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Moustapha Kassem

Publications and source records attributed to Moustapha Kassem.

32 records · Page 2Linked to original sources
PubMed2004

Expression of LRP1 by human osteoblasts: a mechanism for the delivery of lipoproteins and vitamin K1 to bone.

UNLABELLED: Accumulating clinical and experimental data show the importance of dietary lipids and lipophilic vitamins, such as vitamin K1, for bone formation. The molecular mechanism of how they enter the osteoblast is unknown. Here we describe the expression of the multifunctional LRP1 by human osteoblasts in vitro and in vivo. We provide evidence that LRP1 plays an important role in the uptake of postprandial lipoproteins and vitamin K1 by human osteoblasts. INTRODUCTION: Chylomicrons (CM) and their remnants (CR) represent the postprandial plasma carriers of dietary lipids. Dietary vitamin K1 is known to be transported in the circulation as part of CM/CR and is required by osteoblasts as an essential co-factor for the gamma-carboxylation of bone matrix proteins. The molecular mechanisms underlying the delivery of lipophilic substances to bone are not understood. In this study, the expression and function of CM/CR receptors was examined in human osteoblasts. MATERIALS AND METHODS: Four human osteoblast-like cell lines were analyzed: two osteosarcoma lines (MG63, SaOS-2) and two telomerase-immortalized human bone marrow stromal cell lines (hMSC-TERT [4] and [20]) after 1,25(OH)2 vitamin D3 induction of osteoblastic differentiation (hMSC-TERT-OB). Receptor expression was examined by Western blotting and immunohistochemistry of normal human bone sections. Endocytotic receptor function was analyzed by cellular uptake assays using fluorescent and radiolabeled human CR. Vitamin K1-enriched CR (CR-K1) were generated in vivo after oral vitamin administration and vitamin K1 uptake by osteoblasts was measured by HPLC. The effect of CR-K1 uptake on osteocalcin carboxylation was measured by ELISA. RESULTS: Osteoblasts exhibit high levels of protein expression of the CR receptors LRP1 and LDLR. VLDLR is expressed to a lower degree. Immunohistochemistry of normal human bone sections showed strong LRP1 expression by osteoblasts and marrow stromal cells. Uptake of fluorescent CR by osteoblasts resulted in the typical pattern of receptor-mediated endocytosis. CR uptake was stimulated by the exogenous addition of the lipoprotein receptor ligands apolipoprotein E and lipoprotein lipase. Uptake was reduced by the known LRP1 inhibitors RAP, lactoferrin, and suramin, but not by LDL, which exclusively binds to the LDLR. Vitamin K1 uptake by hMSC-TERT-OB after incubation with CR-K1 was also shown to be sensitive to LPL stimulation and the LRP1 specific inhibitor lactoferrin. CR-K1 uptake into osteoblasts stimulated the gamma-carboxylation of osteocalcin. CONCLUSION: Human osteoblasts express receptors of the LDLR family with a capacity for vitamin K1 uptake through CR endocytosis, a novel mechanism for the delivery of dietary lipids and lipophilic vitamins to human bone. The current data suggest that, among the expressed receptors, LRP1 plays a predominant role.

Administration, Oral↗
PubMed2004

Fracture risk in perimenopausal women treated with beta-blockers.

