Friday, December 5, 2008

Redox Reactions and Growth Plates

Initiation of endochondral calcification is related to changes in the redox state of hypertrophic chondrocytes.

"[We] relate the concentrations of NADH and NAD+ to stages of chondrocyte maturation. A dramatic increase was found in the relative concentration of reduced pyridine nucleotides in the hypertrophic zone. On either side of this zone, in proliferating and calcifying cartilage, there was a decrease in NADH fluorescence, and the NADH/NAD+ ratio was depressed. The finding that NADH accumulated in the tissue zone associated with the earliest deposition of bone mineral supports the hypothesis that a change in the redox state initiates tissue mineralization."

Wednesday, December 3, 2008

Mitochondria and chondrocytes

LSJL upregulates these mitochondrial related genes:
Hey2(mitochondrial inner membrane)

LSJL downregulates:
Mfn1(related to mitochondrial fusion)
Cpt1b(related to mitochondrial outer membrane)
Ndufb8(Mitochondrial inner membrane)
Slc25a37(Mitochondrial inner membrane)
Mrpl3(mitochondrial large ribosomal subunit)
Opa1(mitochondrial fission and fusion)
Cul7(mitochondria inner membrane)
Mrpl38(mitochondrial ribosome)
Mrpl11(mitochondrial ribosome)
Prodh2(mitochondrial inner membrane)
Uqcrq(mitochondrial electron transport chain)
Immt(mitochondrial inner membrane)
Cox6a1(mitochondrial membrane)
Slc25a30(mitochondrial inner membrane)
Rhot1(mitochondrial transport along microtubule)
Ucp2(mitochondrial transport)
Mrps14(mitochondrial small ribosomal subunit)
MTERF(involved in terminating mitochondrial transcription)
Fxc1(protein mitochondrial targeting)
Gimap3(mitochondrial outer membrane)
Mrpl30(mitochondrial ribosome)
Gls(mitochondrial inner membrane)
Timm44(mitochondrial inner membrane presequence translocase complex)

Perhaps mitochondrial damage relates to the conditioning effect and it takes time for mitochondria to repair allowing for results with LSJL again.

Monosodium iodoacetate induces apoptosis via the mitochondrial pathway involving ROS production and caspase activation in rat chondrocytes in vitro.

"Monosodium iodoacetate (MIA) is an inhibitor of glyceraldehyde-3-phosphate dehydrogenase activity, and causes dose-dependent cartilage degradation resembling the pathological changes of human osteoarthritis (OA). In this study, we assessed the apoptosis induced by MIA and clarified the underlying mechanisms using the primary rat chondrocytes. The apoptosis of primary rat chondrocytes was analyzed by flow cytometry. The levels of mitochondrial membrane potential (ΔΨm) were evaluated using fluorescence spectrophotometer. The production of reactive oxygen species (ROS) was determined by fluorescence spectrophotometer. Apoptosis-related protein cytochrome c and procaspase-3 expressions were examined by Western blotting. We found that MIA treatment induces apoptosis in chondrocytes, as confirmed by increases in the percent of apoptotic cells, up-regulation of cytochrome c and caspase-3 protein levels. Treatment with MIA increases ROS production and decreases the levels of ΔΨm. The antioxidant, N-acetylcysteine (NAC), significantly prevented the production of ROS, the reduction of ΔΨm, the release of cytochrome c and the activation of caspase-3. NAC completely protected the cells from MIA-induced apoptosis. The mechanisms of MIA-induced apoptosis are primarily via ROS production and mitochondria-mediated caspase-3 activation in primary rat chondrocytes. "

"ROS are also known to activate anti-apoptotic pathways such as NF-κB and the heat shock proteins Hsp70 and Hsp27."

Cytoskeletal dissolution blocks oxidant release and cell death in injured cartilage.

"The linkage of mitochondria to the cytoskeleton suggests that they might release oxidants in response to mechanical strain, an effect that disrupting the cytoskeleton would prevent. To test this we investigated the effects of agents that promote the dissolution of microfilaments (cytochalasin B) or microtubules (nocodazole) on oxidant production and chondrocyte death following impact injury. Osteochondral explants treated with cytochalasin B or nocodazole for 4 h were impacted (7 J/cm(2)) and stained for oxidant production directly after impact and for cell viability 24 h after impact. Surfaces within and outside impact sites were then imaged by confocal microscopy. Both agents significantly reduced impact-induced oxidant release; however, cytochalasin B was more effective than nocodazole (>60% reduction vs. 40% reduction, respectively). Both agents also prevented impact induced cell death. Dissolution of the cytoskeleton by both drugs was confirmed by phalloidin staining and confocal microscopy. Chondrocyte mortality from impact injury depends substantially on mitochondrial-cytoskeletal linkage, suggesting new approaches to stem mechanically induced cartilage degeneration."

"Microfilaments or filamentous actin, f-actin, primarily resist compressive forces applied to the cell. Furthermore, filamentous actin has been shown to be highly concentrated around the cell periphery, thereby maintaining the chondrocyte's shape and structure, and facilitating attachment of the chondrocyte to the surrounding extracellular matrix. F-actin assembly is a highly dynamic process. A readily available pool of globular actin is rapidly assembled into structural f-actin in response to changes in the local mechanical environment. Microtubules have been shown to provide secondary structural support to the f-actin cytoskeleton which is significantly stiffer and less brittle under loading with an intact microtubular lattice."

"F-actin also serves as an important scaffold for mitochondrial attachment."

"mitochondria [binds] to the f-actin cytoskeleton facilitating optimal mitochondrial distribution within the cell and also allowing local intracellular mitochondrial movement. This physical attachment of the mitochondria to the cytoskeleton potentially subjects the mitochondria to external loads encountered by the chondrocyte. Tissue level strains are transduced into cellular level deformations, including deformation of the intracellular mitochondria, primarily via the actin cytoskeleton with support from the microtubules."

The role of mitochondria in osteoarthritis.

"Mitochondrial DNA (mtDNA) mutations and oxidative stresses are known to contribute to aging-related changes. Articular cartilage chondrocytes survive and maintain tissue integrity in an avascular, low-oxygen environment. Mitochondrial dysfunction [is present] in human OA chondrocytes, and analyses of mitochondrial electron transport chain activity in these cells show decreased activity of Complexes I, II and III compared to normal chondrocytes. This mitochondrial dysfunction may affect several pathways that have been implicated in cartilage degradation, including oxidative stress, defective chondrocyte biosynthesis and growth responses, increased cytokine-induced chondrocyte inflammation and matrix catabolism, cartilage matrix calcification, and increased chondrocyte apoptosis. Mitochondrial dysfunction in OA chondrocytes may derive from somatic mutations in the mtDNA or from the direct effects of proinflammatory mediators such as cytokines, prostaglandins, reactive oxygen species and nitric oxide."

"The mitochondrion, found in most eukaryotic cells, is a membrane-enclosed organelle that converts nutritional molecules into ATP via oxidative phosphorylation"

"NADH dehydrogenase (Complex I), succinate dehydrogenase (Complex II), cytochrome c reductase (Complex III) and cytochrome c oxidase (Complex IV) are protein complexes in the inner membrane that perform the transfer and incremental release of energy from the donated electrons, which is used to pump protons (H+) into the intermembrane space. The protons then pass through ATP synthase (sometimes referred to as Complex V) to provide energy for the creation of ATP. While this process is efficient, a small number of electrons may only partially reduce oxygen to form the toxic free radical superoxide, which can cause oxidative damage to the mitochondria that contributes to their impaired function."

"the number of mitochondria is higher in chondrocytes from superficial than deep zone "

"mitochondrial oxidative phosphorylation may account for as much as 25% of the ATP production in cartilage."

"Chondrocyte mitochondria are specialized for the transport of calcium, highlighting their importance in ECM calcification.2 Mineralization has been demonstrated both in matrix vesicles and within the mitochondria themselves: indeed, calcium and phosphorus are present in single mitochondrial granules in chondrocytes from the growth plate and in certain extracellular particles distinct from matrix vesicles. When mitochondrial respiration is directly suppressed, matrix-vesicle-mediated mineralization of chondrocytes is promoted."

"Phosphate is required for terminal differentiation of hypertrophic chondrocytes during postnatal growth plate maturation. In vitro models of chondrocyte differentiation demonstrate that 7 mM phosphate, a concentration analogous to that of the late gestational fetus, activates the mitochondrial apoptotic pathway in hypertrophic chondrocytes. This raises the question as to whether extracellular phosphate modulates chondrocyte differentiation and apoptosis during embryonic endochondral bone formation. To address this question, we performed investigations in the mouse metatarsal culture model that recapitulates in vivo bone development. Metatarsals were cultured for 4, 8, and 12 days with 1.25 and 7 mM phosphate. Metatarsals cultured with 7 mM phosphate showed a decrease in proliferation compared to those cultured in 1.25 mM phosphate. This decrease in proliferation was accompanied by an early enhancement in hypertrophic chondrocyte differentiation, associated with an increase in FGF18 expression. By 8 days in culture, an increase caspase-9 activation and apoptosis of hypertrophic chondrocytes was observed in the metatarsals cultured in 7 mM phosphate. Immunohistochemical analyses of embryonic bones demonstrated activation of caspase-9 in hypertrophic chondrocytes, associated with vascular invasion. Thus, these investigations demonstrate that phosphate promotes chondrocyte differentiation during embryonic development and implicate a physiological role for phosphate activation of the mitochondrial apoptotic pathway during embryonic endochondral bone formation."

