Fayez Safadi - Academia.edu (original) (raw)
Papers by Fayez Safadi
The Journal of biological chemistry, Jan 2, 2015
We previously reported on the importance of osteoactivin (OA/Gpnmb) in osteogenesis. In this stud... more We previously reported on the importance of osteoactivin (OA/Gpnmb) in osteogenesis. In this study, we examined the role of OA in osteoclastogenesis, using mice with a nonsense mutation in Gpnmb gene (D2J) and wild-type controls (D2J/Gpnmb+). In these D2J mice, micro-CT and histomorphometric analyses revealed increased cortical thickness, while total porosity and eroded surface were significantly reduced in D2J mice compared to wild-type controls, and these results were corroborated by lower serum levels of CTX-1. Contrary to these observations, and counterintuitively, temporal gene expression analyses supported up-regulated osteoclastogenesis in D2J mice, and increased osteoclast differentiation rates ex vivo, marked by increased number and size. The finding that MAPK was activated in early differentiating and mature D2J osteoclasts, and survival of D2J osteoclasts was enhanced and mediated by activation of the AKT-GSK3β pathway supports this observation. Furthermore, this was abro...
Journal of Cellular Physiology, 2007
Objective: To evaluate the effect of a thrombospondin 1 (TSP1)-derived peptide on inflammation an... more Objective: To evaluate the effect of a thrombospondin 1 (TSP1)-derived peptide on inflammation and angiogenesis in an animal model of erosive arthritis and to assess the relationship between TSP1 and connective tissue growth factor (CTGF) in the pathophysiology of rheumatoid arthritis. Methods: Erosive arthritis in Lewis rats was induced by peptidoglycan-polysaccharide (PG-PS). Animals were divided into four groups: (1) negative control and groups receiving, (2) no treatment, (3) treatment with a TSP1-derived peptide, and (4) treatment with a scrambled peptide. Samples obtained from ankle joint, spleen and liver were studied using histology, histomorphometry, immunohistochemistry and RT-PCR. Results: Histological data indicated that the TSP1-derived peptide treatment decreased neovascularization, leukocyte infiltration and thickening of the synovial lining of the joint, and reduced granuloma formation in the spleen and liver when compared to control groups. Higher concentrations of CTGF and TSP1 proteins were observed in the affected areas of animals which did not receive TSP1-derived peptide treatment. Also, immunofluorescence and RT-PCR analyses showed an increase in CTGF protein expression and regulation, respectively, in the tissues of untreated animals when compared to the TSP1-derived peptide treated animals. By immunofluorescence, TSP1 expression was decreased in the TSP1-derived peptide treated animals. Moreover, macrophage/monocyte-specific staining revealed a decrease in cell infiltration in the articular tissue of the TSP1-derived peptide treated animals. Conclusion: Both inflammation and angiogenesis were decreased after TSP1-derived peptide treatment indicating a potential pathway by which TSP1 interaction with neutrophils induces CTGF in RA affected tissues.
Critical Reviews in Eukaryotic Gene Expression, 2010
Osteoactivin (OA) protein was discovered in bone cells a decade ago. Recent literature suggests t... more Osteoactivin (OA) protein was discovered in bone cells a decade ago. Recent literature suggests that osteoactivin is crucial for the differentiation and functioning of different cell types, including bone-forming osteoblasts and bone-resorbing osteoclast cells. Here, we review the literature to date on various regulatory functions of osteoactivin, as well as its discovery, structure, expression, and function in different tissues and cells. The transcriptional regulation of osteoactivin and its mechanism of action in normal and diseased conditions with special emphasis on bone are also covered in this review. In addition, we touch on the therapeutic potential of osteoactivin in cancer and bone diseases.
Journal of Cellular Physiology, 2006
Mesenchymal cell (MC) condensation or the aggregation of MCs precedes chondrocyte differentiation... more Mesenchymal cell (MC) condensation or the aggregation of MCs precedes chondrocyte differentiation and is required for subsequent cartilage formation during endochondral ossification. In this study, we used micromass cultures of C3H10T1/2 cells as an in vitro model system for studying MC condensation and the events important for this process. Transforming growth factor b1 (TGF-b1) served as the initiator of MC condensation in our model system and we were interested in determining whether CTGF functions as a downstream mediator of TGF-b1. CTGF is a matricellular protein that has been found to be expressed in MC condensations and in the perichondrium. Micromass cultures of C3H10T1/2 cells condensed under TGF-b1 stimulation concomitant with dramatic up-regulation of CTGF mRNA and protein levels. CTGF silencing by either CTGF siRNA or CTGF antisense oligonucleotide approaches showed that TGF-b1-induced condensation was CTGF dependent. Furthermore, silencing of CTGF expression resulted in significant reductions in cell proliferation and migration, events that are crucial during MC condensation. In addition, up-regulation of Fibronectin (FN) and suppression of Sox9 expression by TGF-b1 was also found to be mediated by CTGF. Immunofluorescence of developing mouse vertebrae showed that CTGF, TGF-b1 and FN were co-expressed in condensations of MCs, while Sox9 expression was low at this stage. During subsequent chondrogenesis, Sox9 expression was high in chondrocytes while CTGF expression was limited to the perichondrium. Thus, CTGF is an essential downstream mediator of T GF-b1-induced MC condensation through its effects on cell proliferation and migration. CTGF is also involved in up-regulating FN and suppressing Sox9 expression during TGF-b1 induced MC condensation.
