Peripheral arterial disease (PAD) is associated with a high risk of cardiovascular events and death and is postulated to be a critical socioeconomic cost in the future. Extracellular vesicles (EVs) have emerged as potential candidates for new biomarker discovery related to their protein and nucleic acid cargo. In search of new prognostic and therapeutic targets in PAD, we determined the prothrombotic activity, the cellular origin and the transcriptomic profile of circulating EVs. This prospective study included control and PAD patients. Coagulation time (Procoag-PPL kit), EVs cellular origin and phosphatidylserine exposure were determined by flow cytometry in plateletfree plasma (n = 45 PAD). Transcriptomic profiles of medium/large EVs were generated using the MARS-Seq RNA-Seq protocol (n = 12/group). The serum concentration of the differentially expressed gene S100A9, in serum calprotectin (S100A8/A9), was validated by ELISA in control (n = 100) and PAD patients (n = 317). S100A9 was also determined in EVs and tissues of human atherosclerotic plaques (n = 3). Circulating EVs of PAD patients were mainly of platelet origin, predominantly Annexin V positive and were associated with the procoagulant activity of plateletfree plasma. Transcriptomic analysis of EVs identified 15 differentially expressed genes. Among them, serum calprotectin was elevated in PAD patients (p < 0.05) and associated with increased amputation risk before and after covariate adjustment (mean follow-up 3.6 years, p < 0.01). The combination of calprotectin with hs-CRP in the multivariate analysis further improved risk stratification (p < 0.01). Furthermore, S100A9 was also expressed in femoral plaque derived EVs and tissues. In summary, we found that PAD patients release EVs, mainly of platelet origin, highly positive for AnnexinV and rich in transcripts related to platelet biology and immune responses. Amputation risk prediction improved with calprotectin and was significantly higher when combined with hs-CRP. Our results suggest that EVs can be a promising component of liquid biopsy to identify the molecular signature of PAD patients.
Angiographic studies were carried out on 21 patients with systemic necrotizing vasculitis. Four basic arterial anomalies were found: (1) saccular microaneurysms appeared in 62% of the patients (2) arterial thrombosis was seen in 81% of patients; (3) arterial stenosis occurred in 81%; and (4) lumen irregularities occurred in 90%. Alterations in the renal vascular flow were also observed in accordance with changes in the cortical medullary differentiation, heterogeneous nephrogram, and prolonged washout. Two patients showed regression of microaneurysms after immunosuppressive therapy. We found angiography to be a low-risk technique of use for diagnosis and followup studies on vasculitis.
Myelodysplastic syndromes (MDS) are clonal hematopoietic stem cell (HSC) malignancies characterized by ineffective hematopoiesis with increased incidence in elderly individuals. Genetic alterations do not fully explain the molecular pathogenesis of the disease, indicating that other types of lesions may play a role in its development. In this work, we analyzed the transcriptional lesions of human HSCs, demonstrating how aging and MDS are characterized by a complex transcriptional rewiring that manifests as diverse linear and non-linear transcriptional dynamisms. While aging-associated lesions seemed to predispose elderly HSCs to myeloid transformation, disease-specific alterations may be involved in triggering MDS development. Among MDS-specific lesions, we detected the overexpression of the transcription factor DDIT3. Exogenous upregulation of DDIT3 in human healthy HSCs induced an MDS-like transcriptional state, and a delay in erythropoiesis, with an accumulation of cells in early stages of erythroid differentiation, as determined by single-cell RNA-sequencing. Increased DDIT3 expression was associated with downregulation of transcription factors required for normal erythropoiesis, such as KLF1, TAL1 or SOX6, and with a failure in the activation of their erythroid transcriptional programs. Finally, DDIT3 knockdown in CD34+ cells from MDS patients was able to restore erythropoiesis, as demonstrated by immunophenotypic and transcriptional profiling. These results demonstrate that DDIT3 may be a driver of MDS transformation, and a potential therapeutic target to restore the inefficient erythropoiesis characterizing these patients.
Background:Over the last years, great effort has been made in characterizing the molecular pathogenesis of hematological malignancies. In myelodysplastic syndromes (MDS), studies have mainly focused on the analysis of the mutational profiles of hematopoietic progenitors, but the fundamental molecular bases of the disease are still incomplete. It is very possible that, as in other hematological diseases, transcriptional and epigenetic mechanisms also play a relevant role.Aims:In the present work, we aimed to characterize the transcriptional profile of the hematopoietic stem cells (HSCs) of MDS patients and healthy donors in order to identify altered genes and pathways that could contribute to the development and/or maintenance of the disease.Methods:HSCs were isolated from an homogeneous group of 10 untreated MDS patients with multilineage dysplasia as well as from healthy aged donors (median of 70 y/o) as controls, with the use of fluorescence activated cell sorting (FACS) (CD34+, CD38‐, CD90+, CD45RA‐). The transcriptional profiles of these cells were determined using low‐input RNA‐sequencing (MARS‐Seq). All statistical analyses were carried out in the R/Bioconductor programming environment. Functional analyses of differentially expressed genes were carried out using gene ontology, ingenuity pathway analysis and gene set enrichment analysis.Results:Unsupervised clustering of the 5% most variable genes among all samples demonstrated that most MDS specimens clustered separately from healthy controls, indicating that differences in the transcriptome of these cells are able to distinguish healthy and pathological HSCs. An unsupervised principal component analysis showed an expected heterogeneity of MDS patients; nevertheless, most of these patients clustered separately from healthy controls, corroborating our previous finding. To identify altered genes and pathways in HSCs from MDS patients we performed differential gene expression analysis, finding 56 and 45 up and down‐regulated genes (p‐value<0.05, FC>2), respectively. Intriguingly, among such genes, and despite the expected heterogeneity observed in MDS patients, we detected a very homogeneous deregulation of a subset of genes, suggesting common pathological mechanisms among patients. Gene ontology analysis showed that upregulated genes were involved in biological processes such as regulation of RNA processing (non‐sense mediated decay and translation initiation), telomere capping and regulation of megakaryocyte differentiation. Among downregulated genes, we found many genes involved in the response to extrinsic signals, including regulators of angiogenesis, cell‐cell signaling, chemokine‐mediated signaling pathways and chemotaxis. Deregulated genes in MDS included the presence of transcriptional regulators, such as transcription factors and chromatin remodelers, which may be responsible for the transcriptional aberrations observed in these patients. Moreover, we also observed an alteration in the expression of many genes involved in the regulation of cell death, a process known to be altered in the hematopoietic progenitors of this disease.Summary/Conclusion:Altogether, our data points towards the deregulation of the HSC transcriptional profile, affecting relevant processes which may alter intrinsic functions as well as their response to the microenvironment in order to favor their survival in MDS patients.
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