"Metatarsals cultured under control conditions (1.25mM phosphate) increased in length at all time points examined , demonstrating a 120 ± 6.7 % increase in length over 12 days. While 7mM phosphate attenuated growth at 4, 8 and 12 days in culture, there was still a significant increase in metatarsal length over 12 days (70 ± 2.6 % increase, "  So in this case inhibiting hypertrophy with less phosphate resulted in the most length increase.  Which is interesting considering that hypertrophy contributes most to length increase.

The higher concentration of phosphate decreased BMP-2 levels.

"7 mM phosphate causes apoptosis of cultured hypertrophic chondrocytes by activating the mitochondrial apoptotic pathway"

Reducing phosphate levels may be a way to grow taller.

Pretreatment of chondrocytes with HA resulted in enhanced mitochondrial function and reduced apoptosis.

"in the mitochondrial fraction, SIRT1 inhibition by siRNA for SIRT1 increased the amount of Bax but reduced the amount of Bcl-2, while resveratrol reduced the amount of Bax but increased the amount of Bcl-2."

Construction of tissue-engineered cartilage with BMP7 gene-transfected chondrocytes.

"the expression of bone morphogenetic protein-7 (BMP7) was investigated in tissue-engineered cartilage constructed using chondrocytes transfected with the BMP7 gene and that constructed using non-transfected chondrocytes after 7, 14, 21 and 28 days. The volume of the BMP7 gene-transfected tissue-engineered cartilage culture after 5 - 7 days was 9 x 9 x 2 mm, while that of the non-transfected tissue-engineered cartilage culture was 8 x 8 x 2 mm. Histomorphological analysis showed that both cultures comprised cartilaginous tissue. Both BMP7 mRNA and BMP7 protein were expressed in BMP7 gene-transfected cartilage culture grown in vitro for 7, 14, 21 or 28 days. The chondrocytes in the BMP7 gene-transfected cartilage culture were active, with increased mitochondria, Golgi bodies and rough endoplasmic reticulum compared with non-transfected cartilage. These results provide an ideal foundation for the study of BMP7 gene-transfected tissue-engineered cartilage transplantation in the repair of cartilaginous defects."

"In a tibia organ culture system, pharmacological inhibition of p38 blocks the anabolic effects of CNP. CNP stimulates endochondral bone growth largely through expansion of the hypertrophic zone of the growth plate, while delaying mineralization. Both effects are reversed by p38 inhibition. Hypertrophic chondrocytes are the main targets of CNP signaling in the growth plate, since many more genes were regulated by CNP in this zone than in the others. While CNP receptors are expressed at similar levels in all three zones, cGMP-dependent kinases I and II, important transducers of CNP signaling, are expressed at much higher levels in hypertrophic cells than in other areas of the tibia, providing a potential explanation for the spatial distribution of CNP effects. CNP induces the expression of NPR3, a decoy receptor for natriuretic peptides, suggesting the existence of a feedback loop to limit CNP signaling. CNP regulates numerous genes involved in BMP signaling and cell adhesion."

"overexpression of CNP results in inhibition of the MEK1/2-ERK1/2 MAP kinase pathway"

"10-8, 10-7 and 10-6M concentrations of CNP caused a 31%, 40%, and 42% increase, respectively, in longitudinal growth of tibiae"

"PDE inhibition stimulated longitudinal growth by 30% when compared to the control."<-Viagra inhibits PDE5.

CNP reduced the mineralizing area of the bone by 30%.

Comparison of CNP genes to LSJL genes to be done.

CNP downregulates mitochondrial ribosomal protein L35 in the mineralized zone.

Peroxynitrite mediates calcium-dependent mitochondrial dysfunction and cell death via activation of calpains.

"a rapid rise in intracellular calcium that lead to mitochondrial dysfunction and cell death. Although, chondrocyte death exhibited several classical hallmarks of apoptosis we did not find evidence for caspase involvement. Additionally, peroxynitrite did not inhibit cellular caspase activity. Furthermore, using other established assays of cell viability, including the MTT assay and release of lactate dehydrogenase, we found that the predominant mode of cell death involved calcium-dependent cysteine proteases, otherwise known as calpains. peroxynitrite induces mitochondrial dysfunction in cells via a calcium-dependent process that leads to caspase-independent apoptosis mediated by calpains."

Nitric oxide from both exogenous and endogenous sources activates mitochondria-dependent events and induces insults to human chondrocytes.

"When treated with exogenous or endogenous NO, the mitochondrial membrane potential time-dependently decreased. Exposure to exogenous or endogenous NO significantly enhanced cellular reactive oxygen species (ROS) and cytochrome c (Cyt c) levels. Administration of exogenous or endogenous NO increased caspase-3 activity and consequently induced DNA fragmentation. Suppression of caspase-3 activation by Z-DEVD-FMK decreased NO-induced DNA fragmentation and cell apoptosis. Similar to SNP, exposure of human chondrocytes to S-nitrosoglutathione (GSNO), another NO donor, caused significant increases in Cyt c levels, caspase-3 activity, and DNA fragmentation, and induced cell apoptosis. Pretreatment with N-monomethyl arginine (NMMA), an inhibitor of NO synthase, significantly decreased cellular NO levels, and lowered endogenous NO-induced alterations in cellular Cyt c amounts, caspase-3 activity, DNA fragmentation, and cell apoptosis. NO from exogenous and endogenous sources can induce apoptotic insults to human chondrocytes via a mitochondria-dependent mechanism."

Hypoxic induction of UCP3 in the growth plate: UCP3 suppresses chondrocyte autophagy.

"Epiphyseal chondrocytes [express] UCP3. In hypoxia, UCP3 mediated regulation of the mitochondrial transmembrane potential (DeltaPsi(m)) was dependent on HIF-1alpha. UCP3 [regulates] the induction of autophagy. Suppression of UCP3 [enhances] the expression of the autophagic phenotype, even in serum-replete media. Mature autophagic chondrocytes were susceptible to an apoptogen challenge. Susceptibility was associated with a lowered expression of the anti-apoptotic proteins Bcl2 and BCL(xL) and a raised baseline expression of cytochrome c in the cytosol. These changes promote sensitivity to apoptogens. In concert with HIF-1alpha, UCP3 regulates the activity of the mitochondrion by modulating the transmembrane potential. [UCP3] inhibits induction of the autophagic response [thereby suppressing] sensitivity to agents that promote chondrocyte deletion from the growth plate."

"low pO2 in the growth plate is sensed by a family of dioxygenases that hydroxylate specific prolyl residues on the transcription factor HIF-1α"

"[Hif1a] promotes the expression and synthesis of many of the enzymes required for anerobic glycolysis while inhibiting the activity of pyruvate dehydrogenase kinase, the enzyme that catalyzes utilization of pyruvate by chondrocyte mitochondria"

" anti-apoptotic proteins Bcl2 and BclxL was significantly reduced in the UCP3 suppressed cells"

Strain-dependent oxidant release in articular cartilage originates from mitochondria.

"lethal amounts of reactive oxygen species (ROS) were liberated from the mitochondria in response to mechanical insult and that chondrocyte deformation may be a source of ROS. To this end, we hypothesized that mechanically induced mitochondrial ROS is related to the magnitude of cartilage deformation. To test this, we measured axial tissue strains in cartilage explants subjected to semi-confined compressive stresses of 0, 0.05, 0.1, 0.25, 0.5, or 1.0 MPa. The presence of ROS was then determined by confocal imaging with dihydroethidium, an oxidant sensitive fluorescent probe. Our results indicated that ROS levels increased linearly relative to the magnitude of axial strains, and significant cell death was observed at strains40 %. By contrast, hydrostatic stress, which causes minimal tissue strain, had no significant effect. Cell-permeable superoxide dismutase mimetic Mn(III)tetrakis (1-methyl-4-pyridyl) porphyrin pentachloride significantly decreased ROS levels at 0.5 and 0.25 MPa. Electron transport chain inhibitor, rotenone, and cytoskeletal inhibitor, cytochalasin B, significantly decreased ROS levels at 0.25 MPa."

"Cartilage is a hypoxic tissue, where mitochondria are scarce and ATP production occurs almost exclusively through anaerobic glycolysis"

"n highly oxygenated tissues, it is estimated that 2–3 % of molecular oxygen is incompletely reduced to reactive oxygen species (ROS) in the mitochondria, specifically superoxide (O⋅2−)"

"Mechanical stimulation of chondrocytes has been shown to reorganize the cytoskeleton and even distort the mitochondria. This mechanical distortion has been linked to mitochondrial ROS release, which is dependent on the condition of the cytoskeleton."

" in vivo contact deformation in the knee on the medial and lateral cartilage was measured at 12.1 and 14.6 %, respectively, after 300 s of full body weight static loading and in a weight-bearing single leg lunge, deformations in the knee will reach 30 %"

"Superoxide release is dependent on mechanical distortion, which at lower strains requires an intact cytoskeletal network to induce mitochondrial superoxide release."

Thursday, November 27, 2008

Impact loading on chondrocytes

If you can prevent cell death by impact loading of chondrocytes, then you may be able to get a little bit of height.  Mechanical impact on cartilage has also been studied in the context of ERK 1/2.

Changes in chondrocyte gene expression following in vitro impaction of porcine articular cartilage in an impact injury model.

