Vittorio Grill

TRAIL promotes the survival, migration and proliferation of vascular smooth muscle cells

AbstractHuman and rat primary sub-cultured vascular smooth muscle cells (VSMCs) showed clear expression of the death receptors TRAIL-R1 and TRAIL-R2; however, recombinant soluble TRAIL did not induce cell death when added to these cells. TRAIL tended to protect rat VSMCs from apoptosis induced either by inflammatory cytokines tumor necrosis factor-α + interleukin-1β + interferon-γ or by prolonged serum withdrawal, and promoted a significant increase in VSMC proliferation and migration. Of note, all the biological effects induced by TRAIL were significantly inhibited by pharmacological inhibitors of the ERK pathway. Western blot analysis consistently showed that TRAIL induced a significant activation of ERK1/2, and a much weaker phosphorylation of Akt, while it did not affect the p38/MAPK pathway. Taken together, these data strengthen the notion that the TRAIL/TRAIL-R system likely plays a role in the biology of the vascular system by affecting the survival, migration and proliferation of VSMCs.

Synergistic cytotoxic activity of recombinant TRAIL plus the non-genotoxic activator of the p53 pathway nutlin-3 in acute myeloid leukemia cells.

To potentiate the response of acute myeloid leukemia (AML) to TRAIL cytotoxicity, we have adopted a strategy of combining nutlin-3, a potent non-genotoxic activator of the p53 pathway, with recombinant TRAIL. The rationale for using such a combination was that deletions and/or mutations of the p53 gene occur in only 5-10% of AML and that TRAIL and nutlin-3 activate the extrinsic and intrinsic pathways of apoptosis, respectively. TRAIL induced a rapid increase of apoptosis when added to OCI M4-type and MOLM M5-type AML cells, carrying a wild-type p53, as well as to NB4 M3-type AML, carrying a mutated p53. On the other hand, the small molecule activator of the p53 pathway nutlin-3 induced p53 accumulation, cell cycle arrest and a slow progressive increase of apoptosis in OCI and MOLM but not in NB4. Of note, nutlin-3 up-regulated the surface expression of TRAIL-R2 and synergized with TRAIL in inducing apoptosis in OCI and MOLM as well as in primary M4-type and M5-type AML blasts, but not in NB4 cells. Moreover, while nutlin-3 up-regulated the expression of cyclin dependent kinase inhibitor p21, a p53-target gene mediating cell cycle block and showing anti-apoptotic activity, the simultaneous addition of TRAIL plus nutlin-3 induced the caspase-dependent cleavage of p21. The relevance of p21 down-regulation for sensitizing AML cells to apoptosis was underscored in knocking-down experiments with small interfering RNAs. Our data suggest that the combined treatment of nutlin-3 plus TRAIL might offer a novel therapeutic strategy for AML.

The nuclear matrix and apoptosis.

Abstract Apoptosis is a form of active cell death, genetically encoded, that plays a key role during several physiological and pathological conditions. During the apoptotic process, striking morphological and biochemical changes take place in the cell nucleus. However, the molecular mechanisms underlying these changes have escaped clarification for many years. Recently, attention has been devoted to identifying the modifications that occur during apoptosis in the nuclear matrix, a mainly proteinaceous framework structure which is thought to play a fundamental role in organizing nuclear structure and function. In this review, we focus our attention on the biochemical and morphological changes detected in the nuclear matrix during the apoptotic process. Particular emphasis will be placed on the proteolysis that some nuclear matrix proteins undergo early during the apoptotic process, as well as on the detachment of DNA loops from the matrix by the action of endonuclease(s). Future research in this field may provide important information about the principal mechanisms that cause nuclear destruction in apoptotic cells.

Human Full-Length Osteoprotegerin Induces the Proliferation of Rodent Vascular Smooth Muscle Cells both in vitro and in vivo

Background/Aims: Since elevated plasma levels of osteoprotegerin (OPG) represent a risk factor for death and heart failure in patients affected by diabetes mellitus and coronary artery disease, this study aimed to elucidate potential roles of OPG in the pathogenesis of atherosclerosis. Methods and Results: Recombinant human full-length OPG, used at concentrations comparable to the elevated levels found in the serum of diabetic patients, significantly increased the proliferation rate of rodent vascular smooth muscle cells (VSMC). To mimic the moderate chronic elevation of OPG observed in diabetic patients, low doses (1 µg/mouse) of full-length human OPG were injected intraperitoneally every 3 weeks in diabetic apolipoprotein E (apoE)-null mice. The group of animals treated for 12 weeks with recombinant OPG showed a small increase in the total aortic plaque area at necropsy in comparison to vehicle-treated animals. Importantly, while no differences in the amount of interstitial collagen or the degree of macrophage infiltration were observed between OPG-treated and vehicle-treated apoE-null diabetic animals, a significant increase in the number of α-actin-positive smooth muscle cells was observed in the plaques of OPG-treated mice. Conclusions: Our data suggest that OPG promotes VSMC proliferation and might be directly involved in pathogenetic aspects of atherosclerosis.

A quantitative study on the spatial and temporal ossification patterns of vertebral centra and neural arches and their relationship to the fetal age

A double-staining technique on 37 human embryos and fetuses (crown-rump length, CRL, between 38 and 116 mm) has been performed to study the ossification patterns of the vertebral column. Different growth sequences for centra and neural arches were observed. The survey of ossified centers suggested it was possible to relate significantly their appearance with the CRL. On the basis of already known data defining the developmental age in relationship to the latter parameter, we suggest their numerical evaluation as a further parameter for the assessment of the fetal age. Therefore, we have worked out a table that may be used either to determine the normal fetal growth, or when other parameters cannot be relied upon (i.e. in morphological diseases) for this aim.