Blood, Nov 16, 2004
Gene expression of CTGF, a proangiogenic molecule, is up-regulated by serine proteases (FXa) and ... more Gene expression of CTGF, a proangiogenic molecule, is up-regulated by serine proteases (FXa) and thrombin (JBC 275, 2000). We have demonstrated that thrombospondin-1 (TSP1) plays a major role in the assembly of the prothrombinase complex on the surface of neutrophils (PMNs) and that PMNs contain the message for CTGF. Rheumatoid arthritis is a chronic inflammatory disease associated with leukocyte adhesion and extravasation with angiogenesis as its hallmark. There is evidence indicating that TSP1 plays a major role in the pathophysiology of RA (JI 171, 2003). In this genetically susceptible rodent model of RA, a single intraperitoneal (i.p.) injection of peptidoglycan-polysaccharide induces an inflammatory immune response characterized by increased joint diameters in a biphasic manner. The acute phase occurs during the first five days with spontaneous reactivation around day 12 resulting in a progressive and irreversible chronic phase. We tested a well-characterized synthetic peptide derived from TSP1-type 3 repeats under three different therapeutic modalities in the experimental model of RA. First, peptide treatment was administered intravenously (i.v.) during the first five days of the experimental protocol representing the acute phase. Second, peptide treatment was administered i.v. during the acute phase but then i.p. every other day during the chronic phase of the experimental protocol. Third, peptide treatment was administered daily i.p. during the acute phase and every other day during the chronic phase. At all times a positive control group was included as well as a negative control group receiving a scrambled peptide. Total RNA was isolated from the ankle joints of the animals and RT-PCR was performed using specific primers for CTGF and G3PDH. Integrated density values were measured and the ratio of CTGF/G3PDH assessed. Immunohistochemical analysis of CTGF was also performed using Bioquant image analysis software to quantitate CTGF immuno expression. In peptide-treated animals CTGF expression in the cells of the synovial lining was significantly decreased when compared to the disease untreated group. Peptide untreated positive control group was associated with upregulation of the CTGF gene expression (2.53±0.33) in the ankle tissue and localized to the synovial membranes as judge by the immunochemical studies (8.7±0.77). In contrast, peptide treatment downregulated CTGF gene expression (0.9±0.41, p
Journal of Cellular Physiology, Aug 24, 2011
Current osteoinductive protein therapy utilizes bolus administration of large doses of bone morph... more Current osteoinductive protein therapy utilizes bolus administration of large doses of bone morphogenetic proteins (BMPs), which is costly, and may not replicate normal bone healing. The limited in vivo biologic activity of BMPs requires the investigation of growth factors that may enhance this activity. In this study, we utilized the C3H10T1/2 murine mesenchymal stem cell line to test the hypotheses that osteoactivin (OA) has comparable osteoinductive effects to bone morphogenetic protein-2 (BMP-2), and that sustained administration of either growth factor would result in increased osteoblastic differentiation as compared to bolus administration. Sustained release biodegradable hydrogels were designed, and C3H10T1/2 cells were grown on hydrogels loaded with BMP-2 or OA. Controls were grown on unloaded hydrogels, and positive controls were exposed to bolus growth factor administration. Cells were harvested at several time points to assess osteoblastic differentiation. Alkaline phosphatase (ALP) staining and activity, and gene expression of ALP and osteocalcin were assessed. Treatment with OA or BMP-2 resulted in comparable effects on osteoblastic marker expression. However, cells grown on hydrogels demonstrated osteoblastic differentiation that was not as robust as cells treated with bolus administration. This study shows that OA has comparable effects to BMP-2 on osteoblastic differentiation using both bolus administration and continuous release, and that bolus administration of OA has a more profound effect than administration using hydrogels for sustained release. This study will lead to a better understanding of appropriate delivery methods of osteogenic growth factors like OA for repair of fractures and segmental bone defects.
Blood, Nov 19, 2010
Abstract 4320 Biologic therapy for rheumatoid arthritis (RA) targets specific molecules that medi... more Abstract 4320 Biologic therapy for rheumatoid arthritis (RA) targets specific molecules that mediate and sustain the clinical manifestations of this complex illness. Compared with the general population, patients with RA are at an increased risk to develop cardiovascular diseases and the precise mechanism(s) of action remain obscure. This laboratory has proposed the existence of a pro-inflammatory axis in RA comprised by thrombospondin-1 (TSP1), transforming growth factor-beta (TGF-b) and CTGF. The present study evaluated plasma levels of TSP1, TGFb and CTGF in patients with RA by ELISA as well as specific cytokines and chemokines. CTGF plasma levels in RA patients (9.2 pg/ml mean, range 3.44–17.08) were found significantly increased…
The FASEB Journal, Apr 1, 2011
This study characterizes the skeletal phenotype in double knockout (dKO) mice using micro-CT and ... more This study characterizes the skeletal phenotype in double knockout (dKO) mice using micro-CT and ex-vivo analysis. We scanned femurs at ages of 4 weeks to 1 year and compared them to matched contro...
Blood, 2011
3275 Background: Diabetic retinopathy (DR) is a progressive disease that affects over 4 million p... more 3275 Background: Diabetic retinopathy (DR) is a progressive disease that affects over 4 million people in the United States and is one of the leading causes of blindness. The pathophysiology of the early events (no evidence of DR) leading to diabetic retinopathy is still not fully understood. Recent studies have proposed that levels of TSP1 (Arch Ophthalmol 127:507, 2009) and CTGF (Diabetes Care 27:758, 2004) may play a role in shifting the angiogenic balance and pathogenesis of DR. We postulate that during acute and chronic inflammation, as seen in type-2 diabetes a pro-inflammatory axis comprised by TSP1, transforming growth factor-beta (TGF-b) and CTGF may play a significant role in the progression of NPDR to PDR. Methods: This is a prospective control study comprised to date of: a) six (6) human subjects with NPDR, b) eleven (11) subjects with PDR, c) five (5) subjects without type-2 diabetes but in need of pars plana vitrectomy, and d) seventeen (17) normal human volunteers. Pl...