"Our objective was to monitor chondrocyte gene expression at 0, 3, 7, and 14 days following in vitro impaction to the articular surface of porcine patellae. Patellar facets were either axially impacted with a cylindrical impactor (25 mm/s loading rate) to a load level of 2,000 N or not impacted to serve as controls. After being placed in organ culture for 0, 3, 7, or 14 days, total RNA was isolated from full thickness cartilage slices and gene expression measured for 17 genes. Targeted genes included those encoding proteins involved with biological stress, inflammation, or anabolism and catabolism of cartilage extracellular matrix. Some gene expression changes were detected on the day of impaction, but most significant changes occurred at 14 days in culture. At 14 days in culture, 10 of the 17 genes were differentially expressed with col1a1 most significantly up-regulated in the impacted samples, suggesting impacted chondrocytes may have reverted to a fibroblast-like phenotype."

"we observed cell necrosis increasing with time, peaking at 7 days post-impaction while apoptosis peaked at 14 days, the longest time point examined."

"On Day 0, col1a1 and mmp3 had a tendency to be up-regulated in impacted samples. At Day 3, only col2a1 showed any difference with a tendency to be up-regulated in impacted samples. At Day 7, col2a1 was significantly down-regulated and col1a1 had a tendency to be up-regulated in the impacted samples. At Day 14, col1a1 was significantly up-regulated at 847-fold, agc was significantly down-regulated, and col2a1 showed a tendency to be up-regulated. Mmp1 was significantly up-regulated, mmp3 down-regulated, and a timp1 had a tendency towards up-regulation."

Clu, Cxcl16, and Ftl were significantly upregulated on day 14.  S100a11 was significantly downregulated on day 14.

"Collagen type I is normally synthesized by fibroblasts, so our results, in conjunction with reduced levels of agc, suggest that chondrocytes found in the impacted cartilage were reverting to a more fibroblast-like phenotype."

Influence of chondrocyte maturation on acute response to impact injury in PEG hydrogels.

"Bovine chondrocytes were selected as model cells and isolated from skeletally immature (juvenile, 1-3 weeks) or mature (adult, 2-3 years) cartilage to represent different aged donors. Juvenile and adult chondrocytes were encapsulated in identical 3D poly(ethylene glycol) hydrogels and subjected to an initial compressive impact load of 25.6±7.5kN/m(2) applied to 50% strain. Under free swelling culture, adult chondrocytes exhibited higher intracellular ROS levels and catabolism, specifically collagen degradation, when compared to juvenile chondrocytes. In response to injurious load, adult chondrocytes responded with higher cell death, while juvenile chondrocytes responded with greater apoptosis and greater increases in intracellular ROS. With respect to anabolism and catabolism in response to injurious load, adult chondrocytes exhibited decreased aggrecan and collagen deposition, while juvenile chondrocytes exhibited decreased proteoglycan synthesis and increased collagen degradation. Overall, chondrocytes responded to injury regardless of age, but exhibited age-dependent responses with respect to anabolism and catabolism."

"Initial impact force was determined to be 25.6±7.5 kN/m2"

"Impact loading immediately lowered the number of live cells by 22.9% and 15.1% for adult and juvenile cells, respectively. Twenty-four hours post-impact, live cell number further dropped by 34% (adult) and 27% (juvenile) compared to controls. For both cell populations, impact loading led to significantly higher active caspase-3 levels at all time points. Caspase-3 activity was the highest at 72 h in adult cells (∼25×103 more apoptotic cells than controls) and at 24 h in the juvenile cells (∼33×103 more apoptotic cells than controls)."

"Proteoglycan synthesis in juvenile chondrocytes was 1.73-fold higher immediately post-impact, but was inhibited by 35% and 45% 2–4 and 4–24 h post-impact, respectively. For adult chondrocytes, impact loading led to increased proteoglycan release 4–24 h post-impact by 1.3-fold. For juvenile chondrocytes, impact loading did not affect proteoglycan release."<-In LSJL, there was no longitudinal analysis of proteoglycan synthesis but histology of the growth plate shows no apoptosis.

"Cell death appears to occur largely by necrosis given the drop in live cells is far greater than the number of cells undergoing apoptosis for both cell populations."

"Post-impact, adult chondrocytes responded with a small but observable increase in intracellular ROS while juvenile chondrocytes responded with a large increase. Oxidant conditioning has been shown to protect chondrocytes from mechanically induced death, suggesting that chronic exposure to ROS may bolster the antioxidant response. Therefore, adult cells may more effectively scavenge intracellular ROS in response to injury"

"Impact loading did not appear to affect collagen II deposition by juvenile chondrocytes, but did inhibit collagen II deposition by adult chondrocytes. Collagen degradation was evident in adult chondrocytes, but not in juvenile chondrocytes under control conditions, suggesting that adult cells are inherently more catabolically active."

Mechanical stress and ATP synthesis are coupled by mitochondrial oxidants in articular cartilage.

"Metabolic adaptation of articular cartilage under joint loading is evident and matrix synthesis seems to be critically tied to ATP. Chondrocytes utilize the glycolytic pathway for energy requirements but seem to require mitochondrial reactive oxygen species (ROS) to sustain ATP synthesis. The role of ROS in regulating ATP reserves under a mechanically active environment is not clear. physiological strains cause deformation of the mitochondria, potentially releasing ROS for energy production. We hypothesized that mechanical loading stimulates ATP synthesis via mitochondrial release of ROS. Bovine osteochondral explants were dynamically loaded at 0.5 Hz with amplitude of 0.25 MPa for 1 h. Cartilage response to mechanical loading was assessed by imaging with dihydroethidium (ROS indicator) and a Luciferase-based ATP assay. Electron transport inhibitor rotenone and mitochondrial ROS scavenger MitoQ significantly suppressed mechanically induced ROS production and ATP synthesis. Our findings indicate that mitochondrial ROS are produced as a result of physiological mechanical strains. Taken together with our previous findings of ROS involvement in blunt impact injuries, mitochondrial ROS are important contributors to cartilage metabolic adaptation and their precise role in the pathogenesis of osteoarthritis warrants further investigation"

"Rotenone elicits its action by binding to the ubiquinone-binding site of mitochondrial Complex I, thereby disrupting the transfer of electrons from iron-sulfur centers in complex I to ubiquinone. This binding of rotenone interferes with NADH during the creation of cellular energy. "

"In vivo, cartilage thinning under normal physical activities is relatively small. For instance, maximum cartilage thickness loss observed after 30 knee bends was 2.8% and squatting at a knee angle of 90° for 20 s lead to a loss of 4.9%. Maximum peak compartmental deformation of 30% was experienced when performing a weight bearing single leg lunge. Preliminary experiments suggested that the magnitude of mechanical stress selected in this study produced cumulative strains <30% of cartilage thickness so as to test our hypothesis under comparable tissue strains. At higher strains, mitochondria can be physically damaged, a process that would lead to increased leakage of oxidants from the electron transport chain. Mitochondrial damage and subsequent apoptosis of cells can result in decrease in matrix synthesis, and also initiate other degenerative cascades that are hallmarks of osteoarthritis."

Single impact cartilage trauma and TNF-α: interactive effects do not increase early cell death and indicate the need for bi-/multidirectional therapeutic approaches.

"TNF-α is increased in synovial fluid early after trauma, potentiates injury-induced proteoglycan degradation and may act proapoptotic under permissive conditions. We asked whether TNF-α also influences chondrocyte death, gene expression of catabolic and anabolic markers and the release of proinflammatory mediators in the early post-traumatic phase. Interactive effects of a defined single impact trauma (0.59 J) and TNF-α (100 ng/ml) on human early-stage osteoarthritic cartilage were investigated in vitro over 24 h. Exposure of traumatized cartilage to TNF-α did not increase chondrocyte death. IL-6-synthesis was augmented by trauma, TNF-α and combined treatment. The impact increased the release of PGE2 and PGD2 in the presence and absence of TNF-α to a similar extent while TNF-α alone showed no effect. In contrast, NOS2A-expression and nitric oxide (NO)-release were not affected by trauma but significantly increased by TNF-α. Expression of OPG and RANKL was not affected by TNF-α but modulated by trauma. TNF-α with and without trauma significantly induced MMP1 gene expression. These results indicate that TNF-α does not potentiate early cell death in early-stage osteoarthritic cartilage after blunt injury. However, trauma and TNF-α showed independent and interactive effects concerning prostaglandin and NO release. TNF-α probably contributes to cartilage degradation after trauma by an early induction of MMP1 gene expression. Our study confirms that an anti-TNF-α therapy may have inhibitory effects on catabolic and, partly, on inflammatory processes after a single impact trauma. As TNF-α does not contribute to the loss of chondrocytes in the initial post-traumatic phase, a combination with pharmaco-therapeutic strategies reducing early cell death could be reasonable."

"In vitro studies with human and bovine cartilage revealed that TNF-α and IL-1α potentiate the effect of cartilage impact"

"Trauma alone had a tendency to elevate COX2 mRNA level"

"TNF-α has previously been reported not to be proapoptotic in chondrocytes unless they were specifically sensitized for apoptosis"

Development of a new biomechanically defined single impact rabbit cartilage trauma model for in vivo-studies.

"We constructed a novel trauma device that allows biomechanically defined force application to the load-bearing region of the medial and lateral femoral condyles in adult rabbits. The fixation to the femur was specially designed to avoid uncontrolled influx of blood into the joint. The device was tested on the articular femoral surface of cadaveric rabbits.
At a lower energy (1.0 J), the tests showed that superficial and partially deep fissuring, partial necrosis of the chondrocytes, and early proteoglycan loss occurred at the region of impact. Subchondral fractures could be excluded by micro CT. At higher energy (≥ 1.4 J), we observed more pronounced deep fissuring and in some cases complete shearing of the articular cartilage from the subchondral bone.
Our model represents an easy to use method to create a biomechanically defined cartilage trauma and offers some advantages with respect to handling under aseptic surgical conditions and prevention of uncontrolled intra-articular bleeding from the bone marrow compartment for pharmacologic studies."

