric oxide-dependent endothelial function.

Background: Angiotensin-converting-enzyme inhibitors (ACEI) and angiotensin II receptor blockers (ARB) are widely used as a first-line therapy for the treatment of cardiovascular disease. Here, ACEI modulate the bradykinin receptor (BDKRB1 and BDKRB2) system and NO-dependent endothelial function, thus determining cardiovascular health and regenerative arteriogenesis. The current study aims at evaluating nitric oxide-dependent endothelial function, and gene expression of bradykinin receptors in peripheral blood mononuclear cells (PBMC) from patients with ACEI or ARB treatment. Patients and methods: The WalkByLab has been established to screen cardiovascular patients for peripheral artery disease and coronary artery disease. In total 177 patients from WalkByLab with heterogenous disease and risk status were randomly selected, divided according to their medication history into the following groups: 1. ACEI group, 2. ARB group or 3. non-ACE/ARB group. Total plasma nitrite/nitrate (NO) levels were measured, endothelial function was evaluated by assessing flow meditated dilation (FMD). PBMC were isolated from peripheral whole blood, and gene expression (qRT-PCR) of bradykinin receptors and angiotensin converting enzyme were assessed. Results: Plasma total NO concentration in the ACEI group (24.66±16.28, µmol/l) was increased as compared to the ARB group (18.57±11.58, µmol/l, P=0.0046) and non-ACE/ARB group (16.83±8.64, µmol/l, P=0.0127) in patients between 40 to 90 years of age. However, FMD values (%) in the ACEI group (7.07±2.40, %) were similar as compared to the ARB (6.35±2.13, %) and non-ACE/ARB group (6.51±2.15, %), but significantly negatively correlated with age. Interestingly, BDKRB1 mRNA level was significantly higher and BDKRB2 mRNA level lower in the ACEI group (BDKRB1 3.88-fold±1.05, BDKRB2 0.22-fold±0.04) as compared to the non-ACE/ARB group (BDKRB1 1.00-fold±0.39, P<0.0001, BDKRB2 1.00-fold±0.45, P=0.0136). Conclusions: ACEI treatment enhances total nitrite/nitrate concentration, furthermore, upregulates BDKRB1 in PBMC, but downregulates BDKRB2 mRNA expression. FMD is a strong determinant of vascular aging and is sensitive to underlying heterogenous cardiovascular diseases.

Hypoxia-induced gene expression results from selective mRNA partitioning to the endoplasmic reticulum.

Protein synthesis is a primary energy-consuming process in the cell. Therefore, under hypoxic conditions, rapid inhibition of global mRNA translation represents a major protective strategy to maintain energy metabolism. How some mRNAs, especially those that encode crucial survival factors, continue to be efficiently translated in hypoxia is not completely understood. By comparing specific transcript levels in ribonucleoprotein complexes, cytoplasmic polysomes and endoplasmic reticulum (ER)-bound ribosomes, we show that the synthesis of proteins encoded by hypoxia marker genes is favoured at the ER in hypoxia. Gene expression profiling revealed that transcripts particularly increased by the HIF-1 transcription factor network show hypoxia-induced enrichment at the ER. We found that mRNAs favourably translated at the ER have higher conservation scores for both the 5'- and 3'-untranslated regions (UTRs) and contain less upstream initiation codons (uAUGs), indicating the significance of these sequence elements for sustained mRNA translation under hypoxic conditions. Furthermore, we found enrichment of specific cis-elements in mRNA 5'- as well as 3'-UTRs that mediate transcript localization to the ER in hypoxia. We conclude that transcriptome partitioning between the cytoplasm and the ER permits selective mRNA translation under conditions of energy shortage.

Amine oxidase copper-containing 1 (AOC1) is a downstream target gene of the Wilms tumor protein, WT1, during kidney development.

