In order to investigate the biologic activity of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) on human erythropoiesis, glycophorin A (GPA)+ erythroid cells were generated in serum-free liquid phase from human cord blood (CB) CD34+ progenitor cells. The surface expression of TRAIL-R1 was weakly detectable in the early-intermediate phase of erythroid differentiation (days 4-6; dim-intermediate GPA expression), whereas a clear-cut expression of TRAIL-R2 was observed through the entire course of erythroid differentiation (up to days 12-14; bright GPA expression). On the other hand, surface TRAIL-R3 and -R4 were not detected at any culture time. Besides inducing a rapid but small increase of apoptotic cell death, which was abrogated by the pan-caspase inhibitor z-VAD-fmk, the addition of recombinant TRAIL at day 6 of culture inhibited the generation of morphologically mature erythroblasts. Among the intracellular pathways investigated, TRAIL significantly stimulated the extracellular signal-regulated kinase 1/2 (ERK1/2) but not the p38/mitogen-activated protein kinase (MAPK) or the c-Jun NH2-terminal kinase (JNK) pathway. Consistently with a key role of ERK1/2 in mediating the negative effects of TRAIL on erythroid maturation, PD98059, a pharmacologic inhibitor of the ERK pathway, but not z-VAD-fmk or SB203580, a pharmacologic inhibitor of p38/MAPK, reverted the antidifferentiative effect of TRAIL on CB-derived erythroblasts.

Endothelial cells express tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) receptors, but the function of TRAIL in endothelial cells is not completely understood. We explored the role of TRAIL in regulation of key intracellular signal pathways in endothelial cells. The addition of TRAIL to primary human endothelial cells increased phosphorylation of endothelial nitric oxide synthase (eNOS), NOS activity, and NO synthesis. Moreover, TRAIL induced cell migration and cytoskeleton reorganization in an NO-dependent manner. TRAIL did not activate the NF-κB or COX-2 pathways in endothelial cells. Instead, TRAIL increased prostanoid production (PGE₂=PGI₂>TXA₂), which was preferentially inhibited by the COX-1 inhibitor SC-560. Because NO and prostanoids play a crucial role in the state of blood vessel vasodilatation and angiogenesis, our data suggest that TRAIL might play an important role in endothelial cell function.

Tissue engineering is widely recognized as a promising approach for bone repair and reconstruction. Several attempts have been made to achieve materials that must be compatible, osteoconductive, and osteointegrative and have mechanical strength to provide a structural support. Composite scaffolds consisting in biodegradable natural polymers are very promising constructs. Hydroxyapatite (HAp) can support alginate as inorganic reinforcement and osteoconductive component of alginate/HAp composite scaffolds. Therefore, HAp-strengthened polymer biocomposites offer a solid system to engineer synthetic bone substitutes. In the present work, HAp was incorporated into an alginate solution and internal gelling was induced by addition of slowly acid-hydrolyzing D-gluconic acid delta-lactone for the direct release of calcium ions from HAp. It has been previously demonstrated that alginate-based composites efficiently support adhesion of cancer bone cell lines. Human dental pulp stem cells (DPSCs) identified in human dental pulp are clonogenic cells capable of differentiating in multiple lineage. Thus, this study is aimed at verifying the mineralization and differentiation potential of human DPSCs seeded onto scaffolds based on alginate and nano-hydroxyapatite. For this purpose, gene expression profile of early and late mineralization-related markers, extracellular matrix components, viability parameters, and oxidative stress occurrence were evaluated and analyzed. In summary, our data show that DPSCs express osteogenic differentiation-related markers and promote calcium deposition and biomineralization when growing onto Alg/HAp scaffolds. These findings confirm the use of Alg/HAp scaffolds as feasible composite materials in tissue engineering, being capable of promoting a specific and successful tissue regeneration as well as mineralized matrix deposition and sustaining natural bone regeneration.

Cytotoxic activity of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL/Apo-2 ligand), used alone or in different combinations with either a low (1.5 Gy) or a high (15 Gy) single dose of ionizing radiation (IR), was investigated on erythroleukemic cells (K562, HEL, Friend, primary leukemic erythroblasts) and on primary CD34⁺-derived normal erythroblasts. Human recombinant TRAIL alone variably affected the survival/growth of erythroleukemic cells; K562 cells were the most sensitive. Moreover, all erythroleukemic cells were radio-resistant, as demonstrated by the fact that cytotoxicity was evident only after treatment with high-dose (15 Gy) IR. Remarkably, when IR and TRAIL were used in combination, an additive effect was noticed in all erythroleukemic cells. Augmentation of TRAIL-induced cell death by IR was observed with both low and high IR doses and required the sequential treatment of IR 3 to 6 hours before the addition of TRAIL. Conversely, both TRAIL and IR showed a moderate cytotoxicity on primary CD34⁺-derived normal erythroblasts when used alone, but their combination did not show any additive effect. Moreover, the cytotoxicity of IR plus TRAIL observed in erythroleukemic cells was accompanied by the selective up-regulation of the surface expression of TRAIL-R1 (DR4), and it was completely blocked by the z-Val-Ala-Asp (OMe)-CH₂ (z-VAD-fmk) caspase inhibitor. On the other hand, the surface expression of TRAIL-R1 in CD34⁺-derived normal erythroblasts was unaffected by IR, which induced the up-regulation of the decoy TRAIL-R3. These data demonstrate that treatment with IR provides an approach to selectively sensitize erythroleukemic cells, but not normal erythroblasts, to TRAIL-induced apoptosis through the functional up-regulation of TRAIL-R1.