Novel methods for the quantification of changes in actin organization in chondrocytes using fluorescent imaging and linear profiling.

"Upon isolation, there was a 49% decrease in total F-actin fluorescence from 1.91 ± 0.16 pixel/μm(3) (Mu) to 0.95 ± 0.55 Mu, whereas upon culture, an apparent increase in total fluorescence was deemed insignificant due to an increase in average cell volume, with a rise, however, in striation units (StU) from 1 ± 1 to 5 ± 1 StU/cell, and a decrease in percentage cortical fluorescence to 30.45% ± 1.52% (P = 7.8 × 10(-5)). Freshly isolated chondrocytes exhibited a decrease in total F-actin fluorescence to 0.61 ± 0.05 Mu and 0.32 ± 0.02 Mu, 10 min posthypertonic and hypotonic challenges, respectively. Regulatory volume decrease was inhibited in the presence of REV5901 with maintenance of actin levels at 1.15 Mu. Following mechanical impact in situ, there was a reduction in total F-actin fluorescence to 0.95 ± 0.08 Mu and 0.74 ± 0.06 Mu under isotonic and hypotonic conditions, respectively, but not under hypertonic conditions."

Hypertonic decreases cell volume.

"maintaining actin organization levels [protects chndrocytes]"

Effect of a solution of hyaluronic acid-chondroitin sulfate-N-acetyl glucosamine on the repair response of cartilage to single-impact load damage.

"Full-thickness cartilage disks were harvested from the third metacarpal bone [of horses]. Cartilage was single-impact loaded (SIL) with 0.175 J at 0.7 m/s and cultured in DMEM plus 1 % (vol/vol) HCNAG[hyaluronic acid-chondroitin sulfate-N-acetyl glucosamine] or fibroblastic growth factor (FGF)-2 (50 ng/mL).
Type II collagen immunoreactivity increased in SIL cartilage, compared with control samples. At days 14 and 28 (day 0 = initiation of culture), control samples had significantly fewer repair cells than did other treatment groups. In control samples and SIL + HCNAG, there was a significant decrease in apoptotic cell number, compared with results for SIL and SIL + FGF-2 samples. At days 14 and 28, there was a significant increase in chondrocytes stained positive for PCNA in the control samples.
1% HCNAG significantly affected apoptotic and repair cell numbers in an SIL damage-repair technique in adult equine articular cartilage. However, HCNAG had no effect on the number of PCNA-positive chondrocytes or on type II collagen immunohistochemical results. The inclusion of 1% HCNAG in lavage solutions administered after arthroscopy may be beneficial to cartilage health by increasing the number of repair cells and decreasing the number of apoptotic cells."

Mechanical injury suppresses autophagy regulators and pharmacologic activation of autophagy results in chondroprotection.

"Bovine and human cartilage explants were subjected to mechanical impact (40% strain for 500 msec). Cell viability, sulfated glycosaminoglycan (sGAG) release, and changes in the levels of the autophagy markers ULK1, beclin 1, and microtubule-associated protein 1 light chain 3 (LC3) were evaluated. Cartilage explants were treated with the mammalian target of rapamycin complex 1 (mTORC-1) inhibitor and the autophagy inducer rapamycin and tested for protective effects against mechanical injury. Explants were also treated with the cell death inducers nitric oxide and tumor necrosis factor α (TNFα) plus actinomycin D, and the proinflammatory cytokine interleukin-1α (IL-1α).
Mechanical injury induced cell death and loss of sGAG in a time-dependent manner. This was associated with significantly decreased ULK1, beclin 1, and LC3 expression in the cartilage superficial zone 48 hours after injury. The levels of LC3-II were increased 24 hours after injury but decreased at 48 and 96 hours. Rapamycin enhanced expression of autophagy regulators and prevented cell death and sGAG loss in mechanically injured explants. Rapamycin also protected against cell death induced by sodium nitroprusside and TNFα plus actinomycin D and prevented sGAG loss induced by IL-1α.
Mechanical injury leads to suppression of autophagy, predominantly in the superficial zone where most of the cell death occurs. Pharmacologic inhibition of mTORC-1, at least in part by enhancement of autophagy, prevents cell and matrix damage, suggesting a novel approach for chondroprotection."

"In articular cartilage, which is characterized by a very low rate of cell turnover, autophagy would appear to be essential to maintain cell survival and function."

"Our findings indicated that there was an increase in LC3-II 24 hours after injury, as an early response to mechanical stress. However, autophagy was suppressed 48 hours and 96 hours after injury. In addition, the levels of LC3-I were decreased 96 hours after injury, indicating a reduction in the expression of autophagy regulators after mechanical stress in articular cartilage. Suppression of autophagy markers, predominantly in the superficial zone, was observed in response to mechanical injury at 48 hours."

"autophagy is suppressed in human OA cartilage as well as in mouse models of joint aging and OA induced by mechanical instability, and this reduction was accompanied by an increase in apoptotic cell death"

Single impact trauma in human early-stage osteoarthritic cartilage: implication of prostaglandin D2 but no additive effect of IL-1β on cell survival.

"Trauma was induced by impacting cartilage explants with a drop-tower system and its effect was measured in terms of cell survival, gene expression and the release of mediators. In addition, the effect of concomitant IL‑1β stimulation and p38MAPK inhibition by SB203580 was investigated. We found a significant decrease in chondrocyte viability after trauma, but no additional effect of IL-1β stimulation. SB203580 had a tendency to improve cell survival suggesting a role for p38 signaling in cell viability after impact in an inflammatory environment. We showed that various mediators are released in response to trauma with or without IL-1β stimulation, differing in composition and time response. Trauma resulted in an increased release of IL-6, whereas TNF-α and IL-1β release was unaffected. Prostaglandin (PG) and NO synthesis pathways were both affected by trauma and/or IL-1β. We demonstrate an elevated release of prostaglandin D2 (PGD2) by human articular cartilage in response to a single mechanical impact. The up-regulation of mediators was time-dependent, with a more early increase of PGD2 compared to prostaglandin E2 (PGE2) and a late induction of NO by co-stimulation with IL-1β between 6 and 24 h."

"an induction of PGD2, PGE2 and IL-6 could be observed by trauma 24 h post-impact whereas concomitant IL-1β stimulation led to elevated levels of PGE2, IL-6, NO, TNF-α and PGD2, with levels of the latter subsiding by 24 h."

"Fresh intact porcine knees were preloaded and impacted at 294 J via a drop track. Osteochondral cores were obtained from the medial and lateral aspects of the femoral condyles and tibial plateau. Chondrocyte viability was assessed at days 0, 3, and 5 postimpact in sham, impacted nonfractured, and impacted fractured joints. Total matrix metalloproteinase (MMP) activity, aggrecanase (ADAMTS-4) activity, and sulfated glycosaminoglycan (S-GAG) release were measured in culture media from days 3 and 5 posttrauma. No differences were observed in chondrocyte viability of impacted nonfractured joints (95.9 ± 6.9%) when compared to sham joints (93.8 ± 7.7%). In impacted fractured joints, viability of the fractured edge was 40.5 ± 27.6% and significantly lower than all other sites, including cartilage adjacent to the fractured edge. MMP and aggrecanase activity and S-GAG release were significantly increased in specimens from the fractured edge. Joint impact resulting in articular fracture significantly decreased chondrocyte viability, increased production of MMPs and aggrecanases, and enhanced S-GAG release, whereas the same level of impact without fracture did not cause such changes."

"the threshold of stress that causes chondrocyte death following acute impact injury is much higher in a closed whole joint model, as compared to an in vitro explant model."

Rotenone prevents impact-induced chondrocyte death.

"Recent reports indicate that antioxidants decrease impact-induced chondrocyte death, but the source(s) of oxidants, the time course of oxidant release, and the identity of the oxidative species generated in response to injury are unknown. we studied the kinetics and distribution of oxidant production in osteochondral explants subjected to a single, blunt-impact injury. We followed superoxide production by measuring the time-dependent accumulation of chondrocyte nuclei stained with the superoxide-sensitive probe dihydroethidium. The percentage of chondrocytes that were dihydroethidium-positive was 35% above baseline 10 min after impact, and 65% above baseline 60 min after impact. Most positive cells were found within and near areas contacted directly by the impact platen. Rotenone, an electron transport chain inhibitor, was used to test the hypothesis that mitochondria contribute to superoxide release. Rotenone treatment significantly reduced dihydroethidium staining, which remained steady at 15% above baseline for up to 60 min postimpact. Moreover, rotenone reduced chondrocyte death in impact sites by more than 40%, even when administered 2 h after injury. much of the acute chondrocyte mortality caused by in vitro impact injuries results from superoxide release from mitochondria, and suggest that brief exposure to free radical scavengers could significantly improve chondrocyte viability following joint injury."

"Both necrosis and apoptosis occur in response to impact loads. Z-VAD-FMK, a cell-permeable fluoromethylketone peptide caspase inhibitor, and P188, a membrane-stabilizing surfactant, reduce impact-related death"

"Impact-induced chondrocyte death in a bovine explant model was blocked by treatment with the intermediate free-radical scavenger n-acetyl cysteine (NAC). NAC is a powerful scavenger of highly damaging hydroxyl and hypochlorous radicals, which are formed in cells following exposure to superoxide. NAC applied within 2 h of impact spared more than 75% of cells that would have otherwise died within 72 h of injury."

