Interleukin‐1α, ‐6, and ‐8 decrease Cdc42 activity resulting in loss of articular chondrocyte phenotype

Small GTPase proteins mediate changes in cellular morphology and other cellular functions. The aim of this study was to examine signaling of the small GTPase Cdc42 by stimulating chondrocytes grown in monolayer with long‐ (96 h) or short‐ (2 and 30 min) term exposure to interleukin‐1α (IL‐1α), IL‐6, or IL‐8. Quantitative PCR was used to determine changes in collagen type IIB (COL2A1), aggrecan (AGG), and matrix metalloproteinase‐13 (MMP‐13) gene expression after prolonged cytokine exposure. Effects of short‐term treatment with IL‐α, IL‐6, or IL‐8 on endogenous GTP‐bound Cdc42 levels were assessed using an affinity assay, and on actin filament organization using confocal microscopy. Cytokine treatments significantly decreased COL2A1 and AGG expression and increased MMP‐13 expression. Short exposure to IL‐1α, IL‐6, or IL‐8 decreased endogenous GTP‐Cdc42 and increased stress fibers, which were reversed with cytochalasin D treatment. These results show that IL‐mediated Cdc42 signaling modifies chondrocyte phenotype and morphology. This may lend insight into the altered chondrocyte phenotype in catabolic conditions such as osteoarthritis. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:246–251, 2012     

The quantitative and functional relation between insulin‐like growth factor‐I (IGF) and IGF‐binding proteins during human osteoarthritis

A previous hypothesis stated that during osteoarthritis (OA) increased insulin‐like growth factor (IGF) binding proteins (IGFBPs) sequester IGFs and limit their access to the cell. The objective of this article was to test this by: (1) quantifying IGF and IGFBP‐3 as well as their ratios in human OA cartilages, and (2) measuring the metabolic responses of diseased cartilage to IGF‐I and its IGFBP‐insensitive analogs. Knee or hip OA cartilages were staged for OA by histology. Cartilage slices were either extracted for assays of IGF proteins, or maintained intact as organ cultures. Proteoglycan (PG) metabolism ± IGFs was measured by use of the ³⁵S‐sulfate precursor. IGFBP‐3 (ng/mg protein) was weakly correlated with OA score by regression analysis (R² = 0.122; p = 0.040; n = 35). IGF‐I (ng/mg protein) was constant across all OA groups (ANOVA; p = .428, n = 18) and the IGF‐I/IGFBP‐3 ratios were > 1 in most samples. All OA cartilages responded to hrIGF‐I by increasing PG synthesis [average 2.29‐fold ± 0.55 (±SD) at saturation, n = 12] irrespective of OA score. The des (1–3) IGF‐I analog (which lacks the three N‐terminal amino acids) had similar maximal effects (average 2.23‐fold stimulation ± 0.71, n = 10), but it was more effective in two out of three samples at suboptimal doses. The effect of hrIGF‐I, des (1–3) IGF‐I, or the B‐chain analog on degradation was minimal. In summary, catabolism was insensitive to IGF‐I, and this was probably not due to IGFBPs. By contrast, IGF‐I exerted a robust stimulation of anabolism at sufficiently high doses, even though IGFBPs could tone down the ligand effect at low doses. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:465–474, 2008     

Association between male Infertility and seminal plasma levels of growth hormone and insulin‐like growth factor‐1

Growth hormone (GH) and insulin‐like growth factor 1 (IGF‐1) have been proposed to play a pivotal role in male infertility due to their anabolic effects. The aim of this study was to investigate possible associations between seminal plasma levels of GH and IGF‐1 and sperm parameters. Fifty men participated in this study. Semen analysis was performed, while cell‐free seminal plasma was collected following sperm centrifugation. Seminal plasma concentrations of IGF‐1 and GH were determined by enzyme‐linked immunosorbent assay (ELISA). Due to the presence of asthenozoospermia in all participants who presented with abnormal sperm parameters, the participants were further subdivided into normal (group A), asthenozoospermic (group B) and asthenozoospermic plus at least one additional abnormal parameter (group C). A marginally nonsignificant statistical difference (p = 0.063) was revealed between the GH levels corresponding to the asthenozoospermic and the normal group with the latter presenting with higher GH levels. A statistically significant positive correlation (p < 0.05) was noted between levels of GH and IGF‐1 in group C. The above relationship has also been observed in men with low sperm concentration, vitality, volume and abnormal morphology. These novel findings require further investigation in order for the biological significance of those associations to be clarified.     

