BackgroundADF/cofilin proteins are key modulators of actin dynamics in metastasis and invasion of cancer cells. Here we focused on the roles of ADF and cofilin-1 individually in the development of polarized migration of rat mammary adenocarcinoma (MTLn3) cells, which express nearly equal amounts of each protein. Small interference RNA (siRNA) technology was used to knockdown (KD) the expression of ADF and cofilin-1 independently.ResultsEither ADF KD or cofilin KD caused cell elongation, a reduction in cell area, a decreased ability to form invadopodia, and a decreased percentage of polarized cells after 180 s of epidermal growth factor stimulation. Moreover, ADF KD or cofilin KD increased the rate of cell migration and the time of lamellipodia protrusion but through different mechanisms: lamellipodia protrude more frequently in ADF KD cells and are more persistent in cofilin KD cells. ADF KD cells showed a significant increase in F-actin aggregates, whereas cofilin KD cells showed a significant increase in prominent F-actin bundles and increased cell adhesion. Focal adhesion area and cell adhesion in cofilin KD cells were returned to control levels by expressing exogenous cofilin but not ADF. Return to control rates of cell migration in ADF KD cells was achieved by expression of exogenous ADF but not cofilin, whereas in cofilin KD cells, expression of cofilin efficiently rescued control migration rates.ConclusionAlthough ADF and cofilin have many redundant functions, each of these isoforms has functional differences that affect F-actin structures, cell adhesion and lamellipodial dynamics, all of which are important determinants of cell migration.
Human platelet lysate (hPL) has been considered as the preferred supplement for the xeno-free stem cell culture for many years. However, the biological effect of hPL on the proliferation and differentiation of dental stem cells combined with the use of medical grade synthetic biomaterial is still under investigation. Thus, the optimal scaffold composition, cell type and specific growth conditions, yet need to be formulated. In this study, we aimed to investigate the regenerative potential of dental stem cells seeded on synthetic scaffolds and maintained in osteogenic media supplemented with either hPL or xeno-derived fetal bovine serum (FBS). Two types of dental stem cells were isolated from human impacted third molars and intact teeth; stem cells of apical papilla (SCAP) and periodontal ligament stem cells (PDLSCs). Cells were expanded in cell culture media supplemented with either hPL or FBS. Consequently, proliferative capacity, immunophenotypic characteristics and multilineage differentiation potential of the derived cells were evaluated on monolayer culture (2D) and on synthetic scaffolds fabricated from poly ’lactic-co-glycolic’ acid (PLGA) (3D). The functionality of the induced cells was examined by measuring the concentration of osteogenic markers ALP, OCN and OPN at different time points. Our results indicate that the isolated dental stem cells showed similar mesenchymal characteristics when cultured on hPL or FBS-containing culture media. Scanning electron microscopy (SEM) and H&E staining revealed the proper adherence of the derived cells on the 3D scaffold cultures. Moreover, the increase in the concentration of osteogenic markers proved that hPL was able to produce functional osteoblasts in both culture conditions (2D and 3D), in a way similar to FBS culture. These results reveal that hPL provides a suitable substitute to the animal-derived serum, for the growth and functionality of both SCAP and PDLSCs. Thus the use of hPL, in combination with PLGA scaffolds, can be useful in future clinical trials for dental regeneration.
Long-standing foot ulcers present a great challenge in diabetes care. Platelet products have been suggested as a possible therapeutic option. However, nor the effect of an injectable form of platelet lysate on the healing of ulcers nor that on primary cells of the epidermis have been studied. In the current study, we present two cases of an ongoing clinical trial showing the positive effect of autologous platelet lysate injected perilesional. Both clinical cases treated with injections of hPL showed complete healing of previously un-healed within 8 weeks of treatment. Further, we describe the in vitro effect of human platelet lysate (hPL) on primary human epidermal keratinocytes (HEK) in terms of chemotaxis, migration and proliferation. In vitro, HEK showed enhanced chemotaxis towards the hPL compared to keratinocyte-defined media (p < 0.0001). Their migration was also stimulated especially at hPL concentration of 10%V/V (p < 0.0001). In contrast, hPL significantly inhibited HEK proliferation measured through MTT assay (p < 0.0001). In conclusion, the findings presented here provide preliminary evidence of an explanatory mechanism for the effect of hPL on primary keratinocytes and therefore of their potential use in a clinical setting. hPL promotes keratinocyte migration and therefore closure of foot ulcers.
Circulating extracellular vesicles (EVs) are recognized as biomarkers and effectors of endothelial dysfunction, the initiating step of cardiovascular abnormalities. Among these EVs, microparticles (MPs) are vesicles directly released from the cytoplasmic membrane of activated cells. MPs were shown to induce endothelial dysfunction through the activation of endoplasmic reticulum (ER) stress. However, it is not known whether ER stress can lead to MPs release from endothelial cells and what biological messages are carried by these MPs. Therefore, we aimed to assess the impact of ER stress on MPs shedding from endothelial cells, and to investigate their effects on endothelial cell function. EA.hy926 endothelial cells or human umbilical vein endothelial cells (HUVECs) were treated for 24 h with ER stress inducers, thapsigargin or dithiothreitol (DTT), in the presence or absence of 4-Phenylbutyric acid (PBA), a chemical chaperone to inhibit ER stress. Then, MPs were isolated and used to treat cells (10–20 μg/mL) for 24–48 h before assessing ER stress response, angiogenic capacity, nitric oxide (NO) release, autophagy and apoptosis. ER stress (thapsigargin or DDT)-generated MPs did not differ quantitatively from controls; however, they carried deleterious messages for endothelial function. Exposure of endothelial cells to ER stress-generated MPs increased mRNA and protein expression of key ER stress markers, indicating a vicious circle activation of ER stress. ER stress (thapsigargin)-generated MPs impaired the angiogenic capacity of HUVECs and reduced NO release, indicating an impaired endothelial function. While ER stress (thapsigargin)-generated MPs altered the release of inflammatory cytokines, they did not, however, affect autophagy or apoptosis in HUVECs. This work enhances the general understanding of the deleterious effects carried out by MPs in medical conditions where ER stress is sustainably activated such as diabetes and metabolic syndrome.
Undiluted autologous platelet lysate, prepared according to a standardized methodology, is a safe and effective adjunct therapy for the treatment of PED.
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