"Increased free radical production is associated with direct oxidative damage to mitochondrial electron transport complexes or to the DNA that encodes them."

"Some 20% of chondrocytes at impact sites produced superoxide in the presence of rotenone. It is possible that NOX, a rotenone-resistant enzyme, contributed to superoxide release at impact sites. "

Effect of impaction sequence on osteochondral graft damage: the role of repeated and varying loads.

"Impacted plugs had significantly lower cell viability than nonimpacted plugs. A dose-response relationship in loss of cell viability with respect to load magnitude was seen immediately and after 4 days but lost after 8 days. Histological analysis revealed intact cartilage surface in all samples (loaded or control), with loaded samples showing alterations in birefringence. While the sulfated GAG release was similar across varying impaction loads, release of nitric oxide increased with increasing impaction magnitudes and time."

Acute repair of chondrocytes in the rabbit tibiofemoral joint following blunt impact using P188 surfactant and a preliminary investigation of its long-term efficacy.

"P188 surfactant has been shown to acutely restore the integrity of damaged chondrocytes; however, its long-term efficacy is unknown. The hypothesis of this study was that a single injection of P188 into a traumatized joint would acutely repair damaged cell membranes and maintain their viability in the long term. Twelve rabbits were divided into two groups, with and without P188, and sacrificed 4 days after tibiofemoral (TF) impact. Another six rabbits were sacrificed after 6 weeks and divided into two groups, with and without P188 treatment immediately posttrauma. Treatment with P188 increased the viable cell density 4 days posttrauma. A higher density of viable cells was also documented 6 weeks posttrauma in the treated versus untreated limb. The results of the current study confirm the acute efficacy of P188 treatment, and may suggest long-term efficacy of treatment, but additional studies are still needed to investigate the chronic implications of the acute repair of cells in the traumatized joint."

"P188 efficacy has also recently been shown on human tali cartilage up to 14 days in both the area of impact and the surrounding cartilage."

"Fresh bovine osteochondral cores were randomly divided into 5 groups: (1) control, (2) 0.35 J, (3) 0.71 J, (4) 1.07 J, and (5) 1.43 J impact energies. 
The highest impact energy fractured the cartilage in all cores (1.43 J, n = 17). Seventy-three percent and 64% of the osteochondral cores remained intact after lower energy impacts of 0.71 J and 1.07 J, respectively. At lower energy levels, fractured cores were thinner than those remaining intact. In cores remaining intact after impact injury, chondrocyte death increased with increasing impact energy  and with greater time after impact. A progressive increase in dead cells near the bone/cartilage interface and at the articular surface was observed."

"Forty-eight hours after impact, the effects on viability of immediate post-impact treatment with N-acetylcysteine were compared with the effects of the caspase inhibitor N-CBZ-Val-Ala-Asp(O-Me) fluoromethyl ketone and those of the cell-membrane-stabilizing surfactant poloxamer 188. The effect of N-acetylcysteine on proteoglycan content was determined at seven and fourteen days post-impact.
Chondrocyte viability declined sharply within an hour and reached a steady state within six to twelve hours after impact. Immediate treatment with N-acetylcysteine doubled the number of viable chondrocytes assayed forty-eight hours after impact, and this effect was significantly greater than that of N-CBZ-Val-Ala-Asp(O-Me) fluoromethyl ketone. Even when N-acetylcysteine treatment was delayed for up to four hours after injury, it still had significant positive effects on cell viability at forty-eight hours. Moreover, N-acetylcysteine treatment significantly improved proteoglycan content at the impact sites at both seven and fourteen days after injury.
Treatment with N-acetylcysteine soon after a blunt impact injury can reduce chondrocyte death and proteoglycan loss measured seven to fourteen days after injury."

The mandibular cartilage metabolism is altered by damaged subchondral bone from traumatic impact loading.

"cytokines released from damaged subchondral bone from impact-loading affect the cartilage catabolism under pathological conditions. An impactor of 200 gw was dropped onto the top of a porcine mandibular condyle. After organ culture for 2 days, we investigated the association between the subchondral bone and cartilage using histological and biochemical experiments. The impact-loading induced the expression of IL-1beta immunohistochemically and prominently up-regulated IL-1alpha and IL-1beta mRNA levels in subchondral bone. We confirmed a significant decrease in type II collagen and aggrecan mRNA expressions in chondrocytes by co-culture with osteoblasts after impact-loading, and significant increase in mRNA and protein expressions of IL-1beta in subchondral osteoblasts from impact-loaded subchondral bone. The mRNA expressions of type II collagen, aggrecan, and type X collagen in chondrocytes were decreased significantly by the co-culture with osteoblasts pre-treated by IL-1beta, -6, and TNF-alpha. Among them, osteoblasts pre-treated by IL-1beta affected chondrocytes most strongly. It was also shown that IL-1beta-treated osteoblasts enhanced the MMP-1 mRNA level most markedly in chondrocytes among the four cytokines. These results suggest that the TMJ subjected to impact-loading can increase directly IL-1beta synthesis in the subchondral region, subsequently altering the metabolism of adjacent cartilage and may eventually resulting in the onset and progression of TMJ-OA."

BMP-7 protects against progression of cartilage degeneration after impact injury.

"In three subsequent studies, bilateral impact injuries were created and one joint received intraarticular injections of 340 microg of rhBMP-7 protein in a collagen particle carrier while the contralateral knee received the vehicle alone. Sheep were allocated to three groups that received intraarticular injections on day 0 (group A), 21 (group B), or 90 (group C) after experimental knee injury. In each group the, joints were evaluated for signs of osteoarthritis progression 90 days after the last treatment using India ink stained area, OARSI histological scoring, cartilage sGAG content, immunostaining for apoptosis (TUNEL), caspase-3, collagen degradation (Col 2 3/4C short collagen epitope), and the endogenous (pro-) form of BMP-7 protein. Knee joints that received rhBMP-7 immediately after injury had small focal lesions at the injury site that did not progress into the surrounding cartilage. Joints that received BMP-7 3 weeks after injury were improved and had limited progression compared to controls, but joints that received the protein 12 weeks after injury had no statistically significant improvement. These studies suggest that BMP-7 may be chondroprotective after traumatic injury in patients if it is administered within 3 to 4 weeks of the index injury. The mechanism of protection after sublethal injury appeared to be an increased survival of chondrocytes that are able to participate in the repair process."

"aged osteoarthritic chondrocytes still respond to BMP-7 with increased anabolic activity and viability."

Single high-energy impact load causes posttraumatic OA in young rabbits via a decrease in cellular metabolism.

"Each rabbit underwent the application of a single, rapid, high-energy impact load to the posterior aspect of their right medial femoral condyle using a previously validated mechanism. At regular intervals (0, 1, 6 months) the injured cartilage was harvested and analyzed for the presence of PTA[post traumatic osteoarthritis]. Each specimen was assessed histologically for cell and tissue morphology and chondrocyte metabolism, including BMP-2 production and synthesis of extracellular matrix (type II procollagen mRNA). Cartilage from the contralateral sham limb, as well as uninjured cartilage from the experimental limb served as internal controls for each animal. Significant changes were found in the morphology of the cartilage including proteoglycan loss along with decreased BMP-2 and type II procollagen mRNA staining. These findings confirm that a single high-energy impact load can cause the development of PTA by disrupting the extracellular matrix and by causing a decrease in chondrocyte metabolism."

Chondrocyte damage and contact pressures following impact on the rabbit tibiofemoral joint.

"Studies from this laboratory document that impacts to the tibiofemoral (TF) joint at 50% of the energy producing gross fracture can generate cartilage damage and microcracks at the interface between calcified cartilage and underlying subchondral bone in the tibial plateau. These injuries are suggestive of the initiation for a long term chronic disease, such as osteoarthritis. The disease process may be further encouraged by acute damage to chondrocytes in the cartilage overlying areas of occult microcracking. The hypothesis of the current study was that significant damage to chondrocytes in tibial plateau cartilage could be generated in areas of high contact pressure by a single impact delivered to the rabbit TF joint, without a gross fracture of bone. Three rabbits received a single, 13 J of energy blunt insult to the TF joint, while another three animals were used as controls. Cell viability analyses compared chondrocyte damage in impacted versus control cartilage. Two additional rabbits were impacted to document contact pressures generated in the TF joint. The study showed high contact pressures in uncovered areas of the plateau, with a trend for higher pressures in the lateral versus medial facets. A significantly higher percentage of damaged chondrocytes existed in impacted versus the opposite, nonimpacted limbs. Additionally, more chondrocyte damage was documented in the superficial zone (top 20% of cartilage thickness) of the cartilage compared to middle (middle 50% of thickness) and deep (bottom 30% of thickness) zones. This study showed that a single blunt insult to the in situ rabbit TF joint, generating large areas of contact pressure exceeding 20 MPa, produces significant chondrocyte damage in the tibial articular cartilage, especially in the superficial zone, without gross fracture of bone. Future studies will be needed to investigate the long term, chronic outcome of this blunt force joint trauma."