Amine oxidase copper-containing 1 (AOC1; formerly known as amiloride-binding protein 1) is a secreted glycoprotein that catalyzes the degradation of putrescine and histamine. Polyamines and their diamine precursor putrescine are ubiquitous to all organisms and fulfill pivotal functions in cell growth and proliferation. Despite the importance of AOC1 in regulating polyamine breakdown, very little is known about the molecular mechanisms that control its expression. We report here that the Wilms tumor protein, WT1, which is necessary for normal kidney development, activates transcription of the AOC1 gene. Expression of a firefly luciferase reporter under control of the proximal AOC1 promoter was significantly enhanced by co-transfection of a WT1 expression construct. Binding of WT1 protein to a cis-regulatory element in the AOC1 promoter was confirmed by electrophoretic mobility shift assay and chromatin immunoprecipitation. Antisense inhibition of WT1 protein translation strongly reduced Aoc1 transcripts in cultured murine embryonic kidneys and gonads. Aoc1 mRNA levels correlated with WT1 protein in several cell lines. Double immunofluorescent staining revealed a co-expression of WT1 and AOC1 proteins in the developing genitourinary system of mice and rats. Strikingly, induced changes in polyamine homeostasis affected branching morphogenesis of cultured murine embryonic kidneys in a developmental stage-specific manner. These findings suggest that WT1-dependent control of polyamine breakdown, which is mediated by changes in AOC1 expression, has a role in kidney organogenesis.

Lipoprotein(a) as a risk factor for atherosclerotic cardiovascular disease in patients in non-metropolitan areas of Brandenburg, Germany.

Background and aims: In the non-metropolitan region of Brandenburg (Germany), which is characterized by high rates of cardiovascular diseases and underserved medical care, there is a lack of awareness regarding lipoprotein(a) [Lp(a)] as a risk factor. In addition, data from patients with atherosclerotic cardiovascular disease (ASCVD) in diverse regional backgrounds, including the understudied Brandenburg cohort, and various healthcare statuses remain insufficient. Methods: In this WalkByLab study, Lp(a) levels were monitored in a non-metropolitan cohort (n = 850) in Brandenburg, Germany, comprising 533 patients at high cardiovascular risk and 317 healthy controls. Patients underwent a comprehensive angiological screening, which included blood serum analysis, assessment of medical and family history, cardiovascular risk, and disease status, and evaluation of lifestyle and quality of life. All parameters were evaluated with regard to two groups based on Lp(a) levels: low (<50 mg/dl) and high (≥50 mg/dl). Results: Brandenburg patients with cardiovascular diseases showed higher Lp(a) levels than healthy controls (24.2% vs. 14.8%, p = 0.001). Logistic regression analysis with different characteristics revealed that Lp(a) was an independent risk factor significantly associated with ASCVD (OR 2.26, 95% CI 1.32-3.95, p = 0.003). The high-Lp(a) group showed a higher proportion of patients with coronary artery disease, peripheral artery disease, or cerebrovascular disease compared to the low-Lp(a) group (50% vs. 36.8%; 57.7% vs. 45.8%; 17.6% vs. 9.2%; p = 0.004); also, a higher percentage of patients in the high-Lp(a) group had heart failure (72.8% vs. 53.2%, p = 0.014) and myocardial infarction (24.7% vs. 13.9%, p = 0.001). The high-Lp(a) group exhibited higher rates of statins (63.1% vs. 50.4%, p = 0.003), ezetimibe (14.8% vs. 5.5.%, p = 0.001), and beta-blockers (55.7% vs. 40.7%, p = 0.001) use. Lp(a) levels were found to be independent of physical activity or smoking behavior and did not change over time (12 months). Conclusions: Our study highlights the significance of elevated Lp(a) levels in Brandenburg cardiovascular patients and identifies them as an independent risk factor for ASCVD, which has implications for addressing cardiovascular health of non-metropolitan populations.

Leukocyte telomere length and mitochondrial DNA copy number associate with endothelial function in aging-related cardiovascular disease.

