TMEM219 regulates the transcription factor expression and proliferation of beta cells.

Pancreatic beta cells replenishment is considered the next therapeutic option for type 1 diabetes; while stimulating endogenous beta cells proliferation is the "holy grail" for those patients with exhausted beta cell mass. Here we are demonstrating that the pro-apoptotic receptor TMEM219 is expressed in fetal pancreas, in beta cell precursors and in in vitro embryonic-derived endocrine progenitors. TMEM219 signaling negatively regulates beta cells at early stages and induces Caspase 8-mediated cell death. Pharmacological blockade of TMEM219 further rescued beta cell precursor and proliferation markers, and decreased cell death, both in islets and in in vitro-derived endocrine progenitors, allowing for beta cell preservation. While addressing the upstream controlling TMEM219 expression, we determined the TMEM219 miRNet; indeed, one of those miRNAs, miR-129-2, is highly expressed in human islets, particularly in patients at risk or with established type 1 diabetes. miR-129-2 mimic downregulated TMEM219 expression in islets, in in vitro embryonic-derived endocrine progenitors and in highly proliferating insulinoma-derived cells. Moreover, miR-129-2 inhibitor induced a TMEM219 overexpression in insulinoma-derived cells, which restored cell proliferation and functional markers, thus acting as endogenous regulator of TMEM219 expression. The TMEM219 upstream regulator miR129-2 controls the fate of beta cell precursors and may unleash their regenerative potentials to replenish beta cells in type 1 diabetes.

Platelet Lysate Induces in Human Osteoblasts Resumption of Cell Proliferation and Activation of Pathways Relevant for Revascularization and Regeneration of Damaged Bone.

To understand the regenerative effect of platelet-released molecules in bone repair one should investigate the cascade of events involving the resident osteoblast population during the reconstructive process. Here the in vitro response of human osteoblasts to a platelet lysate (PL) stimulus is reported. Quiescent or very slow dividing osteoblasts showed a burst of proliferation after PL stimulation and returned to a none or very slow dividing condition when the PL was removed. PL stimulated osteoblasts maintained a differentiation capability in vitro and in vivo when tested in absence of PL. Since angiogenesis plays a crucial role in the bone healing process, we investigated in PL stimulated osteoblasts the activation of hypoxia-inducible factor 1-alpha (HIF-1α) and signal transducer and activator of transcription 3 (STAT3) pathways, involved in both angiogenesis and bone regeneration. We observed phosphorylation of STAT3 and a strong induction, nuclear translocation and DNA binding of HIF-1α. In agreement with the induction of HIF-1α an enhanced secretion of vascular endothelial growth factor (VEGF) occurred. The double effect of the PL on quiescent osteoblasts, i.e., resumption of proliferation and activation of pathways promoting both angiogenesis and bone formation, provides a rationale to the application of PL as therapeutic agent in post-traumatic bone repair.

Platelet Lysate Inhibits NF-κB Activation and Induces Proliferation and an Alert State in Quiescent Human Umbilical Vein Endothelial Cells Retaining Their Differentiation Capability.

: Injured blood vessel repair and blood circulation re-establishment are crucial events for tissue repair. We investigated in primary cultures of human umbilical vein endothelial cells (HUVEC), the effects of platelet lysate (PL), a cocktail of factors released by activated platelets following blood vessel disruption and involved in the wound-healing process triggering. PL exerted a protective effect on HUVEC in an inflammatory milieu by inhibiting IL-1α-activated NF-κB pathway and by inducing the secretion of PGE2, a pro-resolving molecule in the wound microenvironment. Moreover, PL enhanced HUVEC proliferation, without affecting their capability of forming tube-like structures on matrigel, and activated resting quiescent cells to re-enter cell cycle. In agreement with these findings, proliferation-related pathways Akt and ERK1/2 were activated. The expression of the cell-cycle activator Cyclin D1 was also enhanced, as well as the expression of the High Mobility Group Box-1 (HMGB1), a protein of the alarmin group involved in tissue homeostasis, repair, and remodeling. These in vitro data suggest a possible in vivo contribution of PL to new vessel formation after a wound by activation of cells resident in vessel walls. Our biochemical study provides a rationale for the clinical use of PL in the treatment of wound healing-related pathologies.

Electrospun silk fibroin fibers for storage and controlled release of human platelet lysate.

Human platelet lysate (hPL) is a pool of growth factors and cytokines able to induce regeneration of different tissues. Despite its good potentiality as therapeutic tool for regenerative medicine applications, hPL has been only moderately exploited in this field. A more widespread adoption has been limited because of its rapid degradation at room temperature that decreases its functionality. Another limiting factor for its extensive use is the difficulty of handling the hPL gels. In this work, silk fibroin-based patches were developed to address several points: improving the handling of hPL, enabling their delivery in a controlled manner and facilitating their storage by creating a device ready to use with expanded shelf life. Patches of fibroin loaded with hPL were synthesized by electrospinning to take advantage of the fibrous morphology. The release kinetics of the material was characterized and tuned through the control of fibroin crystallinity. Cell viability assays, performed with primary human dermal fibroblasts, demonstrated that fibroin is able to preserve the hPL biological activity and prolong its shelf-life. The strategy of storing and preserving small active molecules within a naturally-derived, protein-based fibrous scaffold was successfully implemented, leading to the design of a biocompatible device, which can potentially simplify the storage and the application of the hPL on a human patient, undergoing medical procedures such as surgery and wound care. STATEMENT OF SIGNIFICANCE: Human platelets lysate (hPL) is a mixture of growth factors and cytokines able to induce the regeneration of damaged tissues. This study aims at enclosing hPL in a silk fibroin electrospun matrix to expand its utilization. Silk fibroin showed the ability to preserve the hPL activity at temperature up to 60 °C and the manipulation of fibroin's crystallinity provided a tool to modulate the hPL release kinetic. This entails the possibility to fabricate the hPL silk fibroin patches in advance and store them, resulting in an easy and fast accessibility and an expanded use of hPL for wound healing.

Platelet-rich plasma-based bioactive membrane as a new advanced wound care tool.

Chronic skin ulcers, consequence of diabetes and other pathological conditions, heavily compromise the patient life quality and represent a high and constantly growing cost for National Health Services. Autologous platelet-rich plasma (PRP), has been proposed to treat these lesions. The absence of guidelines for the PRP production and the need of a fresh preparation for each treatment lead us to develop a protocol for the production of an allogenic PRP-based bioactive membrane (BAM), standardized for platelet concentration and growth factor release. This work compares BAMs obtained starting from two different platelet concentrations. There was no direct correlation between the amount of growth factors released by BAM in vitro and the initial platelet count. However, different release kinetics were noticed for different growth factors, suggesting that they were differently retained by the two BAMs. The angiogenic potential of both BAMs was determined by Luminex Angiogenesis Assay. The biological activity of the factors released by the two BAMs was confirmed by cell proliferation and migration. A diabetic mouse chronic ulcer model was used to define the best PRP therapeutic dose in vivo. Both BAMs induced wound healing by increasing the thickness of the regenerated epidermis and the vessel number. However, a too high platelet concentration resulted in a slowdown of the membrane resorption that interfered with the skin healing. Overall, the results indicate that the BAMs could represent a natural and effective wound healing tool for the treatment of skin ulcers. Copyright © 2016 John Wiley & Sons, Ltd.