"Rat cartilage was single impact loaded (200 g from 8 cm) and cultured for up to 48 hours (n = 72 joints). Histological changes were measured using a semi-quantitative modified Mankin score. Immunolocalisation was used to identify changes in vimentin distribution.
SIL caused damage in 32/36 cartilage samples. Damage included surface fibrillation, fissures, fragmentation, changes in cellularity and loss of proteoglycan. SIL caused a statistically significant increase in modified Mankin score and chondrocyte clusters over time. SIL caused vimentin disassembly (as evidenced by collapse of vimentin around the nucleus).
This study describes a model of SIL damage to rat cartilage. SIL causes changes in histological/chemical parameters which have been measured using a semi-quantitative modified Mankin score. Single impact load also causes changes in the pattern of vimentin immunoreactivity, indicating vimentin dissassembley. Using a semi-quantitative scoring system the disassembly was shown to be statistically significant in SIL damaged cartilage."

"Vimentin intermediate filaments and microtubules form a link between the plasma membrane and the nucleus, with vimentin forming a tighter and finer mesh than microtubules, and these intermediate microfilaments may play a role in the mechanotransduction proces"

"vimentin microfilaments have been shown to contribute to the viscoelastic properties of the chondrocyte"

" ERK was detectable in the cytoplasm of all chondrocytes at t = 0 and t = 1 hour in control and SIL cartilage. At time = 0 there was no pERK within control sections, but in SIL cartilage strong PERK immunoreactivity was detected in the cytoplasm of all of the chondrocytes within the section . By t = 1 hour pERK was detected in both impacted and control sections."

"Caspase proteolysis of vimentin at Asp85 promotes apoptosis by disassembling the vimentin cytoskeleton and amplifying cell death via pro-apoptotic cleavage products "

Gene expression profiling of chondrocytes from a porcine impact injury model.

"Porcine patellae were impacted perpendicular to the articular surface to create an impact injury. Intact patellae (control and impacted) were maintained in culture for 14 days. Total RNA was then extracted from the articular cartilage beneath the impaction.
Thirty-nine SAGE tags were significantly differentially expressed in the impacted and control libraries, representing 30 different annotated pig genes. These genes represented gene products associated with matrix molecules, iron and phosphate transport, protein biosynthesis, skeletal development, cell proliferation, lipid metabolism and the inflammatory response. Twenty-three of the 30 genes were down-regulated in the impacted library and five were up-regulated in the impacted library. 
Damaged chondrocytes may de-differentiate into fibroblast-like cells and proliferate in an attempt to repair themselves."

Genes upregulated in Impacted Carilage versus control at 14 days:
CAPNS1{promotes cell proliferation}
MDK precursor{promotes cell proliferation}

FTH1{negatively regulates cell proliferation}
FTL{negatively regulates cell proliferation}
APOD{inversely related to cell proliferation}

"Cartilage explants were single impact loaded, placed within a scaffold and cultured for up to 20 days +/- BMP-2. 
Following single impact load and culture, chondrocytes were observed in a 3D gelatin scaffold under all culture conditions. Chondrocytes were also observed on the articular surface of the cartilage and extruding out of the parent cartilage and on to the cartilage surface. BMP-2 was demonstrated to quantitatively inhibit these events.
articular chondrocytes can be stimulated to migrate out of parent cartilage following single impact load and culture. The addition of BMP-2 to the culture medium quantitatively reduced the repair response."

" outgrowth of cells into the gelatin scaffold was observed under all experimental conditions i.e. control (no impact), SIL and SIL+BMP."

BMP-2 inhibits FGF-2 which encourages cell migration.

Effect of a glucosamine derivative on impact-induced chondrocyte apoptosis in vitro. A preliminary report.

"A lipophilic derivative of glucosamine, Glu5, is able to prevent impact-induced chondrocyte death by the putative mechanism of reducing mitochondrial depolarisation following a single impact load in vitro."

"When quantified 48 h following impact, a single impact load significantly increased apoptosis from 6.2% in unimpacted controls to 53.5%. Pre-incubation with N-acetyl-glucosamine prior to impact had no effect on chondrocyte viability. The impact-induced increase in cell death was significantly reduced to 22.8% by incubation with Glu5 prior to impact"

"the impact-induced increase in cell death was significantly increased to 86.0% by incubation with Glu11 prior to impact"

"impact-induced chondrocyte death is preceded by impact-induced mitochondrial depolarisation. To determine the molecular mechanism by which the glucosamine derivative Glu5 was able to reduce impact-induced chondrocyte death, the effect of Glu5 on impact-induced mitochondrial depolarisation was investigated. Mitochondrial depolarisation was determined. mitochondrial depolarisation is indicated by an increase in the green:red fluorescence intensity ratio. Cells with an average intensity ratio of 0.9 or above were considered to have depolarised mitochondria. Following culture for 6 h, the percentage of superficial chondrocytes with a green:red fluorescence intensity ratio above 0.9, indicating mitochondrial depolarisation, was 6.7%. This value was significantly increased in explants impacted from 50 mm to 39.4%, but not by impacted explants pre-incubated with Glu5"

" high doses of glucosamine (10–25 mg/ml, the equivalent of 38–96 mM) have previously been found to be detrimental to cartilage matrix and chondrocyte viability, causing apoptosis in up to 90% of chondrocytes. Furthermore, an increase in the intracellular levels of O-linked N-acetyl-glucosamine can lead to cell death following cellular stress"

"Fresh cadaveric canine femoral condyles were subjected to 20-25-MPa impact injury. Condyle explants or dispersed chondrocytes were cultured with autologous blood mononuclear leukocytes (MNLs). Viability of chondrocytes at varying distances from the impact site was assessed by trypan blue exclusion.
Mechanical injury caused a significant loss of viable chondrocytes over 7 days, even in cartilage >10 mm from the impact site. After biomechanical stress, death of cells within 10 mm of the impact could be largely prevented by addition of N(G)-monomethyl-L-arginine to inhibit nitric oxide (NO) generation. Chondrocytes within 10 mm of the impact were also susceptible to killing by living MNLs, but not by incubation with the supernatants of endotoxin-activated MNLs. Chondrocytes in this vulnerable zone expressed intercellular adhesion molecule 1 (ICAM-1) (CD54), facilitating attachment of MNLs that localized adjacent to the chondrocytes. Leukocytes killed dispersed chondrocytes harvested from the impact zone by generation of reactive oxygen species. Leukocyte-mediated killing could be blocked by desferoxamine or by antibodies to CD18, which prevent attachment of leukocytes to ICAM-1-expressing chondrocytes.
after mechanical injury, chondrocytes distant from the site may be killed through the generation of NO. Inflammatory leukocytes further extend the zone of chondrocyte death by adhering to chondrocytes expressing ICAM-1 and by inducing the accumulation of free oxygen radicals in the chondrocyte cytoplasm."

"When activated in vitro, cultured chondrocytes release chemoattractants such as interleukin-8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) that direct the migration of leukocytes. Leukocytes, in turn, are a source of proinflammatory cytokines, such as IL-1 and tumor necrosis factor α (TNFα), that induce apoptosis in both hypertrophic and nonhypertrophic chondrocytes. IL-1 can also induce chondrocytes to produce sufficient nitric oxide (NO) to cause cell death. These proinflammatory cytokines up-regulate the expression of intercellular adhesion molecule 1 (ICAM-1) on chondrocytes, which allows the attachment of leukocytes, and facilitate the accumulation of toxic agents within the chondrocyte cytoplasm. In addition, these cytokines stimulate the release of metalloproteinases by injured chondrocytes and leukocytes, and these metalloproteinases promote the degradation of the extracellular matrix "

"NO and ROS together can generate peroxynitrite, which is highly toxic for chondrocytes"

"attachment to chondrocyte ICAM-1 mediated by leukocyte CD11a/CD18 (LFA-1) or CD11b/CD18 brings leukocytes into close apposition with the chondrocytes. Indeed, as we have shown, treatment of cultured chondrocytes with antibodies to CD18 decreased accumulation of free oxygen radicals in the chondrocytes by 70%. "

Validation of an in vitro single-impact load model of the initiation of osteoarthritis-like changes in articular cartilage.

"Articular cartilage was obtained from seven healthy horses and from four horses diagnosed with OA. Cartilage disks were subjected to a single-impact load (500 g from 25, 50, or 100 mm) using a simple drop-tower device and cultured in vitro for up to 20 days. Cartilage sections were examined histologically to observe surface damage and proteoglycan loss. Following a single-impact load and subsequent culture in vitro, articular cartilage explants demonstrated characteristic surface damage, proteoglycan loss, and chondrocyte death. This closely resembled degenerative changes observed in OA cartilage samples. A kinetic study showed that these degenerative changes (increased weight gain, GAG release into the medium, and chondrocyte death) were initiated within 48 h following impact and increased with recovery time in culture. These parameters were proportional to impact height, that is, impact energy. In conclusion, articular cartilage disks subjected to a single-impact load followed by 48 h of recovery time in culture in vitro developed traumatic OA-like changes."

Distribution of beta-endorphin and substance P in the shoulder joint of the dog before and after a low impact exercise programme.

"On day 0, biopsies of articular cartilage and joint capsule were obtained from the left shoulder joints of dogs receiving limited and regimented exercise. On day 72, biopsies of joint capsule from right and left shoulders and articular cartilage from the right shoulder joint were analysed for the presence of glycosaminoglycans (GAG) and for immunolocalization of substance P and beta-endorphin. Regimented exercise increased the presence of GAGs and immunolocalization of substance P and beta-endorphin in articular cartilage and synovial membrane compared to day 0 biopsies and untreated controls. Localization of beta-endorphin became prominent in and around the chondrocytes. Substance P was increased in chondrocytes and extracellular matrix."