Background: We investigated the association between leukocyte telomere length, mitochondrial DNA copy number, and endothelial function in patients with aging-related cardiovascular disease (CVD). Methods: In total 430 patients with CVD and healthy persons were enrolled in the current study. Peripheral blood was drawn by routine venipuncture procedure. Plasma and peripheral blood mononuclear cells (PBMCs) were collected. Cell-free genomic DNA (cfDNA) and leukocytic genomic DNA (leuDNA) were extracted from plasma and PBMCs, respectively. Relative telomere length (TL) and mitochondrial DNA copy number (mtDNA-CN) were analyzed using quantitative polymerase chain reaction. Endothelial function was evaluated by measuring flow-mediated dilation (FMD). The correlation between TL of cfDNA (cf-TL), mtDNA-CN of cfDNA (cf-mtDNA), TL of leuDNA (leu-TL), mtDNA-CN of leuDNA (leu-mtDNA), age, and FMD were analyzed based on Spearman's rank correlation. The association between cf-TL, cf-mtDNA, leu-TL, leu-mtDNA, age, gender, and FMD were explored using multiple linear regression analysis. Results: cf-TL positively correlated with cf-mtDNA (r = 0.1834, P = 0.0273), and leu-TL positively correlated with leu-mtDNA (r = 0.1244, P = 0.0109). In addition, both leu-TL (r = 0.1489, P = 0.0022) and leu-mtDNA (r = 0.1929, P < 0.0001) positively correlated with FMD. In a multiple linear regression analysis model, both leu-TL (ß = 0.229, P = 0.002) and leu-mtDNA (ß = 0.198, P = 0.008) were positively associated with FMD. In contrast, age was inversely associated with FMD (ß = -0.426, P < 0.0001). Conclusion: TL positively correlates mtDNA-CN in both cfDNA and leuDNA. leu-TL and leu-mtDNA can be regarded as novel biomarkers of endothelial dysfunction.

Iodinated contrast media cause endothelial damage leading to vasoconstriction of human and rat vasa recta.

Contrast-induced acute kidney injury is an important clinical event with a worldwide increasing number of cases. Medullary hypoperfusion and hypoxia due to constriction of vasa recta are main factors in the pathophysiology of acute kidney injury. However, the mechanism of contrast media (CM)-induced vessel constriction is not known. We tested the hypothesis that vasa recta constriction is a consequence of endothelial dysfunction due to the cytotoxicity of CM. Human and rat descending vasa recta (DVR) were isolated and perfused with CM, and the luminal diameter was analyzed. For morphological analysis of the endothelium, renal arteries were CM perfused and then processed for electron microscopy. Transcellular electrical resistance was used to estimate CM-induced changes in the permeability of human umbilical vein endothelial cell (HUVEC) layers. Perfusion with CM constricted human and rat DRV (to 54.3 and 50.9% of initial diameter, respectively). This was blunted by adrenomedullin (77.7 and 77.1%, respectively). The ANG II response was enhanced by CM in rat DVR (reduction to 15.6 and 35.0% of initial diameter, respectively). Adrenomedullin blunted this effect (67.5%). CM led to endothelial damage of renal arteries characterized by a ragged surface, with sharply protruding intimal folds, spindle-like shape, and bulging in the lumen. These phenomena were reduced by adrenomedullin. The permeability of HUVEC cell layers was increased by CM, and this went along with increased myosin light chain phosporylation. Again, adremonedullin reduced the CM effect. Our study suggests that the constrictor effect of CM on the renal medullary microvasculature is a consequence of endothelial cell damage and the resulting endothelial dysfunction.

Granulocyte colony-stimulating factor improves cerebrovascular reserve capacity by enhancing collateral growth in the circle of Willis.

