Biomedical subjects

Melissa A Kacena

Publications and source records attributed to Melissa A Kacena.

7 recordsLinked to original sources

PubMed2006

A reciprocal regulatory interaction between megakaryocytes, bone cells, and hematopoietic stem cells.

A growing body of evidence suggests that megakaryocytes (MK) or their growth factors play a role in skeletal homeostasis. MK have been shown to express and/or secrete several bone-related proteins including osteocalcin, osteonectin, bone sialoprotein, osteopontin, bone morphogenetic proteins, and osteoprotegerin. In addition, at least 3 mouse models have been described in which MK number was significantly elevated with an accompanying marked increase in bone mineral density. Mice overexpressing thrombopoietin, the major MK growth factor, have an osteosclerotic bone phenotype. Mice deficient in transcription factors GATA-1 and NF-E2, which are required for the differentiation of MK, exhibited a strikingly increased bone mass. Importantly, recent studies have demonstrated that MK can stimulate osteoblast (OB) proliferation and differentiation in vitro and that they can also inhibit osteoclast (OC) formation in vitro. These findings suggest that MK play a dual role in skeletal homeostasis by stimulating formation while simultaneously inhibiting resorption. Conversely, cells of the osteoblast lineage support hematopoiesis, including megakaryopoiesis. Postnatal hematopoiesis occurs almost solely in the bone marrow (BM), close to or on endosteal surfaces. This finding, in conjunction with the observed contact of OB with hematopoietic cells, has lead investigators to explore the molecular and cellular interactions between hematopoietic cells and cells of the OB lineage. Importantly, it has been shown that many of the cytokines that are critical for normal hematopoiesis and megakaryopoiesis are produced by OB. Indeed, culturing osteoblasts with CD34+ BM cells significantly enhances hematopoietic cell number by both enhancing the proliferation of long-term culture initiating cells and the proliferation and differentiation of MK. These data are consistent with cells in the OB lineage playing a critical role in the hematopoietic niche. Overall, these observations demonstrate the importance of MK-bone cell interactions in both skeletal homeostasis and hematopoiesis.

Animals↗

PubMed2006

Megakaryocyte-mediated inhibition of osteoclast development.

A growing body of evidence indicates that megakaryocytes (MK) or their growth factors play a role in skeletal homeostasis. We previously identified a novel regulatory pathway that controls bone formation, which is mediated by MK. In vivo megakaryocytosis resulted in massive bone formation. The co-culture of MK with osteoblasts (OB) resulted in increased OB proliferation in vitro, by a mechanism that required direct cell-to-cell contact. Here, we examined a second MK-mediated pathway that regulates osteoclast (OC) development. We have begun examining the unique inhibitory effect of MK on OC development. Spleen or bone marrow (BM) cells from C57BL/6 mice, as a source of OC precursors, were cultured with M-CSF and RANKL to induce OC development. MK were prepared by culturing fetal liver cells with thrombopoietin and separating cells into MK and non-MK populations. MK were titrated into spleen cell cultures and OC were identified as tartrate-resistant acid phosphatase-positive giant cells with >3 nuclei. There was a significant, P < 0.001, up to 10-fold reduction in OC formed when MK were added to the spleen cell cultures. We determined that 30% (vol:vol) MK conditioned media (CM) were able to completely block OC development from precursors, whereas 3% MK CM resulted in up to a 10-fold reduction in OC development, P < 0.001. These data indicate that a soluble factor(s) was responsible, at least in part, for the inhibition. We examined MK CM for known inhibitors of OC formation, using ELISAs. IL-4 was undetectable in MK CM, whereas IL-10 and IFN-gamma levels were similar in MK and non-MK CM. TGFbeta-1 levels were increased 2-fold in MK CM compared to control CM but were not responsible for the inhibition in OC development. Although, we found a significant increase in the levels of osteoprotegerin (OPG) in MK CM, antibody neutralization studies, MK derived from OPG-deficient mice, and tandem mass spectrophotometry, all confirm that OPG was not responsible for the MK-mediated inhibition of OC development. Overall, these data suggest that an unidentified factor(s) is present in MK CM that inhibits OC development. These studies indicate that MK can play a dual role in skeletal homeostasis by stimulating OB proliferation and simultaneously inhibiting OC development.