"Substance P stimulates release of prostaglandin E2, collagenase, interleukin-1, and tumour necrosis factor-α from synoviocytes and chondrocytes, as well as increases synoviocyte proliferation"

"The concomitant immunolocalization of β-endorphin in the territorial matrix of the middle and deep zones of articular cartilage after a low-impact exercise programme suggested that opioids might act on chondrocytes in response to the altered mechanical demands, such as increased and decreased compressive forces, placed on the joint surface."

"Opioids are also anti-inflammatory molecules as they inhibit the release of neuroinflammatory mediators including substance P"

Matrix loss and synthesis following a single impact load on articular cartilage in vitro.

"Articular cartilage biopsies were subjected to a single impact load and the metabolic response of the chondrocytes investigated using radiolabelled precursors for protein ([3H]leucine) and glycosaminoglycan ([35S]sulfate). The severity of the impact was controlled by using different masses and drop heights in a purpose built drop tower. Loss of matrix components was studied by prelabelling prior to loading, the possible repair response by pulse labelling at defined intervals after loading. There was an increase in the loss of both labels from the tissue with increasing severity of impact though the patterns of loss were different. Only 25%-40% of the sulfate was lost over a two week period and the loss increased with the severity of impact. This contrasted with 60% of the leucine being lost over the same period independently of loading. In addition to the loss of synthetic activity caused by cell death, there was a suppression of incorporation immediately following loading. This eventually recovered and increased above control values but the recovery time appeared to depend on the severity of the impact."

Calcium signaling leads to mitochondrial depolarization in impact-induced chondrocyte death in equine articular cartilage explants.

"Articular cartilage explants obtained from healthy horses were subjected to a single impact load (500-gm weight dropped from a height of 50 mm) and cultured in vitro for up to 48 hours. Chondrocyte death was quantified. Release of proteoglycans was determined. Weight change was measured, and mitochondrial depolarization was determined. To assess the role of calcium signaling in impact-induced chondrocyte death, explants were preincubated in culture medium containing various concentrations of calcium. Inhibitors were used to assess the role of individual signaling components in impact-induced chondrocyte death.
Calcium quenching, inhibitors of calpains, calcium/calmodulin-regulated kinase II (CaMKII), and mitochondrial depolarization reduced impact-induced chondrocyte death after 48 hours in culture. Transient mitochondrial depolarization was observed 3-6 hours following a single impact load. Mitochondrial depolarization was prevented by calcium quenching, inhibitors of calpain, CaMKII, permeability transition pore formation, ryanodine receptor, and the mitochondrial uniport transporter. Cathepsin B did not appear to be involved in impact-induced chondrocyte death. The calpain inhibitor prevented proteoglycan loss, but the percentage weight gain and proteoglycan loss were unaffected by all treatments used.
Following a single impact load, calcium is released from the endoplasmic reticulum via the ryanodine receptor and is taken up by the mitochondria via the uniport transporter, causing mitochondrial depolarization and caspase 9 activation. Calpains and CaMKII play important roles in causing mitochondrial depolarization."

"caspase 9 activation occurring upstream of caspase 3 activation leads to apoptosis-like impact-induced chondrocyte death in cartilage explants "

"External calcium enters the cell through various membrane channels in response to extracellular stimuli. Internal calcium is released from intracellular stores through the opening of various channels, such as the inositol-1,4,5-trisphosphate receptor (IP3R) and the ryanodine receptor (RyR). The largest intracellular calcium store is the endoplasmic reticulum (ER), where calcium levels are affected by uptake via sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) and release via IP3R and RyR . Changes in the ER calcium concentration result in disturbance of the normal ER function and in ER stress, causing the activation of calpains and ER membrane-associated caspase, which leads to apoptosis. The mitochondria take up calcium through a uniport transporter and release it through the IP3R or the sodium/calcium exchanger or through the formation of the permeability transition pore (PTP). The accumulation of excessive calcium in the mitochondrial matrix leads to the formation of the PTP. The PTP leads to the collapse of the mitochondrial transmembrane potential, which results in the release of calcium and mitochondrial proteins, such as cytochrome c, and finally, the execution of apoptosis. In the cytoplasm, calcium signals can act indirectly on gene expression through the activation of protein kinases such as calcium/calmodulin-regulated kinase II (CaMKII) to alter the phosphorylation state of transcription factors."

" inhibition of CaMKII protects cells from apoptosis by reducing caspase 2 and caspase 3 activity or by preventing the accumulation of reactive oxygen species second messengers"

"cathepsin B causes the release of mitochondrial cytochrome c, leading to caspase-dependent apoptosis"

"CaMKII has also recently been shown to phosphorylate, and hence inactivate, caspase 2, thereby preventing mitochondrial depolarization"

"The percentage of weight gain of cartilage explants, reflecting increased hydration, is an indirect indicator of loss of collagen II matrix integrity"

"dantrolene (RyR inhibitor) is able to prevent mitochondrial depolarization. Ru360 (a uniport transporter inhibitor) has been reported to inhibit mitochondrial depolarization in neurons"

Chondrogenic progenitor cells respond to cartilage injury.

"Hypocellularity resulting from chondrocyte death in the aftermath of mechanical injury is thought to contribute to posttraumatic osteoarthritis. However, we observed that nonviable areas in cartilage injured by blunt impact were repopulated within 7-14 days by cells that appeared to migrate from the surrounding matrix. The migrating cell population included chondrogenic progenitor cells that were drawn to injured cartilage by alarmins.
Osteochondral explants obtained from mature cattle were injured by blunt impact or scratching, resulting in localized chondrocyte death.
Migrating cells were highly clonogenic and multipotent and expressed markers associated with chondrogenic progenitor cells. Compared with chondrocytes, these cells overexpressed genes involved in proliferation and migration and underexpressed cartilage matrix genes. They were more active than chondrocytes in chemotaxis assays and responded to cell lysates, conditioned medium, and HMGB-1. Glycyrrhizin, a chelator of HMGB-1 and a blocking antibody to receptor for advanced glycation end products (RAGE), inhibited responses to cell debris and conditioned medium and reduced the numbers of migrating cells on injured explants."

"Compared with normal chondrocytes, chondrogenic progenitor cells overexpressed the stem cell–associated factor Notch-1 and the fibronectin receptor α5β1 integrin. The cells also showed enhanced clonality in culture and multipotency when grafted to chick limb buds"

Genes upregulated in Chondrocyte Progenitor Cells versus Normal Chondrocytes also upregulated in LSJL:


Genes upregulated in Chondrocyte Progenitor Cells versus MSCs also upregulated in LSJL:


"putative chondrogenic progenitor cells overexpressed COL2A1, ACAN, PRG4, S100A1, and S100B, all of which are considered to be chondrocyte markers"

"HMGB-1 at 10 nM or 20 nM significantly enhanced chemotaxis[movement in response to chemicals] compared with controls"

A Distinct Catabolic to Anabolic Threshold Due to Single-Cell Static Nanomechanical Stimulation in a Cartilage Biokinetics Model.

"A transition threshold load level [exists] from which the mechanical input causes a shift from a catabolic state to an anabolic state."

"In the articular cartilage, the concentration of lubricating molecules (such as lubricin) is high around the surface, whereas higher concentrations of aggrecans are found in deeper zones"

"fluid-induced stresses on suspended cells ranged from 0.02 Pa to 0.04 Pa during physically limited in vitro experiments. However, the numerical strain analysis indicated the required applied stresses nearing 10.0 Pa to elicit an appreciable strain response depending on the health (and stiffness) of the cell "

"collagen accumulation was less sensitive to lower levels of mechanical stimul (ξ≤0.02) than GAG, requiring greater stimulation to reach homeostatis."

" there is a distinct decrease in matrix molecule accumulation during homeostasis if the static mechanical loading stimulus is very low. static compression significantly inhibits the synthesis of proteoglycans and other protein molecules"

"an increase in growth factors [occurs] with low cytokine levels"

" a higher mechanical stress threshold may dominate the anabolic pathway over catabolic actions"

Wednesday, September 10, 2008

NYRF(chinese herb)

[Regulative effects of Chinese herbs for nourishing yin and removing fire on gene expressions of estrogen receptor alpha, insulin-like growth factor-1 receptor, epithelial growth factor receptor and protein synthesis in epiphyseal growth plate of female pubertal rats].

"To investigate the effect of Chinese herbs for nourishing yin and removing fire (NYRF) on gene expressions of estrogen receptor alpha (ER alpha), insulin-like growth factor-1 receptor (IGF-1R) and epithelial growth factor receptor (EGFR) in the epiphyseal growth plate of the female pubertal rats.
The rats were randomly divided into the control group and the intervened group. Immunohistochemistry and realtime-PCR methods were used to measure the gene expression of ER alpha, IGF-1R and EGFR and their protein synthesis in epiphyseal growth plate.
After being intervened with NYRF, the gene expressions of ER alpha and IGF-1R were down-regulated and their protein synthesis markedly reduced, while those of EGFR were unchanged.
NYRF can modulate the development and maturation of bone by regulating the expressions of ER alpha and IGF-1R in the epiphyseal growth plate."

ERalpha inhibition may result in increased length but IGF1R knockout reduces height.

[Effect of Chinese herbs for nourishing shen-yin and removing xiang-fire on estrogen receptor expression in reproductive organ of rats contaminated with environmental endocrine disruptor].