BACKGROUND AND PURPOSE: Restoration of cerebrovascular reserve capacity (CVRC) depends on the recruitment and positive outward remodeling of preexistent collaterals (arteriogenesis). With this study, we provide functional evidence that granulocyte colony-stimulating factor (G-CSF) augments therapeutic arteriogenesis in two animal models of cerebral hypoperfusion. We identified an effective dosing regimen that improved CVRC and stimulated collateral growth, thereby improving the outcome after experimentally induced stroke. METHODS: We used two established animal models of (a) cerebral hypoperfusion (mouse, common carotid artery ligation) and (b) cerebral arteriogenesis (rat, 3-vessel occlusion). Following therapeutic dose determination, both models received either G-CSF, 40 µg/kg every other day, or vehicle for 1 week. Collateral vessel diameters were measured following latex angiography. Cerebrovascular reserve capacities were assessed after acetazolamide stimulation. Mice with left common carotid artery occlusion (CCAO) were additionally subjected to middle cerebral artery occlusion, and stroke volumes were assessed after triphenyltetrazolium chloride staining. Given the vital role of monocytes in arteriogenesis, we assessed (a) the influence of G-CSF on monocyte migration in vitro and (b) monocyte counts in the adventitial tissues of the growing collaterals in vivo. RESULTS: CVRC was impaired in both animal models 1 week after induction of hypoperfusion. While G-CSF, 40 µg/kg every other day, significantly augmented cerebral arteriogenesis in the rat model, 50 or 150 µg/kg every day did not show any noticeable therapeutic impact. G-CSF restored CVRC in mice (5 ± 2 to 12 ± 6%) and rats (3 ± 4 to 19 ± 12%). Vessel diameters changed accordingly: in rats, the diameters of posterior cerebral arteries (ipsilateral: 209 ± 7-271 ± 57 µm; contralateral: 208 ± 11-252 ± 28 µm) and in mice the diameter of anterior cerebral arteries (185 ± 15-222 ± 12 µm) significantly increased in the G-CSF groups compared to controls. Stroke volume in mice (10 ± 2%) was diminished following CCAO (7 ± 4%) and G-CSF treatment (4 ± 2%). G-CSF significantly increased monocyte migration in vitro and perivascular monocyte numbers in vivo. CONCLUSION: G-CSF augments cerebral collateral artery growth, increases CVRC and protects from experimentally induced ischemic stroke. When comparing three different dosing regimens, a relatively low dosage of G-CSF was most effective, indicating that the common side effects of this cytokine might be significantly reduced or possibly even avoided in this indication.

Angiotensin receptor-neprilysin inhibitor improves coronary collateral perfusion.

Background: We investigated the pleiotropic effects of an angiotensin receptor-neprilysin inhibitor (ARNi) on collateral-dependent myocardial perfusion in a rat model of coronary arteriogenesis, and performed comprehensive analyses to uncover the underlying molecular mechanisms. Methods: A rat model of coronary arteriogenesis was established by implanting an inflatable occluder on the left anterior descending coronary artery followed by a 7-day repetitive occlusion procedure (ROP). Coronary collateral perfusion was measured by using a myocardial particle infusion technique. The putative ARNi-induced pro-arteriogenic effects were further investigated and compared with an angiotensin-converting enzyme inhibitor (ACEi). Expression of the membrane receptors and key enzymes in the natriuretic peptide system (NPS), renin-angiotensin-aldosterone system (RAAS) and kallikrein-kinin system (KKS) were analyzed by quantitative polymerase chain reaction (qPCR) and immunoblot assay, respectively. Protein levels of pro-arteriogenic cytokines were measured by enzyme-linked immunosorbent assay, and mitochondrial DNA copy number was assessed by qPCR due to their roles in arteriogenesis. Furthermore, murine heart endothelial cells (MHEC5-T) were treated with a neprilysin inhibitor (NEPi) alone, or in combination with bradykinin receptor antagonists. MHEC5-T proliferation was analyzed by colorimetric assay. Results: The in vivo study showed that ARNis markedly improved coronary collateral perfusion, regulated the gene expression of KKS, and increased the concentrations of relevant pro-arteriogenic cytokines. The in vitro study demonstrated that NEPis significantly promoted MHEC5-T proliferation, which was diminished by bradykinin receptor antagonists. Conclusion: ARNis improve coronary collateral perfusion and exert pro-arteriogenic effects via the bradykinin receptor signaling pathway.

Angiotensin-converting enzyme inhibitors stimulate cerebral arteriogenesis.