beta2-Adrenergic receptors have been identified on human osteoblastic and osteoclastic cells, raising the question of a sympathetic regulation of bone metabolism. We investigated effects of treatment with beta-adrenergic receptor antagonists (beta-blockers) on bone turnover, bone mineral density (BMD), and fracture risk. Within the Danish Osteoporosis Prevention Study (DOPS) a population based, comprehensive cohort study of 2016 perimenopausal women, associations between treatment with beta-blockers and bone turnover and BMD were assessed in a cross-sectional design at the start of study. Moreover, in a nested case-control design, fracture risk during the subsequent 5 years was assessed in relation to treatment with beta-blockers at baseline. Multiple regression- and logistic regression-analyses were performed. Treatment with beta-blockers was associated with a threefold increased fracture risk (OR(adj) 3.3; 95% CI: 1.1-9.4). Analyses on duration of treatment showed that women who had been treated for more than 8 years had a higher fracture risk (OR(adj) 5.3; 95% CI: 1.1-26.3) than those treated for less than 8 years (OR(adj) 2.4; 95% CI: 0.6-9.5). In addition, cross-sectional data showed 20% lower serum osteocalcin levels (an osteoblastic marker of bone formation) in women treated with beta-blockers compared to untreated women (P < 0.001), whereas BMD at the lumbar spine and femoral neck did not differ between groups. beta-Blockers may decrease the activity of bone-forming cells and thereby increase fracture risk. However, confirmative studies and studies exploring mechanisms of action are needed.

Adrenergic beta-Antagonists↗
PubMed2004

Adult human mesenchymal stem cell as a target for neoplastic transformation.

The neoplastic process may involve a cancer stem cell. This concept has emerged largely from the careful analysis of tumour biopsy systems from haematological, breast and brain tumours. However, the experimental systems necessary to provide the cellular and molecular evidence to support this important concept have been lacking. We have used adult mesenchymal stem cells (hMSC) transduced with the telomerase hTERT gene to investigate the neoplastic potential of adult stem cells. The hTERT-transduced line, hMSC-TERT20 at population doubling level (PDL) 256 showed loss of contact inhibition, anchorage independence and formed tumours in 10/10 mice. hMSC-TERT4 showed loss of contact inhibition at PDL 95, but did not exhibit anchorage independence and did not form tumours in mice. Both lines had a normal karyotype but showed deletion of the Ink4a/ARF locus. At later passage, hMSC-TERT4 also acquired an activating mutation in KRAS. In hMSC-TERT20, expression of the cell cycle-associated gene, DBCCR1 was lost due to promoter hypermethylation. This epigenetic event correlated with acquisition of tumorigenicity. These data suggest that the adult hMSCs can be targets for neoplastic transformation and have implications for the development of novel anticancer therapeutics and for the use of hMSC in tissue engineering and transplantation protocols.

Adult↗
PubMed2004

Regulation of human skeletal stem cells differentiation by Dlk1/Pref-1.

UNLABELLED: Dlk-1/Pref-1 was identified as a novel regulator of human skeletal stem cell differentiation. Dlk1/Pref-1 is expressed in bone and cultured osteoblasts, and its constitutive overexpression led to inhibition of osteoblast and adipocyte differentiation of human marrow stromal cells. INTRODUCTION: Molecular control of human mesenchymal stem cell (hMSC) differentiation into osteoblasts and adipocytes is not known. In this study, we examined the role of delta-like 1/preadipocyte factor-1 (Dlk1/Pref-1) in regulating the differentiation of hMSCs. MATERIALS AND METHODS: As a model for hMSCs, we have stably transduced telomerase-immortalized hMSC (hMSC-TERT) with the full length of human Dlk1/Pref-1 cDNA and tested its effect on hMSC growth and differentiation into osteoblasts or adipocytes as assessed by cytochemical staining, FACS analysis, and real time PCR. Ex vivo calvaria organ cultures assay was used to confirm the in vitro effect of Dlk/Pref-1 on bone formation. RESULTS: Dlk1/Pref-1 was found to be expressed in fetal and adult bone, hMSCs, and some osteoblastic cell lines. A retroviral vector containing the human Dlk1/Pref-1 cDNA was used to create a cell line (hMSC-dlk1) expressing high levels of Dlk1/Pref-1 protein. Overexpression of Dlk1/Pref-1 did not affect the proliferation rate of hMSC, but the ability to form mature adipocytes, mineralized matrix in vitro, and new bone formation in neonatal murine calvariae organ cultures was reduced. These effects were associated with inhibition of gene expression markers of late stages of adipocyte (adipocyte fatty acid-binding protein [aP2], peroxisome proliferator-activated receptor-gamma2 [PPARgamma2], and adiponectin [APM1]) and osteoblast differentiation (alkaline phosphatase [ALP], collagen type I [Col1], and osteocalcin [OC]). Lineage commitment markers for adipocytes (adipocyte determination and differentiation factor -1 [ADD1]) and osteoblasts (core binding factor/runt-related binding factor 2 [Cbfa1/Runx2]) were not affected. CONCLUSION: During hMSC differentiation, Dlk1/Pref-1 maintains the size of the bipotential progenitor cell pool by inhibiting the formation of mature osteoblasts and adipocytes.