BMC Musculoskeletal Disorders, 2016
Background: Expression of the growth factor osteoactivin (OA) increases during tissue degeneratio... more Background: Expression of the growth factor osteoactivin (OA) increases during tissue degeneration and regeneration, fracture repair and after denervation-induced disuse atrophy, concomitant with increased matrix metalloproteinases (MMPs). However, OA's expression with repetitive overuse injuries is unknown. The aim of this study was to evaluate: 1) OA expression in an operant rat model of repetitive overuse; 2) expression of MMPs; 3) inflammatory cytokines indicative of injury or inflammation; and 4) the inducible form of heat shock protein 70 (HSPA1A/HSP72) as the latter is known to increase during metabolic stress and to be involved in cellular repair. Young adult female rats performed a high repetition negligible force (HRNF) food retrieval task for up to 6 weeks and were compared to control rats. Methods: Flexor digitorum muscles and tendons were collected from 22 young adult female rats performing a HRNF reaching task for 3 to 6 weeks, and 12 food restricted control (FRC) rats. OA mRNA levels were assessed by quantitative polymerase chain reaction (qPCR). OA, MMP-1,-2,-3, and-13 and HSP72 protein expression was assayed using Western blotting. Immunohistochemistry and image analysis was used to evaluate OA and HSP72 expression. ELISA was performed for HSP72 and inflammatory cytokines. Results: Flexor digitorum muscles and tendons from 6-week HRNF rats showed increased OA mRNA and protein expression compared to FRC rats. MMP-1,-2 and-3 progressively increased in muscles whereas MMP-1 and-3 increased in tendons with HRNF task performance. HSP72 increased in 6-week HRNF muscles and tendons, compared to controls, and co-localized with OA in the myofiber sarcolemma. IL-1alpha and beta increased transiently in tendons or muscles in HRNF week 3 before resolving in week 6. Conclusion: The simultaneous increases of OA with factors involved in tissue repair (MMPs and HSP72) supports a role of OA in tissue regeneration after repetitive overuse.
Osteoactivin (OA/Gpnmb) was first identified by differential gene display in mutant osteopetrotic... more Osteoactivin (OA/Gpnmb) was first identified by differential gene display in mutant osteopetrotic rats. The role of OA/Gpnmb in osteogenesis has been characterized and reported by our group. A recent study reported on OA/Gpnmb expression during osteoclastogenesis. To specify the critical role and mechanism of action of OA/Gpnmb in osteoclastogenesis, we characterized osteoclast phenotype of three genetically modified mouse models for OA/Gpnmb. These mice are: D2J, mutant for OA/Gpnmb characterized with nonsense loss-of-function mutation generating a truncated protein sequence; D2J wild-type (WT), D2J/Gpnmb+, generated by knocking in OA/Gpnmb alleles into D2J mouse; OA-null mouse (OA-KO), where OA is disrupted within the coding exons; and OA/Gpnmb transgenic mice (OATg), where OA/Gpnmb is overexpressed under the CMV-promoter. We hypothesized that OA/Gpnmb is a negative regulator of osteoclastogenesis. Osteoclastic phenotype examined by m-CT analysis revealed a decrease in bone mass i...
Osteoactivin (OA), a transmembrane protein, has recently emerged as a vital glycoprotein for the ... more Osteoactivin (OA), a transmembrane protein, has recently emerged as a vital glycoprotein for the differentiation and function of bone forming osteoblasts. OA expression has been shown to increase during osteoblast development with maximal expression during the final stages of differentiation. Recent studies showed that OA is also expressed by osteoclasts and plays a role in their differentiation and function. In this study, we used a transgenic mouse model over-expressing OA under the control of CMV promoter (OATg) to determine the mechanisms by which OA contributes to bone formation and remodeling in vivo. Western blot analysis showed a three-fold increase in OA expression in OATg compared to wild-type (WT) osteoblasts. Micro-CT analysis of femurs from 12 week-old OATg showed increased trabecular bone volume (BV/TV) in OATg mice compared to WT mice. Given that OA is also over-expressed in osteoclasts in OATg mice, we evaluated bone resorption by histomophometry and ELISA. Serum RAN...
Thrombosis Research, 2012
Activated factor X (FXa) and thrombin can up-regulate gene expression of connective tissue growth... more Activated factor X (FXa) and thrombin can up-regulate gene expression of connective tissue growth factor (CTGF/CCN2) on fibroblasts. Since tissue factor (TF) is expressed on these cells, we hypothesized that they may assemble the prothrombinase complex leading to CTGF/CCN2 upregulation. In addition, the effect of thrombospondin-1 (TSP1) on this reaction was evaluated. Human foreskin fibroblasts were incubated with purified factor VII (FVII), factor X (FX), factor V (FV), prothrombin and calcium in the presence and absence of TSP1. Generation of FXa and of thrombin were assessed using chromogenic substrates. SMAD pathway phosphorylation was detected via Western-blot analysis. Pre-incubation of fibroblasts with FVII led to its autoactivation by cell-surface expressed TF, which in turn in the presence of FX, FVa, prothrombin and calcium led to FXa (9.7 ± 0.8 nM) and thrombin (7.9 ± 0.04 U/mL × 10-3) generation. Addition of TSP1 significantly enhanced thrombin (23.3 ± 0.7 U/mL × 10-3) but not FXa (8.5 ± 0.6 nM) generation. FXa and thrombin generation leads to upregulation of CTGF/CCN2. TSP1 alone upregulated CTGF/CCN2, an effect mediated via activation of transforming growth factor beta (TGFβ) as showed by phosphorylation of the SMAD pathway an event blunted by using a TGFβ receptor I inhibitor (TGFβRI). FXa-and thrombin-induced upregulation of CTGF/CCN2 was not blocked by TGFβRI.