Developmental pathways of vertebral centra and neural arches in human embryos and fetuses

SummaryThe ossification pathways of both vertebral centra (i.e., vertebral bodies) and neural arches were studied in human embryos and fetuses (CR-length between 38 and 116 mm). A clearing and double-staining method for whole embryo or fetus, using alcian blue and alizarin red S, allowed an easy and precise detection of the morphology of the whole vertebral column and every single vertebra. Both cartilaginous and bony components were clearly visible. Different temporal and topographical patterns of ossification were shown for the centra and arches; the latter were respectively proximaldistal (i.e., bidirectional from a defined starting tract in T10-L1) and cranial-caudal (i.e., monodirectional). The patterns could be related to the morphogenetic processes of other structures (i.e., muscles and nerves). Moreover, the numerical survey of ossification centers provided a possible parameter for the determination of the fetal developmental age. This could be useful in the study of pathological conditions.

Functional expression of TRAIL and TRAIL-R2 during human megakaryocytic development

The expression and function of surface TRAIL and TRAIL receptors were investigated in primary megakaryocytic cells, generated in serum‐free liquid phase from peripheral human CD34+ cells. The surface expression of both TRAIL and “death receptor” TRAIL‐R2 became detectable starting from the early phase of megakaryocytic differentiation (day 6 of culture) and persisted at later (days10–14) culture times. On the other hand, “death receptor” TRAIL‐R1, “decoy receptors” TRAIL‐R3, and TRAIL‐R4 were barely detectable or undetectable at any time point examined. Addition of recombinant TRAIL at day 6 of culture increased the rate of spontaneous apoptosis of CD34+/CD41dim megakaryoblasts and it significantly decreased the total output of mature megakaryocytic cells evaluated after additional 4–8 days of culture. Conversely, addition in culture of TRAIL‐R2‐Fc chimera, which blocked the interaction between endogenous TRAIL and TRAIL‐R2 on the surface of cultured megakaryocytic cells, increased the total megakaryocytic cell count. In addition, recombinant TRAIL promoted a small but reproducible increase of maturation in the surviving megakaryocytic cell population, evaluated by both phenotypic analysis and morphology. A similar pro‐maturation effect was observed when TRAIL was added to bone marrow‐derived CD61+ megakaryocytic cells. Thus, our data suggest a role of TRAIL as a regulator of megakaryocytopoiesis.

Immunolocalisation of bilitranslocase in mucosecretory and parietal cells of the rat gastric mucosa.

SummaryBilitranslocase is a plasma membrane carrier localised at the vascular pole of the rat liver cell, where it mediates uptake of organic anions from the blood into the liver. This carrier is also present in the epithelium of the rat gastric mucosa, with similar molecular mass and functional properties. An immunohistochemical study reveals that both the mucus-secreting cells of the gastric pit and the H+-secreting parietal cells express bilitranslocase. These data point to a possible role of bilitranslocase and of its food-borne substrates (anthocyanins and nicotinic acid) in regulating the function and the permeability of the gastric mucosa.

The influence of dental metal alloys on cell proliferation and fibronectin arrangement in human fibroblast cultures.

The biocompatibility of six single-phase dental metal alloys was studied by determining cell proliferation rates correlated to the arrangement of fibronectin (FN) in fibroblast cultures. Immunocytochemical methods were used to detect cell proliferation by 5-bromodeoxyuridine (BrdU) incorporation, and FN organization [i.e. diffuse in the extracellular matrix and organized in fibrils or in focal adhesions (FA)] in human fibroblast cultures. Cell proliferation rates were related to FN arrangement and in particular a higher percentage of cells in the S-phase was related to a predominance of FA. The greatest difference in behaviour compared to that of the controls was detected after 120 and 168 hr: at these times, as well as at previous ones, the alloy with the highest Au content seemed the most biocompatible among those tested, as it behaved in a very similar way to the controls. In contrast, fibroblasts exposed to the other five alloys showed different behaviours from the controls. It is assumed that a correlation exists between FN organization and the percentage of BrdU-positive cells, and that these features vary in the presence of different alloys. The observation of FN arrangement together with cell proliferation rates could be another useful tool in determining the biocompatibility of dental metal alloys.

TNF-related apoptosis-inducing ligand (TRAIL) up-regulates cyclooxygenase (COX)-1 activity and PGE2 production in cells of the myeloid lineage

Tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) up‐regulated the expression of constitutive cyclooxygenase (COX)‐1 protein in HL‐60 cells without affecting COX‐2. The TRAIL‐mediated COX‐1 up‐regulation was accompanied by a significant increase of the PGE2 synthesis and release, which was suppressed by the COX‐1 inhibitor valeryl salicylate but not by the COX‐2 inhibitor NS‐398. Experiments carried out by adding exogenous PGE2 to HL‐60 cells indicated that PGE2 was not involved in TRAIL cytotoxicity and rather showed a dose‐dependent protection against TRAIL‐induced apoptosis. Importantly, the ability of TRAIL to increase PGE2 production was also observed in normal, human CD34‐derived myeloid cells and in freshly isolated peripheral blood CD14+ monocytes. Moreover, in contrast to HL‐60 cells, primary, normal cells were not susceptible to TRAIL cytotoxicity. These data indicate that the ability of TRAIL to up‐regulate eicosanoid production and release is not confined to malignant leukemic cells, but it may also play a role in normal hematopoiesis.