Adipocytes↗
PubMed2004

Mice deficient in 11beta-hydroxysteroid dehydrogenase type 1 lack bone marrow adipocytes, but maintain normal bone formation.

Glucocorticoids (GCs) exert potent, but poorly characterized, effects on the skeleton. The cellular activity of GCs is regulated at a prereceptor level by 11beta-hydroxysteroid dehydrogenases (11betaHSDs). The type 1 isoform, which predominates in bone, functions as a reductase in intact cells and regenerates active cortisol (corticosterone) from circulating inert 11-keto forms. The aim of the present study was to investigate the role of this intracrine activation of GCs on normal bone physiology in vivo using mice deficient in 11betaHSD1 (HSD1(-/-)). The HSD1(-/-) mice exhibited no significant changes in cortical or trabecular bone mass compared with wild-type (Wt) mice. Aged HSD1(-/-) mice showed age-related bone loss similar to that observed in Wt mice. Histomorphometric analysis showed similar bone formation and bone resorption parameters in HSD1(-/-) and Wt mice. However, examination of bone marrow composition revealed a total absence of marrow adipocytes in HSD1(-/-) mice. Cells from Wt and HSD1(-/-) mice exhibited similar growth rates as well as similar levels of production of osteoblastic markers. The adipocyte-forming capacity of in vitro cultured bone marrow stromal cells and trabecular osteoblasts was similar in HSD1(-/-) and Wt mice. In conclusion, our results suggest that 11betaHSD1 amplification of intracellular GC actions in mice may be required for bone marrow adipocyte formation, but not for bone formation. The clinical relevance of this observation remains to be determined.

11-beta-Hydroxysteroid Dehydrogenase Type 1↗
PubMed2004

Mesenchymal stem cells: biological characteristics and potential clinical applications.

Mesenchymal stem cells (MSC) are clonogenic, non-hematpoietic stem cells present in the bone marrow and are able to differentiate into multiple mesoderm-type cell lineages, for example, osteoblasts, chondrocytes, endothelial-cells and also non-mesoderm-type lineages, for example, neuronal-like cells. Several methods are currently available for isolation of the MSC based on their physical and physico-chemical characteristics, for example, adherence to plastics or other extracellular matrix components. Because of the ease of their isolation and their extensive differentiation potential, MSC are among the first stem cell types to be introduced in the clinic. Several studies have demonstrated the possible use of MSC in systemic transplantation for systemic diseases, local implantation for local tissue defects, as a vehicle for genes in gene therapy protocols or to generate transplantable tissues and organs in tissue engineering protocols. Before their widespread use in therapy, methods allowing the generation of large number of cells without affecting their differentiation potential as well as technologies that overcome immunological rejection (in case allogenic transplantation) must be developed.

Animals↗
PubMed2004

Mesenchymal stem cells: cell biology and potential use in therapy.

