Abstract
Proinflammatory stimuli lead to endothelial injury, which results in pathologies such as cardiovascular diseases, autoimmune diseases, and contributes to alloimmune responses after organ transplantation. Both mesenchymal stromal cells (MSC) and the extracellular vesicles (EV) released by them are widely studied as regenerative therapy for the endothelium. However, for therapeutic application, the manipulation of living MSC and large-scale production of EV are major challenges. Membrane particles (MP) generated from MSC may be an alternative to the use of whole MSC or EV. MP are nanovesicles artificially generated from the membranes of MSC and possess some of the therapeutic properties of MSC. In the present study we investigated whether MP conserve the beneficial MSC effects on endothelial cell repair processes under inflammatory conditions. MP were generated by hypotonic shock and extrusion of MSC membranes. The average size of MP was 120 nm, and they showed a spherical shape. The effects of two ratios of MP (50,000; 100,000 MP per target cell) on human umbilical vein endothelial cells (HUVEC) were tested in a model of inflammation induced by TNFα. Confocal microscopy and flow cytometry showed that within 24 hours >90% of HUVEC had taken up MP. Moreover, MP ended up in the lysosomes of the HUVEC. In a co-culture system of monocytes and TNFα activated HUVEC, MP did not affect monocyte adherence to HUVEC, but reduced the transmigration of monocytes across the endothelial layer from 138 ± 61 monocytes per microscopic field in TNFα activated HUVEC to 61 ± 45 monocytes. TNFα stimulation induced a 2-fold increase in the permeability of the HUVEC monolayer measured by the translocation of FITC-dextran to the lower compartment of a transwell system. At a dose of 1:100,000 MP significantly decreased endothelial permeability (1.5-fold) respect to TNFα Stimulated HUVEC. Finally, MP enhanced the angiogenic potential of HUVEC in an in vitro Matrigel assay by stimulating the formation of angiogenic structures, such as percentage of covered area, total tube length, total branching points, total loops. In conclusion, MP show regenerative effects on endothelial cells, opening a new avenue for treatment of vascular diseases where inflammatory processes damage the endothelium.
Highlights
The endothelium forms an interactive barrier between the circulatory system and the tissues in the body
We previously reported on the generation of large amounts of membrane particles (MP) from human adipose tissue Mesenchymal stromal cells (MSC) (ATMSC) [21]
We have investigated the potential therapeutic effects of Membrane particles (MP) derived from AT-MSC on the barrier integrity of inflamed endothelial cells using a model of TNFa treated human umbilical vein endothelial cells (HUVEC)
Summary
The endothelium forms an interactive barrier between the circulatory system and the tissues in the body. It plays a pivotal role in the regulation of vascular permeability, hemostasis, and immunological processes [1]. Therapies that protect the endothelium from stress and immune factors or enhance the repair processes may be capable of curing or preventing diseases where the endothelium has a key role. The endothelial protective effects of MSC are due to their antiinflammatory and repair properties that have shown substantial therapeutic promise in preclinical models, such as for instance in atherosclerosis [10]. MSC hold great promise for revascularization of tissues as they secrete proangiogenic and anti-apoptotic factors in large amounts [11]
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