Growth Cartilage injury gene expression versus LSJL gene expression

Microarray expression analysis of genes and pathways involved in growth plate cartilage injury responses and bony repair.

"In a rat growth plate injury model, tissue from the injury site was collected across the time-course of bone bridge formation. Four major functional groupings of differentially expressed genes with known roles in skeletal development were identified across the time-course of bone bridge formation, including Wnt signalling (SFRP1, SFRP4, β-catenin, Csnk2a1, Tcf7, Lef1, Fzd1, Fzd2{up}, Wisp1 and Cpz), BMP signalling (BMP-2{up in LSJL}, BMP-6, BMP-7, Chrd, Chrdl2 and Id1), osteoblast differentiation (BMP-2, BMP-6, Chrd, Hgn, Spp1, Axin2, β-catenin, Bglap2) and skeletal development (Chrd, Mmp9, BMP-1, BMP-6, Spp1{up in LSJL as Opn}, Fgfr1{up} and Traf6)."

"An undesirable outcome to growth plate fracture (particularly a Salter–Harris type III or IV fracture) is the bony repair of the injured cartilage at the fractured area."

"studies using a drill-hole injury model in the tibial growth plate of rats have identified sequential inflammatory (days 1–4), fibrogenic (days 3–10), osteogenic (days 7–14) and bone maturation responses (days 14–60) involved in the bony repair of the injured growth plate"

"Twenty-four, 6-week-old male Sprague–Dawley rats underwent experimental drill-hole growth plate injury in the proximal tibia of both hind legs. Under anaesthesia, the growth plate was made accessible after creating a cortical window in the metaphysis using a 2-mm wide dental bur. A central disruption of the growth plate was then made after the bur was passed, via the cortical window, through the entire width of the growth plate, into the epiphyseal region. Six rats were sacrificed by a carbon dioxide overdose 1, 4, 8 and 14 days post injury. "

"genes encoding collagens during the early stages of bone bridge formation (day 4 versus day 8) which were down-regulated [included] Col9a1{up} (− 43.67), Col2a1{up} (− 18.28) and Col9a3{up} (− 8.00)"

"Genes up-regulated at this time with known roles in bone formation included Ltbp2 (9.91), Bglap2 (8.00) and Wisp2{up} (7.51)"

"Clqtnf3 {up} was down-regulated (− 54.05) at day 8 versus day 14"

Genes downregulated on day 4 versus day 8 also downregulated in LSJL:

Ccdc49

Genes upregulated on day 4 versus day 14 also upregulated in LSJL:

THBS4

Cma1

Genes downregulated on day 4 versus day 14 also downregulated in LSJL:

c1qtnf3{up}

Slc38a4{up}

Il1rl1{up}

Bcl11b

Sct

Genes downregulated on day 8 versus day 14 also downregulated in LSJL:

C1qtnf3{up}

Slc38a4{up}

Masp1{up}

Aebp1{up}

"(E) Before dissection of the injury site, with red line denoting the pre-determined area to be excised and collected. (F) Injury site after laser capture microdissection. Dashed lines (green) have traced the uninjured adjacent growth plate."

Injury responses and repair mechanisms of the injured growth plate.

"Following an injury, inflammatory, fibrogenic, osteogenic and bone-bridge maturation repair phases have been observed on days 1-3, 3-7, 7-14 and 10 onwards, respectively. Important roles of several growth factors and cytokines (such as PDGF-BB, FGF-2{up}, TNF-alpha? and IL-1beta) have been highlighted, regulating different phases of growth plate injury repair. While intramembranous ossification is the major mechanism responsible for the bony repair, endochondral ossification, to a lesser extent, also plays a role."

"the gene expression of rat neutrophil chemokine CINC-1 (similar to human interleukin-8) significantly increased during the peak of the inflammatory phase (day 1)"

"p38 mitogen activated protein kinase (MAPK) increased in activation at the injured growth plate"<-lsjl activates p38 MAPK.

"TNF-alpha [has] a strong chemotaxis role for mesenchymal stem cell migration during wound repair"<-TNFa gets stem cells to the wound site.

"neutrophil[involved in inflammatory stage] depletion decreased the expression of chondrogenesis-related genes such as Sox-9 and collagen -2"

"neutrophils [helps initiate] the growth plate injury response and may enhance chondrogenic differentiation"

"Injury-induced inflammatory response[by Cox2 and iNos] in general at the growth plate injury site is necessary for enhancing the chondrogenic differentiation of mesenchymal cells. "<-LSJL upregulates Cox2 as Ptgs2 by 7.63 fold.