Mesenchymal stem cells are clonogenic, non-haematopoietic stem cells present in the bone marrow and are able to differentiate into multiple mesoderm-type cell lineages e.g. osteoblasts, chondrocytes, endothelial-cells and also non-mesoderm-type lineages e.g. neuronal-like cells. Several methods are currently available for isolation of the mesenchymal stem cells based on their physical and immunological characteristics. Because of the ease of their isolation and their extensive differentiation potential, mesenchymal stem cells are among the first stem cell types to be introduced in the clinic. Recent studies have demonstrated that the life span of mesenchymal stem cells in vitro can be extended by increasing the levels of telomerase expression in the cells and thus allowing culture of large number of cells needed for therapy. In addition, it has been shown that it is possible to culture the cells in xeno-free environment without affecting their growth or differentiation potential. Finally, the mesenchymal stem cells seems to be hypoimmunogenic and thus allogenic mesenchymal stem cells transplantation is possible. It is envisaged that mesenchymal stem cells can be used in systemic transplantation for generalized diseases, local implantation for local tissue defects, as a vehicle for genes in gene therapy protocols or to generate transplantable tissues and organs in tissue engineering protocols. The results of these initial trials are very encouraging and several clinical trials are under way to study the efficacy and long-term safety of therapeutics based on mesenchymal stem cells.

Animals↗
PubMed2004

Induction of adipocyte-like phenotype in human mesenchymal stem cells by hypoxia.

Human mesenchymal stem cells (hMSCs) have the capacity to differentiate along several pathways to form bone, cartilage, tendon, muscle, and adipose tissues. The adult hMSCs reside in vivo in the bone marrow in niches where oxygen concentration is far below the ambient air, which is the most commonly encountered laboratory condition. The study reported here was designed to determine whether oxygen has a role in the differentiation of hMSCs into adipocytes. Indeed, when exposed to atmosphere containing only 1% of oxygen, the formation of adipocyte-like phenotype with cytoplasmic lipid inclusions was observed. The effect of hypoxia on the expression of adipocyte-specific genes was determined by real-time reverse transcription polymerase chain reaction. Interestingly, neither of the two central regulators of adipogenesis--the transcription factors peroxisome proliferator-activated receptor gamma2 (PPAR-gamma2) and ADD1/SREBP1c-was induced. Furthermore, hypoxia did not have any effect on the transcription of early (lipoprotein lipase) or late (aP2) marker genes. By the same token, neither of the mature adipocyte-specific genes--leptin and adipophilin--was found responsive to the treatment. High level of induction, however, was observed with the PPAR-gamma-induced angiopoietin-related gene, PGAR. The lack of an adipocyte-specific transcription pattern thus indicates that despite accumulation of the lipid, true adipogenic differentiation did not take place. In conclusion, hypoxia appears to exert a potent lipogenic effect independent of PPAR-gamma2 maturation pathway.

Adipocytes↗
PubMed2003

Aging is associated with decreased maximal life span and accelerated senescence of bone marrow stromal cells.

Age-related decrease in bone formation is well described. However, the cellular causes are not known. Thus, we have established cultures of bone marrow stromal cells (MSC) from young (aged 18-29 years, n = 6) and old (aged 68-81 years, n = 5) donors. MSC were serially passaged until reaching maximal life span. Cell growth, markers of cellular senescence, and osteogenic and adipogenic potential were determined in early-passage and late-passage cells established from young and old donors. MSC from old donors exhibited a decreased maximal life span compared with cells from young donors (24 +/- 11 population doublings [PD] vs 41 +/- 10 PD, P < 0.05) and mean PD rate was lower in old donor cells (0.05 +/- 0.02 PD/day) compared with young donor cells (0.09 +/- 0.02 PD/day) (P < 0.05). No differences were detected in number of senescence-associated beta-galactosidase positive (SA beta-gal+) cells and mean telomere length in early-passage cells obtained from young and old donors. However, MSC from old donors exhibited accelerated senescence evidenced by increased number of SA beta-gal+ cells per PD as compared with young (4% per PD vs 0.4% per PD, respectively). MSC from young and old donors were able to form similar amounts of mineralized matrix in vitro and of normal lamellar bone in vivo. In adipogenic medium similar numbers of adipocytes formed in cultures of young and old donors. In conclusion, aging is associated with decreased proliferative capacity of osteoprogenitor cells, suggesting that decreased osteoblastic cell number, and not function, leads to age-related decrease in bone formation.

Adipocytes↗
PubMed2002

Telomerase expression extends the proliferative life-span and maintains the osteogenic potential of human bone marrow stromal cells.

