Rationale: Acquisition of a procalcific phenotype by resident or circulating cells is important for calcification of atherosclerotic plaques, which is common in diabetes.Objective: We aim to identify and characterize circulating calcifying cells, and to delineate a pathophysiological role for these cells in type 2 diabetes. Methods and Results:

Bone marrow (BM) derived stem and progenitor cells contribute to cardiovascular homeostasis and are affected by cardiovascular risk factors. We devised a clinical data-driven approach to test candidate stem cell mobilizing mechanisms in pre-clinical models. We found that PB and BM CD34+ cell counts were directly correlated, and that most circulating CD34+ cells were viable, non-proliferating and derived from the BM. Thus, we analyzed PB and BM CD34+ cell levels as a two-compartment model in 72 patients with or without cardiovascular disease. Self-organizing maps showed that disturbed compartmentalization of CD34+ cells was associated with aging and cardiovascular risk factors especially diabetes. High activity of DPP-4, a regulator of the mobilizing chemokine SDF-1α, was associated with altered stem cell compartmentalization. For validation of these findings, we assessed the role of DPP-4 in the BM mobilization response of diabetic rats. Diabetes differentially affected DPP-4 activity in PB and BM and impaired stem/progenitor cell mobilization after ischemia or G-CSF administration. DPP-4 activity in the BM was required for the mobilizing effect of G-CSF, while in PB it blunted ischemia-induced mobilization. Indeed, DPP-4 deficiency restored ischemia (but not G-CSF)-induced stem cell mobilization and improved vascular recovery in diabetic animals. In conclusion, the analysis of stem cell compartmentalization in humans led us to discover mechanisms of BM unresponsiveness in diabetes determined by tissue-specific DPP-4 dysregulation.

Aims -Circulating extracellular vesicles (EV) are raising considerable interest as a non-invasive diagnostic tool as they are easily detectable in biological fluids and contain specific set of nucleic acids, proteins, and lipids reflecting pathophysiological conditions. We aimed to investigate differences in plasma-derived EV surface-protein profile as biomarker to be used in combination with endomyocardial biopsies (EMB) for the diagnosis of allograft rejection.Methods and results -Plasma was collected from 90 patients (53 training cohort, 37 validation cohort) prior to EMB. EV concentration was assessed by nanoparticle tracking analysis. EV surface antigens were measured using a multiplex flow cytometry assay comprising 37 fluorescently labelled capture bead populations coated with specific antibodies directed against respective EV surface epitopes. The concentration of EV was significantly increased and their diameter decreased in patients undergoing rejection as compared to negative ones. The trend was highly significant for both antibody-mediated rejection (AMR), and acute cellular rejection (P<0.001). Among EV-surface markers, CD3, CD2, ROR1, SSEA-4, HLA-I, and CD41b were identified as discriminants between controls and ACR, whereas HLA-II, CD326, CD19, CD25, CD20, ROR1, SSEA-4, HLA-I, and CD41b discriminated controls from patients with AMR. ROC curves confirmed a reliable diagnostic performance for each single marker (AUC range 0.727-0.939). According to differential EV-marker expression, a diagnostic model was built and validated in an external cohort of patients. Our model was able to distinguish patients undergoing rejection from those without rejection. The accuracy at validation in an independent external cohort reached 86.5%. Its application for patient management has the potential to reduce the number of EMBs.Further studies in a higher number of patients are required to validate this approach for clinical purpose.Conclusions -Circulating EV are highly promising as new tool to characterize cardiac allograft rejection and to be complementary to EMB monitoring. 2NARRATIVE ABSTRACT -Our study describes a method for detecting and characterising circulating extracellular vesicles (EV) as a minimally invasive, liquid biopsy for the diagnosis of cardiac allograft rejection, and as a complementary tool to EMB monitoring. EV obtained from peripheral blood were profiled to identify rejection and its types in cardiac transplant recipients. A standardized and rapid tool was established using a fluorescent bead-based multiplex assay. We built a diagnostic model based on machine learning algorithms to identify non-rejecting patients who potentially do not require EMBs. EV profiling could represent a tool for non-invasive monitoring of allograft rejection in cardiac transplant recipients.

A SIS-ECM patch allowed the colonization of host endothelial and smooth muscle cells in the graft. This material may be an ideal substitute for reconstructive vascular surgery, and its use could be extended to surgical repair of cardiac defects.

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Background: In cardiorenal syndrome type 2 (CRS2), the role of systemic congestion in heart failure (HF) is still obscure. We studied a model of CRS2 [monocrotaline (MCT)-treated rats] secondary to pulmonary hypertension and right ventricular (RV) failure in order to evaluate the contribution of prevalent congestion to the development of kidney injury. Methods: Ten animals were treated with MCT for 4 weeks until they developed HF. Eleven animals were taken as controls. Signs of hypertrophy and dilatation of the right ventricle demonstrated the occurrence of HF. Brain natriuretic peptide (BNP), serum creatinine (sCreatinine), both kidney and heart neutrophil gelatinase-associated lipocalin (NGAL), matrix metallopeptidase 9 (MMP9), serum cytokines as well as kidney and heart cell death, as assessed by TUNEL, were studied. Results: Rats with HF showed higher BNP levels [chronic HF (CHF) 4.8 ± 0.5 ng/ml; controls 1.5 ± 0.2 ng/ml; p < 0.0001], marked RV hypertrophy and dilatation (RV mass/RV volume: CHF 1.46 ± 0.31, controls 2.41 ± 0.81; p < 0.01) as well as pleural and peritoneal effusions. A significant increase in proinflammatory cytokines and sCreatinine was observed (CHF 3.06 ± 1.3 pg/ml vs. controls 0.54 ± 0.23 pg/ml; p = 0.04). Serum (CHF 562.7 ± 93.34 ng/ml vs. controls 245.3 ± 58.19 ng/ml; p = 0.02) as well as renal and heart tissue NGAL levels [CHF 70,680 ± 4,337 arbitrary units (AU) vs. controls 32,120 ± 4,961 AU; p = 0.001] rose significantly, and they were found to be complexed with MMP9 in CHF rats. A higher number of kidney TUNEL-positive tubular cells was also detected (CHF 114.01 ± 45.93 vs. controls 16.36 ± 11.60 cells/mm²; p = 0.0004). Conclusion: In this model of CHF with prevalent congestion, kidney injury is characterized by tubular damage and systemic inflammation. The upregulated NGAL complexed with MMP9 perpetuates the vicious circle of kidney/heart damage by enhancing the enzymatic activity of MMP9 with extracellular matrix degradation, worsening heart remodeling.

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We have shown greater remodelling of the ventricular myocardial extracellular matrix in patients having a shunt from the right ventricle to the pulmonary arteries when compared to those having a classic arterio-pulmonary shunt, with this remodelling progressing even after the neonatal period. This may influence a later suboptimal ventricular performance.