"To investigate the effect of Chinese herbs for nourishing Shen-yin and removing Xiang-fire (NYRF) on estrogen receptor (ER) expression in uterus and ovary of rats contaminated with nonylphenol (NP) or its bisphenol A (BPA) mixture, for exploring the action mechanism of NYRF in antagonizing the estrogen-mimetic activity of environmental endocrine disruptors (EEDs).
EEDs contaminated female SD rats, 3-week old, were divided into two groups, the treated group fed with NYRF and the control group with corn oil during the same period of contaminating for 15 days. The wet weight (WW) and organ coefficient (OC) of uterus in rats, as well as the ER protein and mRNA expressions in rat's uterus and ovary were detected and compared.
As compared with normal range, WW and OC increased significantly in the contaminated rats of the control group, with significantly down-regulated ER protein expression in uterus, and expressions of ER alpha and ER beta gene and protein in ovary (P<0.05). While in the treated group, the above-mentioned abnormalities of various indictors were markedly reversed to a certain extent (P<0.05).
EEDs show estrogenic-mimetic action on productive organs, which could be antagonized by NYRF, resulting in the down-regulated mRNA and protein expressions of ER in reproductive organs, so as to reduce the sensibility of reproductive organs to EEDs, which is probably one of the acting mechanisms of NYRF."

Effect of nourishing “Yin”-removing “Fire” Chinese herbal mixture on hypothalamic kisspeptin expression in female precocious rats

"Both male and female rats show an obvious increase in Kiss-1 mRNA levels that coincides with the onset of puberty. Pharmacological analyses further set the contention that kisspeptin is likely the most potent elicitor of the GnRH/gonadotropic axis known so far"

"[YNRF] decreased the expression levels of GnRH, FSH and LH mRNAs in female adolescent rats"

"The mixture prescription is mainly composed of 10 medicinal plants: 15 g of Rehmannia glutinosa (Sheng di), 9 g each of Scrophularia buergeriana (Xuan shen), Anemarrhena asphodeloides (Zhi mu), Cortex Phellodendri (Huang bai), Paeonia suffruticosa Andr. (Dan pi), Alisma plantago-aquatica L. var. orientala Sam (Ze xie), Prunella vulgaris L. (Xia ku cao), 12 g of Carapax et Plastrum Testudinis (Gui jia), 30 g of Fructus hordei germinate (Mai ya) and 6 g of Gentiana scabra Bge. (Long dan cao)."

Included in Le Shu Yuan Jin Yin Hua tea.

Saturday, September 6, 2008


Gas1 is upregulated by LSJL.

Functions of the growth arrest specific 1 gene in the development of the mouse embryo.

"The growth arrest specific 1 (gas1) gene is highly expressed in quiescent mammalian cells. Overexpression of gas1 in normal could inhibit G(0)/G(1) transition. The spatial-temporal expression patterns for gas1 were established in 8.5- to 14.5-day-old embryos by immunohistochemical staining and in situ hybridization. Gas1 was found heterogeneously expressed in [the] limb. The antiproliferative effects of gas1 on 10.5 and 12.5 day limb cells were investigated by flow cytometry. In 10.5 day limbs cells, gas1 overexpression could not prevent G(0)/G(1) progression.  Gas1 could only induce growth arrest if p53 was also coexpressed {p53 was not coexpressed significantly in LSJL}. In contrast, gas1 overexpression alone was able to induce growth arrest in 12.5 day limb cells. We also examined the cell cycle profile of gas1-expressing and nonexpressing cells by immunochemistry and flow cytometry. For 10.5 day Gas1-expressing limb cells, we did not find these cells preferentially distributed at G0/G1, as compared with Gas1-negative cells. However, in the 12.5 day limb, we did find significantly more Gas1-expressing cells distributed at G0/G1 phase than Gas1-negative cells. Gas1 alone, during the early stages of development, could not inhibit cell growth. This inhibition was only established when the embryo grew older. We have overexpressed gas1 in subconfluent embryonic limb cells to determine the ability of gas1 to cross-talk with various response elements of important transduction pathways. Specifically, we have examined the interaction of gas1 with Ap-1, NFkappaB, and c-myc responsive elements tagged with a SEAP reporter. In 10.5 day limb cells, gas1 overexpression had little effect on Ap-1, NFkappaB, and c-myc activities. In contrast, gas1 overexpression in 12.5 day limb cells enhanced AP-1 response while it inhibited NFkappaB and c-myc activities. These responses were directly associated with the ability of gas1 to induce growth arrest in embryonic limb cells. In the 12.5 day hindlimb, gas1 was found strongly expressed in the interdigital tissues. We overexpressed gas1 in these tissues and discovered that it promoted interdigital cell death. Our in situ hybridization studies of limb sections and micromass cultures revealed that, during the early stages of chondrogenesis, only cells surrounding the chondrogenic condensations expressed gas1. The gene was only expressed by chondrocytes after the cartilage started to differentiate. To understand the function of gas1 in chondrogenesis, we overexpressed the gene in limb micromass cultures. It was found that cells overexpressing gas1/GFP could not participate in cartilage formation, unlike cells that just express the GFP reporter. We speculated that the reason gas1 was expressed outside the chondrogenic nodules was to restrict cells from being recruited into the nodules and thereby defining the boundary between chondrogenic and nonchondrogenic forming regions."

Several cells were sampled in LSJL so it's likely that the non-chondrogenic cells were the ones expressing Gas1.

"product of gas1 is located in the cell membrane and that it contains a large extracellular domain. This domain contains an arginine– glycine–aspartic acid sequence (RGD), which suggests that it could interact with integrin-type molecules. Therefore, gas1 could be involved in cell– cell and cell–matrix interactions. It is possible that cells expressing gas1 are more anchored to the extracellular matrix and thereby inhibited from participating in chondrogenesis."

Sunday, February 24, 2008


LSJL upregulates MMP3.

Overgrowth with increased proliferation of fibroblast and matrix metalloproteinase activity related to reduced TIMP1: a newly recognized syndrome?

"We report on a child with prenatal onset of overgrowth associated with thick, excessive wrinkled skin and other abnormalities including cleft palate, Chiari malformation and polymicrogyria. His clinical features do not resemble any of the known reported overgrowth syndromes. Genetic evaluations, including karyotype, oligoarray, methylation-sensitive multiplex ligation-dependent probe amplification (MLPA) for 11p11.2 region, CDKN1C sequencing, GPC3 sequencing and dosage analysis, and HRAS sequencing, have been un-revealing. Immunohistochemistry done on the patient's cultured skin fibroblasts showed normally assembled elastic fibers and normal pattern of chondroitin sulfate deposition with defective deposition of Collagen I fibers. In addition, there were high levels of immuno-detectable metalloproteinase 3 (MMP3) and undetectable tissue inhibitor of metalloproteinase 1 (TIMP1){up}. The defective collagen deposition in the fibroblast culture could be reversed by the broad spectrum MMP inhibitor, doxycycline. fibroblasts of this patient have an increased rate of cellular proliferation. We propose that this is a previously unrecognized overgrowth syndrome associated with increased cellular proliferation and defective collagen I deposition due to an imbalance between MMP and TIMP in fibroblasts."

"His birth weight was 4,640 g, length 56.5 cm, and head circumference 38.5 cm (all above 97th centile)."

"patient fibroblasts proliferated faster than normal control counterparts and often formed clusters containing less chondroitin sulfate deposited between individual cells."

It's unclear whether this syndrome induces overgrowth after the neonatal stage.

Since LSJL upregulates MMP3 and TIMP1 perhaps a TIMP1 inhibitor would produce similar overgrowth in the bone.

Thursday, February 14, 2008

The increase in p-Akt in LSJL

It has been found LSJL increases Akt phosphorylation.  Now where does that increase occur?  In the stem, cells, bone or cartilage.  Ideally, we'd want it to be to increased in the stem cells which would be indicative of ectopic chondrossification.

Akt phosphorylation in human chondrocytes is regulated by p53R2 in response to mechanical stress.

"The p53 tumor-suppressor protein p53R2 is activated in response to various stressors that act on cell signaling. When DNA is damaged, phosphorylation of p53 at its Ser 15 residue induces p53R2 production. The role of p53R2 in chondrocytes remains poorly understood. In this study, we evaluated in chondrocytes, p53R2 expression and its regulation in response to mechanical stress. Furthermore, we investigated the function of p53R2 in relation to mechanotransduction.
Osteoarthritis (OA) cartilage obtained from total knee replacements and normal cartilage obtained from femoral neck fractures was used to measure p53R2 expression. The OA chondrocytes were subjected to a high magnitude of cyclical tensile strain by using an FX-2000 Flexercell system. Next, sulfated glycosaminoglycan (sGAG) production was quantified in these cells. Protein expression of p53R2, and phosphorylation of Akt, p38MAPK, ERK1/2, and JNK was also detected. Akt phosphorylation was detected after transfecting the cells with p53R2-specific small interfering RNA (siRNA).
Expression of p53R2 was significantly increased in OA chondrocytes and in chondrocytes after applying 5% tensile strain to the cells. However, Akt phosphorylation was down-regulated in OA chondrocytes after the strain, and was up-regulated after transfection of p53R2. sGAG protein as well as collagen type II and aggrecan mRNA{both up in LSJL} was increased following transfection of p53R2-specific siRNA after 5% tensile strain.
p53R2 could regulate matrix synthesis via Akt phosphorylation during chondrocyte mechanotransduction."

"The ribonucleotide reductase (RR) small subunit 2 homolog p53R2 is directly regulated by p53 during the supply of nucleotides for the repair of damaged DNA "

"p53R2 interacts with MEK2, the extracellular signal-regulated kinase (ERK) kinase 2-mitogen-activated protein kinase (MAPK) 2, and the molecule immediately upstream of ERK in the Ras–Raf–MAPK signaling cascade. p53R2 negatively modulates serum-induced MEK–ERK activity"