Human bone marrow stromal cells (hMSCs) were stably transduced by a retroviral vector containing the gene for the catalytic subunit of human telomerase (hTERT). Transduced cells (hMSC-TERTs) had telomerase activity, and the mean telomere length was increased as compared with that of control cells. The transduced cells have now undergone more than 260 population doublings (PD) and continue to proliferate, whereas control cells underwent senescence-associated proliferation arrest after 26 PD. The cells maintained production of osteoblastic markers and differentiation potential during continuous subculturing, did not form tumors, and had a normal karyotype. When implanted subcutaneously in immunodeficient mice, the transduced cells formed more bone than did normal cells. These results suggest that ectopic expression of telomerase in hMSCs prevents senescence-associated impairment of osteoblast functions.

Animals↗
PubMed2002

Involvement of the MEN1 gene locus in familial isolated hyperparathyroidism.

BACKGROUND: Familial isolated hyperparathyroidism (FIHP) is a hereditary disorder characterised by uni- or multiglandular parathyroid disease. A subset of families are likely to be genetic variants of other familial tumour syndromes in which PHPT is the main feature, for example multiple endocrine neoplasia type 1 (MEN 1) and the hyperparathyroidism-jaw tumour syndrome (HPT-JT). OBJECTIVE: To investigate seven families diagnosed with FIHP, each with two to eight affected family members, to clarify the underlying genetic mechanism. METHODS: The entire MEN1 gene was sequenced for germline mutations and, in addition, tumour specimens were analysed in comparative genomic hybridisation and loss of heterozygosity studies. RESULTS: Two families exhibited MEN1 mutations, L112V and 1658delG, which were associated with loss of the wild-type 11q13 alleles in all tumours analysed. In the remaining five families, no MEN1 mutation was identified. CONCLUSION: These results support the involvement of the MEN1 tumour suppressor gene in the pathogenesis of some of the FIHP kindreds. However, loss on chromosome 11 was seen in all tumours exhibiting somatic deletions, although in two families the tumour deletions involved 11q distal to MEN1. We conclude that the altered MEN1 gene function is of importance in the development of FIHP.

Adult↗
PubMedDate not supplied

Subcutaneous adipocytes can differentiate into bone-forming cells in vitro and in vivo.

Interconversion of bone marrow osteoblasts and adipocytes has been reported previously. However, the osteogenic potential of extramedullary adipocytes is not known. Thus, we incubated a pure culture of human subcutaneous adipocytes in control medium for 1-2 weeks. Afterward, the cells were incubated in either osteoblast medium (OB medium) containing various combinations of calcitriol, dexamethasone, ascorbic acid, and beta-glycerophosphate or in adipocyte medium (AD medium) containing HEPES, biotin, pantothenate, insulin, triiodothyronine, dexamethasone, and isobutylmethylxanthine for 4 weeks. Expression of osteoblastic and adipocytic phenotypes was examined by determination of lineage-specific mRNA markers and in vitro adipocyte and osteoblast formation. Cells were also implanted, mixed with hydroxyapatite-tricalcium phosphate powder, in the subcutaneous tissue of immunodeficient mice in order to assess in vivo bone formation potential. One week after incubation in control medium, cells formed fusiform elongated fibroblast-like cells. In OB medium, cells stained positive for alkaline phosphatase (AP) and expressed mRNAs encoding Cbfa1/Runx2, AP, and osteocalcin. In AD medium cells reacquired adipocyte morphology with multilocular lipid-filled cells. Also, the cells expressed adipocyte-specific mRNA markers: lipoprotein lipase and peroxisome proliferator-activated receptor gamma2. Bone was formed only in the in vivo implants of cells incubated in OB medium. In conclusion, extramedullary adipocytes can transdifferentiate to bone-forming cells. Because of their ease of isolation, adipocytes may be good candidates for tissue-engineering protocols aimed at creating bone tissue for the repair of nonunion fractures and large bone defects.

Adipocytes↗