Animals↗

PubMed2006

The role of megakaryocytes in skeletal homeostasis and rheumatoid arthritis.

PURPOSE OF REVIEW: This review provides an update on the role of megakaryocytes in skeletal homeostasis, and discusses these findings in the context of rheumatoid arthritis. RECENT FINDINGS: Thrombocytosis is a common complication of rheumatoid arthritis, and is presumably caused by an up-regulation in megakaryocytopoiesis. In general, patients with rheumatoid arthritis exhibit localized joint bone erosion with systemic bone loss, and rheumatoid arthritis patients with thrombocytosis tend to have more severe disease. Interestingly, in addition to their role in rheumatoid arthritis with thrombocytosis, it has been demonstrated recently that megakaryocytes play a dual role in regulating skeletal mass by inhibiting bone resorption while simultaneously stimulating bone formation. This seeming contradiction in the putative role of megakaryocytes in skeletal regulation and rheumatoid arthritis is the focus of this review. SUMMARY: In rheumatoid arthritis there are substantial increases in the levels of several pro-inflammatory pleiotropic cytokines. As would be expected, in addition to their role in inflammation, these cytokines play a critical role in the megakaryocytopoiesis seen in patients who develop reactive thrombocytosis, and these cytokines also are known to regulate osteoclastogenesis. Thus, it appears that in rheumatoid arthritis with reactive thrombocytosis, the ability of the cytokines to enhance osteoclastogenesis outweighs the ability of megakaryocytes to inhibit osteoclastogenesis.

Arthritis, Rheumatoid↗

PubMed2004

Experiments with osteoblasts cultured under hypergravity conditions.

To understand further the role of gravity in osteoblast attachment, osteoblasts were subjected to hypergravity conditions in vitro. Scanning electron microscopy of all confluent coverslips from FPA units show that the number of attached osteoblasts was similar among gravitational levels and growth durations (~90 cells/microscopic field). Specifically, confluent 1.0 G control cultures contained an average of 91 +/- 8 cells/field, 3.3 G samples had 88 +/- 8 cells/field, and 4.0 G cultures averaged 90 +/- 7 cells/field. The sparsely plated cultures assessed by immunohistochemistry also had similar numbers of cells at each time point (l.0 G was similar to 3.3 and 4.0 G), but cell number changed from one time point to the next as those cells proliferated. Immunohistochemistry of centrifuged samples showed an increase in number (up to 160% increase) and thickness (up to 49% increase) of actin fibers, a decrease in intensity of fibronectin fluorescence (18-23% decrease) and an increase in number of vinculin bulbs (202-374% increase in number of vinculin bulbs/area). While hypergravity exposure did not alter the number of attached osteoblasts, it did result in altered actin, fibronectin, and vinculin elements, changing some aspects of osteoblast- substrate adhesion.

Actins↗

PubMed2003

Megakaryocyte-osteoblast interaction revealed in mice deficient in transcription factors GATA-1 and NF-E2.