Journal of Cellular Physiology, 2007
Objective: To evaluate the effect of a thrombospondin 1 (TSP1)-derived peptide on inflammation an... more Objective: To evaluate the effect of a thrombospondin 1 (TSP1)-derived peptide on inflammation and angiogenesis in an animal model of erosive arthritis and to assess the relationship between TSP1 and connective tissue growth factor (CTGF) in the pathophysiology of rheumatoid arthritis. Methods: Erosive arthritis in Lewis rats was induced by peptidoglycan-polysaccharide (PG-PS). Animals were divided into four groups: (1) negative control and groups receiving, (2) no treatment, (3) treatment with a TSP1-derived peptide, and (4) treatment with a scrambled peptide. Samples obtained from ankle joint, spleen and liver were studied using histology, histomorphometry, immunohistochemistry and RT-PCR. Results: Histological data indicated that the TSP1-derived peptide treatment decreased neovascularization, leukocyte infiltration and thickening of the synovial lining of the joint, and reduced granuloma formation in the spleen and liver when compared to control groups. Higher concentrations of CTGF and TSP1 proteins were observed in the affected areas of animals which did not receive TSP1-derived peptide treatment. Also, immunofluorescence and RT-PCR analyses showed an increase in CTGF protein expression and regulation, respectively, in the tissues of untreated animals when compared to the TSP1-derived peptide treated animals. By immunofluorescence, TSP1 expression was decreased in the TSP1-derived peptide treated animals. Moreover, macrophage/monocyte-specific staining revealed a decrease in cell infiltration in the articular tissue of the TSP1-derived peptide treated animals. Conclusion: Both inflammation and angiogenesis were decreased after TSP1-derived peptide treatment indicating a potential pathway by which TSP1 interaction with neutrophils induces CTGF in RA affected tissues.
Journal of Cellular Physiology, 2006
Connective tissue growth factor (CTGF/CCN2) is a cysteine‐rich, extracellular matrix (ECM) protei... more Connective tissue growth factor (CTGF/CCN2) is a cysteine‐rich, extracellular matrix (ECM) protein that acts as an anabolic growth factor to regulate osteoblast differentiation and function. Recent studies have identified CTGF as a downstream effector of transforming growth factor‐β1 (TGF‐β1) for certain functions in specific cell types. In this study, we examined the role of CTGF as a downstream mediator of TGF‐β1‐induced ECM production and cell growth in osteoblasts. Using primary cultures, we demonstrated that TGF‐β1 is a potent inducer of CTGF expression in osteoblasts, and that this induction occurred at all stages of osteoblast differentiation from the proliferative through mineralization stages. TGF‐β1 treatment of osteoblasts increased the expression and synthesis of the ECM components, collagen and fibronectin. When CTGF‐specific siRNA was used to prevent TGF‐β1 induction of CTGF expression, it also inhibited collagen and fibronectin production, thereby demonstrating the re...
Journal of Cellular Physiology, 2006
Mesenchymal cell (MC) condensation or the aggregation of MCs precedes chondrocyte differentiation... more Mesenchymal cell (MC) condensation or the aggregation of MCs precedes chondrocyte differentiation and is required for subsequent cartilage formation during endochondral ossification. In this study, we used micromass cultures of C3H10T1/2 cells as an in vitro model system for studying MC condensation and the events important for this process. Transforming growth factor β1 (TGF‐β1) served as the initiator of MC condensation in our model system and we were interested in determining whether CTGF functions as a downstream mediator of TGF‐β1. CTGF is a matricellular protein that has been found to be expressed in MC condensations and in the perichondrium. Micromass cultures of C3H10T1/2 cells condensed under TGF‐β1 stimulation concomitant with dramatic up‐regulation of CTGF mRNA and protein levels. CTGF silencing by either CTGF siRNA or CTGF antisense oligonucleotide approaches showed that TGF‐β1‐induced condensation was CTGF dependent. Furthermore, silencing of CTGF expression resulted in...
Journal of Cellular Physiology, 2011
Current osteoinductive protein therapy utilizes bolus administration of large doses of bone morph... more Current osteoinductive protein therapy utilizes bolus administration of large doses of bone morphogenetic proteins (BMPs), which is costly, and may not replicate normal bone healing. The limited in vivo biologic activity of BMPs requires the investigation of growth factors that may enhance this activity. In this study, we utilized the C3H10T1/2 murine mesenchymal stem cell line to test the hypotheses that osteoactivin (OA) has comparable osteoinductive effects to bone morphogenetic protein‐2 (BMP‐2), and that sustained administration of either growth factor would result in increased osteoblastic differentiation as compared to bolus administration. Sustained release biodegradable hydrogels were designed, and C3H10T1/2 cells were grown on hydrogels loaded with BMP‐2 or OA. Controls were grown on unloaded hydrogels, and positive controls were exposed to bolus growth factor administration. Cells were harvested at several time points to assess osteoblastic differentiation. Alkaline phosp...
