Congenital Factor VII (FVII) deficiency can be divided into two groups: cases of "true" deficiency, or cross-reactive material (CRM) negative and variants that are cross-reactive material ...positive.The first form is commonly recognized as Type I condition whereas the second one is known as Type II. FVII deficiency has been occasionally associated with thrombotic events, mainly venous. The reasons underlying this peculiar manifestation are unknown even though in the majority of associated patients thrombotic risk factors are present. The purpose of the present study was to investigate if a thrombotic event was more frequent in Type I or in Type II defect.The majority of patients with FVII deficiency and thrombosis belong to Type II defects. In the following paper we discuss the possible role of the dysfunctional FVII cross-reaction material as a contributory cause for the occurrence of thrombosis.
Abstract
Background/Introduction
Protective bone marrow (BM) CD34+ hematopoietic stem/progenitor cells (HSPCs) are released in the bloodstream after cardiomacrovascular (CV) tissue damage such as ...myocardial infarction1. Diabetes, an high risk condition to develop CV diseases, results in BM damage2-4 and detrimental pro-apoptotic and pro-oxidant shifts in BM-derived CD34+HSPCs5,6. Being able to classify and study the mechanisms of CD34+HSPC-vascular cell interaction would be a great step forward in the CV research field. However, nowadays there is no available primary human cell-based in vitro model to study the BM vascular niche.
Purpose
We aim to develop a human-based in vitro model to reproduce the BM vascular niche that allows the analysis of molecular mechanisms involved in CD34+HSPC function. This model will be relevant to identify molecular targets beyond hypoglycemic treatment and potentiate personalized medicine applications for diabetic complications.
Method
Human endothelial cells and pericytes were isolated from femoral head of patient who underwent hip replacement or from ad hoc BM aspirate by magnetic bead separation and characterized by immunofluorescence staining and flow cytometry. A culture of endothelial and pericytic cells to replicate the BM vascular niche was developed on a chip. CD34+HSPC subpopulation alteration in diabetes was preliminarily evaluated through single cell sequencing analysis (scRNAseq) on CD34+HSPC cells isolated from BM of patients with or without diabetes (N=1).
Results
We successfully set up a new method to efficiently isolate autologous BM endothelial cells and pericytes derived from patients (N=3). The cells expressing typically endothelial (CD31, ZO-1, and VE-cadherin) and pericytic (CD146, CD140b) markers were cultured in a 2D chip model. ScRNAseq reveals 15 clusters of CD34+HSPCs and important differences between the non-diabetic and diabetic samples. In particular, cluster 2 is highly represented in the non-diabetic and completely disappears in the diabetic sample, while on the contrary cluster 9 is over-represented in the diabetic compared to the non-diabetic sample. Interestingly, though the trajectory analysis cluster 2 shows the highest probability of turning into cluster 9. Through Ingenuity Canonical Pathways analysis we identified the anti-oxidant pathway as the most affected in diabetes-derived CD34+HSPCs.
Conclusions
In vitro human vascular niche model could be the gold standard to study BM cell crosstalk. In our interest, cluster 2 completely disappears in diabetes-derived CD34+HSPCs, potentially representing the CV-protective CD34+HSPC fractions more prone to be mobilized. Developing of our in vitro model would be useful to validate this hypothesis.Figure 1:scRNAseq cluster UMAPFigure 2:Endothelial cells IF staining
Abstract
Background/Introduction
The annual rate of restenosis, amputation, and death of subjects with type 2 diabetes (T2DM) and critical limb ischemia (CLI) is very high even after successful ...revascularization. We have recently demonstrated that the in-vitro migratory ability of proangiogenic bone marrow (BM)-derived CD45dimKDR+CXCR4+ CD34+ cells predicts cardiovascular mortality at 18 months after percutaneous angioplasty (PTA) in people with T2DM and CLI.
Purpose
To verify the predictor at a longer follow-up time and to investigate the underpinning mechanism.
Methods
We analyzed the rate of cardiovascular mortality in the previously studied cohort of 119 subjects at a six-year follow-up. In addition, we compared apoptosis and angiogenic capability of MAC-sorted BM-CD34+ cells in a newly-recruited series of patients with T2DM and CLI and control nondiabetic subjects (CTRL). Following the screening of a spectrum of microRNA (miR) using PCR-based Exiqon technology, we validated the modulated miRs in BM-CD34+ cells from 6 new donors per group. Finally, we focused on miR-21 and its inhibitory targets, by assessing the effect of miR-21 modulation of functional outcomes, including apoptosis (caspase assay and Annexin V positive staining) and the induction of in-vitro endothelial networking on Matrigel.
Results
Multivariable regression model analysis confirmed that CD34+ cell migration forecasts cardiovascular mortality after revascularization. BM-CD34+ cells isolated from T2DM-CLI donors undergo apoptosis in a higher percentage and are less proangiogenic in-vitro than cells from CTRL. Of the 56 microRNAs commonly expressed in CD34+ cells from the two studied groups, 6 miRs were differentially expressed. Biological validation confirmed a significant down-modulation of miR-21 in CD34+ cells from T2DM-CLI patients. The expression of the miR-21 inhibitory target, PDCD4, a regulator of apoptosis, was increased in CD34+ cells from T2DM-CLI patients compared to CTRL (1.6±0.6 vs. 74.4±14.9 average±SE 2ddCt via PCR). Silencing miR-21 in CD34+ cells from CTRL phenocopied the T2DM-CLI behavior, resulting in significantly increased apoptosis and inhibition of EC networking. Likewise, exposure of endothelial cells to patient-derived CD34+ CCM induced apoptosis and reduced the network formation ability on Matrigel, with these functional liabilities being associated with miR-21 inhibition and PDCD4 upregulation.
Conclusions
Migratory activity of CD34+ cells predicts cardiovascular mortality at 6 years follow-up after PTA in subjects with T2DM and CLI. Complicated T2DM is associated with a specific miR signature in BM CD34+ cells which could negatively impact on vascular cell survival and angiogenic capacity, thereby possibly contributing to adverse cardiovascular outcomes. The miR-21/ PDCD4 duo may represent a therapeutic target to rescue diabetes-associated complications.
Acknowledgement/Funding
British Heart Foundation grant RG/13/17/30545, Cariplo Foundation (code: 2016-0922)
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