LSJL upregulates these mitochondrial related genes:
Me3
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)
Ears2
Cul7(mitochondria inner membrane)
Mrpl38(mitochondrial ribosome)
Mrpl11(mitochondrial ribosome)
Mtus1
Prodh2(mitochondrial inner membrane)
Uqcrq(mitochondrial electron transport chain)
Grpel1
Immt(mitochondrial inner membrane)
Cox6a1(mitochondrial membrane)
Slc25a30(mitochondrial inner membrane)
Mrpl40
Rhot1(mitochondrial transport along microtubule)
Ucp2(mitochondrial transport)
Mrps14(mitochondrial small ribosomal subunit)
Mrpl4
MTERF(involved in terminating mitochondrial transcription)
Fxc1(protein mitochondrial targeting)
Gimap3(mitochondrial outer membrane)
Mrpl30(mitochondrial ribosome)
Gls(mitochondrial inner membrane)
Mrps17
mt-Atp6
Tfb1m
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.
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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.
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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."