Autologous protein solution inhibits MMP‐13 production by IL‐1β and TNFα‐stimulated human articular chondrocytes

Catabolic inflammatory cytokines are prevalent in osteoarthritis (OA). The purpose of this study was to evaluate an autologous protein solution (APS) as a potential chondroprotective agent for OA therapy. APS was prepared from platelet‐rich plasma (PRP). The APS solution contained both anabolic (bFGF, TGF‐β1, TGF‐β2, EGF, IGF‐1, PDGF‐AB, PDGF‐BB, and VEGF) and anti‐inflammatory (IL‐1ra, sTNF‐RI, sTNF‐RII, IL‐4, IL‐10, IL‐13, and IFNγ) cytokines but low concentrations of catabolic cytokines (IL‐1α, IL‐1β, TNFα, IL‐6, IL‐8, IL‐17, and IL‐18). Human articular chondrocytes were pre‐incubated with the antagonists IL‐1ra, sTNF‐RI, or APS prior to the addition of recombinant human IL‐1β or TNFα. Following exposure to inflammatory cytokines, the levels of MMP‐13 in the culture medium were evaluated by ELISA. MMP‐13 production stimulated in chondrocytes by IL‐1β or TNFα was reduced by rhIL‐1ra and sTNF‐RI to near basal levels. APS was also capable of inhibiting the production of MMP‐13 induced by both IL‐1β and TNFα. The combination of anabolic and anti‐inflammatory cytokines in the APS created from PRP may render this formulation to be a potential candidate for the treatment of inflammation in patients at early stages of OA. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1320–1326, 2011     

Interleukin‐1β stimulates stromal‐derived factor‐1α expression in human subacromial bursa

Chemokines produced by synoviocytes of the subacromial bursa are up‐regulated in subacromial bursitis and rotator cuff disease. We hypothesized that SDF‐1α production in bursal synoviocytes may be induced by local cytokines such as interleukin IL‐1β and IL‐6. Subacromial bursa specimens were obtained from patients undergoing shoulder surgery. Bursal specimens were stained with anti‐human antibodies to IL‐1, IL‐6, and SDF‐1α by immunohistochemistry and compared to normal and rheumatoid controls. Bursal cells were also isolated from specimens and cultured. Early passaged cells were then treated with cytokines (IL‐1β and IL‐6) and SDF‐1α expression was measured by ELISA and RT‐PCR. SDF‐1α, IL‐1β, and IL‐6 were expressed at high levels in bursitis specimens from human subacromial bursa compared to normal controls. In cultured bursal synoviocytes, there was a dose‐dependent increase in SDF‐1α production in the supernatants of cells treated with IL‐1β. SDF‐1α mRNA expression was also increased in bursal cells treated with IL‐1β. IL‐6 caused a minimal but not statistically significant increase in SDF‐1α expression. SDF‐1α, IL‐1β, and IL‐6 are expressed in the inflamed human subacromial bursal tissues in patients with subacromial bursitis. In cultured bursal synoviocytes, SDF‐1α gene expression and protein production are stimulated by IL‐1β. IL‐1β produced by bursal syvoviocytes and inflammatory cells in the human subacromial bursa is an important signal in the inflammatory response that occurs in subacromial bursitis and rotator cuff disease. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:1695–1699, 2011     

Modulation of Na+‐H+ exchange isoforms NHE1 and NHE3 by insulin‐like growth factor‐1 in isolated bovine articular chondrocytes

Incubation with serum modulates the transporters that regulate intracellular pH (pH_i) in articular chondrocytes, upregulating acid extrusion by Na⁺‐H⁺ exchange (NHE). There is stimulation of NHE1, together with induction of NHE3 activity. These isoforms exhibit differential responses to components of mechanical load experienced by chondrocytes during joint loading. The identity of the component(s) of serum responsible is unknown. A possibility, however, is insulin‐like growth factor‐1 (IGF‐1), present in normal cartilage and found at enhanced levels in osteoarthritic tissue. In the present study, the effects of IGF‐1 on pH_i regulation have been characterized using fluorescence measurements of bovine articular chondrocytes, and the sensitivity of pH_i regulation to hyperosmotic shock and raised hydrostatic pressure determined. For cells isolated in the absence of IGF‐1, pH_i recovery following acidification was predominantly mediated by NHE1. Recovery was enhanced when cells were incubated for 18 h with 20 ng mL^?1 IGF; this effect represented increased acid extrusion by NHE1, supplemented by NHE3 activity. NHE3 activity was not detected in IGF‐1‐treated cells that had been incubated with the protein synthesis inhibitor cycloheximide, although NHE1 activity was unaffected. In the absence of IGF‐1, suspension in hyperosmotic solutions or raised hydrostatic pressure enhanced pH_i recovery of acidified cells. This response was missing in cells incubated with IGF‐1. Unresponsiveness to hyperosmotic shock represented inhibition of NHE3 activity, and was prevented using the protein kinase A inhibitor KT5720. For raised hydrostatic pressure, a decrease in NHE1 activity was responsible, and was prevented by the protein kinase C inhibitor chelerythrine. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:1428–1433, 2008     