The Journal of biological chemistry, Jan 2, 2015
We previously reported on the importance of osteoactivin (OA/Gpnmb) in osteogenesis. In this stud... more We previously reported on the importance of osteoactivin (OA/Gpnmb) in osteogenesis. In this study, we examined the role of OA in osteoclastogenesis, using mice with a nonsense mutation in Gpnmb gene (D2J) and wild-type controls (D2J/Gpnmb+). In these D2J mice, micro-CT and histomorphometric analyses revealed increased cortical thickness, while total porosity and eroded surface were significantly reduced in D2J mice compared to wild-type controls, and these results were corroborated by lower serum levels of CTX-1. Contrary to these observations, and counterintuitively, temporal gene expression analyses supported up-regulated osteoclastogenesis in D2J mice, and increased osteoclast differentiation rates ex vivo, marked by increased number and size. The finding that MAPK was activated in early differentiating and mature D2J osteoclasts, and survival of D2J osteoclasts was enhanced and mediated by activation of the AKT-GSK3β pathway supports this observation. Furthermore, this was abro...
Journal of Cellular Physiology, 2007
Objective: To evaluate the effect of a thrombospondin 1 (TSP1)-derived peptide on inflammation an... more Objective: To evaluate the effect of a thrombospondin 1 (TSP1)-derived peptide on inflammation and angiogenesis in an animal model of erosive arthritis and to assess the relationship between TSP1 and connective tissue growth factor (CTGF) in the pathophysiology of rheumatoid arthritis. Methods: Erosive arthritis in Lewis rats was induced by peptidoglycan-polysaccharide (PG-PS). Animals were divided into four groups: (1) negative control and groups receiving, (2) no treatment, (3) treatment with a TSP1-derived peptide, and (4) treatment with a scrambled peptide. Samples obtained from ankle joint, spleen and liver were studied using histology, histomorphometry, immunohistochemistry and RT-PCR. Results: Histological data indicated that the TSP1-derived peptide treatment decreased neovascularization, leukocyte infiltration and thickening of the synovial lining of the joint, and reduced granuloma formation in the spleen and liver when compared to control groups. Higher concentrations of CTGF and TSP1 proteins were observed in the affected areas of animals which did not receive TSP1-derived peptide treatment. Also, immunofluorescence and RT-PCR analyses showed an increase in CTGF protein expression and regulation, respectively, in the tissues of untreated animals when compared to the TSP1-derived peptide treated animals. By immunofluorescence, TSP1 expression was decreased in the TSP1-derived peptide treated animals. Moreover, macrophage/monocyte-specific staining revealed a decrease in cell infiltration in the articular tissue of the TSP1-derived peptide treated animals. Conclusion: Both inflammation and angiogenesis were decreased after TSP1-derived peptide treatment indicating a potential pathway by which TSP1 interaction with neutrophils induces CTGF in RA affected tissues.
Critical Reviews in Eukaryotic Gene Expression, 2010
Osteoactivin (OA) protein was discovered in bone cells a decade ago. Recent literature suggests t... more Osteoactivin (OA) protein was discovered in bone cells a decade ago. Recent literature suggests that osteoactivin is crucial for the differentiation and functioning of different cell types, including bone-forming osteoblasts and bone-resorbing osteoclast cells. Here, we review the literature to date on various regulatory functions of osteoactivin, as well as its discovery, structure, expression, and function in different tissues and cells. The transcriptional regulation of osteoactivin and its mechanism of action in normal and diseased conditions with special emphasis on bone are also covered in this review. In addition, we touch on the therapeutic potential of osteoactivin in cancer and bone diseases.
Journal of Cellular Physiology, 2006
Mesenchymal cell (MC) condensation or the aggregation of MCs precedes chondrocyte differentiation... more Mesenchymal cell (MC) condensation or the aggregation of MCs precedes chondrocyte differentiation and is required for subsequent cartilage formation during endochondral ossification. In this study, we used micromass cultures of C3H10T1/2 cells as an in vitro model system for studying MC condensation and the events important for this process. Transforming growth factor b1 (TGF-b1) served as the initiator of MC condensation in our model system and we were interested in determining whether CTGF functions as a downstream mediator of TGF-b1. CTGF is a matricellular protein that has been found to be expressed in MC condensations and in the perichondrium. Micromass cultures of C3H10T1/2 cells condensed under TGF-b1 stimulation concomitant with dramatic up-regulation of CTGF mRNA and protein levels. CTGF silencing by either CTGF siRNA or CTGF antisense oligonucleotide approaches showed that TGF-b1-induced condensation was CTGF dependent. Furthermore, silencing of CTGF expression resulted in significant reductions in cell proliferation and migration, events that are crucial during MC condensation. In addition, up-regulation of Fibronectin (FN) and suppression of Sox9 expression by TGF-b1 was also found to be mediated by CTGF. Immunofluorescence of developing mouse vertebrae showed that CTGF, TGF-b1 and FN were co-expressed in condensations of MCs, while Sox9 expression was low at this stage. During subsequent chondrogenesis, Sox9 expression was high in chondrocytes while CTGF expression was limited to the perichondrium. Thus, CTGF is an essential downstream mediator of T GF-b1-induced MC condensation through its effects on cell proliferation and migration. CTGF is also involved in up-regulating FN and suppressing Sox9 expression during TGF-b1 induced MC condensation.