"During the influx of fibrogenic cells in both injured growth plate and bone, mRNA levels of growth factors FGF-2 and PDGF-BB [were] significantly upregulated, indicating the possible involvement of both growth factors in regulating this mesenchymal reaction phase in both bone or growth plate injury repair "<-LSJL upregulates FGF2.

"During bone fracture repair, PDGFs have been found to be essential for triggering the initial events leading to the migration and proliferation of fibroblasts and osteoblasts "

Microarray analysis of irradiated growth plate zones following laser microdissection shows later importance of differentially expressed genes during radiorecovery.

"5-week-old male Sprague-Dawley rats underwent fractionated irradiation to the right tibiae over 5 days totaling 17.5 Gy and were then killed at 7, 11, and 16 days following the first radiotherapy fraction. The growth plates were collected from the proximal tibiae bilaterally and subsequently underwent laser microdissection to separate reserve, perichondral, proliferative, and hypertrophic zones.

The reserve zone showed the greatest number of differentially expressed genes and enriched pathways: 259 and 134, respectively. Differentially expressed genes included: Timp3, Gpx1, Gas6, Notch2, VEGF, and HIF-1. Enriched pathways included the developmental processes of regeneration, antiapoptosis, developmental growth, tissue regeneration, mesenchymal cell proliferation, negative regulation of immune response, and determination of symmetry. The reserve zone late upregulation of genes was validated using real-time PCR for Mgp, Gas6, and Eef1a1{down}.

A significant difference in late upregulated genes between growth plate zones exists. The reserve zone shows the greatest change, containing a 10-fold increase in the total number of genes differentially expressed between days 7 and 16. These findings suggest that reserve zone chondrocytes may play a later role in growth plate recovery response following irradiation."

Genes upregulated at day 16(when radiation recovery occurred) that are also upregulated by LSJL:

Col3a1

MMP2

Col12a1

Agc1

Lox

Col10a1

Bgn

Col6a3

Eef1a1{down}

Kras{down}

Col16a1

23.7% of genes upregulated on day 16 were also expressed by LSJL.  The genes above are associated with the reserve zone.

Proliferative Zone:

Bsp

Col5a2

Col1a1

Lum

Kifc1{down}

ADAMTS1

56%.

Hypertrophic Zone:

Rsad2{down}

Ccng2{down}

"blocking KDR{up by LSJL} resulted in the inhibition of VEGF, supporting the concept that the migration and invasion of endothelial cells are regulated by VEGF produced by chondrocytes via a paracrine angiogenic loop"

"Ednra has been shown to bind to endothelin 1{up in LSJL} and induce an increase in intracellular calcium"

Articular cartilage injury:

FGF2 drives changes in gene expression following cartilage injury in vitro and in vivo.

"One important injury-activated pathway involves the release of pericellular fibroblast growth factor-2 (FGF2) from the articular cartilage. Using a novel model of murine cartilage injury, and joints from surgically destabilized mice we examined the extent to which FGF2 contributes to the cellular gene response to injury. Femoral epiphyses from 5 week old wild type mice were avulsed into serum-free medium. Explant lysates were western blotted for phospho-ERK, phospho-p38 and phospho-JNK or were fixed for immunohistochemistry for nuclear translocation of p65 (indicative of NFκB activation). RNA was extracted from injured explants, rested explants stimulated with recombinant FGF2 or FGF18, or whole joints of either wild type or Fgf2-/- mice. RT-PCR was performed for a number of inflammatory response genes previously identified from a microarray analysis. Murine cartilage avulsion injury resulted in the rapid activation of the three mitogen activated kinase pathways as well as NFκB. Almost all genes identified in murine joints following surgical destabilization were also regulated in cartilage explants upon injury. Many of these genes, including activin A, TNF-stimulated gene 6, MMP19, tissue inhibitor of metalloproteinase 1 and podoplanin were significantly FGF2 dependent following injury to cartilage in vitro and to joint tissues in vivo.  FGF2-dependent gene expression occurs in vitro and in vivo in response to cartilage/joint injury."

"inhibin βA (the subunit of activin A), TNF-stimulated gene 6 (Tsg6) and arginase 1, [are inducted] over the first 4h following cartilage injury."