Decrease of the insulin‐like growth factor‐1 bioavailability in spontaneously hypertensive rats with erectile dysfunction

We investigated the role of insulin‐like growth factor‐1 (IGF‐1) in spontaneously hypertensive rats with erectile dysfunction. Firstly, we evaluated intracavernous pressure. The bioavailability of IGF‐1 at both mRNA and protein levels were measured by quantitative real‐time PCR and Western blot respectively. Then, cavernous cyclic guanosine monophosphate concentrations were detected by enzyme‐linked immunosorbent assay. The cavernosal pressure was significantly decreased in the hypertensive and the propranolol treatment groups compared to the normal control group (P < 0.01). Cavernous IGF‐1 bioavailability and the concentrations of cavernous cyclic guanosine monophosphate were both significantly decreased in the hypertensive and the propranolol treatment groups compared to the normal control group (P < 0.01). This study suggests that an obvious decrease in cavernous IGF‐1 levels might play an important role in spontaneously hypertensive rats with erectile dysfunction.     

Biphasic effects of interleukin‐1β on osteoblast differentiation in vitro

A rat calvarial cell model of osteoblast differentiation using the formation of bone nodules in vitro as an endpoint was used to assess the effects of IL‐1β on osteoblast differentiation. Short‐term treatment (2 days) with IL‐1β early in culture resulted in increased nodule number and size as well as calcium content in contrast to long‐term treatment (6 days) in cultures assessed at 10–12 days. This increase in bone formation was blocked by IL‐1 receptor antagonists. Short‐term treatment increased COX‐2, prostaglandin (PGE₂), and iNOS production. Exogenous PGE₂ with IL‐1β enhanced this effect. COX‐2 inhibitors, indomethacin and N‐39, blocked 50% of nodule formation. NO donor did not modify effects of IL‐1β, but iNOS inhibitor (1400W) partially blocked the effects. However, PGE₂ and NO donors could not rescue the decreased nodule number resulting from long‐term IL‐1β treatment. The results of this study suggest a biphasic effect of IL‐1β on bone nodule formation activated by IL‐1β binding with IL‐1 receptors, and the anabolic effect of early short‐term treatment with IL‐1β is likely mediated by PGE without ruling out nitric oxide. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:958–964, 2010     

Concerted actions of insulin‐like growth factor 1, testosterone,and estradiol on peripubertal bone growth: A 7‐year longitudinal study

A better understanding of how bone growth is regulated during peripuberty is important for optimizing the attainment of peak bone mass and for the prevention of osteoporosis in later life. In this report we used hierarchical models to evaluate the associations of insulin‐like growth factor 1 (IGF‐1), estradiol (E₂), and testosterone (T) with peripubertal bone growth in a 7‐year longitudinal study. Two‐hundred and fifty‐eight healthy girls were assessed at baseline (mean age 11.2 years) and at 1, 2, 3.5, and 7 years. Serum concentrations of IGF‐1, E₂, and T were determined. Musculoskeletal properties in the left lower leg were measured using peripheral quantitative computed tomography (pQCT). Serum levels of IGF‐1, E₂, and T increased dramatically before menarche, whereas they decreased, plateaued, or increased at a lower rate, respectively, after menarche. IGF‐1 level was positively associated with periosteal circumference (PC) and total bone mineral content (tBMC) throughout peripuberty but not after adjustment for muscle cross‐sectional area (mCSA). On the other hand, IGF‐1 was associated with tibial length (TL) independently of mCSA before menarche. T was positively associated with TL, PC, tBMC, and cortical volumetric bone mineral density, independent of mCSA, before menarche but not after. E₂ was associated with TL positively before menarche but negatively after menarche. These findings suggest that during puberty, circulating IGF‐1 promotes bone periosteal apposition and mass accrual indirectly, probably through stimulating muscle growth, whereas the effects of sex steroids on bone growth differ before and after menarche, presenting a biphasic pattern. Hence the concerted actions of these hormones are essential for optimal bone development in peripuberty. © 2011 American Society for Bone and Mineral Research     