Blood, Nov 16, 2004
Gene expression of CTGF, a proangiogenic molecule, is up-regulated by serine proteases (FXa) and ... more Gene expression of CTGF, a proangiogenic molecule, is up-regulated by serine proteases (FXa) and thrombin (JBC 275, 2000). We have demonstrated that thrombospondin-1 (TSP1) plays a major role in the assembly of the prothrombinase complex on the surface of neutrophils (PMNs) and that PMNs contain the message for CTGF. Rheumatoid arthritis is a chronic inflammatory disease associated with leukocyte adhesion and extravasation with angiogenesis as its hallmark. There is evidence indicating that TSP1 plays a major role in the pathophysiology of RA (JI 171, 2003). In this genetically susceptible rodent model of RA, a single intraperitoneal (i.p.) injection of peptidoglycan-polysaccharide induces an inflammatory immune response characterized by increased joint diameters in a biphasic manner. The acute phase occurs during the first five days with spontaneous reactivation around day 12 resulting in a progressive and irreversible chronic phase. We tested a well-characterized synthetic peptide derived from TSP1-type 3 repeats under three different therapeutic modalities in the experimental model of RA. First, peptide treatment was administered intravenously (i.v.) during the first five days of the experimental protocol representing the acute phase. Second, peptide treatment was administered i.v. during the acute phase but then i.p. every other day during the chronic phase of the experimental protocol. Third, peptide treatment was administered daily i.p. during the acute phase and every other day during the chronic phase. At all times a positive control group was included as well as a negative control group receiving a scrambled peptide. Total RNA was isolated from the ankle joints of the animals and RT-PCR was performed using specific primers for CTGF and G3PDH. Integrated density values were measured and the ratio of CTGF/G3PDH assessed. Immunohistochemical analysis of CTGF was also performed using Bioquant image analysis software to quantitate CTGF immuno expression. In peptide-treated animals CTGF expression in the cells of the synovial lining was significantly decreased when compared to the disease untreated group. Peptide untreated positive control group was associated with upregulation of the CTGF gene expression (2.53±0.33) in the ankle tissue and localized to the synovial membranes as judge by the immunochemical studies (8.7±0.77). In contrast, peptide treatment downregulated CTGF gene expression (0.9±0.41, p
Journal of Cellular Physiology, Aug 24, 2011
Current osteoinductive protein therapy utilizes bolus administration of large doses of bone morph... more Current osteoinductive protein therapy utilizes bolus administration of large doses of bone morphogenetic proteins (BMPs), which is costly, and may not replicate normal bone healing. The limited in vivo biologic activity of BMPs requires the investigation of growth factors that may enhance this activity. In this study, we utilized the C3H10T1/2 murine mesenchymal stem cell line to test the hypotheses that osteoactivin (OA) has comparable osteoinductive effects to bone morphogenetic protein-2 (BMP-2), and that sustained administration of either growth factor would result in increased osteoblastic differentiation as compared to bolus administration. Sustained release biodegradable hydrogels were designed, and C3H10T1/2 cells were grown on hydrogels loaded with BMP-2 or OA. Controls were grown on unloaded hydrogels, and positive controls were exposed to bolus growth factor administration. Cells were harvested at several time points to assess osteoblastic differentiation. Alkaline phosphatase (ALP) staining and activity, and gene expression of ALP and osteocalcin were assessed. Treatment with OA or BMP-2 resulted in comparable effects on osteoblastic marker expression. However, cells grown on hydrogels demonstrated osteoblastic differentiation that was not as robust as cells treated with bolus administration. This study shows that OA has comparable effects to BMP-2 on osteoblastic differentiation using both bolus administration and continuous release, and that bolus administration of OA has a more profound effect than administration using hydrogels for sustained release. This study will lead to a better understanding of appropriate delivery methods of osteogenic growth factors like OA for repair of fractures and segmental bone defects.
Blood, Nov 19, 2010
Abstract 4320 Biologic therapy for rheumatoid arthritis (RA) targets specific molecules that medi... more Abstract 4320 Biologic therapy for rheumatoid arthritis (RA) targets specific molecules that mediate and sustain the clinical manifestations of this complex illness. Compared with the general population, patients with RA are at an increased risk to develop cardiovascular diseases and the precise mechanism(s) of action remain obscure. This laboratory has proposed the existence of a pro-inflammatory axis in RA comprised by thrombospondin-1 (TSP1), transforming growth factor-beta (TGF-b) and CTGF. The present study evaluated plasma levels of TSP1, TGFb and CTGF in patients with RA by ELISA as well as specific cytokines and chemokines. CTGF plasma levels in RA patients (9.2 pg/ml mean, range 3.44–17.08) were found significantly increased…
The FASEB Journal, Apr 1, 2011
This study characterizes the skeletal phenotype in double knockout (dKO) mice using micro-CT and ... more This study characterizes the skeletal phenotype in double knockout (dKO) mice using micro-CT and ex-vivo analysis. We scanned femurs at ages of 4 weeks to 1 year and compared them to matched contro...
Blood, 2011
3275 Background: Diabetic retinopathy (DR) is a progressive disease that affects over 4 million p... more 3275 Background: Diabetic retinopathy (DR) is a progressive disease that affects over 4 million people in the United States and is one of the leading causes of blindness. The pathophysiology of the early events (no evidence of DR) leading to diabetic retinopathy is still not fully understood. Recent studies have proposed that levels of TSP1 (Arch Ophthalmol 127:507, 2009) and CTGF (Diabetes Care 27:758, 2004) may play a role in shifting the angiogenic balance and pathogenesis of DR. We postulate that during acute and chronic inflammation, as seen in type-2 diabetes a pro-inflammatory axis comprised by TSP1, transforming growth factor-beta (TGF-b) and CTGF may play a significant role in the progression of NPDR to PDR. Methods: This is a prospective control study comprised to date of: a) six (6) human subjects with NPDR, b) eleven (11) subjects with PDR, c) five (5) subjects without type-2 diabetes but in need of pars plana vitrectomy, and d) seventeen (17) normal human volunteers. Pl...