UNLABELLED: Mice deficient in GATA-1 or NF-E2 have a 200-300% increase in bone volume and formation parameters. Osteoblasts and osteoclasts generated in vitro from mutant and control animals were similar in number and function. Osteoblast proliferation increased up to 6-fold when cultured with megakaryocytes. A megakaryocyte-osteoblast interaction plays a role in the increased bone formation in these mice. INTRODUCTION: GATA-1 and NF-E2 are transcription factors required for the differentiation of megakaryocytes. Mice deficient in these factors have phenotypes characterized by markedly increased numbers of immature megakaryocytes, a concomitant drastic reduction of platelets, and a striking increased bone mass. The similar bone phenotype in both animal models led us to explore the interaction between osteoblasts and megakaryocytes. MATERIALS AND METHODS: Histomorphometry, microCT, and serum and urine biochemistries were used to assess the bone phenotype in these mice. Wildtype and mutant osteoblasts were examined for differences in proliferation, alkaline phosphatase activity, and osteocalcin secretion. In vitro osteoclast numbers and resorption were measured. Because mutant osteoblasts and osteoclasts were similar to control cells, and because of the similar bone phenotype, we explored the interaction between cells of the osteoblast lineage and megakaryocytes. RESULTS: A marked 2- to 3-fold increase in trabecular bone volume and bone formation indices were observed in these mice. A 20- to 150-fold increase in trabecular bone volume was measured for the entire femoral medullary canal. The increased bone mass phenotype in these animals was not caused by osteoclast defects, because osteoclast number and function were not compromised in vitro or in vivo. In contrast, in vivo osteoblast number and bone formation parameters were significantly elevated. When wildtype or mutant osteoblasts were cultured with megakaryocytes from GATA-1- or NF-E2-deficient mice, osteoblast proliferation increased over 3- to 6-fold by a mechanism that required cell-to-cell contact. CONCLUSIONS: These observations show an interaction between megakaryocytes and osteoblasts, which results in osteoblast proliferation and increased bone mass, and may represent heretofore unrecognized anabolic pathways in bone.

Alkaline Phosphatase↗

PubMed2003

The effect of increased peripheral suture purchase on the strength of flexor tendon repairs.

PURPOSE: Previous studies have hypothesized unequal load sharing between peripheral and core sutures in flexor tendon repairs. Most commonly peripheral sutures are placed very near the repair site and characteristically fail before the core strands. We hypothesized that placement of the peripheral sutures farther from the repair site would better optimize load sharing and resist suture pullout, yielding a stronger overall repair. METHODS: To test the hypothesis we developed a mathematical model of the load sharing between core and peripheral sutures. By using this model we predicted that placement of peripheral sutures 2 mm from the repair site would optimize the balance of load between core and peripheral sutures. We then divided and repaired 27 flexor digitorum profundus tendons in 6 ways (core plus peripheral or peripheral sutures only at 1 mm, 2 mm, or 3 mm from the repair site). Tendons were clamped to a custom-built linear loading machine and distracted to failure. RESULTS: There was a clinically and statistically significant increase in strength with an increased distance of the peripheral suture from the repair site showing that core sutures augmented by a 2-mm peripheral repair were stronger than those performed with 1-mm peripheral repairs (50.8 vs 37.1 N). CONCLUSIONS: A peripheral stitch placement approximately 2 mm from the repair site represents a simple modification that can significantly increase the ultimate strength of flexor tendon repairs.

Cadaver↗

PubMedDate not supplied

Osteoblasts subjected to spaceflight and simulated space shuttle launch conditions.

To understand further the effects of spaceflight on osteoblast-enriched cultures, normal chicken calvarial osteoblasts were flown aboard shuttle flight STS-77, and the total number of attached cells was determined. Spaceflight and control cultures were chemically fixed 3 h and 3 d after launch. These fixed cultures were processed for scanning electron microscopy (SEM). The SEM analysis showed that with just 3 d of exposure to spaceflight, coverslip cultures contained 300 +/- 100 cells/mm2, whereas 1G control samples contained a confluent monolayer of cells (2400 +/- 200 cells/mm2). Although the cultures flown in space experienced a drastic decline in cell number in just 3 d, without further experimentation it was impossible to determine whether the decline was a result of microgravity, the harsh launch environment, or some combination of these factors. Therefore, this research attempted to address the effect of launch by subjecting osteoblasts to conditions simulating shuttle launch accelerations, noise, and vibrations. No differences, compared with controls, were seen in the number of total or viable cells after exposure to these various launch conditions. Taken together, these data indicate that the magnitude of gravitational loading (3G maximum) and vibration (7.83G rms maximum) resulting from launch does not adversely affect osteoblasts in terms of total or viable cell number immediately, but launch conditions, or the microgravity environment itself, may start a cascade of events that over several d contributes to cell loss.

Animals↗