Selective tyrosine kinase inhibition of insulin‐like growth factor‐1 receptor inhibits human and mouse breast cancer–induced bone cell activity,bone remodeling,and osteolysis

Insulin‐like growth factor 1 (IGF‐1) plays an important role in both bone metabolism and breast cancer. In this study, we investigated the effects of the novel IGF‐1 receptor tyrosine kinase inhibitor cis‐3‐[3‐(4‐methyl‐piperazin‐l‐yl)‐cyclobutyl]‐1‐(2‐phenyl‐quinolin‐7‐yl)‐imidazo[1,5‐a]pyrazin‐8‐ylamine (PQIP) on osteolytic bone disease associated with breast cancer. Human MDA‐MB‐231 and mouse 4T1 breast cancer cells enhanced osteoclast formation in receptor activator of NF‐κB ligand (RANKL) and macrophage colony‐stimulating factor (M‐CSF) stimulated bone marrow cultures, and these effects were significantly inhibited by PQIP. Functional studies in osteoclasts showed that PQIP inhibited both IGF‐1 and conditioned medium–induced osteoclast formation by preventing phosphatidylinositol 3‐kinase (PI3K)/protein kinase B (Akt) activation without interfering with RANKL or M‐CSF signaling. Treatment of osteoblasts with PQIP significantly inhibited the increase in RANKL/osteoprotegerin (OPG) ratio by IGF‐1 and conditioned medium and totally prevented conditioned medium–induced osteoclast formation in osteoblast–bone marrow (BM) cell cocultures, thereby suggesting an inhibitory effect on osteoblast–osteoclast coupling. PQIP also inhibited IGF‐1–induced osteoblast differentiation, spreading, migration, and bone nodule formation. Treatment with PQIP significantly reduced MDA‐MB‐231 conditioned medium–induced osteolytic bone loss in a mouse calvarial organ culture system ex vivo and in adult mice in vivo. Moreover, once daily oral administration of PQIP significantly decreased trabecular bone loss and reduced the size of osteolytic bone lesions following 4T1 intratibial injection in mice. Quantitative histomorphometry showed a significant reduction in bone resorption and formation indices, indicative of a reduced rate of cancer‐associated bone turnover. We conclude that inhibition of IGF‐1 receptor tyrosine kinase activity by PQIP suppresses breast cancer–induced bone turnover and osteolysis. Therefore, PQIP, and its novel derivatives that are currently in advanced clinical development for the treatment of a number of solid tumors, may be of value in the treatment of osteolytic bone disease associated with breast cancer. © 2013 American Society for Bone and Mineral Research.     

Mesenchymal stem cells and insulin‐like growth factor‐I gene‐enhanced mesenchymal stem cells improve structural aspects of healing in equine flexor digitorum superficialis tendons

Tendinitis remains a catastrophic injury among athletes. Mesenchymal stem cells (MSCs) have recently been investigated for use in the treatment of tendinitis. Previous work has demonstrated the value of insulin‐like growth factor‐I (IGF‐I) to stimulate cellular proliferation and tendon fiber deposition in the core lesion of tendinitis. This study examined the effects of MSCs, as well as IGF‐I gene‐enhanced MSCs (AdIGF‐MSCs) on tendon healing in vivo. Collagenase‐induced bilateral tendinitis lesions were created in equine flexor digitorum superficialis tendons (SDFT). Tendons were treated with 10 × 10⁶ MSCs or 10 × 10⁶ AdIGF‐MSCs. Control limbs were injected with 1 mL of phosphate‐buffered saline (PBS). Ultrasound examinations were performed at t = 0, 2, 4, 6, and 8 weeks. Horses were euthanized at 8 weeks and SDFTs were mechanically tested to failure and evaluated for biochemical composition and histologic characteristics. Expression of collagen types I and III, IGF‐I, cartilage oligomeric matrix protein (COMP), matrix metalloproteinase‐3 (MMP‐3), matrix metalloproteinase‐13 (MMP‐13), and aggrecanase‐1 (ADAMTS‐4) were similar in MSC and control tendons. Both MSC and AdIGF‐MSC injection resulted in significantly improved tendon histological scores. These findings indicate a benefit to the use of MSCs and AdIGF‐MSCs for the treatment of tendinitis. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:1392–1398, 2009     

The overexpression of SIRT1 inhibited osteoarthritic gene expression changes induced by interleukin‐1β in human chondrocytes