BMC Musculoskeletal Disorders, 2016
Background: Expression of the growth factor osteoactivin (OA) increases during tissue degeneratio... more Background: Expression of the growth factor osteoactivin (OA) increases during tissue degeneration and regeneration, fracture repair and after denervation-induced disuse atrophy, concomitant with increased matrix metalloproteinases (MMPs). However, OA's expression with repetitive overuse injuries is unknown. The aim of this study was to evaluate: 1) OA expression in an operant rat model of repetitive overuse; 2) expression of MMPs; 3) inflammatory cytokines indicative of injury or inflammation; and 4) the inducible form of heat shock protein 70 (HSPA1A/HSP72) as the latter is known to increase during metabolic stress and to be involved in cellular repair. Young adult female rats performed a high repetition negligible force (HRNF) food retrieval task for up to 6 weeks and were compared to control rats. Methods: Flexor digitorum muscles and tendons were collected from 22 young adult female rats performing a HRNF reaching task for 3 to 6 weeks, and 12 food restricted control (FRC) rats. OA mRNA levels were assessed by quantitative polymerase chain reaction (qPCR). OA, MMP-1,-2,-3, and-13 and HSP72 protein expression was assayed using Western blotting. Immunohistochemistry and image analysis was used to evaluate OA and HSP72 expression. ELISA was performed for HSP72 and inflammatory cytokines. Results: Flexor digitorum muscles and tendons from 6-week HRNF rats showed increased OA mRNA and protein expression compared to FRC rats. MMP-1,-2 and-3 progressively increased in muscles whereas MMP-1 and-3 increased in tendons with HRNF task performance. HSP72 increased in 6-week HRNF muscles and tendons, compared to controls, and co-localized with OA in the myofiber sarcolemma. IL-1alpha and beta increased transiently in tendons or muscles in HRNF week 3 before resolving in week 6. Conclusion: The simultaneous increases of OA with factors involved in tissue repair (MMPs and HSP72) supports a role of OA in tissue regeneration after repetitive overuse.
Osteoactivin (OA/Gpnmb) was first identified by differential gene display in mutant osteopetrotic... more Osteoactivin (OA/Gpnmb) was first identified by differential gene display in mutant osteopetrotic rats. The role of OA/Gpnmb in osteogenesis has been characterized and reported by our group. A recent study reported on OA/Gpnmb expression during osteoclastogenesis. To specify the critical role and mechanism of action of OA/Gpnmb in osteoclastogenesis, we characterized osteoclast phenotype of three genetically modified mouse models for OA/Gpnmb. These mice are: D2J, mutant for OA/Gpnmb characterized with nonsense loss-of-function mutation generating a truncated protein sequence; D2J wild-type (WT), D2J/Gpnmb+, generated by knocking in OA/Gpnmb alleles into D2J mouse; OA-null mouse (OA-KO), where OA is disrupted within the coding exons; and OA/Gpnmb transgenic mice (OATg), where OA/Gpnmb is overexpressed under the CMV-promoter. We hypothesized that OA/Gpnmb is a negative regulator of osteoclastogenesis. Osteoclastic phenotype examined by m-CT analysis revealed a decrease in bone mass i...
Osteoactivin (OA), a transmembrane protein, has recently emerged as a vital glycoprotein for the ... more Osteoactivin (OA), a transmembrane protein, has recently emerged as a vital glycoprotein for the differentiation and function of bone forming osteoblasts. OA expression has been shown to increase during osteoblast development with maximal expression during the final stages of differentiation. Recent studies showed that OA is also expressed by osteoclasts and plays a role in their differentiation and function. In this study, we used a transgenic mouse model over-expressing OA under the control of CMV promoter (OATg) to determine the mechanisms by which OA contributes to bone formation and remodeling in vivo. Western blot analysis showed a three-fold increase in OA expression in OATg compared to wild-type (WT) osteoblasts. Micro-CT analysis of femurs from 12 week-old OATg showed increased trabecular bone volume (BV/TV) in OATg mice compared to WT mice. Given that OA is also over-expressed in osteoclasts in OATg mice, we evaluated bone resorption by histomophometry and ELISA. Serum RAN...
Thrombosis Research, 2012
Activated factor X (FXa) and thrombin can up-regulate gene expression of connective tissue growth... more Activated factor X (FXa) and thrombin can up-regulate gene expression of connective tissue growth factor (CTGF/CCN2) on fibroblasts. Since tissue factor (TF) is expressed on these cells, we hypothesized that they may assemble the prothrombinase complex leading to CTGF/CCN2 upregulation. In addition, the effect of thrombospondin-1 (TSP1) on this reaction was evaluated. Human foreskin fibroblasts were incubated with purified factor VII (FVII), factor X (FX), factor V (FV), prothrombin and calcium in the presence and absence of TSP1. Generation of FXa and of thrombin were assessed using chromogenic substrates. SMAD pathway phosphorylation was detected via Western-blot analysis. Pre-incubation of fibroblasts with FVII led to its autoactivation by cell-surface expressed TF, which in turn in the presence of FX, FVa, prothrombin and calcium led to FXa (9.7 ± 0.8 nM) and thrombin (7.9 ± 0.04 U/mL × 10-3) generation. Addition of TSP1 significantly enhanced thrombin (23.3 ± 0.7 U/mL × 10-3) but not FXa (8.5 ± 0.6 nM) generation. FXa and thrombin generation leads to upregulation of CTGF/CCN2. TSP1 alone upregulated CTGF/CCN2, an effect mediated via activation of transforming growth factor beta (TGFβ) as showed by phosphorylation of the SMAD pathway an event blunted by using a TGFβ receptor I inhibitor (TGFβRI). FXa-and thrombin-induced upregulation of CTGF/CCN2 was not blocked by TGFβRI.