AIM: Arteriogenesis constitutes the most efficient endogenous rescue mechanism in cases of cerebral ischaemia. The aim of this work was to investigate whether angiotensin-converting enzyme inhibitors (ACEi) stimulates, and angiotensin II receptor type 1 blockers (ARB) inhibits cerebral collateral growth by applying a three-vessel occlusion (3-VO) model in rat. METHODS: Cerebral collateral growth was measured post 3-VO (1) by assessing blood flow using the cerebrovascular reserve capacity (CVRC) technique, and (2) by assessing vessel diameters in the posterior cerebral artery (PCA) via the evaluation of latex angiographies. A stimulatory effect on arteriogenesis was investigated for ACEi administration ± bradykinin receptor 1 (B1R) and 2 (B2R) blockers, and an inhibitory effect was analysed for ARB administration. Results were validated by immunohistochemical analysis and mechanistic data were collected by human umbilical vein endothelial cell (HUVEC) viability or scratch assay and monocyte (THP-1) migration assay. RESULTS: An inhibitory effect of ARB on arteriogenesis could not be demonstrated. However, collateral growth measurements demonstrated a significantly increased CVRC and PCA diameters in the ACEi group. ACEi stimulates cell viability and migration, which could be partially reduced by additional administration of bradykinin receptor 1 inhibitor (B1Ri). ACEi inhibits the degradation of pro-arteriogenic bradykinin derivatives, but combined ACEi + B1Ri + B1Ri (BRB) treatment did not reverse the stimulatory effect. Yet, co-administration of ACEi + BRB enhances arteriogenesis and cell migration. CONCLUSION: We demonstrate a potent stimulatory effect of ACEi on cerebral arteriogenesis in rats, presumable via B1R. However, results imply a pleiotropic and compensatory effect of ACEi on bradykinin receptor-stimulated arteriogenesis.

Recombinant surface proteomics as a tool to analyze humoral immune responses in bovines infected by Mycoplasma mycoides subsp. mycoides small colony type.

A systematic approach to characterize the surface proteome of Mycoplasma mycoides subspecies mycoides small colony type (M. mycoides SC), the causative agent of contagious bovine pleuropneumonia (CBPP) in cattle, is presented. Humoral immune responses in 242 CBPP-affected cattle and controls were monitored against one-third of the surface proteins of M. mycoides SC in a high throughput magnetic bead-based assay. Initially, 64 surface proteins were selected from the genome sequence of M. mycoides SC and expressed as recombinant proteins in Escherichia coli. Binding of antibodies to each individual protein could then be analyzed simultaneously in minute sample volumes with the Luminex suspension array technology. The assay was optimized on Namibian CBPP-positive sera and Swedish negative controls to allow detection and 20-fold mean signal separation between CBPP-positive and -negative sera. Signals were proven to be protein-specific by inhibition experiments, and results agreed with Western blot experiments. The potential of the assay to monitor IgG, IgM, and IgA responses over time was shown in a proof-of-concept study with 116 sera from eight animals in a CBPP vaccine study. In conclusion, a toolbox with recombinant proteins and a flexible suspension array assay that allows multiplex analysis of humoral immune responses to M. mycoides SC has been created.

A global view of protein expression in human cells, tissues, and organs.

Defining the protein profiles of tissues and organs is critical to understanding the unique characteristics of the various cell types in the human body. In this study, we report on an anatomically comprehensive analysis of 4842 protein profiles in 48 human tissues and 45 human cell lines. A detailed analysis of over 2 million manually annotated, high-resolution, immunohistochemistry-based images showed a high fraction (>65%) of expressed proteins in most cells and tissues, with very few proteins (<2%) detected in any single cell type. Similarly, confocal microscopy in three human cell lines detected expression of more than 70% of the analyzed proteins. Despite this ubiquitous expression, hierarchical clustering analysis, based on global protein expression patterns, shows that the analyzed cells can be still subdivided into groups according to the current concepts of histology and cellular differentiation. This study suggests that tissue specificity is achieved by precise regulation of protein levels in space and time, and that different tissues in the body acquire their unique characteristics by controlling not which proteins are expressed but how much of each is produced.

Increased levels of recombinant human proteins with the Escherichia coli strain Rosetta(DE3).

The effect of two Escherichiacoli expression strains on the production of recombinant human protein fragments was evaluated. High-throughput protein production projects, such as the Swedish Human Protein Atlas project, are dependent on high protein yield and purity. By changing strain from E. coli BL21(DE3) to E. coli Rosetta(DE3) the overall success rate of the protein production has increased dramatically. The Rosetta(DE3) strain compensates for a number of rare codons. Here, we describe how the protein expression of human gene fragments in E. coli strains BL21(DE3) and Rosetta(DE3) was evaluated in two stages. Initially a test set of 68 recombinant proteins that previously had been expressed in BL21(DE3) was retransformed and expressed in Rosetta(DE3). The test set generated very positive results with an improved expression yield and a significantly better purity of the protein product which prompted us to implement the Rosetta(DE3) strain in the high-throughput protein production. Except for analysis of protein yield and purity the sequences were also analyzed regarding number of rare codons and rare codon clusters. The content of rare codons showed to have a significant effect on the protein purity. Based on the results of this study the atlas project permanently changed expression strain to Rosetta(DE3).