In this study, we examined the effects of overexpression of SIRT1 on IL‐1β‐induced gene expression changes in human chondrocytes to explore a protective role of SIRT1 in human chondrocytes. SIRT1 was overexpressed in human chondrocytes by expression plasmid under stimulation with IL‐1β. SIRT1 was also inhibited by siRNA under stimulation with IL‐1β. Gene expression changes were examined by real‐time PCR. The interaction of SIRT1 and p65 (NF‐κB) were examined by Western blotting. SIRT1, MMP‐13, and ADAMTS‐5 expressions in human cartilage were examined by immunohistochemistry. IL‐1β stimulation significantly up‐regulated MMP‐1, 2, 9, and 13 and ADAMTS‐5. Overexpression of SIRT1 significantly inhibited the up‐regulation of those genes caused by IL‐1β while the inhibition of SIRT1 further increased them. In addition, the overexpression of SIRT1 markedly reduced the IL‐1β‐induced acetylation of p65. SIRT1 expression was clearly detected in the non‐OA cartilage while MMP‐13 and ADAMTS‐5 were undetectable. In contrast, in the OA cartilage, SIRT1 expression was decreased while MMP‐13 and ADAMTS‐5 were increased. Our observations suggested that SIRT1 can play a protective role by suppressing IL‐1β‐induced expressions of cartilage‐degrading enzymes partially through the modulation of the NF‐κB pathway. SIRT1 overexpression might be a new therapeutic approach for OA. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 531–537, 2013     

Additive effects of hyperbaric oxygen and platelet‐derived growth factor‐BB in chondrocyte transplantation via up‐regulation expression of platelet‐derived growth factor‐β receptor

The present study investigated the effects of hyperbaric oxygen (HBO) and platelet‐derived growth factor‐BB (PDGF‐BB) in chondrocyte transplantation. In vitro, chondrocytes were treated with HBO, PDGF‐BB, and HBO combined with PDGF‐BB (H+P). Cell growth was analyzed using cell counting, MTT assay, and FACS analysis. mRNA expression of the PDGF‐α receptor (PDGFR‐α) and β receptor (PDGFR‐β) was detected by RT‐PCR. Protein expression of PDGFR‐β was detected by Western blotting. In vivo, chondrocytes and PDGF‐BB were suspended in alginate as a transplantation system. Cartilage defects were grafted with this system and with or without HBO treatment. Released PDGF‐BB concentration was quantified by ELISA. After 8 weeks, animals were sacrificed and the repaired tissues were examined. In vitro data suggested that each treatment increased cell growth via the up‐regulated mRNA expression of PDGFR‐α and increased cell accumulation in the S‐phase. The H+P treatment was more additive in cell growth and in mRNA and protein expression of PDGFR‐β than HBO or PDGF‐BB. In vivo results suggested that PDGF‐BB delivery lasted for more than 5 weeks. Scoring results showed that each treatment significantly increased the cartilage repair. Safranin‐O and type II collagen staining confirmed the hyaline‐like cartilage regeneration in the repaired tissues. In situ up‐regulation of PDGFR‐β expression partially explains the additive effect of H+P treatment in cartilage repair. Accordingly, H+P offers a potential treatment method for cartilage repair. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:1439–1446, 2009     

Insulin‐like growth factor‐I improves chondrogenesis of predifferentiated human umbilical cord mesenchymal stromal cells

Human umbilical cord mesenchymal stromal cells (hUCMSCs) are an attractive cell source for tissue engineering with numerous advantages over other adult stem cell sources, such as great expansion ability in vitro and extensive availability. The objective of this 6‐week study was to test the hypothesis that switching from chondrogenic transforming growth factor‐beta3 (TGF‐β3) to anabolic insulin‐like growth factor‐I (IGF‐I) at the 3‐week time point would produce more cartilage‐like matrix than TGF‐β3 alone. hUCMSCs were seeded into polyglycolic acid (PGA) scaffolds and then cultured in chondrogenic medium containing TGF‐β3 for 3 weeks. The TGF‐β3‐treated hUCMSCs were then exposed for 3 more weeks to one of four different conditions: (1) continued in chondrogenic medium, (2) control medium (no TGF‐β3), (3) control medium with 10 ng/ml IGF‐I, or (4) control medium with 100 ng/ml IGF‐I. Compared to continuing with TGF‐β3, switching to IGF‐I increased collagen production, and furthermore increased both collagen type II gene expression and immunostaining. In conclusion, the shift from TGF‐β3 to IGF‐I at week 3 resulted in a significant increase of cartilage‐like extracellular matrix, confirming our hypothesis. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 1109–1115, 2009