Journal of Cellular Physiology, 2007
Objective: To evaluate the effect of a thrombospondin 1 (TSP1)-derived peptide on inflammation an... more Objective: To evaluate the effect of a thrombospondin 1 (TSP1)-derived peptide on inflammation and angiogenesis in an animal model of erosive arthritis and to assess the relationship between TSP1 and connective tissue growth factor (CTGF) in the pathophysiology of rheumatoid arthritis. Methods: Erosive arthritis in Lewis rats was induced by peptidoglycan-polysaccharide (PG-PS). Animals were divided into four groups: (1) negative control and groups receiving, (2) no treatment, (3) treatment with a TSP1-derived peptide, and (4) treatment with a scrambled peptide. Samples obtained from ankle joint, spleen and liver were studied using histology, histomorphometry, immunohistochemistry and RT-PCR. Results: Histological data indicated that the TSP1-derived peptide treatment decreased neovascularization, leukocyte infiltration and thickening of the synovial lining of the joint, and reduced granuloma formation in the spleen and liver when compared to control groups. Higher concentrations of CTGF and TSP1 proteins were observed in the affected areas of animals which did not receive TSP1-derived peptide treatment. Also, immunofluorescence and RT-PCR analyses showed an increase in CTGF protein expression and regulation, respectively, in the tissues of untreated animals when compared to the TSP1-derived peptide treated animals. By immunofluorescence, TSP1 expression was decreased in the TSP1-derived peptide treated animals. Moreover, macrophage/monocyte-specific staining revealed a decrease in cell infiltration in the articular tissue of the TSP1-derived peptide treated animals. Conclusion: Both inflammation and angiogenesis were decreased after TSP1-derived peptide treatment indicating a potential pathway by which TSP1 interaction with neutrophils induces CTGF in RA affected tissues.
Journal of Cellular Physiology, 2006
Connective tissue growth factor (CTGF/CCN2) is a cysteine‐rich, extracellular matrix (ECM) protei... more Connective tissue growth factor (CTGF/CCN2) is a cysteine‐rich, extracellular matrix (ECM) protein that acts as an anabolic growth factor to regulate osteoblast differentiation and function. Recent studies have identified CTGF as a downstream effector of transforming growth factor‐β1 (TGF‐β1) for certain functions in specific cell types. In this study, we examined the role of CTGF as a downstream mediator of TGF‐β1‐induced ECM production and cell growth in osteoblasts. Using primary cultures, we demonstrated that TGF‐β1 is a potent inducer of CTGF expression in osteoblasts, and that this induction occurred at all stages of osteoblast differentiation from the proliferative through mineralization stages. TGF‐β1 treatment of osteoblasts increased the expression and synthesis of the ECM components, collagen and fibronectin. When CTGF‐specific siRNA was used to prevent TGF‐β1 induction of CTGF expression, it also inhibited collagen and fibronectin production, thereby demonstrating the re...
Journal of Cellular Physiology, 2006
Mesenchymal cell (MC) condensation or the aggregation of MCs precedes chondrocyte differentiation... more Mesenchymal cell (MC) condensation or the aggregation of MCs precedes chondrocyte differentiation and is required for subsequent cartilage formation during endochondral ossification. In this study, we used micromass cultures of C3H10T1/2 cells as an in vitro model system for studying MC condensation and the events important for this process. Transforming growth factor β1 (TGF‐β1) served as the initiator of MC condensation in our model system and we were interested in determining whether CTGF functions as a downstream mediator of TGF‐β1. CTGF is a matricellular protein that has been found to be expressed in MC condensations and in the perichondrium. Micromass cultures of C3H10T1/2 cells condensed under TGF‐β1 stimulation concomitant with dramatic up‐regulation of CTGF mRNA and protein levels. CTGF silencing by either CTGF siRNA or CTGF antisense oligonucleotide approaches showed that TGF‐β1‐induced condensation was CTGF dependent. Furthermore, silencing of CTGF expression resulted in...
Journal of Cellular Physiology, 2011
Current osteoinductive protein therapy utilizes bolus administration of large doses of bone morph... more Current osteoinductive protein therapy utilizes bolus administration of large doses of bone morphogenetic proteins (BMPs), which is costly, and may not replicate normal bone healing. The limited in vivo biologic activity of BMPs requires the investigation of growth factors that may enhance this activity. In this study, we utilized the C3H10T1/2 murine mesenchymal stem cell line to test the hypotheses that osteoactivin (OA) has comparable osteoinductive effects to bone morphogenetic protein‐2 (BMP‐2), and that sustained administration of either growth factor would result in increased osteoblastic differentiation as compared to bolus administration. Sustained release biodegradable hydrogels were designed, and C3H10T1/2 cells were grown on hydrogels loaded with BMP‐2 or OA. Controls were grown on unloaded hydrogels, and positive controls were exposed to bolus growth factor administration. Cells were harvested at several time points to assess osteoblastic differentiation. Alkaline phosp...