A genecentric Human Protein Atlas for expression profiles based on antibodies.

An attractive path forward in proteomics is to experimentally annotate the human protein complement of the genome in a genecentric manner. Using antibodies, it might be possible to design protein-specific probes for a representative protein from every protein-coding gene and to subsequently use the antibodies for systematical analysis of cellular distribution and subcellular localization of proteins in normal and disease tissues. A new version (4.0) of the Human Protein Atlas has been developed in a genecentric manner with the inclusion of all human genes and splice variants predicted from genome efforts together with a visualization of each protein with characteristics such as predicted membrane regions, signal peptide, and protein domains and new plots showing the uniqueness (sequence similarity) of every fraction of each protein toward all other human proteins. The new version is based on tissue profiles generated from 6120 antibodies with more than five million immunohistochemistry-based images covering 5067 human genes, corresponding to approximately 25% of the human genome. Version 4.0 includes a putative list of members in various protein classes, both functional classes, such as kinases, transcription factors, G-protein-coupled receptors, etc., and project-related classes, such as candidate genes for cancer or cardiovascular diseases. The exact antigen sequence for the internally generated antibodies has also been released together with a visualization of the application-specific validation performed for each antibody, including a protein array assay, Western blot analysis, immunohistochemistry, and, for a large fraction, immunofluorescence-based confocal microscopy. New search functionalities have been added to allow complex queries regarding protein expression profiles, protein classes, and chromosome location. The new version of the protein atlas thus is a resource for many areas of biomedical research, including protein science and biomarker discovery.

Longitudinal serum biomarker screening identifies malate dehydrogenase 2 as candidate prognostic biomarker for Duchenne muscular dystrophy.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a fatal disease for which no cure is available. Clinical trials have shown to be largely underpowered due to inter-individual variability and noisy outcome measures. The availability of biomarkers able to anticipate clinical benefit is highly needed to improve clinical trial design and facilitate drug development. METHODS: In this study, we aimed to appraise the value of protein biomarkers to predict prognosis and monitor disease progression or treatment outcome in patients affected by DMD. We collected clinical data and 303 blood samples from 157 DMD patients in three clinical centres; 78 patients contributed multiple blood samples over time, with a median follow-up time of 2 years. We employed linear mixed models to identify biomarkers that are associated with disease progression, wheelchair dependency, and treatment with corticosteroids and performed survival analysis to find biomarkers whose levels are associated with time to loss of ambulation. RESULTS: Our analysis led to the identification of 21 proteins whose levels significantly decrease with age and nine proteins whose levels significantly increase. Seven of these proteins are also differentially expressed in non-ambulant patients, and three proteins are differentially expressed in patients treated with glucocorticosteroids. Treatment with corticosteroids was found to partly counteract the effect of disease progression on two biomarkers, namely, malate dehydrogenase 2 (MDH2, P = 0.0003) and ankyrin repeat domain 2 (P = 0.0005); however, patients treated with corticosteroids experienced a further reduction on collagen 1 serum levels (P = 0.0003), especially following administration of deflazacort. A time to event analysis allowed to further support the use of MDH2 as a prognostic biomarker as it was associated with an increased risk of wheelchair dependence (P = 0.0003). The obtained data support the prospective evaluation of the identified biomarkers in natural history and clinical trials as exploratory biomarkers. CONCLUSIONS: We identified a number of serum biomarkers associated with disease progression, loss of ambulation, and treatment with corticosteroids. The identified biomarkers are promising candidate prognostic and surrogate biomarkers, which may support drug developers if confirmed in prospective studies. The serum levels of MDH2 are of particular interest, as they correlate with disease stage and response to treatment with corticosteroids, and are also associated with the risk of wheelchair dependency and pulmonary function.